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Sample records for gene arg kinase

  1. Loss of dendrite stabilization by the Abl-related gene (Arg) kinase regulates behavioral flexibility and sensitivity to cocaine.

    PubMed

    Gourley, Shannon L; Koleske, Anthony J; Taylor, Jane R

    2009-09-29

    Adolescence is characterized by increased vulnerability to developing neuropsychiatric disorders and involves a period of prefrontal cortical dendritic refinement and synaptic pruning that culminates in cytoskeletal stabilization in adulthood. The Abl-related gene (Arg) acts through p190RhoGAP to inhibit the RhoA GTPase and stabilize cortical dendritic arbors beginning in adolescence. Cortical axons, dendrites, and synapses develop normally in Arg-deficient (arg(-/-)) mice, but adult dendrites destabilize and regress; thus, arg(-/-) mice present a model of adolescent-onset dendritic simplification. We show that arg(-/-) mice are impaired in a reversal task and that deficits are grossly exacerbated by low-dose cocaine administration. Although ventral prefrontal dopamine D2 receptor levels predict "perseverative" error counts in wild-type mice, no such relationship is found in arg(-/-) mice. Moreover, arg(-/-) mice are insensitive to the disruptive effects of the D2/D3 antagonist haloperidol in reversal but show normal sensitivity to its locomotor-depressant actions. Arg deficiency and orbitofrontal cortical Arg inhibition via STI-571 infusion also enhance the psychomotor stimulant actions of cocaine. These findings provide evidence that stabilization of dendritic structure beginning in adolescence is critical for the development of adaptive and flexible behavior after cocaine exposure.

  2. Regulation of actin polymerization and adhesion-dependent cell edge protrusion by the Abl-related gene (Arg) tyrosine kinase and N-WASp.

    PubMed

    Miller, Matthew M; Lapetina, Stefanie; MacGrath, Stacey M; Sfakianos, Mindan K; Pollard, Thomas D; Koleske, Anthony J

    2010-03-16

    Extracellular cues stimulate the Abl family nonreceptor tyrosine kinase Arg to promote actin-based cell edge protrusions. Several Arg-interacting proteins are potential links to the actin cytoskeleton, but exactly how Arg stimulates actin polymerization and cellular protrusion has not yet been fully elucidated. We used affinity purification to identify N-WASp as a novel binding partner of Arg. N-WASp activates the Arp2/3 complex and is an effector of Abl. We find that the Arg SH3 domain binds directly to N-WASp. Arg phosphorylates N-WASp on Y256, modestly increasing the affinity of Arg for N-WASp, an interaction that does not require the Arg SH2 domain. The Arg SH3 domain stimulates N-WASp-dependent actin polymerization in vitro, and Arg phosphorylation of N-WASp weakly stimulates this effect. Arg and N-WASp colocalize to adhesion-dependent cell edge protrusions in vivo. The cell edge protrusion defects of arg-/- fibroblasts can be complemented by re-expression of an Arg-yellow fluorescent protein (YFP) fusion, but not by an N-WASp binding-deficient Arg SH3 domain point mutant. These results suggest that Arg promotes actin-based protrusions in response to extracellular stimuli through phosphorylation of and physical interactions with N-WASp.

  3. Structure of the ABL2/ARG kinase in complex with dasatinib.

    PubMed

    Ha, Byung Hak; Simpson, Mark Adam; Koleske, Anthony J; Boggon, Titus J

    2015-04-01

    ABL2/ARG (ABL-related gene) belongs to the ABL (Abelson tyrosine-protein kinase) family of tyrosine kinases. ARG plays important roles in cell morphogenesis, motility, growth and survival, and many of these biological roles overlap with the cellular functions of the ABL kinase. Chronic myeloid leukemia (CML) is associated with constitutive ABL kinase activation resulting from fusion between parts of the breakpoint cluster region (BCR) and ABL1 genes. Similarly, fusion of the ETV6 (Tel) and ARG genes drives some forms of T-cell acute lymphoblastic leukemia (T-ALL) and acute myeloid leukemia (AML). Dasatinib is a tyrosine kinase inhibitor used for the treatment of CML by inhibiting ABL, and while it also inhibits ARG, there is currently no structure of ARG in complex with dasatinib. Here, the co-crystal structure of the mouse ARG catalytic domain with dasatinib at 2.5 Å resolution is reported. Dasatinib-bound ARG is found in the DFG-in conformation although it is nonphosphorylated on the activation-loop tyrosine. In this structure the glycine-rich P-loop is found in a relatively open conformation compared with other known ABL family-inhibitor complex structures.

  4. Direct interactions with the integrin β1 cytoplasmic tail activate the Abl2/Arg kinase.

    PubMed

    Simpson, Mark A; Bradley, William D; Harburger, David; Parsons, Maddy; Calderwood, David A; Koleske, Anthony J

    2015-03-27

    Integrins are heterodimeric α/β extracellular matrix adhesion receptors that couple physically to the actin cytoskeleton and regulate kinase signaling pathways to control cytoskeletal remodeling and adhesion complex formation and disassembly. β1 integrins signal through the Abl2/Arg (Abl-related gene) nonreceptor tyrosine kinase to control fibroblast cell motility, neuronal dendrite morphogenesis and stability, and cancer cell invasiveness, but the molecular mechanisms by which integrin β1 activates Arg are unknown. We report here that the Arg kinase domain interacts directly with a lysine-rich membrane-proximal segment in the integrin β1 cytoplasmic tail, that Arg phosphorylates the membrane-proximal Tyr-783 in the β1 tail, and that the Arg Src homology domain then engages this phosphorylated region in the tail. We show that these interactions mediate direct binding between integrin β1 and Arg in vitro and in cells and activate Arg kinase activity. These findings provide a model for understanding how β1-containing integrins interact with and activate Abl family kinases.

  5. Arg kinase regulates prefrontal dendritic spine refinement and cocaine-induced plasticity.

    PubMed

    Gourley, Shannon L; Olevska, Anastasia; Warren, M Sloan; Taylor, Jane R; Koleske, Anthony J

    2012-02-15

    Adolescence is characterized by vulnerability to the development of neuropsychiatric disorders including drug addiction, as well as prefrontal cortical refinement that culminates in structural stability in adulthood. Neuronal refinement and stabilization are hypothesized to confer resilience to poor decision making and addictive-like behaviors, although intracellular mechanisms are largely unknown. We characterized layer V prefrontal dendritic spine development and refinement in adolescent wild-type mice and mice lacking the cytoskeletal regulatory protein Abl-related gene (Arg) kinase. Relative to hippocampal CA1 pyramidal neurons, which exhibited a nearly linear increase in spine density up to postnatal day 60 (P60), wild-type prefrontal spine density peaked at P31, and then declined by 18% by P56-P60. In contrast, dendritic spines in mice lacking Arg destabilized by P31, leading to a net loss in both structures. Destabilization corresponded temporally to the emergence of exaggerated psychomotor sensitivity to cocaine. Moreover, cocaine reduced dendritic spine density in wild-type orbitofrontal cortex and enlarged remaining spine heads, but arg(-/-) spines were unresponsive. Local application of Arg or actin polymerization inhibitors exaggerated cocaine sensitization, as did reduced gene dosage of the Arg substrate, p190RhoGAP. Genetic and pharmacological Arg inhibition also retarded instrumental reversal learning and potentiated responding for reward-related cues, providing evidence that Arg regulates both psychomotor sensitization and decision-making processes implicated in addiction. These findings also indicate that structural refinement in the adolescent orbitofrontal cortex mitigates psychostimulant sensitivity and support the emerging perspective that the structural response to cocaine may, at any age, have behaviorally protective consequences.

  6. The cytoplasmic tyrosine kinase Arg regulates gastrulation via control of actin organization.

    PubMed

    Bonacci, Gustavo; Fletcher, Jason; Devani, Madhav; Dwivedi, Harsh; Keller, Ray; Chang, Chenbei

    2012-04-01

    Coordinated cell movements are crucial for vertebrate gastrulation and are controlled by multiple signals. Although many factors are shown to mediate non-canonical Wnt pathways to regulate cell polarity and intercalation during gastrulation, signaling molecules acting in other pathways are less investigated and the connections between various signals and cytoskeleton are not well understood. In this study, we show that the cytoplasmic tyrosine kinase Arg modulates gastrulation movements through control of actin remodeling. Arg is expressed in the dorsal mesoderm at the onset of gastrulation, and both gain- and loss-of-function of Arg disrupted axial development in Xenopus embryos. Arg controlled migration of anterior mesendoderm, influenced cell decision on individual versus collective migration, and modulated spreading and protrusive activities of anterior mesendodermal cells. Arg also regulated convergent extension of the trunk mesoderm by influencing cell intercalation behaviors. Arg modulated actin organization to control dynamic F-actin distribution at the cell-cell contact or in membrane protrusions. The functions of Arg required an intact tyrosine kinase domain but not the actin-binding motifs in its carboxyl terminus. Arg acted downstream of receptor tyrosine kinases to regulate phosphorylation of endogenous CrkII and paxillin, adaptor proteins involved in activation of Rho family GTPases and actin reorganization. Our data demonstrate that Arg is a crucial cytoplasmic signaling molecule that controls dynamic actin remodeling and mesodermal cell behaviors during Xenopus gastrulation.

  7. Arg kinase signaling in dendrite and synapse stabilization pathways: memory, cocaine sensitivity, and stress.

    PubMed

    Kerrisk, Meghan E; Koleske, Anthony J

    2013-11-01

    The Abl2/Arg nonreceptor tyrosine kinase is enriched in dendritic spines where it is essential for maintaining dendrite and synapse stability in the postnatal mouse brain. Arg is activated downstream of integrin α3β1 receptors and it regulates the neuronal actin cytoskeleton by directly binding F-actin and via phosphorylation of substrates including p190RhoGAP and cortactin. Neurons in mice lacking Arg or integrin α3β1 develop normally through postnatal day 21 (P21), however by P42 mice exhibit major reductions in dendrite arbor size and complexity, and lose dendritic spines and synapses. As a result, mice with loss of Arg and Arg-dependent signaling pathways have impairments in memory tasks, heightened sensitivity to cocaine, and vulnerability to corticosteroid-induced neuronal remodeling. Therefore, understanding the molecular mechanisms of Arg regulation may lead to therapeutic approaches to treat human psychiatric and neurodegenerative diseases in which neuronal structure is destabilized.

  8. Assessment of the Role of MAP Kinase in Mediating Activity-Dependent Transcriptional Activation of the Immediate Early Gene "Arc/Arg3.1" in the Dentate Gyrus in Vivo

    ERIC Educational Resources Information Center

    Chotiner, Jennifer K.; Nielson, Jessica; Farris, Shannon; Lewandowski, Gail; Huang, Fen; Banos, Karla; de Leon, Ray; Steward, Oswald

    2010-01-01

    Different physiological and behavioral events activate transcription of "Arc/Arg3.1" in neurons in vivo, but the signal transduction pathways that mediate induction in particular situations remain to be defined. Here, we explore the relationships between induction of "Arc/Arg3.1" transcription in dentate granule cells in vivo and activation of…

  9. Constitutively active ABL family kinases, TEL/ABL and TEL/ARG, harbor distinct leukemogenic activities in vivo.

    PubMed

    Yokota, A; Hirai, H; Shoji, T; Maekawa, T; Okuda, K

    2017-04-07

    ABL (ABL1) and ARG (ABL2) are highly homologous to each other in overall domain structure and amino acid sequence, with the exception of their C-termini. As with ABL, translocations that fuse ARG to ETV6/TEL have been identified in patients with leukemia. To assess the in vivo leukemogenic activity of constitutively active ABL and ARG, we generated a bone marrow (BM) transplantation model using the chimeric forms TEL/ABL and TEL/ARG, which have comparable kinase activities. TEL/ABL rapidly induced fatal myeloid leukemia in recipient mice, whereas recipients of TEL/ARG-transduced cells did not develop myeloid leukemia; instead, they succumbed to a long-latency infiltrative mastocytosis that could be adoptively transferred to secondary recipients. Swapping of the C-termini of ABL and ARG altered disease latency and phenotypes. In a detailed in vitro study, TEL/ARG strongly promoted mast cell differentiation in response to SCF or IL-3, whereas TEL/ABL preferentially induced myeloid differentiation of hematopoietic stem/progenitor cells. These results indicate that ABL and ARG kinase activate distinct differentiation pathways to induce specific diseases in vivo, i.e., myeloid leukemia and mastocytosis, respectively. Further elucidation of the differences in their properties should provide important insight into the pathogenic mechanisms of oncogenes of the ABL kinase family.Leukemia accepted article preview online, 07 April 2017. doi:10.1038/leu.2017.114.

  10. One isoform of Arg/Abl2 tyrosine kinase is nuclear and the other seven cytosolic isoforms differently modulate cell morphology, motility and the cytoskeleton

    SciTech Connect

    Bianchi, Cristina; Torsello, Barbara; Di Stefano, Vitalba; Zipeto, Maria A.; Facchetti, Rita; Bombelli, Silvia; Perego, Roberto A.

    2013-08-01

    The non-receptor tyrosine kinase Abelson related gene (Arg/Abl2) regulates cell migration and morphogenesis by modulating the cytoskeleton. Arg promotes actin-based cell protrusions and spreading, and inhibits cell migration by attenuating stress fiber formation and contractility via activation of the RhoA inhibitor, p190RhoGAP, and by regulating focal adhesion dynamics also via CrkII phosphorylation. Eight full-length Arg isoforms with different N- and C-termini are endogenously expressed in human cells. In this paper, the eight Arg isoforms, subcloned in the pFLAG-CMV2 vector, were transfected in COS-7 cells in order to study their subcellular distribution and role in cell morphology, migration and cytoskeletal modulation. The transfected 1BSCTS Arg isoform has a nuclear distribution and phosphorylates CrkII in the nucleus, whilst the other isoforms are detected in the cytoplasm. The 1BLCTL, 1BSCTL, 1ASCTS isoforms were able to significantly decrease stress fibers, induce cell shrinkage and filopodia-like protrusions with a significant increase in p190RhoGAP phosphorylation. In contrast, 1ALCTL, 1ALCTS, 1ASCTL and 1BLCTS isoforms do not significantly decrease stress fibers and induce the formation of retraction tail-like protrusions. The 1BLCTL and 1ALCTL isoforms have different effects on cell migration and focal adhesions. All these data may open new perspectives to study the mechanisms of cell invasiveness. -Highlights: • Each of the eight Arg isoforms was transfected in COS-7 cells. • Only the 1BSCTS Arg isoform has a nuclear distribution in transfected cells. • The cytoplasmic isoforms and F-actin colocalize cortically and in cell protrusions. • Arg isoforms differently phosphorylate p190RhoGAP and CrkII. • Arg isoforms differently modulate stress fibers, cell protrusions and motility.

  11. Arg tyrosine kinase modulates TGF-β1 production in human renal tubular cells under high-glucose conditions.

    PubMed

    Torsello, Barbara; Bianchi, Cristina; Meregalli, Chiara; Di Stefano, Vitalba; Invernizzi, Lara; De Marco, Sofia; Bovo, Giorgio; Brivio, Rinaldo; Strada, Guido; Bombelli, Silvia; Perego, Roberto A

    2016-08-01

    Renal tubular cells are involved in the tubular interstitial fibrosis observed in diabetic nephropathy. It is debated whether epithelial-mesenchymal transition (EMT) affects tubular cells, which under high-glucose conditions overproduce transforming growth factor-β (TGF-β), a fibrogenic cytokine involved in interstitial fibrosis development. Our study investigated the involvement of non-receptor tyrosine kinase Arg (also called Abl2) in TGF-β production. Human primary tubular cell cultures exposed to high-glucose conditions were used. These cells showed an elongated morphology, stress fibers and vimentin increment but maintained most of the epithelial marker expression and distribution. In these cells exposed to high glucose, which overexpressed and secreted active TGF-β1, Arg protein and activity was downregulated. A further TGF-β1 increase was induced by Arg silencing with siRNA, as with the Arg tyrosine kinase inhibitor Imatinib. In the cells exposed to high glucose, reactive oxygen species (ROS)-dependent Arg kinase downregulation induced both RhoA activation, through p190RhoGAPA (also known as ARHGAP35) modulation, and proteasome activity inhibition. These data evidence a new specific involvement of Arg kinase into the regulation of TGF-β1 expression in tubular cells under high-glucose conditions and provide cues for new translational approaches in diabetic nephropathy.

  12. One isoform of Arg/Abl2 tyrosine kinase is nuclear and the other seven cytosolic isoforms differently modulate cell morphology, motility and the cytoskeleton.

    PubMed

    Bianchi, Cristina; Torsello, Barbara; Di Stefano, Vitalba; Zipeto, Maria A; Facchetti, Rita; Bombelli, Silvia; Perego, Roberto A

    2013-08-01

    The non-receptor tyrosine kinase Abelson related gene (Arg/Abl2) regulates cell migration and morphogenesis by modulating the cytoskeleton. Arg promotes actin-based cell protrusions and spreading, and inhibits cell migration by attenuating stress fiber formation and contractility via activation of the RhoA inhibitor, p190RhoGAP, and by regulating focal adhesion dynamics also via CrkII phosphorylation. Eight full-length Arg isoforms with different N- and C-termini are endogenously expressed in human cells. In this paper, the eight Arg isoforms, subcloned in the pFLAG-CMV2 vector, were transfected in COS-7 cells in order to study their subcellular distribution and role in cell morphology, migration and cytoskeletal modulation. The transfected 1BSCTS Arg isoform has a nuclear distribution and phosphorylates CrkII in the nucleus, whilst the other isoforms are detected in the cytoplasm. The 1BLCTL, 1BSCTL, 1ASCTS isoforms were able to significantly decrease stress fibers, induce cell shrinkage and filopodia-like protrusions with a significant increase in p190RhoGAP phosphorylation. In contrast, 1ALCTL, 1ALCTS, 1ASCTL and 1BLCTS isoforms do not significantly decrease stress fibers and induce the formation of retraction tail-like protrusions. The 1BLCTL and 1ALCTL isoforms have different effects on cell migration and focal adhesions. All these data may open new perspectives to study the mechanisms of cell invasiveness.

  13. Phospholipase C of Cryptococcus neoformans regulates homeostasis and virulence by providing inositol trisphosphate as a substrate for Arg1 kinase.

    PubMed

    Lev, Sophie; Desmarini, Desmarini; Li, Cecilia; Chayakulkeeree, Methee; Traven, Ana; Sorrell, Tania C; Djordjevic, Julianne T

    2013-04-01

    Phospholipase C (PLC) of Cryptococcus neoformans (CnPlc1) is crucial for virulence of this fungal pathogen. To investigate the mechanism of CnPlc1-mediated signaling, we established that phosphatidylinositol 4,5-bisphosphate (PIP(2)) is a major CnPlc1 substrate, which is hydrolyzed to produce inositol trisphosphate (IP(3)). In Saccharomyces cerevisiae, Plc1-derived IP(3) is a substrate for the inositol polyphosphate kinase Arg82, which converts IP(3) to more complex inositol polyphosphates. In this study, we show that in C. neoformans, the enzyme encoded by ARG1 is the major IP(3) kinase, and we further demonstrate that catalytic activity of Arg1 is essential for cellular homeostasis and virulence in the Galleria mellonella infection model. IP(3) content was reduced in the CnΔplc1 mutant and markedly increased in the CnΔarg1 mutant, while PIP(2) was increased in both mutants. The CnΔplc1 and CnΔarg1 mutants shared significant phenotypic similarity, including impaired thermotolerance, compromised cell walls, reduced capsule production and melanization, defective cell separation, and the inability to form mating filaments. In contrast to the S. cerevisiae ARG82 deletion mutant (ScΔarg82) strain, the CnΔarg1 mutant exhibited dramatically enlarged vacuoles indicative of excessive vacuolar fusion. In mammalian cells, PLC-derived IP(3) causes Ca(2+) release and calcineurin activation. Our data show that, unlike mammalian PLCs, CnPlc1 does not contribute significantly to calcineurin activation. Collectively, our findings provide the first evidence that the inositol polyphosphate anabolic pathway is essential for virulence of C. neoformans and further show that production of IP(3) as a precursor for synthesis of more complex inositol polyphosphates is the key biochemical function of CnPlc1.

  14. Phospholipase C of Cryptococcus neoformans Regulates Homeostasis and Virulence by Providing Inositol Trisphosphate as a Substrate for Arg1 Kinase

    PubMed Central

    Lev, Sophie; Desmarini, Desmarini; Li, Cecilia; Chayakulkeeree, Methee; Traven, Ana; Sorrell, Tania C.

    2013-01-01

    Phospholipase C (PLC) of Cryptococcus neoformans (CnPlc1) is crucial for virulence of this fungal pathogen. To investigate the mechanism of CnPlc1-mediated signaling, we established that phosphatidylinositol 4,5-bisphosphate (PIP2) is a major CnPlc1 substrate, which is hydrolyzed to produce inositol trisphosphate (IP3). In Saccharomyces cerevisiae, Plc1-derived IP3 is a substrate for the inositol polyphosphate kinase Arg82, which converts IP3 to more complex inositol polyphosphates. In this study, we show that in C. neoformans, the enzyme encoded by ARG1 is the major IP3 kinase, and we further demonstrate that catalytic activity of Arg1 is essential for cellular homeostasis and virulence in the Galleria mellonella infection model. IP3 content was reduced in the CnΔplc1 mutant and markedly increased in the CnΔarg1 mutant, while PIP2 was increased in both mutants. The CnΔplc1 and CnΔarg1 mutants shared significant phenotypic similarity, including impaired thermotolerance, compromised cell walls, reduced capsule production and melanization, defective cell separation, and the inability to form mating filaments. In contrast to the S. cerevisiae ARG82 deletion mutant (ScΔarg82) strain, the CnΔarg1 mutant exhibited dramatically enlarged vacuoles indicative of excessive vacuolar fusion. In mammalian cells, PLC-derived IP3 causes Ca2+ release and calcineurin activation. Our data show that, unlike mammalian PLCs, CnPlc1 does not contribute significantly to calcineurin activation. Collectively, our findings provide the first evidence that the inositol polyphosphate anabolic pathway is essential for virulence of C. neoformans and further show that production of IP3 as a precursor for synthesis of more complex inositol polyphosphates is the key biochemical function of CnPlc1. PMID:23381992

  15. The Abl and Arg kinases mediate distinct modes of phagocytosis and are required for maximal Leishmania infection.

    PubMed

    Wetzel, Dawn M; McMahon-Pratt, Diane; Koleske, Anthony J

    2012-08-01

    Leishmania, an obligate intracellular parasite, binds several receptors to trigger engulfment by phagocytes, leading to cutaneous or visceral disease. These receptors include complement receptor 3 (CR3), used by promastigotes, and the Fc receptor (FcR), used by amastigotes. The mechanisms mediating uptake are not well understood. Here we show that Abl family kinases mediate both phagocytosis and the uptake of Leishmania amazonensis by macrophages (Ms). Imatinib, an Abl/Arg kinase inhibitor, decreases opsonized polystyrene bead phagocytosis and Leishmania uptake. Interestingly, phagocytosis of IgG-coated beads is decreased in Arg-deficient Ms, while that of C3bi-coated beads is unaffected. Conversely, uptake of C3bi-coated beads is decreased in Abl-deficient Ms, but that of IgG-coated beads is unaffected. Consistent with these results, Abl-deficient Ms are inefficient at C3bi-opsonized promastigote uptake, and Arg-deficient Ms are defective in IgG1-opsonized amastigote uptake. Finally, genetic loss of Abl or Arg reduces infection severity in murine cutaneous leishmaniasis, and imatinib treatment results in smaller lesions with fewer parasites than in controls. Our studies are the first to demonstrate that efficient phagocytosis and maximal Leishmania infection require Abl family kinases. These results highlight Abl family kinase-mediated signaling pathways as potential therapeutic targets for leishmaniasis.

  16. Polycyclic aromatic hydrocarbons (PAHs) enriching antibiotic resistance genes (ARGs) in the soils.

    PubMed

    Chen, Baowei; He, Rong; Yuan, Ke; Chen, Enzhong; Lin, Lan; Chen, Xin; Sha, Sha; Zhong, Jianan; Lin, Li; Yang, Lihua; Yang, Ying; Wang, Xiaowei; Zou, Shichun; Luan, Tiangang

    2017-01-01

    The prevalence of antibiotic resistance genes (ARGs) in modern environment raises an emerging global health concern. In this study, soil samples were collected from three sites in petrochemical plant that represented different pollution levels of polycyclic aromatic hydrocarbons (PAHs). Metagenomic profiling of these soils demonstrated that ARGs in the PAHs-contaminated soils were approximately 15 times more abundant than those in the less-contaminated ones, with Proteobacterial being the preponderant phylum. Resistance profile of ARGs in the PAHs-polluted soils was characterized by the dominance of efflux pump-encoding ARGs associated with aromatic antibiotics (e.g., fluoroquinolones and acriflavine) that accounted for more than 70% of the total ARGs, which was significantly different from representative sources of ARG pollution due to wide use of antibiotics. Most of ARGs enriched in the PAHs-contaminated soils were not carried by plasmids, indicating the low possibilities of them being transferred between bacteria. Significant correlation was observed between the total abundance of ARGs and that of Proteobacteria in the soils. Proteobacteria selected by PAHs led to simultaneously enriching of ARGs carried by them in the soils. Our results suggested that PAHs could serve as one of selective stresses for greatly enriching of ARGs in the human-impacted environment.

  17. Arabidopsis inositol polyphosphate 6-/3-kinase is a nuclear protein that complements a yeast mutant lacking a functional ArgR-Mcm1 transcription complex.

    PubMed

    Xia, Hui-Jun; Brearley, Charles; Elge, Stephan; Kaplan, Boaz; Fromm, Hillel; Mueller-Roeber, Bernd

    2003-02-01

    Inositol 1,4,5-trisphosphate 3-kinase, and more generally inositol polyphosphate kinases (Ipk), play important roles in signal transduction in animal cells; however, their functions in plant cells remain to be elucidated. Here, we report the molecular cloning of a cDNA (AtIpk2beta) from a higher plant, Arabidopsis. Arabidopsis AtIpk2beta is a 33-kD protein that exhibits weak homology ( approximately 25% identical amino acids) with Ipk proteins from animals and yeast and lacks a calmodulin binding site, as revealed by sequence analysis and calmodulin binding assays. However, recombinant AtIpk2beta phosphorylates inositol 1,4,5-trisphosphate to inositol 1,4,5,6-tetrakisphosphate and also converts it to inositol 1,3,4,5,6-pentakisphosphate [Ins(1,3,4,5,6)P(5)]. AtIpk2beta also phosphorylates inositol 1,3,4,5-tetrakisphosphate to Ins(1,3,4,5,6)P(5). Thus, the enzyme is a D3/D6 dual-specificity inositol phosphate kinase. AtIpk2beta complements a yeast ARG82/IPK2 mutant lacking a functional ArgR-Mcm1 transcription complex. This complex is involved in regulating Arg metabolism-related gene expression and requires inositol polyphosphate kinase activity to function. AtIpk2beta was found to be located predominantly in the nucleus of plant cells, as demonstrated by immunolocalization and fusion to green fluorescent protein. RNA gel blot analysis and promoter-beta-glucuronidase reporter gene studies demonstrated AtIpk2beta gene expression in various organs tested. These data suggest a role for AtIpk2beta as a transcriptional control mediator in plants.

  18. ArgR-dependent repression of arginine and histidine transport genes in Escherichia coli K-12.

    PubMed

    Caldara, Marina; Minh, Phu Nguyen Le; Bostoen, Sophie; Massant, Jan; Charlier, Daniel

    2007-10-19

    In Escherichia coli L-arginine is taken up by three periplasmic binding protein-dependent transport systems that are encoded by two genetic loci: the artPIQM-artJ and argT-hisJQMP gene clusters. The transcription of the artJ, artPIQM and hisJQMP genes and operons is repressed by liganded ArgR, whereas argT, encoding the LAO (lysine, arginine, ornithine) periplasmic binding protein, is insensitive to the repressor. Here we characterize the repressible Esigma70 P artJ, P artP and P hisJ promoters and demonstrate that the cognate operators consist of two 18 bp ARG boxes separated by 3 bp. Determination of the energy landscape of the ArgR-operator contacts by missing contact probing and mutant studies indicated that each box of a pair contributes to complex formation in vitro and to the repressibility in vivo, but to a different extent. The organization of the ARG boxes and promoter elements in the control regions of the uptake genes is distinct from that of the arginine biosynthetic genes. The hisJQMP operon is the first member of the E. coli ArgR regulon, directly repressed by liganded ArgR, where none of the core promoter elements overlaps the ARG boxes. Single round in vitro transcription assays and DNase I footprinting experiments indicate that liganded ArgR inhibits P artJ and P artP promoter activity by steric exclusion of the RNA polymerase. In contrast, ArgR-mediated repression of P hisJ by inhibition of RNA polymerase binding appears to occur through topological changes of the promoter region.

  19. The Src kinases Hck, Fgr and Lyn activate Arg to facilitate IgG-mediated phagocytosis and Leishmania infection.

    PubMed

    Wetzel, Dawn M; Rhodes, Emma L; Li, Shaoguang; McMahon-Pratt, Diane; Koleske, Anthony J

    2016-08-15

    Leishmaniasis is a devastating disease that disfigures or kills nearly two million people each year. Establishment and persistence of infection by the obligate intracellular parasite Leishmania requires repeated uptake by macrophages and other phagocytes. Therefore, preventing uptake could be a novel therapeutic strategy for leishmaniasis. Amastigotes, the life cycle stage found in the human host, bind Fc receptors and enter macrophages primarily through immunoglobulin-mediated phagocytosis. However, the host machinery that mediates amastigote uptake is poorly understood. We have previously shown that the Arg (also known as Abl2) non-receptor tyrosine kinase facilitates L. amazonensis amastigote uptake by macrophages. Using small-molecule inhibitors and primary macrophages lacking specific Src family kinases, we now demonstrate that the Hck, Fgr and Lyn kinases are also necessary for amastigote uptake by macrophages. Src-mediated Arg activation is required for efficient uptake. Interestingly, the dual Arg and Src kinase inhibitor bosutinib, which is approved to treat cancer, not only decreases amastigote uptake, but also significantly reduces disease severity and parasite burden in Leishmania-infected mice. Our results suggest that leishmaniasis could potentially be treated with host-cell-active agents such as kinase inhibitors.

  20. ArgR-Regulated Genes Are Derepressed in the Legionella-Containing Vacuole▿ †

    PubMed Central

    Hovel-Miner, Galadriel; Faucher, Sebastien P.; Charpentier, Xavier; Shuman, Howard A.

    2010-01-01

    Legionella pneumophila is an intracellular pathogen that infects protozoa in aquatic environments and when inhaled by susceptible human hosts replicates in alveolar macrophages and can result in the often fatal pneumonia called Legionnaires' disease. The ability of L. pneumophila to replicate within host cells requires the establishment of a specialized compartment that evades normal phagolysosome fusion called the Legionella-containing vacuole (LCV). Elucidation of the biochemical composition of the LCV and the identification of the regulatory signals sensed during intracellular replication are inherently challenging. l-Arginine is a critical nutrient in the metabolism of both prokaryotic and eukaryotic organisms. We showed that the L. pneumophila arginine repressor homolog, ArgR, is required for maximal intracellular growth in the unicellular host Acanthamoeba castellanii. In this study, we present evidence that the concentration of l-arginine in the LCV is sensed by ArgR to produce an intracellular transcriptional response. We characterized the L. pneumophila ArgR regulon by global gene expression analysis, identified genes highly affected by ArgR, showed that ArgR repression is dependent upon the presence of l-arginine, and demonstrated that ArgR-regulated genes are derepressed during intracellular growth. Additional targets of ArgR that may account for the argR mutant's intracellular multiplication defect are discussed. These results suggest that l-arginine availability functions as a regulatory signal during Legionella intracellular growth. PMID:20622069

  1. Arg-Pro-X-Ser/Thr is a Consensus Phosphoacceptor Sequence for the Meiosis-Specific Ime2 Protein Kinase in Saccharomyces cerevisiae†

    PubMed Central

    Moore, Michael; Shin, Marcus; Bruning, Adrian; Schindler, Karen; Vershon, Andrew; Winter, Edward

    2008-01-01

    Ime2 is a meiosis-specific protein kinase in Saccharomyces cerevisiae that is functionally related to cyclin-dependent kinase. Although Ime2 regulates multiple steps in meiosis, only a few of its substrates have been identified. Here we show that Ime2 phosphorylates Sum1, a repressor of meiotic gene transcription, on Thr-306. Ime2 protein kinase assays on Sum1 mutants and synthetic peptides define a consensus motif Arg-Pro-X-Ser/Thr that is required for efficient phosphorylation by Ime2. The carboxyl residue adjacent to the phosphoacceptor (+1 position) also influences the efficiency of Ime2 phosphorylation with alanine being a preferred residue. This information has predictive value in identifying new potential Ime2 targets as shown by the ability of Ime2 to phosphorylate Sgs1 and Gip1 in vitro, and could be important in differentiating mitotic and meiotic regulatory pathways. PMID:17198398

  2. A conserved Glu-Arg salt bridge connects co-evolved motifs that define the eukaryote protein kinase fold

    PubMed Central

    Yang, Jie; Wu, Jian; Steichen, Jon M.; Kornev, Alexandr P.; Deal, Michael S.; Li, Sheng; Sankaran, Banumathi; Woods, Virgil L.; Taylor, Susan S.

    2012-01-01

    Eukaryotic protein kinases (EPK)feature two co-evolved structural segments, the Activation segment which starts with the Asp-Phe-Gly (DFG) and ends with the Ala-Pro-Glu (APE) motifs, and the helical GHI-subdomain that comprises αG-αH-αI helices. Eukaryotic-like kinases have a much shorter Activation segment and lack the GHI-subdomain. They thus lack the conserved salt bridge interaction between the APE Glu and an Arg from the GHI-subdomain, a hallmark signature of EPKs. Although the conservation of this salt bridge in EPKs is well known and its implication in diseases has been illustrated by polymorphism analysis, its function has not been carefully studied. In this work, we use murine cAMP dependent protein kinase (PKA) as the model enzyme (Glu208 and Arg280) to examine the role of these two residues. We showed that Ala replacement of either residue caused a 40–120 fold decrease in catalytic efficiency of the enzyme due to an increase in Km(ATP) and a decrease in kcat. Crystal structures, as well as solution studies, also demonstratethat this ion pair contributes to the hydrophobic network and stability of the enzyme. We show that mutation of either Glu or Arg to Ala renders bothmutant proteins less effective substrates for upstream kinase phosphoinositide dependent kinase 1. We propose that the Glu208-Arg280 pair serves as a center hub of connectivity between these two structurally conserved elements in EPKs. Mutations of either residue disrupt communication not only between the two segments but also within the rest of the molecule leading to altered catalytic activity and enzyme regulation. PMID:22138346

  3. Sequence diversity within the argF, fbp and recA genes of natural isolates of Neisseria meningitidis: interspecies recombination within the argF gene.

    PubMed

    Zhou, J; Spratt, B G

    1992-08-01

    Studies of natural populations of Neisseria meningitidis using multilocus enzyme electrophoresis have shown extensive genetic variation within this species, which, it has been proposed, implies a level of sequence diversity within meningococci that is greater than that normally considered as the criterion for species limits in bacteria. To obtain a direct measure of the sequence diversity among meningococci, we obtained the nucleotide sequences of most of the argF, recA and fbp genes of eight meningococci of widely differing electrophoretic type (from the reference collection of Caugant). Sequence variation between the meningococcal strains ranged from 0-0.6% for fbp, 0-1.3% for argF, and 0-3.3% for recA. These levels of diversity are no greater than those found within Escherichia coli 'housekeeping' genes and suggest that multilocus enzyme electrophoresis may overestimate the extent of nucleotide sequence diversity within meningococci. The average sequence divergence between the Neisseria meningitidis strains and N. gonorrhoeae strain FA19 was 1.0% for fbp and 1.6% for recA. The argF gene, although very uniform among the eight meningococcal isolates, had a striking mosaic structure when compared with the gonococcal argF gene: two regions of the gene differed by greater than 13% in nucleotide sequence between meningococci and gonococci, whereas the rest of the gene differed by less than 1.7%. One of the diverged regions was shown to have been introduced from the argF gene of a commensal Neisseria species that is closely related to Neisseria cinerea. The source of the other region was unclear.

  4. Lack of Arg972 polymorphism in the IRS1 gene in Parakanã Brazilian Indians.

    PubMed

    Bezerra, Rosângela M N; Chadid, Thiago T; Altemani, Claúdia M; Sales, Teresa S I; Menezes, Raimundo; Soares, Manoel C P; Saad, Sara T O; Saad, Mario J A

    2004-02-01

    Several polymorphisms in the insulin receptor substrate-1 (IRS1) gene have been reported in the last years. The most common IRS1 variant, a Gly --> Arg substitution at codon 972 (Arg972 IRS1), is more prevalent among subjects who have features of insulin resistance syndrome associated, or not, with type 2 diabetes in European populations. To determine whether the absence of IRS1 polymorphism is a more general characteristic of Paleo-Indian-derived populations, we examined the Arg972 IRS1 polymorphism in Parakanã Indians and found a lack of this polymorphism in the Parakanã population.

  5. [Effects of Thermophilic Composting on Antibiotic Resistance Genes (ARGs) of Swine Manure Source].

    PubMed

    Zheng, Ning-guo; Huang, Nan; Wang, Wei-wei; Yu, Man; Chen, Xiao-yang; Yao, Yan-lai; Wang, Wei-ping; Hong, Chun-lai

    2016-05-15

    To investigate the effects of thermophilic composting process on antibiotic resistance genes (ARGs) of swine manure source at a field scale, the abundance of four erythromycin resistance genes (ermA, ermB, ermC and ermF), three β-lactam resistance genes (blaTEM, blaCTX and blaSHV) and two quinolone resistance genes (qnrA and qnrS) were quantified by quantitative PCR ( qPCR) during the composting process. The results suggested that the erm genes' copy numbers were significantly higher than those of the bla and qnr genes in the early stage of composting (P < 0.01). The maximum abundance of erm genes was ermB (9.88 x 10⁸ copies · g⁻¹), following by ermF (9.4 x 10⁸ copies · g⁻¹). At the end of the composting process, bla and qnr genes were at low levels, while erm genes were still at high levels. Even through ermF was proliferated comparing with the initial copies. These results indicated that thermophilic composting process could not effectively remove all ARGs. For some ARGs, compost may be a good bioreactor resulting in their proliferation. Application of composting products on farmland may cause transference of ARGs.

  6. Dissemination of antibiotic resistance genes in representative broiler feedlots environments: identification of indicator ARGs and correlations with environmental variables.

    PubMed

    He, Liang-Ying; Liu, You-Sheng; Su, Hao-Chang; Zhao, Jian-Liang; Liu, Shuang-Shuang; Chen, Jun; Liu, Wang-Rong; Ying, Guang-Guo

    2014-11-18

    Livestock operations are known to harbor elevated levels of antibiotic resistance genes (ARGs) that may pose a threat to public health. Broiler feedlots may represent an important source of ARGs in the environment. However, the prevalence and dissemination mechanisms of various types of ARGs in the environment of broiler feedlots have not previously been identified. We examined the occurrence, abundance and variation of ARGs conferring resistance to chloramphenicols, sulfonamides and tetracyclines in the environments of two representative types of broiler feedlots (free range and indoor) by quantitative PCR, and assessed their dissemination mechanisms. The results showed the prevalence of various types of ARGs in the environmental samples of the broiler feedlots including manure/litter, soil, sediment, and water samples, with the first report of five chloramphenicol resistance genes (cmlA, floR, fexA, cfr, and fexB) in broiler feedlots. Overall, chloramphenicol resistance genes and sulfonamides sul genes were more abundant than tetracyclines tet genes. The ARG abundances in the samples from indoor boiler feedlots were generally different to the free range feedlots, suggesting the importance of feeding operations in ARG dissemination. Pearson correlation analysis showed significant correlations between ARGs and mobile genetic element genes (int1 and int2), and between the different classes of ARGs themselves, revealing the roles of horizontal gene transfer and coselection for ARG dissemination in the environment. Further regression analysis revealed that fexA, sul1 and tetW could be reliable indicator genes to surrogate anthropogenic sources of ARGs in boiler feedlots (correlations of fexA, sul1 and tetW to all ARGs: R = 0.95, 0.96 and 0.86, p < 0.01). Meanwhile, significant correlations were also identified between indicator ARGs and their corresponding antibiotics. In addition, some ARGs were significantly correlated with typical metals (e.g., Cu, Zn, and As with

  7. Mutation of the Erwinia amylovora argD gene causes arginine auxotrophy, nonpathogenicity in apples, and reduced virulence in pears.

    PubMed

    Ramos, Laura S; Lehman, Brian L; Peter, Kari A; McNellis, Timothy W

    2014-11-01

    Fire blight is caused by Erwinia amylovora and is the most destructive bacterial disease of apples and pears worldwide. In this study, we found that E. amylovora argD(1000)::Tn5, an argD Tn5 transposon mutant that has the Tn5 transposon inserted after nucleotide 999 in the argD gene-coding region, was an arginine auxotroph that did not cause fire blight in apple and had reduced virulence in immature pear fruits. The E. amylovora argD gene encodes a predicted N-acetylornithine aminotransferase enzyme, which is involved in the production of the amino acid arginine. A plasmid-borne copy of the wild-type argD gene complemented both the nonpathogenic and the arginine auxotrophic phenotypes of the argD(1000)::Tn5 mutant. However, even when mixed with virulent E. amylovora cells and inoculated onto immature apple fruit, the argD(1000)::Tn5 mutant still failed to grow, while the virulent strain grew and caused disease. Furthermore, the pCR2.1-argD complementation plasmid was stably maintained in the argD(1000)::Tn5 mutant growing in host tissues without any antibiotic selection. Therefore, the pCR2.1-argD complementation plasmid could be useful for the expression of genes, markers, and reporters in E. amylovora growing in planta, without concern about losing the plasmid over time. The ArgD protein cannot be considered an E. amylovora virulence factor because the argD(1000)::Tn5 mutant was auxotrophic and had a primary metabolism defect. Nevertheless, these results are informative about the parasitic nature of the fire blight disease interaction, since they indicate that E. amylovora cannot obtain sufficient arginine from apple and pear fruit tissues or from apple vegetative tissues, either at the beginning of the infection process or after the infection has progressed to an advanced state.

  8. Role of Arg228 in the phosphorylation of galactokinase: the mechanism of GHMP kinases by quantum mechanics/molecular mechanics studies.

    PubMed

    Huang, Meilan; Li, Xiaozhou; Zou, Jian-Wei; Timson, David J

    2013-07-16

    GHMP kinases are a group of structurally related small molecule kinases. They have been found in all kingdoms of life and are mostly responsible for catalyzing the ATP-dependent phosphorylation of intermediary metabolites. Although the GHMP kinases are of clinical, pharmaceutical, and biotechnological importance, the mechanism of GHMP kinases is controversial. A catalytic base mechanism was suggested for mevalonate kinase that has a structural feature of the γ-phosphate of ATP close to an aspartate residue; however, for one GHMP family member, homoserine kinase, where the residue acting as general base is absent, a direct phosphorylation mechanism was suggested. Furthermore, it was proposed by some authors that all the GHMP kinases function by a similar mechanism. This controversy in mechanism has limited our ability to exploit these enzymes as drug targets and in biotechnology. Here the phosphorylation reaction mechanism of the human galactokinase, a member of the GHMP kinase family, was investigated using molecular dynamics simulations and density functional theory-based quantum mechanics/molecular mechanics calculations (B3LYP-D/AMBER99). The reaction coordinates were localized by potential energy scan using an adiabatic mapping method. Our results indicate that a highly conserved Glu174 captures Arg105 in the proximity of the α-phosphate of ATP, forming a H-bond network; therefore, the mobility of ATP in the large oxyanion hole is restricted. Arg228 functions to stabilize the negative charge developed at the β,γ-bridging oxygen of the ATP during bond cleavage. The reaction occurs via a direct phosphorylation mechanism, and the Asp186 in the proximity of ATP does not directly participate in the reaction pathway. Because Arg228 is not conserved among GHMP kinases, reagents which form interactions with Arg228, and therefore can interrupt its function in phosphorylation, may be developed into potential selective inhibitors for galactokinase.

  9. Antibiotic resistance genes (ARGs) in duck and fish production ponds with integrated or non-integrated mode.

    PubMed

    Huang, Lu; Xu, Yan-Bin; Xu, Jia-Xin; Ling, Jia-Yin; Chen, Jin-Liang; Zhou, Jia-Le; Zheng, Li; Du, Qing-Ping

    2017-02-01

    Antibiotic resistance genes (ARGs) are emerging micropollutants with environmental persistence. Aquaculture environments are considered as potential reservoirs for ARGs pollution and horizontal gene transfer (HGT). This study analyzed water and sediment from eight culture ponds (integrated culture: duck-fish pond; monoculture: duck pond and fish pond) and a control pond (without any aquaculture activity) in Zhongshan, South China. Seventeen types of ARGs were detected in all ponds, which conferring resistance to four classes of antibiotics including tetracycline (tetA, tetB, tetC, tetE, tetG, tetL, tetA-P, tetM, tetO, tetS, tetW and tetX), AmpC beta-lactamase products (EBC and FOX), sulfonamide (sul1 and sul2) and erythromycin (ermA), with class 1 integron (intI1) as motility gene. The total concentrations of detected ARGs in culture pond water were much higher than control (about 1.6-4.0 times). Integrated culture showed lowest absolute abundance of ∑ARGs in water (3.686 × 10(7) copies mL(-1)) and the highest in sediment (4.574 × 10(8) copies g(-1)). Monoculture ponds showed higher relative abundance of ∑ARGs both in water (fish pond: 0.5149) and sediment (duck pond: 0.4919). As the main contributor to the ARGs abundance and significant correlations with ∑tet, ∑ARGs and intI1 (P < 0.01), tetA was suggested to be a potential indicator for the abundance of tetracycline resistance genes in these classes of aquaculture modes in the Pearl River Delta. This study provides a case for the ARGs abundance in aquaculture and as a reference for the upcoming health risk assessment in aquatic environment.

  10. The Abl and Arg non-receptor tyrosine kinases regulate different zones of stress fiber, focal adhesion, and contractile network localization in spreading fibroblasts.

    PubMed

    Peacock, Justin G; Couch, Brian A; Koleske, Anthony J

    2010-10-01

    Directed cell migration requires precise spatial control of F-actin-based leading edge protrusion, focal adhesion (FA) dynamics, and actomyosin contractility. In spreading fibroblasts, the Abl family kinases, Abl and Arg, primarily localize to the nucleus and cell periphery, respectively. Here we provide evidence that Abl and Arg exert different spatial regulation on cellular contractile and adhesive structures. Loss of Abl function reduces FA, F-actin, and phosphorylated myosin light chain (pMLC) staining at the cell periphery, shifting the distribution of these elements more to the center of the cell than in wild-type (WT) and arg(-/-) cells. Conversely, loss of Arg function shifts the distribution of these contractile and adhesion elements more to the cell periphery relative to WT and abl(-/-) cells. Abl/Arg-dependent phosphorylation of p190RhoGAP (p190) promotes its binding to p120RasGAP (p120) to form a functional RhoA GTPase inhibitory complex, which attenuates RhoA activity and downstream pMLC and FA formation. p120 and p190 colocalize both in the central region and at the cell periphery in WT cells. This p120:p190 colocalization redistributes to a more peripheral distribution in abl(-/-) cells and to a more centralized distribution in arg(-/-) cells, and these altered distributions can be restored to WT patterns via re-expression of Abl or Arg, respectively. Thus, the altered p120:p190 distribution in the mutant cells correlates inversely with the redistribution in adhesions, actin, and pMLC staining in these cells. Our studies suggest that Abl and Arg exert different spatial regulation on actomyosin contractility and focal adhesions within cells.

  11. ARG-ANNOT, a New Bioinformatic Tool To Discover Antibiotic Resistance Genes in Bacterial Genomes

    PubMed Central

    Gupta, Sushim Kumar; Padmanabhan, Babu Roshan; Diene, Seydina M.; Lopez-Rojas, Rafael; Kempf, Marie; Landraud, Luce

    2014-01-01

    ARG-ANNOT (Antibiotic Resistance Gene-ANNOTation) is a new bioinformatic tool that was created to detect existing and putative new antibiotic resistance (AR) genes in bacterial genomes. ARG-ANNOT uses a local BLAST program in Bio-Edit software that allows the user to analyze sequences without a Web interface. All AR genetic determinants were collected from published works and online resources; nucleotide and protein sequences were retrieved from the NCBI GenBank database. After building a database that includes 1,689 antibiotic resistance genes, the software was tested in a blind manner using 100 random sequences selected from the database to verify that the sensitivity and specificity were at 100% even when partial sequences were queried. Notably, BLAST analysis results obtained using the rmtF gene sequence (a new aminoglycoside-modifying enzyme gene sequence that is not included in the database) as a query revealed that the tool was able to link this sequence to short sequences (17 to 40 bp) found in other genes of the rmt family with significant E values. Finally, the analysis of 178 Acinetobacter baumannii and 20 Staphylococcus aureus genomes allowed the detection of a significantly higher number of AR genes than the Resfinder gene analyzer and 11 point mutations in target genes known to be associated with AR. The average time for the analysis of a genome was 3.35 ± 0.13 min. We have created a concise database for BLAST using a Bio-Edit interface that can detect AR genetic determinants in bacterial genomes and can rapidly and easily discover putative new AR genetic determinants. PMID:24145532

  12. Molecular analysis of the Trichosporon cutaneum DSM 70698 argA gene and its use for DNA-mediated transformations.

    PubMed Central

    Reiser, J; Glumoff, V; Ochsner, U A; Fiechter, A

    1994-01-01

    Genomic clones capable of complementing a previously isolated arginine auxotrophic mutant strain of the filamentous yeast Trichosporon cutaneum DSM 70698 have been identified by DNA-mediated transformation, and a complementing 4,082-bp subfragment was sequenced. This analysis revealed an intact gene (arg4) showing a high degree of homology with the Saccharomyces cerevisiae CPA2 gene encoding the large subunit of carbamoyl-phosphate synthetase (CPS-A). The inferred amino acid sequence of the T. cutaneum argA-encoded protein contains 1,168 residues showing 62% identity with the sequence of the S. cerevisiae CPA2 protein, and the comparison of the two sequences uncovered a putative intron sequence of 81 nucleotides close to the 5' end of the coding region of the T. cutaneum argA gene. The presence of this intron was confirmed by nuclease protection studies and by direct DNA sequence analysis of a cDNA fragment which had been obtained by PCR amplification. The T. cutaneum intron shares the general characteristics of introns found in yeasts and filamentous fungi. A major transcript of around 4 kb was found in Northern (RNA) blots. The T. cutaneum argA coding region was expressed in Escherichia coli under the control of the regulatable tac promoter. A roughly 130-kDa protein which was found to cross-react with an anti-rat CPS antibody in Western blots (immunoblots) was observed. Two putative ATP-binding domains were identified, one in the amino-terminal half of the argA-encoded protein and the other in the carboxy-terminal half. These domains are highly conserved among the known CPS-A sequences from S. cerevisiae, E. coli, and the rat. From these results we conclude that the T. cutaneum argA gene encodes the large subunit of CPS. This is the first gene to be identified and analyzed in the T. cutaneum DSM 70698 strain. Images PMID:8188603

  13. Effect of river landscape on the sediment concentrations of antibiotics and corresponding antibiotic resistance genes (ARG).

    PubMed

    Pei, Ruoting; Kim, Sung-Chul; Carlson, Kenneth H; Pruden, Amy

    2006-07-01

    The purpose of this study was to quantify antibiotic resistance genes (ARG) in the sediments of the mixed-landscape Cache La Poudre River, which has previously been studied and shown to have high concentrations of antibiotics related to urban and agricultural activities. River sediments were sampled during two events (high-flow and low-flow) from five sites with varying urban and agricultural impact levels. Polymerase-chain-reaction (PCR) detection assays were conducted for four sulfonamide resistance gene families, using newly designed primers, and five tetracycline resistance gene families, using previously published primers. Sul(I), sul(II), tet(W), and tet(O) gene families were further quantified by real-time quantitative polymerase chain reaction (Q-PCR). Resistance to four classes of antibiotics (tetracyclines, sulfonamides, ionophores, and macrolides) was also investigated using a culture-based approach. The quantities of resistance genes normalized to the 16S gene copy number were significantly different between the sites, with higher resistance gene concentrations at the impacted sites than at the pristine site. Total resistant CFUs were over an order of magnitude lower at the pristine site, but differences were less apparent when normalized to the total CFUs. Six tetracyclines and six sulfonamides were also quantified in the sediments and were found to be highest at sites impacted by urban and agricultural activity, with no antibiotics detected at the pristine sit. To the knowledge of the authors, this study is the first to demonstrate a relationship between urban and agricultural activity and microbial resistance in river sediments using quantitative molecular tools.

  14. Distribution of antibiotic resistance genes (ARGs) in anaerobic digestion and land application of swine wastewater.

    PubMed

    Sui, Qianwen; Zhang, Junya; Chen, Meixue; Tong, Juan; Wang, Rui; Wei, Yuansong

    2016-06-01

    Swine farm and the adjacent farmland are hot spots of ARGs. However, few studies have investigated the on-site occurrence of ARGs distributed in the process of anaerobic digestion (AD) followed by land application of swine wastewater. Two typical swine farms, in southern and northern China respectively, with AD along with land application were explored on ARG distributions. ARGs were highly abundant in raw swine wastewater, AD effectively reduced the copy number of all detected ARGs (0.21-1.34 logs removal), but the relative abundance with different resistance mechanisms showed distinctive variation trends. The reduction efficiency of ARGs was improved by stable operational temperature and longer solid retention time (SRT) of AD. ARGs in soil characterized the contamination from the irrigation of the digested liquor. The total ARGs quantity in soil fell down by 1.66 logs in idle period of winter compared to application period of summer in the northern region, whereas the total amount was steady with whole-year application in south. Some persistent (sul1 and sul2) and elevated ARGs (tetG and ereA) in AD and land application need more attention.

  15. Cerebral arteriopathy associated with heterozygous Arg179Cys mutation in the ACTA2 gene: Report in 2 newborn siblings.

    PubMed

    de Grazia, Jose; Delgado, Ignacio; Sanchez-Montanez, Angel; Boronat, Susana; Del Campo, Miguel; Vazquez, Elida

    2017-01-01

    Mutations in the ACTA2 gene lead to a multisystemic smooth muscle dysfunction syndrome that causes vascular disease, congenital mydriasis, and variable presentation of urinary and gastrointestinal problems. The heterozygous Arg179 mutation is associated with a distinctive cerebrovascular phenotype. We report the cases of two newborn siblings with heterozygous ACTA2 Arg179Cys substitution and provide neuroimaging exams that demonstrate the distinctive cerebrovascular phenotype, also associated with variable degree of hypoplasia of the vertebro-basilar circulation as well as hypoxic-ischemic lesions.

  16. Association between TP53 gene Arg72Pro polymorphism and Wilms’ tumor risk in a Chinese population

    PubMed Central

    Fu, Wen; Zhuo, Zhen-Jian; Jia, Wei; Zhu, Jinhong; Zhu, Shi-Bo; Lin, Ze-Feng; Wang, Feng-Hua; Xia, Huimin; He, Jing; Liu, Guo-Chang

    2017-01-01

    Wilms’ tumor is one of the most prevalent pediatric malignancies, ranking fourth in childhood cancer worldwide. TP53 is a critical tumor suppressor gene, which encodes a 53 kDa protein, p53. The p53 functions to protect against cancer by regulating cell cycle and apoptosis and maintaining DNA integrity. TP53 gene is highly polymorphic. Several TP53 gene polymorphisms have been considered to be associated with cancer risk. Of them, a nonsynonymous polymorphism, Arg72Pro (rs1042522 C>G), has been most extensively studied for the association with cancer risk; however, few studies have investigated its effect on Wilms’ tumor. Because of the central role of p53 in cell cycle control, the TP53 gene Arg72Pro polymorphism is also a good potential candidate predisposition locus for this pediatric cancer. We genotyped this polymorphism in 145 patients and 531 cancer-free controls recruited from Chinese children by Taqman methodology. Overall, our result suggested a lack of association between the TP53 gene Arg72Pro polymorphism and Wilms’ tumor. In the stratified analysis, we found that carriers of CG/GG genotypes had a significantly increased Wilms’ tumor risk in children not older than 18 months (adjusted odds ratio =2.04, 95% confidence interval =1.003–4.13, P=0.049) compared with CC genotype carriers. Our study indicated that the TP53 gene Arg72Pro polymorphism may have a weak, age-related effect on Wilms’ tumor risk in Chinese children. These findings need further validations in other populations with larger sample size. PMID:28260929

  17. Association between TP53 gene Arg72Pro polymorphism and Wilms' tumor risk in a Chinese population.

    PubMed

    Fu, Wen; Zhuo, Zhen-Jian; Jia, Wei; Zhu, Jinhong; Zhu, Shi-Bo; Lin, Ze-Feng; Wang, Feng-Hua; Xia, Huimin; He, Jing; Liu, Guo-Chang

    2017-01-01

    Wilms' tumor is one of the most prevalent pediatric malignancies, ranking fourth in childhood cancer worldwide. TP53 is a critical tumor suppressor gene, which encodes a 53 kDa protein, p53. The p53 functions to protect against cancer by regulating cell cycle and apoptosis and maintaining DNA integrity. TP53 gene is highly polymorphic. Several TP53 gene polymorphisms have been considered to be associated with cancer risk. Of them, a nonsynonymous polymorphism, Arg72Pro (rs1042522 C>G), has been most extensively studied for the association with cancer risk; however, few studies have investigated its effect on Wilms' tumor. Because of the central role of p53 in cell cycle control, the TP53 gene Arg72Pro polymorphism is also a good potential candidate predisposition locus for this pediatric cancer. We genotyped this polymorphism in 145 patients and 531 cancer-free controls recruited from Chinese children by Taqman methodology. Overall, our result suggested a lack of association between the TP53 gene Arg72Pro polymorphism and Wilms' tumor. In the stratified analysis, we found that carriers of CG/GG genotypes had a significantly increased Wilms' tumor risk in children not older than 18 months (adjusted odds ratio =2.04, 95% confidence interval =1.003-4.13, P=0.049) compared with CC genotype carriers. Our study indicated that the TP53 gene Arg72Pro polymorphism may have a weak, age-related effect on Wilms' tumor risk in Chinese children. These findings need further validations in other populations with larger sample size.

  18. The Arg160Trp allele of melanocortin-1 receptor gene might protect against vitiligo.

    PubMed

    Széll, Márta; Baltás, Eszter; Bodai, László; Bata-Csörgo, Zsuzsanna; Nagy, Nikoletta; Dallos, Attila; Pourfarzi, Reza; Simics, Eniko; Kondorosi, Ildikó; Szalai, Zsuzsanna; Tóth, Gábor K; Hunyadi, János; Dobozy, Attila; Kemény, Lajos

    2008-01-01

    Melanocortin-1 receptor (MC1R) and agouti signaling protein (ASIP) play pivotal roles in the regulation of human pigmentation. We aimed to study whether single nucleotide polymorphisms (SNPs) of the MC1R and ASIP genes contribute to the pathogenesis of the polygenic pigment skin disorder, vitiligo. The PCR-amplified, full-length MC1R gene was studied with sequence analysis, and the 3' untranslated region (3' UTR) SNP of ASIP was detected using restriction fragment length polymorphism. The allele frequency of the ASIP SNP did not show any difference between the skin type, hair color and eye color-matched 97 vitiligo patients and the 59 healthy control individuals. As one of the MC1R polymorphisms showed significantly higher incidence among fair-skinned individuals (Fitzpatrick I+II, n=140) than among dark-skinned individuals (Fitzpatrick III+IV, n=90), both vitiligo patients and controls were divided into two groups and the frequency of the MC1R alleles was studied separately in fair-skinned and dark-skinned subgroups of diseased and healthy groups. C478T, one of the MC1R SNPs studied in 108 fair-skinned vitiligo patients and in 70 fair-skinned healthy control individuals, showed a significant difference (P=0.0262, odds ratio [95% confidence interval]=3.6 [0.0046-0.1003]) in allele frequency between the two groups: the allele frequency was higher in the control group, suggesting protection against vitiligo. Computer prediction of antigenicity has revealed that the Arg160Trp amino acid change caused by this SNP results in a decrease in antigenicity of the affected peptide epitope.

  19. Arg/Abl2 promotes invasion and attenuates proliferation of breast cancer in vivo.

    PubMed

    Gil-Henn, H; Patsialou, A; Wang, Y; Warren, M S; Condeelis, J S; Koleske, A J

    2013-05-23

    Tumor progression is a complex, multistep process involving accumulation of genetic aberrations and alterations in gene expression patterns leading to uncontrolled cell division, invasion into surrounding tissue and finally dissemination and metastasis. We have previously shown that the Arg/Abl2 non-receptor tyrosine kinase acts downstream of the EGF receptor and Src tyrosine kinases to promote invadopodium function in breast cancer cells, thereby promoting their invasiveness. However, whether and how Arg contributes to tumor development and dissemination in vivo has never been investigated. Using a mouse xenograft model, we show that knocking down Arg in breast cancer cells leads to increased tumor cell proliferation and significantly enlarged tumor size. Despite having larger tumors, the Arg-knockdown (Arg KD) tumor-bearing mice exhibit significant reductions in tumor cell invasion, intravasation into blood vessels and spontaneous metastasis to lungs. Interestingly, we found that proliferation-associated genes in the Ras-MAPK (mitogen-activated protein kinase) pathway are upregulated in Arg KD breast cancer cells, as is Ras-MAPK signaling, while invasion-associated genes are significantly downregulated. These data suggest that Arg promotes tumor cell invasion and dissemination, while simultaneously inhibiting tumor growth. We propose that Arg acts as a switch in metastatic cancer cells that governs the decision to 'grow or go' (divide or invade).

  20. The Polymorphisms of Ser49Gly and Gly389Arg in Beta-1-Adrenergic Receptor Gene in Major Depression

    PubMed Central

    KOKUT, Süleyman; ATAY, İnci Meltem; UZ, Efkan; AKPINAR, Abdullah; DEMİRDAŞ, Arif

    2015-01-01

    Introduction It was reported that the genetic susceptibility of major depressive disorder (MDD) is related with genetic polymorphisms. The aim of this study was to investigate the possible association of the genotype and allele frequencies of Ser49Gly and Arg389Gly polymorphisms in MDD by comparing them with healthy subjects. Methods A total of 144 patients with MDD diagnosed according to Diagnostic and Statistical Manual of Mental Disorders, 4th Edition (DSM-IV) criteria and 105 healthy controls were included in the study. Polymerase chain reaction (PCR) with restriction fragment length polymorphism (RFLP) was used for genotyping. Results Of the 144 participants in the MDD group, 77 (53.5%) had homozygous wild type (AA), 57 (39.6%) had heterozygous type (AG), and 10 (6.9%) had mutant (GG) genotype for Ser49Gly, whereas 75 (52.1%) had homozygous wild type (GG), 59 (41.0%) had heterozygous (GC) type, and 10 (6.9%) had mutant homozygous (CC) genotype for Gly386Arg. There were no significant difference in the allele and genotype frequencies of the beta-1-adrenergic receptor (ADRB1) gene for Ser49Gly and Arg389Gly polymorphisms after comparing with healthy controls (p=0.626; p=0.863 and p=0.625; p=0.914). Conclusion The results of our study did not reveal a major effect of the polymorphism of Ser49Gly and Gly389Arg in the ADRB1 gene in MDD. Further studies with larger sample size are required to elucidate the role of other beta-1 adrenergic gene polymorphisms in MDD. PMID:28360691

  1. Arg399Gln polymorphism of XRCC1 gene and risk of colorectal cancer in Kashmir: A case control study.

    PubMed

    Nissar, Saniya; Lone, Tufail Ahmad; Banday, Mujeeb Zafar; Rasool, Roohi; Chowdri, Nissar A; Parray, Fazl Q; Abdullah, Safiya; Sameer, Aga Syed

    2013-03-01

    The aim of this study was to investigate the role of the XRCC1 Arg399Gln polymorphism in the susceptibility of a Kashmiri population to colorectal cancer (CRC). We investigated the genotype distribution of the XRCC1 gene in 130 CRC cases in comparison with that of 150 healthy subjects. There was no direct significant association between the XRCC1 genotypes and CRC; however, the Arg/Gln genotype was associated with an elevated risk of CRC (OR>1.47) and the Gln/Gln variant genotype was associated with an increased risk of CRC in various clinicopathological parameters. This study suggests that the XRCC1 polymorphism is associated with an increased risk of CRC.

  2. Arg399Gln polymorphism of XRCC1 gene and risk of colorectal cancer in Kashmir: A case control study

    PubMed Central

    NISSAR, SANIYA; LONE, TUFAIL AHMAD; BANDAY, MUJEEB ZAFAR; RASOOL, ROOHI; CHOWDRI, NISSAR A.; PARRAY, FAZL Q.; ABDULLAH, SAFIYA; SAMEER, AGA SYED

    2013-01-01

    The aim of this study was to investigate the role of the XRCC1 Arg399Gln polymorphism in the susceptibility of a Kashmiri population to colorectal cancer (CRC). We investigated the genotype distribution of the XRCC1 gene in 130 CRC cases in comparison with that of 150 healthy subjects. There was no direct significant association between the XRCC1 genotypes and CRC; however, the Arg/Gln genotype was associated with an elevated risk of CRC (OR>1.47) and the Gln/Gln variant genotype was associated with an increased risk of CRC in various clinicopathological parameters. This study suggests that the XRCC1 polymorphism is associated with an increased risk of CRC. PMID:23426866

  3. The immediate early gene arc/arg3.1: regulation, mechanisms, and function.

    PubMed

    Bramham, Clive R; Worley, Paul F; Moore, Melissa J; Guzowski, John F

    2008-11-12

    In a manner unique among activity-regulated immediate early genes (IEGs), mRNA encoded by Arc (also known as Arg3.1) undergoes rapid transport to dendrites and local synaptic translation. Despite this intrinsic appeal, relatively little is known about the neuronal and behavioral functions of Arc or its molecular mechanisms of action. Here, we attempt to distill recent advances on Arc spanning its transcriptional and translational regulation, the functions of the Arc protein in multiple forms of neuronal plasticity [long-term potentiation (LTP), long-term depression (LTD), and homeostatic plasticity], and its broader role in neural networks of behaving animals. Worley and colleagues have shown that Arc interacts with endophilin and dynamin, creating a postsynaptic trafficking endosome that selectively modifies the expression of AMPA-type glutamate receptors at the excitatory synapses. Both LTD and homeostatic plasticity in the hippocampus are critically dependent on Arc-mediated endocytosis of AMPA receptors. LTD evoked by activation of metabotropic glutamate receptors depends on rapid Arc translation controlled by elongation factor 2. Bramham and colleagues have shown that sustained translation of newly induced Arc mRNA is necessary for cofilin phosphorylation and stable expansion of the F-actin cytoskeleton underlying LTP consolidation in the dentate gyrus of live rats. In addition to regulating F-actin, Arc synthesis maintains the activity of key translation factors during LTP consolidation. This process of Arc-dependent consolidation is activated by the secretory neurotrophin, BDNF. Moore and colleagues have shown that Arc mRNA is a natural target for nonsense-mediated mRNA decay (NMD) by virtue of its two conserved 3'-UTR introns. NMD and other related translation-dependent mRNA decay mechanisms may serve as critical brakes on protein expression that contribute to the fine spatial-temporal control of Arc synthesis. In studies in behaving rats, Guzowski and

  4. A novel hypothyroid dwarfism due to the missense mutation Arg479Cys of the thyroid peroxidase gene in the mouse.

    PubMed

    Takabayashi, Shuji; Umeki, Kazumi; Yamamoto, Etsuko; Suzuki, Tohru; Okayama, Akihiko; Katoh, Hideki

    2006-10-01

    Recently, we found a novel dwarf mutation in an ICR closed colony. This mutation was governed by a single autosomal recessive gene. In novel dwarf mice, plasma levels of the thyroid hormones, T3 and T4, were reduced; however, TSH was elevated. Their thyroid glands showed a diffuse goiter exhibiting colloid deficiency and abnormal follicle epithelium. The dwarfism was improved by adding thyroid hormone in the diet. Gene mapping revealed that the dwarf mutation was closely linked to the thyroid peroxidase (Tpo) gene on chromosome 12. Sequencing of the Tpo gene of the dwarf mice demonstrated a C to T substitution at position 1508 causing an amino acid change from arginine (Arg) to cysteine (Cys) at codon 479 (Arg479Cys). Western blotting revealed that TPO protein of the dwarf mice was detected in a microsomal fraction of thyroid tissue, but peroxidase activity was not detected. These findings suggested that the dwarf mutation caused a primary congenital hypothyroidism by TPO deficiency, resulting in a defect of thyroid hormone synthesis.

  5. Effects of aged garlic extract and FruArg on gene expression and signaling pathways in lipopolysaccharide-activated microglial cells

    PubMed Central

    Song, Hailong; Lu, Yuan; Qu, Zhe; Mossine, Valeri V.; Martin, Matthew B.; Hou, Jie; Cui, Jiankun; Peculis, Brenda A.; Mawhinney, Thomas P.; Cheng, Jianlin; Greenlief, C. Michael; Fritsche, Kevin; Schmidt, Francis J.; Walter, Ronald B.; Lubahn, Dennis B.; Sun, Grace Y.; Gu, Zezong

    2016-01-01

    Aged garlic extract (AGE) is widely used as a dietary supplement on account of its protective effects against oxidative stress and inflammation. But less is known about specific molecular targets of AGE and its bioactive components, including N-α-(1-deoxy-D-fructos-1-yl)-L-arginine (FruArg). Our recent study showed that both AGE and FruArg significantly attenuate lipopolysaccharide (LPS)-induced neuroinflammatory responses in BV-2 microglial cells. This study aims to unveil effects of AGE and FruArg on gene expression regulation in LPS stimulated BV-2 cells. Results showed that LPS treatment significantly altered mRNA levels from 2563 genes. AGE reversed 67% of the transcriptome alteration induced by LPS, whereas FruArg accounted for the protective effect by reversing expression levels of 55% of genes altered by LPS. Key pro-inflammatory canonical pathways induced by the LPS stimulation included toll-like receptor signaling, IL-6 signaling, and Nrf2-mediated oxidative stress pathway, along with elevated expression levels of genes, such as Il6, Cd14, Casp3, Nfkb1, Hmox1, and Tnf. These effects could be modulated by treatment with both AGE and FruArg. These findings suggests that AGE and FruArg are capable of alleviating oxidative stress and neuroinflammatory responses stimulated by LPS in BV-2 cells. PMID:27734935

  6. Arg interacts with cortactin to promote adhesion-dependent cell edge protrusion.

    PubMed

    Lapetina, Stefanie; Mader, Christopher C; Machida, Kazuya; Mayer, Bruce J; Koleske, Anthony J

    2009-05-04

    The molecular mechanisms by which the Abelson (Abl) or Abl-related gene (Arg) kinases interface with the actin polymerization machinery to promote cell edge protrusions during cell-matrix adhesion are unclear. In this study, we show that interactions between Arg and the Arp2/3 complex regulator cortactin are essential to mediate actin-based cell edge protrusion during fibroblast adhesion to fibronectin. Arg-deficient and cortactin knockdown fibroblasts exhibit similar defects in adhesion-dependent cell edge protrusion, which can be restored via reexpression of Arg and cortactin. Arg interacts with cortactin via both binding and catalytic events. The cortactin Src homology (SH) 3 domain binds to a Pro-rich motif in the Arg C terminus. Arg mediates adhesion-dependent phosphorylation of cortactin, creating an additional binding site for the Arg SH2 domain. Mutation of residues that mediate Arg-cortactin interactions abrogate the abilities of both proteins to support protrusions, and the Nck adapter, which binds phosphocortactin, is also required. These results demonstrate that interactions between Arg, cortactin, and Nck1 are critical to promote adhesion-dependent cell edge protrusions.

  7. Receptor-like kinases from Arabidopsis form a monophyletic gene family related to animal receptor kinases

    PubMed Central

    Shiu, Shin-Han; Bleecker, Anthony B.

    2001-01-01

    Plant receptor-like kinases (RLKs) are proteins with a predicted signal sequence, single transmembrane region, and cytoplasmic kinase domain. Receptor-like kinases belong to a large gene family with at least 610 members that represent nearly 2.5% of Arabidopsis protein coding genes. We have categorized members of this family into subfamilies based on both the identity of the extracellular domains and the phylogenetic relationships between the kinase domains of subfamily members. Surprisingly, this structurally defined group of genes is monophyletic with respect to kinase domains when compared with the other eukaryotic kinase families. In an extended analysis, animal receptor kinases, Raf kinases, plant RLKs, and animal receptor tyrosine kinases form a well supported group sharing a common origin within the superfamily of serine/threonine/tyrosine kinases. Among animal kinase sequences, Drosophila Pelle and related cytoplasmic kinases fall within the plant RLK clade, which we now define as the RLK/Pelle family. A survey of expressed sequence tag records for land plants reveals that mosses, ferns, conifers, and flowering plants have similar percentages of expressed sequence tags representing RLK/Pelle homologs, suggesting that the size of this gene family may have been close to the present-day level before the diversification of land plant lineages. The distribution pattern of four RLK subfamilies on Arabidopsis chromosomes indicates that the expansion of this gene family is partly a consequence of duplication and reshuffling of the Arabidopsis genome and of the generation of tandem repeats. PMID:11526204

  8. Congenital erythropoietic porphyria with two mutations of the uroporphyrinogen III synthase gene (Cys73Arg, Thr228Met).

    PubMed

    Gucev, Zoran; Slavevska, Nevenka; Tasic, Velibor; Laban, Nevenka; Pop-Jordanova, Nada; Danilovski, Dragan; Woolf, Jacqueline; Cole, Duncan

    2011-05-01

    Congenital erythropoietic porphyria (CEP) is an autosomal recessive inborn error of metabolism that results from the markedly deficient activity of uroporphyrinogen III synthase (UROS). We describe a 14-year-old girl with red urine since infancy, progressive blistering and scarring of the skin, and moderate hemolytic anemia. After years of skin damage, her face is mutilated; she has a bald patch on the scalp, hypertrichosis of the neck, areas of skin darkening, and limited joint movements of the hands. Total urine excretion and fecal total porphyrin were both markedly raised above normal levels. Sequencing of the UROS gene identified two mutations causing CEP (Cys73Arg, Thr228Met). The patient lesions are progressing. Bone marrow transplantation and/or gene therapy are proposed as the next steps in her treatment. In brief, we describe a CEP with confirmed two pathogenic mutations, severe phenotype and discuss the various treatment options available.

  9. Putative modifier genes in mevalonate kinase deficiency.

    PubMed

    Marcuzzi, Annalisa; Vozzi, Diego; Girardelli, Martina; Tricarico, Paola Maura; Knowles, Alessandra; Crovella, Sergio; Vuch, Josef; Tommasini, Alberto; Piscianz, Elisa; Bianco, Anna Monica

    2016-04-01

    Mevalonate kinase deficiency (MKD) is an autosomal recessive auto‑inflammatory disease, caused by impairment of the mevalonate pathway. Although the molecular mechanism remains to be elucidated, there is clinical evidence suggesting that other regulatory genes may be involved in determining the phenotype. The identification of novel target genes may explain non‑homogeneous genotype‑phenotype correlations, and provide evidence in support of the hypothesis that novel regulatory genes predispose or amplify deregulation of the mevalonate pathway in this orphan disease. In the present study, DNA samples were obtained from five patients with MKD, which were then analyzed using whole exome sequencing. A missense variation in the PEX11γ gene was observed in homozygosis in P2, possibly correlating with visual blurring. The UNG rare gene variant was detected in homozygosis in P5, without correlating with a specific clinical phenotype. A number of other variants were found in the five analyzed DNA samples from the MKD patients, however no correlation with the phenotype was established. The results of the presents study suggested that further analysis, using next generation sequencing approaches, is required on a larger sample size of patients with MKD, who share the same MVK mutations and exhibit 'extreme' clinical phenotypes. As MVK mutations may be associated with MKD, the identification of specific modifier genes may assist in providing an earlier diagnosis.

  10. p.Pro4Arg mutation in LMNA gene: a new atypical progeria phenotype without metabolism abnormalities.

    PubMed

    Guo, Hong; Luo, Na; Hao, Fei; Bai, Yun

    2014-08-01

    Hutchinson-Gilford progeria syndrome (HGPS) is a typical presenile disorder, with mutation in the LMNA gene. Besides HGPS, mutations in LMNA gene have also been reported in atypical progeroid syndrome (APS). The objective of the study was to investigate the phenotype and molecular basis of APS in a Chinese family. LMNA gene mutations were also reviewed to identify the phenotypic and pathogenic differences among APS. Two siblings in a non-consanguineous Chinese family with atypical progeria were reported. The clinical features were observed, including presenile manifestations such as bird-like facial appearance, generalized lipodystrophy involving the extremities and mottled hyperpigmentation on the trunk and extremities. A heterozygous mutation c.11C>G (p.Pro4Arg) of the LMNA gene was detected in the two patients. 28 different variants of the LMNA gene have been reported in APS families, spreading over almost all the 12 exons of the LMNA gene with some hot-spot regions. This is the first detailed description of an APS family without metabolism abnormalities. APS patients share most of the clinical features, but there may be some distinct features in different ethnic groups.

  11. Homozygous Pro74-->Arg mutation in the platelet glycoprotein Ibbeta gene associated with Bernard-Soulier syndrome.

    PubMed

    Kunishima, S; Tomiyama, Y; Honda, S; Fukunishi, M; Hara, J; Inoue, C; Kamiya, T; Saito, H

    2000-07-01

    Bernard-Soulier syndrome (BSS) is an autosomal recessive bleeding disorder due to quantitative or qualitative abnormalities in the glycoprotein (GP) Ib/IX/V complex, the platelet receptor for von Willebrand factor. This complex is composed of four subunits, GPIbalpha, GPIbbeta, GPIX and GPV. We describe here the genetic basis of the disorder in a patient with BSS. Flow cytometric analysis of the patient's platelets showed greatly reduced GPIbalpha and GPIX surface expression. Immunoblot analysis disclosed absence of GPIbalpha, GPIbbeta and GPIX in the platelets. DNA sequencing analysis revealed a novel missense mutation in the GPIbbeta gene that converts Pro (CCG) to Arg (CGG) at residue 74. Homozygosity of the mutation was confirmed by allele-specific restriction analysis, chromosome 22 microsatellite analysis and quantitative Southern blotting. The mutant GPIbbeta was normally transcribed. Transient transfection studies confirmed that mutant GPIbbeta impairs surface expression of GPIb/IX, showing that the mutation is responsible for a BSS phenotype observed in the patient.

  12. Gene looping facilitates TFIIH kinase-mediated termination of transcription

    PubMed Central

    Medler, Scott; Ansari, Athar

    2015-01-01

    TFIIH is a general transcription factor with kinase and helicase activities. The kinase activity resides in the Kin28 subunit of TFIIH. The role of Kin28 kinase in the early steps of transcription is well established. Here we report a novel role of Kin28 in the termination of transcription. We show that RNAPII reads through a termination signal upon kinase inhibition. Furthermore, the recruitment of termination factors towards the 3′ end of a gene was compromised in the kinase mutant, thus confirming the termination defect. A concomitant decrease in crosslinking of termination factors near the 5′ end of genes was also observed in the kinase-defective mutant. Simultaneous presence of termination factors towards both the ends of a gene is indicative of gene looping; while the loss of termination factor occupancy from the distal ends suggest the abolition of a looped gene conformation. Accordingly, CCC analysis revealed that the looped architecture of genes was severely compromised in the Kin28 kinase mutant. In a looping defective sua7-1 mutant, even the enzymatically active Kin28 kinase could not rescue the termination defect. These results strongly suggest a crucial role of Kin28 kinase-dependent gene looping in the termination of transcription in budding yeast. PMID:26286112

  13. Methylation of Gata3 protein at Arg-261 regulates transactivation of the Il5 gene in T helper 2 cells.

    PubMed

    Hosokawa, Hiroyuki; Kato, Miki; Tohyama, Hiroyuki; Tamaki, Yuuki; Endo, Yusuke; Kimura, Motoko Y; Tumes, Damon John; Motohashi, Shinichiro; Matsumoto, Masaki; Nakayama, Keiichi I; Tanaka, Tomoaki; Nakayama, Toshinori

    2015-05-22

    Gata3 acts as a master regulator for T helper 2 (Th2) cell differentiation by inducing chromatin remodeling of the Th2 cytokine loci, accelerating Th2 cell proliferation, and repressing Th1 cell differentiation. Gata3 also directly transactivates the interleukin-5 (Il5) gene via additional mechanisms that have not been fully elucidated. We herein identified a mechanism whereby the methylation of Gata3 at Arg-261 regulates the transcriptional activation of the Il5 gene in Th2 cells. Although the methylation-mimicking Gata3 mutant retained the ability to induce IL-4 and repress IFNγ production, the IL-5 production was selectively impaired. We also demonstrated that heat shock protein (Hsp) 60 strongly associates with the methylation-mimicking Gata3 mutant and negatively regulates elongation of the Il5 transcript by RNA polymerase II. Thus, arginine methylation appears to play a pivotal role in the organization of Gata3 complexes and the target gene specificity of Gata3.

  14. Synthetic genes for human muscle-type adenylate kinase in Escherichia coli.

    PubMed

    Kim, H J; Nishikawa, S; Tanaka, T; Uesugi, S; Takenaka, H; Hamada, M; Kuby, S A

    1989-01-01

    An artificial gene coding for the human muscle-type cytosolic adenylate kinase (hAK1) was chemically synthesized and directly expressed in Escherichia coli under the control of trp promoter. The DNA duplex of 596 bp was designed and constructed from 40 oligonucleotide fragments of typically 30 nucleotides in length. Twelve unique restriction sites were fairly evenly spaced in the synthetic gene to facilitate site-specific mutagenesis at any part of this recombinant protein. The genes for mutant hAK1 (Tyr 95----Phe 95, Y95F hAK1; Arg 97----Ala 97, R97A hAK1) were constructed by cassette mutagenesis and utilized restriction sites incorporated in the hAK1 gene. The recombinant hAK1 was purified to homogeneity by a two-step chromatographic procedure with a good yield, and showed the same adenylate kinase activity as that of authentic hAK1. Preliminary kinetic studies show that the enzymatic activity (Vmax app,cor/Et) of Y95F hAK1 was slightly greater than that of recombinant hAK1, whereas R97A hAK1 still possessed approximately 4% of recombinant hAK1 activity. These results suggest that the Arg-97 residue is important but not essential for catalytic activity, and that Tyr-95 can be replaced by phenylalanine without substantial effects on the enzymatic activity. Moreover, preliminary estimates of the apparent kinetic parameters suggest that these residues are not required for MgATP binding, and therefore they do not appear to be part of the MgATP binding site.

  15. The ARG1-LIKE2 gene of Arabidopsis functions in a gravity signal transduction pathway that is genetically distinct from the PGM pathway.

    PubMed

    Guan, Changhui; Rosen, Elizabeth S; Boonsirichai, Kanokporn; Poff, Kenneth L; Masson, Patrick H

    2003-09-01

    The arl2 mutants of Arabidopsis display altered root and hypocotyl gravitropism, whereas their inflorescence stems are fully gravitropic. Interestingly, mutant roots respond like the wild type to phytohormones and an inhibitor of polar auxin transport. Also, their cap columella cells accumulate starch similarly to wild-type cells, and mutant hypocotyls display strong phototropic responses to lateral light stimulation. The ARL2 gene encodes a DnaJ-like protein similar to ARG1, another protein previously implicated in gravity signal transduction in Arabidopsis seedlings. ARL2 is expressed at low levels in all organs of seedlings and plants. arl2-1 arg1-2 double mutant roots display kinetics of gravitropism similar to those of single mutants. However, double mutants carrying both arl2-1 and pgm-1 (a mutation in the starch-biosynthetic gene PHOSPHOGLUCOMUTASE) at the homozygous state display a more pronounced root gravitropic defect than the single mutants. On the other hand, seedlings with a null mutation in ARL1, a paralog of ARG1 and ARL2, behave similarly to the wild type in gravitropism and other related assays. Taken together, the results suggest that ARG1 and ARL2 function in the same gravity signal transduction pathway in the hypocotyl and root of Arabidopsis seedlings, distinct from the pathway involving PGM.

  16. The ARG1-LIKE2 gene of Arabidopsis functions in a gravity signal transduction pathway that is genetically distinct from the PGM pathway

    NASA Technical Reports Server (NTRS)

    Guan, Changhui; Rosen, Elizabeth S.; Boonsirichai, Kanokporn; Poff, Kenneth L.; Masson, Patrick H.

    2003-01-01

    The arl2 mutants of Arabidopsis display altered root and hypocotyl gravitropism, whereas their inflorescence stems are fully gravitropic. Interestingly, mutant roots respond like the wild type to phytohormones and an inhibitor of polar auxin transport. Also, their cap columella cells accumulate starch similarly to wild-type cells, and mutant hypocotyls display strong phototropic responses to lateral light stimulation. The ARL2 gene encodes a DnaJ-like protein similar to ARG1, another protein previously implicated in gravity signal transduction in Arabidopsis seedlings. ARL2 is expressed at low levels in all organs of seedlings and plants. arl2-1 arg1-2 double mutant roots display kinetics of gravitropism similar to those of single mutants. However, double mutants carrying both arl2-1 and pgm-1 (a mutation in the starch-biosynthetic gene PHOSPHOGLUCOMUTASE) at the homozygous state display a more pronounced root gravitropic defect than the single mutants. On the other hand, seedlings with a null mutation in ARL1, a paralog of ARG1 and ARL2, behave similarly to the wild type in gravitropism and other related assays. Taken together, the results suggest that ARG1 and ARL2 function in the same gravity signal transduction pathway in the hypocotyl and root of Arabidopsis seedlings, distinct from the pathway involving PGM.

  17. Factor XI deficiency in French Basques is caused predominantly by an ancestral Cys38Arg mutation in the factor XI gene.

    PubMed

    Zivelin, Ariella; Bauduer, Frederic; Ducout, Louis; Peretz, Hava; Rosenberg, Nurit; Yatuv, Rivka; Seligsohn, Uri

    2002-04-01

    Inherited factor XI deficiency is an injury-related bleeding disorder that is rare in most populations except for Jews, in whom 2 mutations, a stop mutation in exon 5 (type II) and a missense mutation in exon 9 (type III), predominate. Recently, a cluster of 39 factor XI-deficient patients was described in the Basque population of Southwestern France. In this study, we determined the molecular basis of factor XI deficiency in 16 patients belonging to 12 unrelated families of French Basque origin. In 8 families, a nucleotide 209T>C transition in exon 3 was detected that predicts a Cys38Arg substitution. Four additional novel mutations in the factor XI gene, Cys237Tyr, Tyr493His, codon 285delG, and IVS6 + 3A>G, were identified in 4 families. Expression studies showed that Cys38Arg and Cys237Tyr factor XI were produced in transfected baby hamster kidney cells, but their secretion was impaired. Cells transfected with Tyr493His contained reduced amounts of factor XI and displayed decreased secretion. A survey of 206 French Basque controls for Cys38Arg revealed that the prevalence of the mutant allele was 0.005. Haplotype analysis based on the study of 10 intragenic polymorphisms was consistent with a common ancestry (a founder effect) for the Cys38Arg mutation.

  18. Development and dissection of diagnostic SNP markers for the downy mildew resistance genes Pl Arg and Pl 8 and maker-assisted gene pyramiding in sunflower (Helianthus annuus L.).

    PubMed

    Qi, L L; Talukder, Z I; Hulke, B S; Foley, M E

    2017-02-03

    Diagnostic DNA markers are an invaluable resource in breeding programs for successful introgression and pyramiding of disease resistance genes. Resistance to downy mildew (DM) disease in sunflower is mediated by Pl genes which are known to be effective against the causal fungus, Plasmopara halstedii. Two DM resistance genes, Pl Arg and Pl 8 , are highly effective against P. halstedii races in the USA, and have been previously mapped to the sunflower linkage groups (LGs) 1 and 13, respectively, using simple sequence repeat (SSR) markers. In this study, we developed high-density single nucleotide polymorphism (SNP) maps encompassing the Pl arg and Pl 8 genes and identified diagnostic SNP markers closely linked to these genes. The specificity of the diagnostic markers was validated in a highly diverse panel of 548 sunflower lines. Dissection of a large marker cluster co-segregated with Pl Arg revealed that the closest SNP markers NSA_007595 and NSA_001835 delimited Pl Arg to an interval of 2.83 Mb on the LG1 physical map. The SNP markers SFW01497 and SFW06597 delimited Pl 8 to an interval of 2.85 Mb on the LG13 physical map. We also developed sunflower lines with homozygous, three gene pyramids carrying Pl Arg , Pl 8 , and the sunflower rust resistance gene R 12 using the linked SNP markers from a segregating F2 population of RHA 340 (carrying Pl 8 )/RHA 464 (carrying Pl Arg and R 12 ). The high-throughput diagnostic SNP markers developed in this study will facilitate marker-assisted selection breeding, and the pyramided sunflower lines will provide durable resistance to downy mildew and rust diseases.

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

    PubMed

    Kim, Pora; Jia, Peilin; Zhao, Zhongming

    2016-12-24

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

  20. Missense mutations in the gene encoding prothrombin corresponding to Arg596 cause antithrombin resistance and thrombomodulin resistance.

    PubMed

    Takagi, Yuki; Murata, Moe; Kozuka, Toshihiro; Nakata, Yukiko; Hasebe, Ryo; Tamura, Shogo; Takagi, Akira; Matsushita, Tadashi; Saito, Hidehiko; Kojima, Tetsuhito

    2016-11-30

    Antithrombin (AT) and thrombomodulin (TM) play important roles in the process of natural anticoagulation in vivo. Recently, we reported that the prothrombin Yukuhashi mutation (p.Arg596Leu) was associated with AT and TM resistance-related thrombophilia. To assess the AT and TM resistances associated with other missense mutations by single base substitution in the Arg596 codon, we generated recombinant variants (596Gln, 596Trp, 596Gly, and 596Pro) and investigated the effects on AT and TM anticoagulant functions. All variants except 596Pro were secreted in amounts comparable to that of the wild-type but exhibited variable procoagulant activities. After a 30-minute inactivation by AT, the relative residual activity of wild-type thrombin decreased to 15 ± 4.0 %, in contrast to values of all variants were maintained at above 80 %. The thrombin-AT complex formation, as determined by enzyme-linked immunosorbent assay, was reduced with all tested variants in the presence and absence of heparin. In the presence of soluble TM (sTM), the relative fibrinogen clotting activity of wild-type thrombin decreased to 16 ± 0.12 %, whereas that of tested variants was 37 %-56 %. In a surface plasmon resonance assay, missense Arg596 mutations reduced thrombin-TM affinity to an extent similar to the reduction of fibrinogen clotting inhibition. In the presence of sTM or cultured endothelial-like cells, APC generation was enhanced differently by variant thrombins in a thrombin-TM affinity-dependent manner. These data indicate that prothrombin Arg596 missense mutations lead to AT and TM resistance in the variant thrombins and suggest that prothrombin Arg596 is important for AT- and TM-mediated anticoagulation.

  1. The actinophage RP3 DNA integrates site-specifically into the putative tRNA(Arg)(AGG) gene of Streptomyces rimosus.

    PubMed Central

    Gabriel, K; Schmid, H; Schmidt, U; Rausch, H

    1995-01-01

    The temperate actinophage RP3 integrates site-specifically into the chromosome of Streptomyces rimosus R6-554. The phage attachment site attP and the hybrid attachment sites of the integrated prophage--attL and attR--were cloned and sequenced. The 54nt core sequence, common to all RP3 related attachment sites, comprises the 3' terminal end of a putative tRNA(Arg)(AGG) gene. AttB bears the complete tRNA gene which is restored in attL after integration. A 7.5kb HindIII fragment, bearing attP, was used to construct an integrative plasmid to simulate the integration process in vivo and to localize the phage genes necessary for site specific integration. The int and xis genes were sequenced and compared to other recombination genes. PMID:7870591

  2. [Real-time PCR detection and quantification of emerging waterborne pathogens (EWPs) and antibiotic resistance genes (ARGs) in the downstream area of Jiulong River].

    PubMed

    Wang, Qing; Lin, Hui-rong; Zhang, Shu-ting; Yu, Xin

    2012-08-01

    The emerging waterborne pathogens (EWPs) and antibiotic resistance genes (ARGs) are important for drinking water safety. The detection and quantification of 7 EWPs and 4 ARGs were carried out in Jiulong River, which is the main water source of southwestern Fujian Province. The water samples were collected from four sites of the Jiulong River downstream area and a drinking water treatment plant nearby. DNA was extracted and quantified by real-time (SYBR Green) PCR methods after the samples were filtered through 0.22 pim membranes. The results showed that the amount of Salmonella enterica (Salmonella spp.), Legionella pneumophila (L. pneumophila) and Pseudomonas aeruginosa (P. aeruginosa) could reach up to 10(3), 10(4) and 10(5) copies x mL(-1), respectively. The concentration of organic matter in water may affect the copy numbers significantly. The water plant could effectively remove most EWPs and ARGs except Salmonella spp.. Therefore, more efforts should be made on water pollution source control, water treatment technology and point-of-use system to make sure the safety of drinking water.

  3. Fibroblast growth factor receptor 4 gene (FGFR4) 388Arg allele predicts prolonged survival and platinum sensitivity in advanced ovarian cancer.

    PubMed

    Marmé, Frederik; Hielscher, Thomas; Hug, Sarah; Bondong, Sandra; Zeillinger, Robert; Castillo-Tong, Dan Cacsire; Sehouli, Jalid; Braicu, Ioana; Vergote, Ignace; Isabella, Cadron; Mahner, Sven; Ferschke, Irmgard; Rom, Joachim; Sohn, Christof; Schneeweiss, Andreas; Altevogt, Peter

    2012-08-15

    FGFR4 has been shown to play an important role in the etiology and progression of solid tumors. A single nucleotide polymorphism (SNP) within the FGFR4 gene has previously been linked to prognosis and response to chemotherapy in breast cancer and other malignancies. This study evaluates the relevance of this SNP in advanced ovarian cancer. FGFR4-genotype was analyzed in 236 patients recruited as part of the OVCAD project. Genotyping was performed on germ-line DNA using a TaqMan based genotyping assay. Results were correlated with clinicopathological variables and survival. The FGFR4 388Arg genotype was significantly associated with prolonged progression-free and overall survival (univariate: HR 0.68, p = 0.017; HR 0.49, p = 0.005; multivariate: HR 0.69, p = 0.025; HR 0.49, p = 0.006) though the positive prognostic value was restricted to patients without postoperative residual tumor. Indeed, there was a significant interaction between FGFR4 genotype and residual tumor for overall survival. Furthermore, the FGFR4 388Arg genotype significantly correlated with platinum sensitivity in the same subgroup (multivariate OR 3.81 p = 0.004). FGFR4 Arg388Gly genotype is an independent and strong context specific prognostic factor in patients with advanced ovarian cancer and could be used to predict platinum-sensitivity.

  4. Synergistic activation of Arg1 gene by retinoic acid and IL-4 involves chromatin remodeling for transcription initiation and elongation coupling

    PubMed Central

    Lee, Bomi; Wu, Cheng-Ying; Lin, Yi-Wei; Park, Sung Wook; Wei, Li-Na

    2016-01-01

    All-trans Retinoic acid (RA) and its derivatives are potent therapeutics for immunological functions including wound repair. However, the molecular mechanism of RA modulation in innate immunity is poorly understood, especially in macrophages. We found that topical application of RA significantly improves wound healing and that RA and IL-4 synergistically activate Arg1, a critical gene for tissue repair, in M2 polarized macrophages. This involves feed forward regulation of Raldh2, a rate-limiting enzyme for RA biosynthesis, and requires Med25 to coordinate RAR, STAT6 and chromatin remodeler, Brg1 to remodel the +1 nucleosome of Arg1 for transcription initiation. By recruiting elongation factor TFIIS, Med25 also facilitates transcriptional initiation-elongation coupling. This study uncovers synergistic activation of Arg1 by RA and IL-4 in M2 macrophages that involves feed forward regulation of RA synthesis and dual functions of Med25 in nucleosome remodeling and transcription initiation-elongation coupling that underlies robust modulatory activity of RA in innate immunity. PMID:27166374

  5. The Wall-associated Kinase gene family in rice genomes.

    PubMed

    de Oliveira, Luiz Felipe Valter; Christoff, Ana Paula; de Lima, Júlio Cesar; de Ross, Bruno Comparsi Feijó; Sachetto-Martins, Gilberto; Margis-Pinheiro, Marcia; Margis, Rogerio

    2014-12-01

    The environment is a dynamic system in which life forms adapt. Wall-Associated Kinases (WAK) are a subfamily of receptor-like kinases associated with the cell wall. These genes have been suggested as sensors of the extracellular environment and triggers of intracellular signals. They belong to the ePK superfamily with or without a conserved arginine before the catalytic subdomain VIB, which characterizes RD and non-RD WAKs. WAK is a large subfamily in rice. We performed an extensive comparison of WAK genes from A. thaliana (AtWAK), O. sativa japonica and indica subspecies (OsWAK). Phylogenetic studies and WAK domain characterization allowed for the identification of two distinct groups of WAK genes in Arabidopsis and rice. One group corresponds to a cluster containing only OsWAKs that most likely expanded after the monocot-dicot separation, which evolved into a non-RD kinase class. The other group comprises classical RD-kinases with both AtWAK and OsWAK representatives. Clusterization analysis using extracellular and kinase domains demonstrated putative functional redundancy for some genes, but also highlighted genes that could recognize similar extracellular stimuli and activate different cascades. The gene expression pattern of WAKs in response to cold suggests differences in the regulation of the OsWAK genes in the indica and japonica subspecies. Our results also confirm the hypothesis of functional diversification between A. thaliana and O. sativa WAK genes. Furthermore, we propose that plant WAKs constitute two evolutionarily related but independent subfamilies: WAK-RD and WAK-nonRD. Recognition of this structural division will further provide insights to understanding WAK functions and regulations.

  6. Effects of Arc/Arg3.1 gene deletion on rhythmic synchronization of hippocampal CA1 neurons during locomotor activity and sleep.

    PubMed

    Malkki, Hemi A I; Mertens, Paul E C; Lankelma, Jan V; Vinck, Martin; van Schalkwijk, Frank J; van Mourik-Donga, Laura B; Battaglia, Francesco P; Mahlke, Claudia; Kuhl, Dietmar; Pennartz, Cyriel M A

    2016-05-01

    The activity-regulated cytoskeletal-associated protein/activity regulated gene (Arc/Arg3.1) is crucial for long-term synaptic plasticity and memory formation. However, the neurophysiological substrates of memory deficits occurring in the absence of Arc/Arg3.1 are unknown. We compared hippocampal CA1 single-unit and local field potential (LFP) activity in Arc/Arg3.1 knockout and wild-type mice during track running and flanking sleep periods. Locomotor activity, basic firing and spatial coding properties of CA1 cells in knockout mice were not different from wild-type mice. During active behavior, however, knockout animals showed a significantly shifted balance in LFP power, with a relative loss in high-frequency (beta-2 and gamma) bands compared to low-frequency bands. Moreover, during track-running, knockout mice showed a decrease in phase locking of spiking activity to LFP oscillations in theta, beta and gamma bands. Sleep architecture in knockout mice was not grossly abnormal. Sharp-wave ripples, which have been associated with memory consolidation and replay, showed only minor differences in dynamics and amplitude. Altogether, these findings suggest that Arc/Arg3.1 effects on memory formation are not only manifested at the level of molecular pathways regulating synaptic plasticity, but also at the systems level. The disrupted power balance in theta, beta and gamma rhythmicity and concomitant loss of spike-field phase locking may affect memory encoding during initial storage and memory consolidation stages.

  7. Presence and distribution of Macrolides-Lincosamide-Streptogramin resistance genes and potential indicator ARGs in the university ponds in Guangzhou, China.

    PubMed

    Wang, Mianzhi; Sun, Jing; Zhong, Weixin; Xiong, Wenguang; Zeng, Zhenling; Sun, Yongxue

    2016-11-01

    This study aimed to determine the occurrence, abundance, and variation of seven Macrolides-Lincosamide-Streptogramin (MLS) resistance genes (ereB, ermA, ermB, ermF, mefA, vatB, mphA) and six potential indicator ARGs (tet (B), sul1, qnrS, fexA, IntI1, ermB) from three ponds at university by quantitative PCR and assess the impacts on the surroundings. Solid samples (fish feces, soil and sediment) and water samples were tested. All the genes were found at low levels in soil samples. For the MLS resistance genes, only two MLS genes (ermB, ermF) were detected in all samples and significant correlations between ermB and Σ MLS (R = 0.91 in solid samples; R = 0.86 in water samples, p < 0.01) were found. For the potential indicators, intl1 and sul1 were present at high levels in the three different ponds while the other genes showed varying levels. These findings show that the ermB gene can probably be served as an indicator to evaluate the overall level of MLS resistance genes. The fairly low abundance of all the tested resistance genes in soil samples and the moderate levels in other samples suggests that the university ponds kept a good state and did not have a significant impact on their surroundings.

  8. DNA Repair Gene (XRCC1) Polymorphism (Arg399Gln) Associated with Schizophrenia in South Indian Population: A Genotypic and Molecular Dynamics Study

    PubMed Central

    Sujitha, S. P.; Kumar, D. Thirumal; Doss, C. George Priya; Aavula, K.; Ramesh, R.; Lakshmanan, S.; Gunasekaran, S.; Anilkumar, G.

    2016-01-01

    This paper depicts the first report from an Indian population on the association between the variant Arg399Gln of XRCC1 locus in the DNA repair system and schizophrenia, the debilitating disease that affects 1% of the world population. Genotypic analysis of a total of 523 subjects (260 patients and 263 controls) revealed an overwhelming presence of Gln399Gln in the case subjects against the controls (P < 0.0068), indicating significant level of association of this nsSNP with schizophrenia; the Gln399 allele frequency was also perceptibly more in cases than in controls (p < 0.003; OR = 1.448). The results of the genotypic studies were further validated using pathogenicity and stability prediction analysis employing computational tools [I-Mutant Suite, iStable, PolyPhen2, SNAP, and PROVEAN], with a view toassess the magnitude of deleteriousness of the mutation. The pathogenicity analysis reveals that the nsSNP could be deleterious inasmuch as it could affect the functionality of the gene, and interfere with protein function. Molecular dynamics simulation of 60ns was performed using GROMACS to analyse structural change due to a mutation (Arg399Gln) that was never examined before. RMSD, RMSF, hydrogen bonds, radius of gyration and SASA analysis showedthe existence of asignificant difference between the native and the mutant protein. The present study gives astrong indication that the XRCC1 locus deserves serious attention, as it could be a potential candidatecontributing to the etio-pathogenesis of the disease. PMID:26824244

  9. The Effect of a Novel c.820C>T (Arg274Trp) Mutation in the Mitofusin 2 Gene on Fibroblast Metabolism and Clinical Manifestation in a Patient

    PubMed Central

    Kawalec, Maria; Kabzińska, Dagmara; Kochański, Andrzej; Krzyśko, Krystiana A.; Zabłocka, Barbara

    2017-01-01

    Charcot-Marie-Tooth disease type 2A (CMT2A) is an autosomal dominant axonal peripheral neuropathy caused by mutations in the mitofusin 2 gene (MFN2). Mitofusin 2 is a GTPase protein present in the outer mitochondrial membrane and responsible for regulation of mitochondrial network architecture via the fusion of mitochondria. As that fusion process is known to be strongly dependent on the GTPase activity of mitofusin 2, it is postulated that the MFN2 mutation within the GTPase domain may lead to impaired GTPase activity, and in turn to mitochondrial dysfunction. The work described here has therefore sought to verify the effects of MFN2 mutation within its GTPase domain on mitochondrial and endoplasmic reticulum morphology, as well as the mtDNA content in a cultured primary fibroblast obtained from a CMT2A patient harboring a de novo Arg274Trp mutation. In fact, all the parameters studied were affected significantly by the presence of the mutant MFN2 protein. However, using the stable model for mitofusin 2 obtained by us, we were next able to determine that the Arg274Trp mutation does not impact directly upon GTP binding. Such results were also confirmed for GTP-hydrolysis activity of MFN2 protein in patient fibroblast. We therefore suggest that the biological malfunctions observable with the disease are not consequences of impaired GTPase activity, but rather reflect an impaired contribution of the GTPase domain to other MFN2 activities involving that region, for example protein-protein interactions. PMID:28076385

  10. Leptin Receptor Gene Gln223Arg Polymorphism Is Not Associated with Hypertension: A Preliminary Population-Based Cross-Sectional Study

    PubMed Central

    Pena, Geórgia das Graças; Guimarães, Andre L. S.; Veloso, Rosângela R.; Reis, Tatiana C.; Gomes, Crizian S.; Neto, João F. R.; Velasquez-Melendez, Gustavo

    2014-01-01

    Hypertension is responsible for high morbidity and mortality as one of the most important cardiometabolic risk factors. The aim of the study was to investigate whether the Gln223Arg in the leptin receptor (LEPR) influences the prevalence of hypertension. A cross-sectional study was carried out in individuals aged ≥ 18 years. Polymorphism identification was performed using PCR-RFLP analysis. Participants with blood pressure ≥ 140/90 mmHg or medication use were considered hypertensive. Frequencies, means, cross-tabulations, and multivariate models were produced to study differences in hypertension prevalence by genotypes. The study includes 470 participants. The frequency of GG polymorphism variant was 10.43%, 46.81% AG, and 42.77% AA. The distribution of hypertension frequency by LEPR genotypes was the following: AA 43.8%, AG 40.4%, and GG 40.8%; there were no significant differences between groups. Comparative analysis which used multivariate Poisson regression adjusted by many potential confounders (age, sex, schooling, smoking, alcohol intake, obesity, and family history of parental obesity) did not modify this result. In this large sample of population-based study, the association of the LEPR Gln223Arg gene polymorphism with hypertension was not observed. PMID:24772364

  11. A novel Gypsy founder mutation, p.Arg1109X in the CMT4C gene, causes variable peripheral neuropathy phenotypes

    PubMed Central

    Gooding, R; Colomer, J; King, R; Angelicheva, D; Marns, L; Parman, Y; Chandler, D; Bertranpetit, J; Kalaydjieva, L

    2005-01-01

    Background: Linkage, haplotype and sequencing analysis in a large Spanish Gypsy kindred with multiple members affected by autosomal recessive peripheral neuropathy led to the identification of a novel mutation, p.Arg1109X, in the CMT4C gene. The screening of further unrelated patients, and of a panel of ethnically matched controls, showed that p.Arg1109X is an ancestral mutation which occurs in Gypsy populations across Europe and is the most common cause of autosomal recessive Charcot–Marie–Tooth disease in Spanish Gypsies. Objective: To report the identification of a novel Gypsy founder mutation causing autosomal recessive CMT4C disease in a sample of homozygous affected individuals. Results: The mutation was associated with a surprisingly broad spectrum of neuropathy phenotypes, with variation in the age at onset, rate of progression, severity of muscle and sensory involvement, the presence of scoliosis, and cranial nerve involvement. Conclusions: Ascertainment and further studies of CMT4C patients in this population will provide a unique opportunity for characterising the full range of clinical manifestations of the disease in a genetically homogeneous sample. PMID:16326826

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

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

  13. A Causal Gene for Seed Dormancy on Wheat Chromosome 4A Encodes a MAP Kinase Kinase.

    PubMed

    Torada, Atsushi; Koike, Michiya; Ogawa, Taiichi; Takenouchi, Yu; Tadamura, Kazuki; Wu, Jianzhong; Matsumoto, Takashi; Kawaura, Kanako; Ogihara, Yasunari

    2016-03-21

    Seed germination under the appropriate environmental conditions is important both for plant species survival and for successful agriculture. Seed dormancy, which controls germination time, is one of the adaptation mechanisms and domestication traits [1]. Seed dormancy is generally defined as the absence of germination of a viable seed under conditions that are favorable for germination [2]. The seed dormancy of cultivated plants has generally been reduced during domestication [3]. Bread wheat (Triticum aestivum L.) is one of the most widely grown crops in the world. Weak dormancy may be an advantage for the productivity due to uniform emergence and a disadvantage for the risks of pre-harvest sprouting (PHS), which decreases grain quality and yield [4]. A number of quantitative trait loci (QTLs) controlling natural variation of seed dormancy have been identified on various chromosomes [5]. A major QTL for seed dormancy has been consistently detected on chromosome 4A [6-13]. The QTL was designated as a major gene, Phs1, which could be precisely mapped within a 2.6 cM region [14]. Here, we identified a mitogen-activated protein kinase kinase 3 (MKK3) gene (designated TaMKK3-A) by a map-based approach as a candidate gene for the seed dormancy locus Phs1 on chromosome 4A in bread wheat. Complementation analysis showed that transformation of a dormant wheat cultivar with the TaMKK3-A allele from a nondormant cultivar clearly reduced seed dormancy. Cultivars differing in dormancy had a single nonsynonymous amino acid substitution in the kinase domain of the predicted MKK3 protein sequence, which may be associated with the length of seed dormancy.

  14. Bacteriophage T4 deoxynucleotide kinase: gene cloning and enzyme purification.

    PubMed Central

    Brush, G S; Bhatnagar, S K; Bessman, M J

    1990-01-01

    Gene 1 of bacteriophage T4 has been cloned into a lambda pL expression vector, resulting in the overproduction of deoxynucleotide kinase. A procedure that includes affinity chromatography on Cibacron Blue F3GA-agarose has been used to purify milligram quantities of enzymes from a 2-liter culture. The enzyme has been partially characterized in vitro and in vivo, and it appears to be identical to the deoxynucleotide kinase isolated from T4-infected Escherichia coli. These results prove the earlier contention that the phosphorylation of three dissimilar deoxynucleotides (5-hydroxymethyldeoxycytidylate, dTMP, and dGMP), to the exclusion of most others, is catalyzed by a single protein. Images PMID:2160930

  15. ArgR of Streptomyces coelicolor Is a Versatile Regulator

    PubMed Central

    Pérez-Redondo, Rosario; Rodríguez-García, Antonio; Botas, Alma; Santamarta, Irene; Martín, Juan F.; Liras, Paloma

    2012-01-01

    ArgR is the regulator of arginine biosynthesis genes in Streptomyces species. Transcriptomic comparison by microarrays has been made between Streptomyces coelicolor M145 and its mutant S. coelicolor ΔargR under control, unsupplemented conditions, and in the presence of arginine. Expression of 459 genes was different in transcriptomic assays, but only 27 genes were affected by arginine supplementation. Arginine and pyrimidine biosynthesis genes were derepressed by the lack of ArgR, while no strong effect on expression resulted on arginine supplementation. Several nitrogen metabolism genes expression as glnK, glnA and glnII, were downregulated in S. coelicolor ΔargR. In addition, downregulation of genes for the yellow type I polyketide CPK antibiotic and for the antibiotic regulatory genes afsS and scbR was observed. The transcriptomic data were validated by either reverse transcription-PCR, expression of the gene-promoter coupled to the luciferase gene, proteomic or by electrophoresis mobility shift assay (EMSA) using pure Strep-tagged ArgR. Two ARG-boxes in the arginine operon genes suggest that these genes are more tightly controlled. Other genes, including genes encoding regulatory proteins, possess a DNA sequence formed by a single ARG-box which responds to ArgR, as validated by EMSA. PMID:22403700

  16. Epigenetic Perturbations by Arg882-Mutated DNMT3A Potentiate Aberrant Stem Cell Gene-Expression Program and Acute Leukemia Development.

    PubMed

    Lu, Rui; Wang, Ping; Parton, Trevor; Zhou, Yang; Chrysovergis, Kaliopi; Rockowitz, Shira; Chen, Wei-Yi; Abdel-Wahab, Omar; Wade, Paul A; Zheng, Deyou; Wang, Gang Greg

    2016-07-11

    DNA methyltransferase 3A (DNMT3A) is frequently mutated in hematological cancers; however, the underlying oncogenic mechanism remains elusive. Here, we report that the DNMT3A mutational hotspot at Arg882 (DNMT3A(R882H)) cooperates with NRAS mutation to transform hematopoietic stem/progenitor cells and induce acute leukemia development. Mechanistically, DNMT3A(R882H) directly binds to and potentiates transactivation of stemness genes critical for leukemogenicity including Meis1, Mn1, and Hoxa gene cluster. DNMT3A(R882H) induces focal epigenetic alterations, including CpG hypomethylation and concurrent gain of active histone modifications, at cis-regulatory elements such as enhancers to facilitate gene transcription. CRISPR/Cas9-mediated ablation of a putative Meis1 enhancer carrying DNMT3A(R882H)-induced DNA hypomethylation impairs Meis1 expression. Importantly, DNMT3A(R882H)-induced gene-expression programs can be repressed through Dot1l inhibition, providing an attractive therapeutic strategy for DNMT3A-mutated leukemias.

  17. Identification of an Arg35X mutation in the PDCD10 gene in a patient with cerebral and multiple spinal cavernous malformations.

    PubMed

    Lee, Seung-Tae; Choi, Ki-Whan; Yeo, Hyung-Tae; Kim, Jong-Won; Ki, Chang-Seok; Cho, Young-Dae

    2008-04-15

    Although cerebral cavernous malformations (CCMs) are not uncommon, the concurrent finding of cavernous malformations (CMs) both in the brain and spinal cord is quite rare. Furthermore, multiple spinal cord CMs are extremely rare with only a few cases being reported thus far. Recently, we encountered a 33-year-old Korean male with both CCM and multiple spinal intramedullary CMs. The patient complained of seizure and right chest paresthesia. The lesions were located throughout the neuraxis including the cerebral hemisphere, brain stem, and cervical and thoracic spinal cords. Molecular analysis of the KRIT1 (CCM1), CCM2, and PDCD10 (CCM3) genes identified a heterozygous nonsense mutation (c.103C>T; Arg35X) in the PDCD10 gene, which was reported previously in a CCM family. The patient denied a family history, however, his daughter had an identical mutation, but was asymptomatic. Three months later, after identifying the mutation in the father and the daughter, the daughter presented with seizure. To the best of our knowledge, this is the first report of an association between a mutation in the PDCD10 gene and spinal CMs.

  18. The ANKK1 kinase gene and psychiatric disorders.

    PubMed

    Ponce, Guillermo; Pérez-González, Rocío; Aragüés, María; Palomo, Tomás; Rodríguez-Jiménez, Roberto; Jiménez-Arriero, Miguel Angel; Hoenicka, Janet

    2009-07-01

    The TaqIA single nucleotide polymorphism (SNP, rs1800497), which is located in the gene that codes for the putative kinase ANKK1 (ANKK1) near the termination codon of the D2 dopamine receptor gene (DRD2; chromosome 11q22-q23), is the most studied genetic variation in a broad range of psychiatric disorders and personality traits. A large number of individual genetic association studies have found that the TaqIA SNP is linked to alcoholism and antisocial traits. In addition, it has also been related to other conditions such as schizophrenia, eating disorders, and some behavioral childhood disorders. The TaqIA A1 allele is mainly associated with addictions, antisocial disorders, eating disorders, and attention-deficit/hyperactivity disorders, while the A2 allele occurs more frequently in schizophrenic and obsessive-compulsive patients. Current data show that the TaqIA polymorphism may be a marker of both DRD2 and ANKK1 genetic variants. ANKK1 would belong to a family of kinases involved in signal transduction. This raises the question of whether signaling players intervene in the pathophysiology of psychiatric disorders. Basic research on the ANKK1 protein and its putative interaction with the D2 dopamine receptor could shed light on this issue.

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

    SciTech Connect

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

    2005-07-05

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

  20. Nutrient and hormonal regulation of pyruvate kinase gene expression.

    PubMed

    Yamada, K; Noguchi, T

    1999-01-01

    Mammalian pyruvate kinase (PK), a key glycolytic enzyme, has two genes named PKL and PKM, which produce the L- and R-type isoenzymes by means of alternative promoters, and the M1-and M2-types by mutually exclusive alternative splicing respectively. The expression of these genes is tissue-specific and under developmental, dietary and hormonal control. The L-type isoenzyme (L-PK) gene contains multiple regulatory elements necessary for regulation in the 5' flanking region, up to position -170. Both L-II and L-III elements are required for stimulation of L-PK gene transcription by carbohydrates such as glucose and fructose, although the L-III element is itself responsive to carbohydrates. The L-II element is also responsible for the gene regulation by polyunsaturated fatty acids. Nuclear factor-1 proteins and hepatocyte nuclear factor 4, which bind to the L-II element, may also be involved in carbohydrate and polyunsaturated fatty acid regulation of the L-PK gene respectively. However, the L-III-element-binding protein that is involved in carbohydrate regulation remains to be clarified, although involvement by an upstream stimulating factor has been proposed. Available evidence suggests that the carbohydrate signalling pathway to the L-PK gene includes a glucose metabolite, possibly glucose 6-phosphate or xylulose 5-phosphate, as well as phosphorylation and dephosphorylation mechanisms. In addition, at least five regulatory elements have been identified in the 5' flanking region of the PKM gene up to position -279. Sp1-family proteins bind to two proximal elements, but the binding of proteins to other elements have not yet been clarified. Glucose may stimulate the transcription of the PKM gene via hexosamine derivatives. Sp1 may be involved in this regulation via its dephosphorylation, although the carbohydrate response element has not been determined precisely in the PKM gene. Thus glucose stimulates transcription of the PKM gene by the mechanism which is probably

  1. A double-mutant collection targeting MAP kinase related genes in Arabidopsis for studying genetic interactions.

    PubMed

    Su, Shih-Heng; Krysan, Patrick J

    2016-12-01

    Mitogen-activated protein kinase cascades are conserved in all eukaryotes. In Arabidopsis thaliana there are approximately 80 genes encoding MAP kinase kinase kinases (MAP3K), 10 genes encoding MAP kinase kinases (MAP2K), and 20 genes encoding MAP kinases (MAPK). Reverse genetic analysis has failed to reveal abnormal phenotypes for a majority of these genes. One strategy for uncovering gene function when single-mutant lines do not produce an informative phenotype is to perform a systematic genetic interaction screen whereby double-mutants are created from a large library of single-mutant lines. Here we describe a new collection of 275 double-mutant lines derived from a library of single-mutants targeting genes related to MAP kinase signaling. To facilitate this study, we developed a high-throughput double-mutant generating pipeline using a system for growing Arabidopsis seedlings in 96-well plates. A quantitative root growth assay was used to screen for evidence of genetic interactions in this double-mutant collection. Our screen revealed four genetic interactions, all of which caused synthetic enhancement of the root growth defects observed in a MAP kinase 4 (MPK4) single-mutant line. Seeds for this double-mutant collection are publicly available through the Arabidopsis Biological Resource Center. Scientists interested in diverse biological processes can now screen this double-mutant collection under a wide range of growth conditions in order to search for additional genetic interactions that may provide new insights into MAP kinase signaling.

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

    DOEpatents

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

    1996-01-01

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

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

    NASA Technical Reports Server (NTRS)

    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.

  4. Isolation of Drosophila genes encoding G protein-coupled receptor kinases.

    PubMed Central

    Cassill, J A; Whitney, M; Joazeiro, C A; Becker, A; Zuker, C S

    1991-01-01

    G protein-coupled receptors are regulated via phosphorylation by a variety of protein kinases. Recently, termination of the active state of two such receptors, the beta-adrenergic receptor and rhodopsin, has been shown to be mediated by agonist- or light-dependent phosphorylation of the receptor by members of a family of protein-serine/threonine kinases (here referred to as G protein-coupled receptor kinases). We now report the isolation of a family of genes encoding a set of Drosophila protein kinases that appear to code for G protein-coupled receptor kinases. These proteins share a high degree of sequence homology with the bovine beta-adrenergic receptor kinase. The presence of a conserved family of G protein-coupled receptor kinases in vertebrates and invertebrates points to the central role of these kinases in signal transduction cascades. Images PMID:1662381

  5. Yeast Pho85 kinase is required for proper gene expression during the diauxic shift.

    PubMed

    Nishizawa, Masafumi; Katou, Yuki; Shirahige, Katsuhiko; Toh-e, Akio

    2004-08-01

    The budding yeast Saccharomyces cerevisiae changes its gene expression profile when environmental nutritional conditions are changed. Protein kinases including cyclic AMP-dependent kinase, Snf1 and Tor kinases play important roles in this process. Pho85 kinase, a member of the yeast cyclin-dependent kinase family, is involved in the regulation of phosphate metabolism and reserve carbohydrates, and thus is implicated to function as a nutrient-sensing kinase. Upon depletion of glucose in the medium, yeast cells undergo a diauxic shift, accompanied by a carbon metabolic pathway shift, stimulation of mitochondrial function and downregulation of ribosome biogenesis and protein synthesis. We analysed the effect of a pho85Delta mutation on the expression profiles of the genes in this process to investigate whether Pho85 kinase participates in the yeast diauxy. We found that, in the absence of PHO85, a majority of mitochondrial genes were not properly induced, that proteasome-related and chaperonin genes were more repressed, and that, when glucose was still present in the medium, a certain class of genes involved in ribosome biogenesis (ribosomal protein and rRNA processing genes) was repressed, whereas those involved in gluconeogenesis and the glyoxylate cycle were induced. We also found that PHO85 is required for proper expression of several metal sensor genes and their regulatory genes. These results suggest that Pho85 is required for proper onset of changes in expression profiles of genes responsible for the diauxic shift.

  6. Separate nuclear genes encode cytosolic and mitochondrial nucleoside diphosphate kinase in Dictyostelium discoideum.

    PubMed

    Troll, H; Winckler, T; Lascu, I; Müller, N; Saurin, W; Véron, M; Mutzel, R

    1993-12-05

    We have previously isolated cDNA clones for the gip17 gene encoding the cytosolic nucleoside diphosphate (NDP) kinase from Dictyostelium discoideum, and partial cDNAs for guk, a second member of the NDP kinase gene family (Wallet, V., Mutzel, R., Troll, H., Barzu, O., Wurster, B., Véron, M., and Lacombe, M. L. (1990) J. Natl. Cancer Inst. 80, 1199-1202). We now characterize genomic DNA clones for both NDP kinase genes, and we show that guk defines a nuclear-encoded mitochondrial NDP kinase. Isolated D. discoideum mitochondria contain 3% of the total cellular NDP kinase activity. Antibodies which specifically recognize and inhibit the activity of either cytosolic or mitochondrial NDP kinase unambiguously distinguish between these activities. The nascent mitochondrial NDP kinase contains a presequence of 57 amino acids that is removed during import into the organelle as shown by determination of the NH2 terminus of the mature protein from mitochondria. The genes for mitochondrial and cytosolic NDP kinases contain four and two introns, respectively. The positions of the of the introns in the gene for the cytosolic enzyme match exactly the positions of the second and fourth introns in the coding region of its mitochondrial homologue. From these results we conclude that the isozymes diverged from a common ancestor, and we discuss possible phylogenetic pathways for the evolution of cytosolic and organelle NDP kinases.

  7. Comparative functional characterization of novel non-syndromic GJB2 gene variant p.Gly45Arg and lethal syndromic variant p.Gly45Glu

    PubMed Central

    Gordhandas, Jeenal A.; Pique, Lynn

    2016-01-01

    We characterized a novel GJB2 missense variant, c.133G>A, p.Gly45Arg, and compared it with the only other variant at the same amino acid position of the connexin 26 protein (Cx26) reported to date: c.134G>A, p.Gly45Glu. Whereas both variants are associated with hearing loss and are dominantly inherited, p.Gly45Glu has been implicated in the rare fatal keratitis-ichthyosis-deafness (KID) syndrome, which results in cutaneous infections and septicemia with premature demise in the first year of life. In contrast, p.Gly45Arg appears to be non-syndromic. Subcellular localization experiments in transiently co-transfected HeLa cells demonstrated that Cx26-WT (wild-type) and p.Gly45Arg form gap junctions, whereas Cx26-WT with p.Gly45Glu protein does not. The substitution of a nonpolar amino acid glycine in wildtype Cx26 at position 45 with a negatively charged glutamic acid (acidic) has previously been shown to interfere with Ca2+ regulation of hemichannel gating and to inhibit the formation of gap junctions, resulting in cell death. The novel variant p.Gly45Arg, however, changes this glycine to a positively charged arginine (basic), resulting in the formation of dysfunctional gap junctions that selectively affect the permeation of negatively charged inositol 1,4,5-trisphosphate (IP3) and contribute to hearing loss. Cx26 p.Gly45Arg transfected cells, unlike cells transfected with p.Gly45Glu, thrived at physiologic Ca2+ concentrations, suggesting that Ca2+ regulation of hemichannel gating is unaffected in Cx26 p.Gly45Arg transfected cells. Thus, the two oppositely charged amino acids that replace the highly conserved uncharged glycine in p.Gly45Glu and p.Gly45Arg, respectively, produce strikingly different effects on the structure and function of the Cx26 protein. PMID:27761313

  8. Cadmium activates a mitogen-activated protein kinase gene and MBP kinases in rice.

    PubMed

    Yeh, Chuan-Ming; Hsiao, Lin-June; Huang, Hao-Jen

    2004-09-01

    Mitogen-activated protein kinase (MAPK) pathways are modules involved in the transduction of extracellular signals to intracellular targets in all eukaryotes. In plants, it has been evidenced that MAPKs play a role in the signaling of biotic and abiotic stresses, plant hormones, and cell cycle cues. However, the effect of heavy metals on plant MAPKs has not been well examined. The Northern blot analysis of OsMAPK mRNA levels has shown that only OsMAPK2, but not OsMAPK3 and OsMAPK4, expressed in suspension-cultured cells in response to 100-400 microM Cd treatments. The OsMAPK2 transcripts increased within 12 h upon 400 microM Cd treatment. In addition, we found that 42- and 50-kDa MBP kinases were significantly activated by Cd treatment in rice suspension-cultured cells. And 40-, 42-, 50- and 64-kDa MBP kinases were activated in rice roots. Furthermore, GSH inhibits Cd-induced 40-kDa MBP kinase activation. By immunoblot analysis and immunoprecipitation followed by in-gel kinase assay, we confirmed that Cd-activated 42-kDa MBP kinase is a MAP kinase. Our results suggest that a MAP kinase cascade may function in the Cd-signalling pathway in rice.

  9. Comparative analysis of Pseudomonas syringae pv. actinidiae and pv. phaseolicola based on phaseolotoxin-resistant ornithine carbamoyltransferase gene (argK) and 16S-23S rRNA intergenic spacer sequences.

    PubMed

    Sawada, H; Takeuchi, T; Matsuda, I

    1997-01-01

    Pseudomonas syringae pv. phaseolicola, which causes halo blight on various legumes, and pv. actinidiae, responsible for canker or leaf spot on actinidia plants, are known as phaseolotoxin producers, and the former possesses phaseolotoxin-resistant ornithine carbamoyltransferase (ROCT) which confers resistance to the toxin. We confirmed that the latter is also resistant to phaseolotoxin and possesses ROCT, and we compared the two pathovars by using sequence data of the ROCT gene and the intergenic spacer region located between the 16S and 23S rRNA genes (16S-23S spacer region) as an index. It was found that the identical ROCT gene (argK) is contained not only in bean isolates of P. syringae pv. phaseolicola in Mexico and the United States but also in bean isolates in Japan and Canada, and that it is also distributed in the kudzu (Pueraria lobata) isolates of P. syringae pv. phaseolicola. Moreover, the kiwifruit and tara vine isolates of P. syringae pv. actinidiae were also found to possess the identical argK. On the contrary, the 16S-23S spacer regions showed a significant level of sequence variation between P. syringae pv. actinidiae and pv. phaseolicola, suggesting that these two pathovars evolved differently from each other in the phylogenetic development. The fact that even synonymous substitution has not occurred in argK among these strains despite their extreme differences in phylogenetic evolution and geographical distribution suggests that it was only recently in evolutionary time that argK was transferred from its origin to P. syringae pv. actinidiae and/or pv. phaseolicola.

  10. Comparative analysis of Pseudomonas syringae pv. actinidiae and pv. phaseolicola based on phaseolotoxin-resistant ornithine carbamoyltransferase gene (argK) and 16S-23S rRNA intergenic spacer sequences.

    PubMed Central

    Sawada, H; Takeuchi, T; Matsuda, I

    1997-01-01

    Pseudomonas syringae pv. phaseolicola, which causes halo blight on various legumes, and pv. actinidiae, responsible for canker or leaf spot on actinidia plants, are known as phaseolotoxin producers, and the former possesses phaseolotoxin-resistant ornithine carbamoyltransferase (ROCT) which confers resistance to the toxin. We confirmed that the latter is also resistant to phaseolotoxin and possesses ROCT, and we compared the two pathovars by using sequence data of the ROCT gene and the intergenic spacer region located between the 16S and 23S rRNA genes (16S-23S spacer region) as an index. It was found that the identical ROCT gene (argK) is contained not only in bean isolates of P. syringae pv. phaseolicola in Mexico and the United States but also in bean isolates in Japan and Canada, and that it is also distributed in the kudzu (Pueraria lobata) isolates of P. syringae pv. phaseolicola. Moreover, the kiwifruit and tara vine isolates of P. syringae pv. actinidiae were also found to possess the identical argK. On the contrary, the 16S-23S spacer regions showed a significant level of sequence variation between P. syringae pv. actinidiae and pv. phaseolicola, suggesting that these two pathovars evolved differently from each other in the phylogenetic development. The fact that even synonymous substitution has not occurred in argK among these strains despite their extreme differences in phylogenetic evolution and geographical distribution suggests that it was only recently in evolutionary time that argK was transferred from its origin to P. syringae pv. actinidiae and/or pv. phaseolicola. PMID:8979356

  11. The Trp64Arg mutation of the beta3 adrenergic receptor gene has no effect on obesity phenotypes in the Québec Family Study and Swedish Obese Subjects cohorts.

    PubMed Central

    Gagnon, J; Mauriège, P; Roy, S; Sjöström, D; Chagnon, Y C; Dionne, F T; Oppert, J M; Pérusse, L; Sjöström, L; Bouchard, C

    1996-01-01

    The beta adrenergic system plays a key role in regulating energy balance through the stimulation of both thermogenesis and lipid mobilization in brown and white adipose tissues in human and various animal models. Recent studies have suggested that a missense Trp64Arg mutation in the beta3 adrenergic receptor (ADRB3) gene was involved in obesity and insulin resistance. We have investigated the effect of this mutation on obesity-related phenotypes in two cohorts: the Québec Family Study (QFS) and the Swedish Obese Subjects (SOS). In QFS, no association was found between this mutation and body mass index (BMI), body fat including abdominal visceral fat, resting metabolic rate, various diabetes and cardiovascular risk factors, and changes in body weight and body fat over a 12-yr period. With the exception of RMR (P = 0.04), no evidence of linkage was detected between the mutation and phenotypes of QFS based on sib-pair data. In SOS, the frequency of the Trp64Arg allele was not significantly different between nonobese and obese female subjects and no association was found between the mutation and body weight gain over time. These findings do not support the view that there is an association between the Trp64Arg mutation in the ADRB3 gene and obesity. PMID:8903328

  12. Global Analysis of Serine-Threonine Protein Kinase Genes in Neurospora crassa ▿ †

    PubMed Central

    Park, Gyungsoon; Servin, Jacqueline A.; Turner, Gloria E.; Altamirano, Lorena; Colot, Hildur V.; Collopy, Patrick; Litvinkova, Liubov; Li, Liande; Jones, Carol A.; Diala, Fitz-Gerald; Dunlap, Jay C.; Borkovich, Katherine A.

    2011-01-01

    Serine/threonine (S/T) protein kinases are crucial components of diverse signaling pathways in eukaryotes, including the model filamentous fungus Neurospora crassa. In order to assess the importance of S/T kinases to Neurospora biology, we embarked on a global analysis of 86 S/T kinase genes in Neurospora. We were able to isolate viable mutants for 77 of the 86 kinase genes. Of these, 57% exhibited at least one growth or developmental phenotype, with a relatively large fraction (40%) possessing a defect in more than one trait. S/T kinase knockouts were subjected to chemical screening using a panel of eight chemical treatments, with 25 mutants exhibiting sensitivity or resistance to at least one chemical. This brought the total percentage of S/T mutants with phenotypes in our study to 71%. Mutants lacking apg-1, an S/T kinase required for autophagy in other organisms, possessed the greatest number of phenotypes, with defects in asexual and sexual growth and development and in altered sensitivity to five chemical treatments. We showed that NCU02245/stk-19 is required for chemotropic interactions between female and male cells during mating. Finally, we demonstrated allelism between the S/T kinase gene NCU00406 and velvet (vel), encoding a p21-activated protein kinase (PAK) gene important for asexual and sexual growth and development in Neurospora. PMID:21965514

  13. Identification of a novel nonsense mutation of the neurotrophic tyrosine kinase receptor type 1 gene in two siblings with congenital insensitivity to pain with anhidrosis.

    PubMed

    Wang, Ting; Li, Haibo; Xiang, Jingjing; Wei, Bin; Zhang, Qin; Zhu, Qin; Liu, Minjuan; Sun, Miao; Li, Hong

    2017-01-01

    Objective To explore the aetiology of congenital insensitivity to pain with anhidrosis (CIPA) in two Chinese siblings with typical CIPA symptoms including insensitivity to pain, inability to sweat, and self-mutilating behaviours. Methods Clinical examination and genetic testing were conducted of all available family members, and the findings were used to create a pedigree. Mutation screening using PCR amplification and DNA Sanger sequencing of the entire neurotrophic tyrosine kinase receptor type 1 gene ( NTRK1) including intron-exon boundaries was used to identify mutations associated with CIPA. Results A novel nonsense mutation (c.7C > T, p. Arg3Ter) and a known splice-site mutation (c.851-33 T > A) were detected in NTRK1 and shown to be associated with CIPA. Conclusion Our findings expand the known mutation spectrum of NTRK1 and provide insights into the aetiology of CIPA.

  14. Rapid detection of mutations by conformation sensitive gel electrophoresis: Application to the identification of three new mutations in the type II procollagen gene and a fourth family with the Arg{sub 519}{yields}Cys base substitution

    SciTech Connect

    Williams, C.J.; Rock, M.; McCarron, S.

    1994-09-01

    Conformation sensitive gel electrophoresis (CSGE) detects differences as small as a single base mismatch in DNA heteroduplexes of polymerase chain reaction (PCR) products. The altered migration of heteroduplexes versus homoduplexes is resolved in a polyacrylamide-based gel electrophoresis system. The technique was used here to detect conformational changes in the type II procollagen gene (COL2A1) in patients with growth plate defects. PCR products which displayed heteroduplex species were directly sequenced and all revealed either base substitutions or base deletions. Three of the base substitutions resulted in the identification of new mutations. These include a Gly{sub 691}{yields}Arg substitution in a proband with hypochondrogenesis, a Gly{sub 975}{yields}Ser base substitution in a family with late-onset spondyloepiphyseal dysplasia (SEDT) and precocious osteoarthritis (POA), and a Gly{sub 988}{yields}Arg mutation in another patient with hypochondrogenesis. A fourth substitution was found to be the fourth example of an Arg{sub 519}{yields}Cys point mutation in a family with SEDT and POA. All mutations were confirmed by restriction site analysis. These results illustrate the utility of the CSGE method for the rapid detection of mutations in PCR products without the need for special equipment, primers or sample preparation.

  15. Concerted transcriptional activation of the low density lipoprotein receptor gene by insulin and luteinizing hormone in cultured porcine granulosa-luteal cells: possible convergence of protein kinase a, phosphatidylinositol 3-kinase, and mitogen-activated protein kinase signaling pathways.

    PubMed

    Sekar, N; Veldhuis, J D

    2001-07-01

    -repressive region in this gene. Non-LH receptor-dependent agonists of protein kinase A (PKA), 8-bromo-cAMP (1 mM), and forskolin (10 microM) with or without insulin/IGF-I costimulation likewise augmented LDL receptor promoter expression with similar strong dependency on the -255 to -139 bp 5'-upstream region. To assess more specific PKA-dependent mediation of LH's contribution to combined hormonal drive, the LDL receptor (-1076 to +11 bp) reporter plasmid was cotransfected with a full-sequence rabbit muscle protein kinase inhibitor (PKI) minigene driven constitutively by a Rous sarcoma virus promoter. Expression of the latter PKA antagonist blocked transcriptional stimulation by LH alone as well as that by LH combined with insulin (or IGF-I) by 70-85% without reducing basal transcriptional activity. Transfection of a mutant inactive (Arg to Gly) Rous sarcoma virus/PKI gene confirmed the specificity of the PKI effect. To investigate the convergent role of the insulin/IGF-I effector pathway mediating bihormonal stimulation of LDL receptor promoter expression, transfected granulosa-luteal cells were pretreated for 30 min with two specific inhibitors of phophatidylinositol 3-kinase, wortmannin (100 nM) and LY 294002 (10 microM), or of mitogen-activated protein kinase kinase, PD 98059 (50 microM), U0126 (10 microM), or the latter's inactive derivative, U0124 (10 microM). Both classes of antagonists impeded the ability of insulin or IGF-I to enhance LH-stimulated LDL receptor promoter expression by 60-80%. In conclusion, the present analyses indicate that LH and insulin (or IGF-I) can up-regulate LDL receptor transcriptional activity supraadditively in porcine granulosa-luteal cells 1) via one or more agonistic cis-acting DNA regions located between -255 and -139 bp 5'- upstream of the transcriptional start site, 2) without abrogating sterol-sensitive repressive of this promoter, and 3) by way of intracellular mechanisms that include the PKA, phophatidylinositol 3-kinase, and mitogen

  16. The S-locus receptor kinase gene in a self-incompatible Brassica napus line encodes a functional serine/threonine kinase.

    PubMed Central

    Goring, D R; Rothstein, S J

    1992-01-01

    An S-receptor kinase (SRK) cDNA, SRK-910, from the active S-locus in a self-incompatible Brassica napus W1 line has been isolated and characterized. The SRK-910 gene is predominantly expressed in pistils and segregates with the W1 self-incompatibility phenotype in an F2 population derived from a cross between the self-incompatible W1 line and a self-compatible Westar line. Analysis of the predicted amino acid sequence demonstrated that the extracellular receptor domain is highly homologous to S-locus glycoproteins, whereas the cytoplasmic kinase domain contains conserved amino acids present in serine/threonine kinases. An SRK-910 kinase protein fusion was produced in Escherichia coli and found to contain kinase activity. Phosphoamino acid analysis confirmed that only serine and threonine residues were phosphorylated. Thus, the SRK-910 gene encodes a functional serine/threonine receptor kinase. PMID:1332796

  17. The equine herpes virus 4 thymidine kinase is a better suicide gene than the human herpes virus 1 thymidine kinase.

    PubMed

    Loubière, L; Tiraby, M; Cazaux, C; Brisson, E; Grisoni, M; Zhao-Emonet, J; Tiraby, G; Klatzmann, D

    1999-09-01

    The herpes simplex virus type 1 thymidine kinase suicide gene (HSV1tk) together with ganciclovir (GCV) have been successfully used for in vivo treatment of various experimental tumors, and many clinical trials using this system have been launched. With the aim to improve this therapeutic system, we compared the potential efficacy of different herpes virus derived thymidine kinases (HSV1, varicella-zoster virus, equine herpes virus type-4 and Epstein-Barr virus) as suicide genes in association with the nucleoside analogs acyclovir, ganciclovir and bromovinyldeoxyur- idine. Using various murine and human cell lines expressing these viral tk, we show that HSV1- and EHV4tk are the more efficient suicide genes for the different nucleoside analogs tested. Moreover, EHV4tk expressing murine and human cells were three- to 12-fold more sensitive to GCV than HSV1tk expressing cells. This was correlated with the presence of five-fold higher amounts of the toxic triphosphated-GCV in EHV4- versus HSV1tk expressing cells. Altogether, these experiments underline the potential advantages of the EHV4tk as a suicide gene.

  18. 3pK, a new mitogen-activated protein kinase-activated protein kinase located in the small cell lung cancer tumor suppressor gene region.

    PubMed Central

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

    1996-01-01

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

  19. Emdogain-regulated gene expression in palatal fibroblasts requires TGF-βRI kinase signaling.

    PubMed

    Stähli, Alexandra; Bosshardt, Dieter; Sculean, Anton; Gruber, Reinhard

    2014-01-01

    Genome-wide microarrays have suggested that Emdogain regulates TGF-β target genes in gingival and palatal fibroblasts. However, definitive support for this contention and the extent to which TGF-β signaling contributes to the effects of Emdogain has remained elusive. We therefore studied the role of the TGF-β receptor I (TGF-βRI) kinase to mediate the effect of Emdogain on palatal fibroblasts. Palatal fibroblasts were exposed to Emdogain with and without the inhibitor for TGF-βRI kinase, SB431542. Emdogain caused 39 coding genes to be differentially expressed in palatal fibroblasts by microarray analysis (p<0.05; >10-fold). Importantly, in the presence of the TGF-βRI kinase inhibitor SB431542, Emdogain failed to cause any significant changes in gene expression. Consistent with this mechanism, three independent TGF-βRI kinase inhibitors and a TGF-β neutralizing antibody abrogated the increased expression of IL-11, a selected Emdogain target gene. The MAPK inhibitors SB203580 and U0126 lowered the impact of Emdogain on IL-11 expression. The data support that TGF-βRI kinase activity is necessary to mediate the effects of Emdogain on gene expression in vitro.

  20. Genome-wide identification and expression analysis of WNK kinase gene family in rice.

    PubMed

    Manuka, Rakesh; Saddhe, Ankush Ashok; Kumar, Kundan

    2015-12-01

    Eukaryotic protein kinases represent one of the largest gene families involved in diverse regulatory functions. WNK (With No Lysine) kinases are members of ser/thr protein kinase family, which lack conserved catalytic lysine (K) residue at protein kinase subdomain II and is replaced by either asparagine, serine or glycine residues. They are involved in regulation of flowering time, circadian rhythms and abiotic stresses in Arabidopsis thaliana. In the present study, we have identified 9 members of WNK in rice, showed resemblance to Arabidopsis and human WNK and clustered into five main clades phylogenetically. The predicted genes structure, bonafide conserved signature motif and domains strongly support their identity, as members of WNK kinase family. We have analyzed their chromosomal distribution, physio-chemical properties, subcellular localizations and cis-elements in the promoter regions in silico. Further, transcript analysis of OsWNK by qRT-PCR revealed their differential regulation in tissue specific and abiotic stresses libraries. In conclusion, the identification of nine OsWNK and transcript level expression pattern under abiotic stress using qRT-PCR in rice will significantly contribute towards the understanding of WNK genes in monocots and thus provide a set up for functional genomics studies of WNK protein kinases.

  1. Extent of Genetic Lesions of the Arginine and Pyrimidine Biosynthetic Pathways in Lactobacillus plantarum, L. paraplantarum, L. pentosus, and L. casei: Prevalence of CO2-Dependent Auxotrophs and Characterization of Deficient arg Genes in L. plantarum

    PubMed Central

    Bringel, Françoise; Hubert, Jean-Claude

    2003-01-01

    Lactic acid bacteria require rich media since, due to mutations in their biosynthetic genes, they are unable to synthesize numerous amino acids and nucleobases. Arginine biosynthesis and pyrimidine biosynthesis have a common intermediate, carbamoyl phosphate (CP), whose synthesis requires CO2. We investigated the extent of genetic lesions in both the arginine biosynthesis and pyrimidine biosynthesis pathways in a collection of lactobacilli, including 150 strains of Lactobacillus plantarum, 32 strains of L. pentosus, 15 strains of L. paraplantarum, and 10 strains of L. casei. The distribution of prototroph and auxotroph phenotypes varied between species. All L. casei strains, no L. paraplantarum strains, two L. pentosus strains, and seven L. plantarum strains required arginine for growth. Arginine auxotrophs were more frequently found in L. plantarum isolated from milk products than in L. plantarum isolated from fermented plant products or humans; association with dairy products might favor arginine auxotrophy. In L. plantarum the argCJBDF genes were functional in most strains, and when they were inactive, only one gene was mutated in more than one-half of the arginine auxotrophs. Random mutation may have generated these auxotrophs since different arg genes were inactivated (there were single point mutations in three auxotrophs and nonrevertible genetic lesions in four auxotrophs). These data support the hypothesis that lactic acid bacteria evolve by progressively loosing unnecessary genes upon adaptation to specific habitats, with genome evolution towards cumulative DNA degeneration. Although auxotrophy for only uracil was found in one L. pentosus strain, a high CO2 requirement (HCR) for arginine and pyrimidine was common; it was found in 74 of 207 Lactobacillus strains tested. These HCR auxotrophs may have had their CP cellular pool-related genes altered or deregulated. PMID:12732536

  2. Sequence of the canine herpesvirus thymidine kinase gene: taxon-preferred amino acid residues in the alphaherpesviral thymidine kinases.

    PubMed

    Rémond, M; Sheldrick, P; Lebreton, F; Foulon, T

    1995-12-01

    Multiple sequence alignments of evolutionarily related proteins are finding increasing use as indicators of critical amino acid residues necessary for structural stability or involved in functional domains responsible for catalytic activities. In the past, a number of alignments have provided such information for the herpesviral thymidine kinases, for which three-dimensional structures are not yet available. We have sequenced the thymidine kinase gene of a canine herpesvirus, and with a multiple alignment have identified amino acids preferentially conserved in either of two taxons, the genera Varicellovirus and Simplexvirus, of the subfamily Alphaherpesvirinae. Since some regions of the thymidine kinases show otherwise elevated levels of substitutional tolerance, these conserved amino acids are candidates for critical residues which have become fixed through selection during the evolutionary divergence of these enzymes. Several pairs with distinctive patterns of distribution among the various viruses occur in or near highly conserved sequence motifs previously proposed to form the catalytic site, and we speculate that they may represent interacting, co-ordinately variable residues.

  3. Calcium-Dependent Protein Kinase Genes in Corn Roots

    NASA Technical Reports Server (NTRS)

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

    1996-01-01

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

  4. The Medicago truncatula lysin [corrected] motif-receptor-like kinase gene family includes NFP and new nodule-expressed genes.

    PubMed

    Arrighi, Jean-François; Barre, Annick; Ben Amor, Besma; Bersoult, Anne; Soriano, Lidia Campos; Mirabella, Rossana; de Carvalho-Niebel, Fernanda; Journet, Etienne-Pascal; Ghérardi, Michèle; Huguet, Thierry; Geurts, René; Dénarié, Jean; Rougé, Pierre; Gough, Clare

    2006-09-01

    Rhizobial Nod factors are key symbiotic signals responsible for starting the nodulation process in host legume plants. Of the six Medicago truncatula genes controlling a Nod factor signaling pathway, Nod Factor Perception (NFP) was reported as a candidate Nod factor receptor gene. Here, we provide further evidence for this by showing that NFP is a lysin [corrected] motif (LysM)-receptor-like kinase (RLK). NFP was shown both to be expressed in association with infection thread development and to be involved in the infection process. Consistent with deviations from conserved kinase domain sequences, NFP did not show autophosphorylation activity, suggesting that NFP needs to associate with an active kinase or has unusual functional characteristics different from classical kinases. Identification of nine new M. truncatula LysM-RLK genes revealed a larger family than in the nonlegumes Arabidopsis (Arabidopsis thaliana) or rice (Oryza sativa) of at least 17 members that can be divided into three subfamilies. Three LysM domains could be structurally predicted for all M. truncatula LysM-RLK proteins, whereas one subfamily, which includes NFP, was characterized by deviations from conserved kinase sequences. Most of the newly identified genes were found to be expressed in roots and nodules, suggesting this class of receptors may be more extensively involved in nodulation than was previously known.

  5. Sixteen-kinase gene expression identifies luminal breast cancers with poor prognosis.

    PubMed

    Finetti, Pascal; Cervera, Nathalie; Charafe-Jauffret, Emmanuelle; Chabannon, Christian; Charpin, Colette; Chaffanet, Max; Jacquemier, Jocelyne; Viens, Patrice; Birnbaum, Daniel; Bertucci, François

    2008-02-01

    Breast cancer is a heterogeneous disease made of various molecular subtypes with different prognosis. However, evolution remains difficult to predict within some subtypes, such as luminal A, and treatment is not as adapted as it should be. Refinement of prognostic classification and identification of new therapeutic targets are needed. Using oligonucleotide microarrays, we profiled 227 breast cancers. We focused our analysis on two major breast cancer subtypes with opposite prognosis, luminal A (n = 80) and basal (n = 58), and on genes encoding protein kinases. Whole-kinome expression separated luminal A and basal tumors. The expression (measured by a kinase score) of 16 genes encoding serine/threonine kinases involved in mitosis distinguished two subgroups of luminal A tumors: Aa, of good prognosis and Ab, of poor prognosis. This classification and its prognostic effect were validated in 276 luminal A cases from three independent series profiled across different microarray platforms. The classification outperformed the current prognostic factors in univariate and multivariate analyses in both training and validation sets. The luminal Ab subgroup, characterized by high mitotic activity compared with luminal Aa tumors, displayed clinical characteristics and a kinase score intermediate between the luminal Aa subgroup and the luminal B subtype, suggesting a continuum in luminal tumors. Some of the mitotic kinases of the signature represent therapeutic targets under investigation. The identification of luminal A cases of poor prognosis should help select appropriate treatment, whereas the identification of a relevant kinase set provides potential targets.

  6. Genetic and biochemical characterization of the thymidine kinase gene from herpesvirus of turkeys.

    PubMed Central

    Martin, S L; Aparisio, D I; Bandyopadhyay, P K

    1989-01-01

    The thymidine kinase gene encoded by herpesvirus of turkeys has been identified and characterized. A viral mutant (ATR0) resistant to 1-beta-D-arabinofuranosylthymine was isolated. This mutant was also resistant to 1-(2-fluoro-2-deoxy-beta-D-arabinofuronosyl)-5-methyluracil and was unable to incorporate [125I]deoxycytidine into DNA. The mutant phenotype was rescued by a cloned region of the turkey herpesvirus genome whose DNA sequence was found to contain an open reading frame similar to that for known thymidine kinases from other viruses. When expressed in Escherichia coli, this open reading frame complemented a thymidine kinase-deficient strain and resulted in thymidine kinase activity in extracts assayed in vitro. Images PMID:2724415

  7. The Arabidopsis ERECTA gene encodes a putative receptor protein kinase with extracellular leucine-rich repeats.

    PubMed Central

    Torii, K U; Mitsukawa, N; Oosumi, T; Matsuura, Y; Yokoyama, R; Whittier, R F; Komeda, Y

    1996-01-01

    Arabidopsis Landsberg erecta is one of the most popular ecotypes and is used widely for both molecular and genetic studies. It harbors the erecta (er) mutation, which confers a compact inflorescence, blunt fruits, and short petioles. We have identified five er mutant alleles from ecotypes Columbia and Wassilewskija. Phenotypic characterization of the mutant alleles suggests a role for the ER gene in regulating the shape of organs originating from the shoot apical meristem. We cloned the ER gene, and here, we report that it encodes a putative receptor protein kinases. The deduced ER protein contains a cytoplasmic protein kinase catalytic domain, a transmembrane region, and an extracellular domain consisting of leucine-rich repeats, which are thought to interact with other macromolecules. Our results suggest that cell-cell communication mediated by a receptor kinase has an important role in plant morphogenesis. PMID:8624444

  8. Genome-wide identification and transcriptional expression analysis of mitogen-activated protein kinase and mitogen-activated protein kinase kinase genes in Capsicum annuum

    PubMed Central

    Liu, Zhiqin; Shi, Lanping; Liu, Yanyan; Tang, Qian; Shen, Lei; Yang, Sheng; Cai, Jinsen; Yu, Huanxin; Wang, Rongzhang; Wen, Jiayu; Lin, Youquan; Hu, Jiong; Liu, Cailing; Zhang, Yangwen; Mou, Shaoliang; He, Shuilin

    2015-01-01

    The tripartite mitogen-activated protein kinase (MAPK) signaling cascades have been implicated in plant growth, development, and environment adaptation, but a comprehensive understanding of MAPK signaling at genome-wide level is limited in Capsicum annuum. Herein, genome-wide identification and transcriptional expression analysis of MAPK and MAPK kinase (MAPKK) were performed in pepper. A total of 19 pepper MAPK (CaMAPKs) genes and five MAPKK (CaMAPKKs) genes were identified. Phylogenetic analysis indicated that CaMAPKs and CaMAPKKs could be classified into four groups and each group contains similar exon-intron structures. However, significant divergences were also found. Notably, five members of the pepper MAPKK family were much less conserved than those found in Arabidopsis, and 9 Arabidopsis MAPKs did not have orthologs in pepper. Additionally, 7 MAPKs in Arabidopsis had either two or three orthologs in the pepper genome, and six pepper MAPKs and one MAPKK differing in sequence were found in three pepper varieties. Quantitative real-time RT-PCR analysis showed that the majority of MAPK and MAPKK genes were ubiquitously expressed and transcriptionally modified in pepper leaves after treatments with heat, salt, and Ralstonia solanacearum inoculation as well as exogenously applied salicylic acid, methyl jasmonate, ethephon, and abscisic acid. The MAPKK-MAPK interactome was tested by yeast two-hybrid assay, the results showed that one MAPKK might interact with multiple MAPKs, one MAPK might also interact with more than one MAPKKs, constituting MAPK signaling networks which may collaborate in transmitting upstream signals into appropriate downstream cellular responses and processes. These results will facilitate future functional characterization of MAPK cascades in pepper. PMID:26442088

  9. P53 codon 72 Arg/Pro polymorphism and glioma risk: an updated meta-analysis.

    PubMed

    He, Fang; Xia, Yi; Liu, Huafeng; Li, Jin; Wang, Chao

    2013-10-01

    P53 codon 72 Arg/Pro is a C/G variation upstream of the p53 gene on human chromosome 17p13. Many case-control studies have investigated the association between p53 codon 72 Arg/Pro polymorphism and glioma risk but provided inconsistent findings. To better understand the pathogenesis of glioma, we performed the current meta-analysis by pooling data from all available individual studies. According to the inclusion criteria, ten independent publications with 11 case-control studies were included into this meta-analysis. The pooled odds ratio (OR) with 95 % confidence interval (95 % CI) was calculated to estimate the effect of p53 codon 72 Arg/Pro variant on the development of glioma. Overall, no appreciable correlation was observed among the total studies in all gene models (ORPro allele vs. Arg allele = 1.04, 95 % CI = 0.90-1.20, P OR = 0.581; ORPro/Pro vs. Arg/Arg = 0.95, 95 % CI = 0.80-1.14, P OR = 0.614; ORPro/Arg vs. Arg/Arg = 1.01, 95 % CI = 0.79-1.29, P OR = 0.993; ORPro/Arg + Pro/Pro vs. Arg/Arg = 1.03, 95 % CI = 0.82-1.29, P OR = 0.799; ORPro/Pro vs. Arg/Arg + Pro/Arg = 1.02, 95 % CI = 0.86-1.22, P OR = 0.785). In stratified analyses by ethnicity, source of controls, and glioma subtypes, the p53 codon 72 Arg/Pro polymorphism did not alter the risk for glioma in population-based, hospital-based, astrocytoma, and oligodendroglioma studies among Caucasian. Interestingly, the Pro/Pro genotype seemed to be negatively associated with the glioma risk among patients with glioblastoma (ORPro/Pro vs. Arg/Arg = 0.68, 95 % CI = 0.48-0.95, P OR = 0.026). Our study suggests that the polymorphism of p53 codon 72 Arg/Pro may play a protective role in the development of glioblastoma. The relationship of p53 codon 72 Arg/Pro polymorphism with the susceptibility to glioma needs further estimation by more individual studies with high quality across ethnicities.

  10. Mediator Kinase Inhibition Further Activates Super-Enhancer Associated Genes in AML

    PubMed Central

    Nitulescu, Ioana I.; Tangpeerachaikul, Anupong; Poss, Zachary C.; Da Silva, Diogo H.; Caruso, Brittany T.; Arefolov, Alexander; Fadeyi, Olugbeminiyi; Christie, Amanda L.; Du, Karrie; Banka, Deepti; Schneider, Elisabeth V.; Jestel, Anja; Zou, Ge; Si, Chong; Ebmeier, Christopher C.; Bronson, Roderick T.; Krivtsov, Andrei V.; Myers, Andrew G.; Kohl, Nancy E.; Kung, Andrew L.; Armstrong, Scott A.; Lemieux, Madeleine E.; Taatjes, Dylan J.; Shair, Matthew D.

    2015-01-01

    Super-enhancers (SEs), which are composed of large clusters of enhancers densely loaded with the Mediator complex, transcription factors (TFs), and chromatin regulators, drive high expression of genes implicated in cell identity and disease, such as lineage-controlling TFs and oncogenes 1, 2. BRD4 and CDK7 are positive regulators of SE-mediated transcription3,4,5. In contrast, negative regulators of SE-associated genes have not been well described. Here we report that Mediator-associated kinases cyclin-dependent kinase 8 (CDK8) and CDK19 restrain increased activation of key SE-associated genes in acute myeloid leukaemia (AML) cells. We determined that the natural product cortistatin A (CA) selectively inhibited Mediator kinases, had antileukaemic activity in vitro and in vivo, and disproportionately induced upregulation of SE-associated genes in CA-sensitive AML cell lines but not in CA-insensitive cell lines. In AML cells, CA upregulated SE-associated genes with tumour suppressor and lineage-controlling functions, including the TFs CEBPA, IRF8, IRF1 and ETV6 6, 7, 8. The BRD4 inhibitor I-BET151 downregulated these SE-associated genes, yet also has antileukaemic activity. Individually increasing or decreasing expression of these TFs suppressed AML cell growth, providing evidence that leukaemia cells are sensitive to dosage of SE-associated genes. Our results demonstrate that Mediator kinases can negatively regulate SE-associated gene expression in specific cell types and can be pharmacologically targeted as a therapeutic approach to AML. PMID:26416749

  11. Programmed cell death genes are linked to elevated creatine kinase levels in unhealthy male nonagenarians

    PubMed Central

    Kim, Sangkyu; Simon, Eric; Myers, Leann; Hamm, L. Lee; Jazwinski, S. Michal

    2016-01-01

    Declining health in the oldest-old takes an energy toll for simple maintenance of body functions. The underlying mechanisms, however, differ in males and females. In females, the declines are explained by loss of muscle mass, but this is not the case in males in whom they are associated with increased levels of circulating creatine kinase. This relationship raises the possibility that muscle damage rather than muscle loss is the cause of the increased energy demands of unhealthy aging in males. We have now examined factors that contribute to the increase in creatine kinase. Much of it (60%) can be explained by a history of cardiac problems and lower kidney function, while being mitigated by moderate physical activity, reinforcing the notion that tissue damage is a likely source. In a search for genetic risk factors associated with elevated creatine kinase, the Ku70 gene XRCC6 and the ceramide synthase gene LASS1 were investigated because of their roles in telomere length and longevity and healthy aging, respectively. Single-nucleotide polymorphisms in these two genes were independently associated with creatine kinase levels. The XRCC6 variant was epistatic to one of the LASS1 variants but not to the other. These gene variants have potential regulatory activity. Ku70 is an inhibitor of the pro-apoptotic Bax, while the product of Lass1, ceramide, operates in both caspase-dependent and independent pathways of programmed cell death, providing a potential cellular mechanism for the effects of these genes on tissue damage and circulating creatine kinase. PMID:26913518

  12. Aurora kinase B activity is modulated by thyroid hormone during transcriptional activation of pituitary genes.

    PubMed

    Tardáguila, Manuel; González-Gugel, Elena; Sánchez-Pacheco, Aurora

    2011-03-01

    Covalent histone modifications clearly play an essential role in ligand-dependent transcriptional regulation by nuclear receptors. One of the predominant mechanisms used by nuclear receptors to activate or repress target-gene transcription is the recruitment of coregulatory factors capable of covalently modify the amino terminal ends of histones. Here we show that the thyroid hormone (T3) produces a rapid increase in histone H3Ser10 phosphorylation (H3Ser10ph) concomitant to the rapid displacement of the heterochromatin protein 1β (HP1β) to the nuclear periphery. Moreover, we found that T3-mediated pituitary gene transcription is associated with an increase in H3Ser10ph. Interestingly, the Aurora kinase B inhibitor ZM443979 abolishes the effect of T3 on H3Ser10ph, blocks HP1β delocalization, and significantly reduces ligand-dependent transactivation. Similar effects were shown when Aurora kinase B expression was abrogated in small interfering RNA assays. In an effort to understand the underlying mechanism by which T3 increases H3Ser10ph, we demonstrate that liganded thyroid hormone receptor directly interacts with Aurora kinase B, increasing its kinase activity. Moreover, using chromatin immunoprecipitation assays, we have shown that Aurora kinase B participates of a mechanism that displaces HP1β from promoter region, thus preparing the chromatin for the transcriptional activation of T3 regulated genes. Our findings reveal a novel role for Aurora kinase B during transcriptional initiation in GO/G1, apart from its well-known mitotic activity.

  13. Comprehensive gene expression atlas for the Arabidopsis MAP kinase signalling pathways.

    PubMed

    Menges, Margit; Dóczi, Róbert; Okrész, László; Morandini, Piero; Mizzi, Luca; Soloviev, Mikhail; Murray, James A H; Bögre, László

    2008-01-01

    * Mitogen activated protein kinase (MAPK) pathways are signal transduction modules with layers of protein kinases having c. 120 genes in Arabidopsis, but only a few have been linked experimentally to functions. * We analysed microarray expression data for 114 MAPK signalling genes represented on the ATH1 Affymetrix arrays; determined their expression patterns during development, and in a wide range of time-course microarray experiments for their signal-dependent transcriptional regulation and their coregulation with other signalling components and transcription factors. * Global expression correlation of the MAPK genes with each of the represented 21 692 Arabidopsis genes was determined by calculating Pearson correlation coefficients. To group MAPK signalling genes based on similarities in global regulation, we performed hierarchical clustering on the pairwise correlation values. This should allow inferring functional information from well-studied MAPK components to functionally uncharacterized ones. Statistical overrepresentation of specific gene ontology (GO) categories in the gene lists showing high expression correlation values with each of the MAPK components predicted biological themes for the gene functions. * The combination of these methods provides functional information for many uncharacterized MAPK genes, and a framework for complementary future experimental dissection of the function of this complex family.

  14. Mutations in the pantothenate kinase gene PANK2 are not associated with Parkinson disease.

    PubMed

    Klopstock, Thomas; Elstner, Matthias; Lücking, Christoph B; Müller-Myhsok, Bertram; Gasser, Thomas; Botz, Evelyn; Lichtner, Peter; Hörtnagel, Konstanze

    2005-05-13

    Pantothenate kinase-associated neurodegeneration (PKAN) may serve as a model for Parkinson disease (PD) since many PKAN patients suffer from parkinsonism and both conditions lead to iron accumulation in the basal ganglia. We screened the gene coding for pantothenate kinase 2 (PANK2) for sequence variants in PD. We found no mutations in 67 PD patients with affected sibs or early-onset disease. Moreover, PANK2 polymorphisms were not associated with late-onset idiopathic PD in 339 patients. We conclude that PANK2 variants exert, if any, only a very small effect in the genetic risk of PD.

  15. Dictyostelium discoideum has a single diacylglycerol kinase gene with similarity to mammalian theta isoforms.

    PubMed Central

    De La Roche, Marc A; Smith, Janet L; Rico, Maribel; Carrasco, Silvia; Merida, Isabel; Licate, Lucila; Côté, Graham P; Egelhoff, Thomas T

    2002-01-01

    Diacylglycerol kinases (DGKs) phosphorylate the neutral lipid diacylglycerol (DG) to produce phosphatidic acid (PA). In mammalian systems DGKs are a complex family of at least nine isoforms that are thought to participate in down-regulation of DG-based signalling pathways and perhaps activation of PA-stimulated signalling events. We report here that the simple protozoan amoeba Dictyostelium discoideum appears to contain a single gene encoding a DGK enzyme. This gene, dgkA, encodes a deduced protein that contains three C1-type cysteine-rich repeats, a DGK catalytic domain most closely related to the theta subtype of mammalian DGKs and a C-terminal segment containing a proline/glutamine-rich region and a large aspargine-repeat region. This gene corresponds to a previously reported myosin II heavy chain kinase designated myosin heavy chain-protein kinase C (MHC-PKC), but our analysis clearly demonstrates that this protein does not, as suggested by earlier data, contain a protein kinase catalytic domain. A FLAG-tagged version of DgkA expressed in Dictyostelium displayed robust DGK activity. Earlier studies indicating that disruption of this locus alters myosin II assembly levels in Dictyostelium raise the intriguing possibility that DG and/or PA metabolism may play a role in controlling myosin II assembly in this system. PMID:12296770

  16. Identification of new protein kinase-related genes in three herpesviruses, herpes simplex virus, varicella-zoster virus, and Epstein-Barr virus.

    PubMed Central

    Smith, R F; Smith, T F

    1989-01-01

    By using amino acid sequence patterns (motifs) diagnostic of conserved regions within the catalytic domains of protein kinases, homologous open reading frames of three herpesviruses were identified as protein kinase-related genes. The three sequences, herpes simplex virus gene UL13, varicella-zoster virus gene 47, and Epstein-Barr virus gene BGLF4, resemble serine/threonine kinases rather than tyrosine kinases. PMID:2535748

  17. Case–control study and meta-analysis of SULT1A1 Arg213His polymorphism for gene, ethnicity and environment interaction for cancer risk

    PubMed Central

    Kotnis, A; Kannan, S; Sarin, R; Mulherkar, R

    2008-01-01

    Cytosolic sulphotransferase SULT1A1 plays a dual role in the activation of some carcinogens and inactivation of others. A functional polymorphism leading to Arg213His substitution (SULT1A1*2) affects its catalytic activity and thermostability. To study the association of SULT1A1*2 polymorphism with tobacco-related cancers (TRCs), a case–control study comprising 132 patients with multiple primary neoplasm (MPN) involving TRC and 198 cancer-free controls was carried out. One hundred and thirteen MPN patients had at least one cancer in upper aerodigestive tract including lung (UADT-MPN). SULT1A1*2 showed significant risk association with UADT-MPN (odds ratio (OR)=5.50, 95% confidence interval (CI): 1.09, 27.7). Meta-analysis was conducted combining the data with 34 published studies that included 11 962 cancer cases and 14 673 controls in diverse cancers. The SULT1A1*2 revealed contrasting risk association for UADT cancers (OR=1.62, 95% CI: 1.12, 2.34) and genitourinary cancers (OR=0.73, 95% CI: 0.58, 0.92). Furthermore, although SULT1A1*2 conferred significant increased risk of breast cancer to Asian women (OR=1.91, 95% CI: 1.08, 3.40), it did not confer increased risk to Caucasian women (OR=0.92, 95% CI: 0.71, 1.18). Thus risk for different cancers in distinct ethnic groups could be modulated by interaction between genetic variants and different endogenous and exogenous carcinogens. PMID:18854828

  18. Pantothenate kinase-associated neurodegeneration in two Chinese children: identification of a novel PANK2 gene mutation.

    PubMed

    Chan, K Y; Lam, C W; Lee, L P; Tong, S F; Yuen, Y P

    2008-02-01

    Pantothenate kinase-associated neurodegeneration (formerly Hallervorden-Spatz syndrome), the most prevalent form of neurodegeneration with brain iron accumulation, is a rare degenerative brain disease characterised by predominantly extrapyramidal dysfunction resulting from mutations in the PANK2 (pantothenate kinase 2) gene. Using DNA mutation analysis, the authors identified a novel missense mutation (P354L) in exon 4 of the PANK2 gene in an adolescent with classic pantothenate kinase-associated neurodegeneration. DNA-based diagnosis of pantothenate kinase-associated neurodegeneration plays a key role in determination, and can make the diagnosis more simply, directly, and economically because it obviates the need for unnecessary biochemical tests. Once pantothenate kinase-associated neurodegeneration-like symptoms are identified, mutation analysis and target screening for the family of the proband can provide efficient and accurate evidence of pantothenate kinase-associated neurodegeneration inheritance.

  19. Cloning and characterization of a protein kinase C gene from the spruce budworm, Choristoneura fumiferana.

    PubMed

    Quan, Guo-Xing; Doucet, Daniel; Ladd, Tim; Krell, Peter J; Arif, Basil M

    2008-11-01

    Recent studies have implicated protein kinase C (PKC) in the control of 20-hydroxyecdysone (20E)-dependent gene expression during molting and metamorphosis in insects. To further understand the role of this kinase in 20E signal transduction, we cloned a homolog of mammalian PKC by RT-PCR and 5'/3'-RACE from adult of the moth Choristoneura fumiferana. The full-length cDNA of the C. fumiferana PKC (CfPKC1) is 2.3 kb with an open reading frame encoding a protein of 669 amino acids. The deduced amino acid sequence contains all the characteristic features of the classical protein kinase C subfamily. Northern and Western blot analysis showed that CfPKC1 was distributed ubiquitously in various tissues and at different developmental stages. Activation of CfPKC1 with the PKC activator phorbol 12-myristate 13-acetate (PMA) resulted in a rapid redistribution of the protein from the cytosol to the plasma membrane. Knock-down of the CfPKC1 gene by double-stranded RNA interference or treatment of the CF-203 cells with PKC-specific inhibitors reduces the expression of the 20E-responsive genes CHR3 and E75. This data suggests that CfPKC1 is involved in the 20E-response gene expression in C. fumiferana.

  20. Transcriptional upregulation of the human MRP2 gene expression by serine/threonine protein kinase inhibitors.

    PubMed

    Pułaski, L; Szemraj, J; Uchiumi, T; Kuwano, M; Bartosz, G

    2005-01-01

    Transcriptional regulation by cellular signalling pathways of multidrug resistance proteins that pump anticancer drugs out of cells is one of key issues in the development of the multidrug resistance phenotype. In our study, we have used the reporter gene approach as well as determination of mRNA levels in two cancer cell lines of human origin, MCF-7 and A549, to study the regulation of multidrug resistance proteins 2 and 3 (MRP2 AND MRP3) by serine/threonine protein kinases. Since a prototypic PKC inducer, PMA, caused a marked upregulation of transcription from both human MRP2 and MRP3 promoters, a role for PKC isoforms in positive control of expression of these proteins could be postulated. Interestingly, broad-spectrum serine-threonine protein kinase inhibitors which also inhibit PKC, staurosporine and H-7, stimulated expression from the MRP2 promoter instead of inhibiting it. This effect was not seen for MRP3. MRP2 induction by staurosporine and H-7 was shown to have phenotypic consequences in whole cells, rendering them more resistant to etoposide and increasing their ability to export calcein through the plasma membrane. These results point to the involvement of serine/threonine protein kinases in negative regulation of the human MRP2 gene and to the necessity of testing novel anti-cancer drugs acting as protein kinase inhibitors with regard to their potential ability to induce multidrug resistance.

  1. p.Arg82Leu von Hippel-Lindau (VHL) Gene Mutation among Three Members of a Family with Familial Bilateral Pheochromocytoma in India: Molecular Analysis and In Silico Characterization

    PubMed Central

    John, Anulekha Mary; C, George Priya Doss; Ebenazer, Andrew; Seshadri, Mandalam Subramaniam; Nair, Aravindan; Rajaratnam, Simon; Pai, Rekha

    2013-01-01

    Various missense mutations in the VHL gene have been reported among patients with familial bilateral pheochromocytoma. However, the p.Arg82Leu mutation in the VHL gene described here among patients with familial bilateral pheochromocytoma, has never been reported previously in a germline configuration. Interestingly, long-term follow-up of these patients indicated that the mutation might have had little impact on the normal function of the VHL gene, since all of them have remained asymptomatic. We further attempted to correlate this information with the results obtained by in silico analysis of this mutation using SIFT, PhD-SNP SVM profile, MutPred, PolyPhen2, and SNPs&GO prediction tools. To gain, new mechanistic insight into the structural effect, we mapped the mutation on to 3D structure (PDB ID 1LM8). Further, we analyzed the structural level changes in time scale level with respect to native and mutant protein complexes by using 12 ns molecular dynamics simulation method. Though these methods predict the mutation to have a pathogenic potential, it remains to be seen if these patients will eventually develop symptomatic disease. PMID:23626751

  2. P.Arg82Leu von Hippel-Lindau (VHL) gene mutation among three members of a family with familial bilateral pheochromocytoma in India: molecular analysis and in silico characterization.

    PubMed

    John, Anulekha Mary; C, George Priya Doss; Ebenazer, Andrew; Seshadri, Mandalam Subramaniam; Nair, Aravindan; Rajaratnam, Simon; Pai, Rekha

    2013-01-01

    Various missense mutations in the VHL gene have been reported among patients with familial bilateral pheochromocytoma. However, the p.Arg82Leu mutation in the VHL gene described here among patients with familial bilateral pheochromocytoma, has never been reported previously in a germline configuration. Interestingly, long-term follow-up of these patients indicated that the mutation might have had little impact on the normal function of the VHL gene, since all of them have remained asymptomatic. We further attempted to correlate this information with the results obtained by in silico analysis of this mutation using SIFT, PhD-SNP SVM profile, MutPred, PolyPhen2, and SNPs&GO prediction tools. To gain, new mechanistic insight into the structural effect, we mapped the mutation on to 3D structure (PDB ID 1LM8). Further, we analyzed the structural level changes in time scale level with respect to native and mutant protein complexes by using 12 ns molecular dynamics simulation method. Though these methods predict the mutation to have a pathogenic potential, it remains to be seen if these patients will eventually develop symptomatic disease.

  3. Analysis of kinase gene expression in the frontal cortex of suicide victims: implications of fear and stress.

    PubMed

    Choi, Kwang; Le, Thien; Xing, Guoqiang; Johnson, Luke R; Ursano, Robert J

    2011-01-01

    Suicide is a serious public health issue that results from an interaction between multiple risk factors including individual vulnerabilities to complex feelings of hopelessness, fear, and stress. Although kinase genes have been implicated in fear and stress, including the consolidation and extinction of fearful memories, expression profiles of those genes in the brain of suicide victims are less clear. Using gene expression microarray data from the Online Stanley Genomics Database and a quantitative PCR, we investigated the expression profiles of multiple kinase genes including the calcium calmodulin-dependent kinase (CAMK), the cyclin-dependent kinase, the mitogen-activated protein kinase (MAPK), and the protein kinase C (PKC) in the prefrontal cortex (PFC) of mood disorder patients died with suicide (N = 45) and without suicide (N = 38). We also investigated the expression pattern of the same genes in the PFC of developing humans ranging in age from birth to 49 year (N = 46). The expression levels of CAMK2B, CDK5, MAPK9, and PRKCI were increased in the PFC of suicide victims as compared to non-suicide controls (false discovery rate, FDR-adjusted p < 0.05, fold change >1.1). Those genes also showed changes in expression pattern during the postnatal development (FDR-adjusted p < 0.05). These results suggest that multiple kinase genes undergo age-dependent changes in normal brains as well as pathological changes in suicide brains. These findings may provide an important link to protein kinases known to be important for the development of fear memory, stress associated neural plasticity, and up-regulation in the PFC of suicide victims. More research is needed to better understand the functional role of these kinase genes that may be associated with the pathophysiology of suicide.

  4. Identification of expression profiles of tapping panel dryness (TPD) associated genes from the latex of rubber tree (Hevea brasiliensis Muell. Arg.).

    PubMed

    Venkatachalam, Perumal; Thulaseedharan, Arjunan; Raghothama, Kashchandra

    2007-07-01

    Tapping panel dryness (TPD) occurrence in high latex yielding rubber tree (Hevea brasiliensis) is characterized by the partial or complete cessation of latex flow upon tapping leading to severe loss in natural rubber production around the world. The goal of this study was to identify genes whose mRNA transcript levels are differentially regulated in rubber tree during the onset of TPD. To isolate TPD responsive genes, two cDNA libraries (forward and reverse) from total RNA isolated from latex of healthy and TPD trees were constructed using suppression subtractive hybridization (SSH) method. In total, 1,079 EST clones were obtained from two cDNA libraries and screened by reverse Northern blot analysis. Screening results revealed that about 352 clones were differentially regulated and they were selected for sequencing. Based on the nucleotide sequence data, the putative functions of cDNA clones were predicted by BLASTX/BLASTN analysis. Among these, 64 were genes whose function had been previously identified while the remaining clones were genes with either unknown protein function or insignificant similarity to other protein/DNA/EST sequences in existing databases. RT-PCR analysis was carried out to validate the up-regulated genes from both the libraries. Among them, two genes were strongly down-regulated in TPD trees. The level of mRNA transcripts of these two genes was further examined by conventional Northern and RT-PCR analysis. Results indicated that the expression level of two genes was significantly lower in TPD trees compared to healthy trees. Many TPD associated genes were also up-regulated in TPD trees suggesting that they may be involved in triggering programmed cell death (PCD) during the onset of TPD syndrome. The results presented here demonstrate that SSH technique provides a powerful complementary approach for the identification of TPD related genes from rubber tree.

  5. The human liver-type pyruvate kinase (PKL) gene is on chromosome 1 at band q21.

    PubMed

    Satoh, H; Tani, K; Yoshida, M C; Sasaki, M; Miwa, S; Fujii, H

    1988-01-01

    Pyruvate kinase (PK) is an important enzyme for ATP production in the glycolytic pathway. Deficiency of this enzyme in erythrocytes is characterized by hemolytic anemia. Using in situ hybridization, we have mapped the human liver-type pyruvate kinase gene (PKL) to band q21 of chromosome 1.

  6. Expression and characterization of the thymidine kinase gene of African swine fever virus.

    PubMed Central

    Martin Hernandez, A M; Tabares, E

    1991-01-01

    The thymidine kinase (TK) gene of African swine fever virus (ASFV) was located within the viral genome by using two degenerate oligonucleotide probes derived from sequences of the vaccinia virus and cellular TK genes. The TK gene was mapped within a 0.72-kbp BglII-XhoI fragment (0.242 to 0.246 map units) derived from a 23.9-kbp SalI-B fragment of the ASFV genome. Identification of this region as the ASFV TK gene was confirmed by expression of TK in Escherichia coli and by the synthesis of active TK in a cell-free system programmed with RNA synthesized in vitro. The sequenced gene for TK includes an open reading frame of 588 nucleotides encoding a protein of 196 amino acids. The deduced amino acid sequence shows 32.4% identity with the TK of vaccinia virus. Images PMID:1987368

  7. Myxococcus xanthus sasS encodes a sensor histidine kinase required for early developmental gene expression.

    PubMed Central

    Yang, C; Kaplan, H B

    1997-01-01

    Initiation of Myxococcus xanthus multicellular development requires integration of information concerning the cells' nutrient status and density. A gain-of-function mutation, sasB7, that bypasses both the starvation and high cell density requirements for developmental expression of the 4521 reporter gene, maps to the sasS gene. The wild-type sasS gene was cloned and sequenced. This gene is predicted to encode a sensor histidine protein kinase that appears to be a key element in the transduction of starvation and cell density inputs. The sasS null mutants express 4521 at a basal level, form defective fruiting bodies, and exhibit reduced sporulation efficiencies. These data indicate that the wild-type sasS gene product functions as a positive regulator of 4521 expression and participates in M. xanthus development. The N terminus of SasS is predicted to contain two transmembrane domains that would locate the protein to the cytoplasmic membrane. The sasB7 mutation, an E139K missense mutation, maps to the predicted N-terminal periplasmic region. The C terminus of SasS contains all of the conserved residues typical of the sensor histidine protein kinases. SasS is predicted to be the sensor protein in a two-component system that integrates information required for M. xanthus developmental gene expression. PMID:9401035

  8. A Kinase-Independent Activity of Cdk9 Modulates Glucocorticoid Receptor-Mediated Gene Induction

    PubMed Central

    2015-01-01

    A gene induction competition assay has recently uncovered new inhibitory activities of two transcriptional cofactors, NELF-A and NELF-B, in glucocorticoid-regulated transactivation. NELF-A and -B are also components of the NELF complex, which participates in RNA polymerase II pausing shortly after the initiation of gene transcription. We therefore asked if cofactors (Cdk9 and ELL) best known to affect paused polymerase could reverse the effects of NELF-A and -B. Unexpectedly, Cdk9 and ELL augmented, rather than prevented, the effects of NELF-A and -B. Furthermore, Cdk9 actions are not blocked either by Ckd9 inhibitors (DRB or flavopiridol) or by two Cdk9 mutants defective in kinase activity. The mode and site of action of NELF-A and -B mutants with an altered NELF domain are similarly affected by wild-type and kinase-dead Cdk9. We conclude that Cdk9 is a new modulator of GR action, that Ckd9 and ELL have novel activities in GR-regulated gene expression, that NELF-A and -B can act separately from the NELF complex, and that Cdk9 possesses activities that are independent of Cdk9 kinase activity. Finally, the competition assay has succeeded in ordering the site of action of several cofactors of GR transactivation. Extension of this methodology should be helpful in determining the site and mode of action of numerous additional cofactors and in reducing unwanted side effects. PMID:24559102

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

    PubMed Central

    Muchir, Antoine; Worman, Howard J.

    2016-01-01

    The most frequently occurring mutations in the gene encoding nuclear lamin A and nuclear lamin C cause striated muscle diseases virtually always involving the heart. In this review, we describe the approaches and methods used to discover that cardiomyopathy-causing lamin A/C gene mutations increase MAP kinase signaling in the heart and that this plays a role in disease pathogenesis. We review different mouse models of cardiomyopathy caused by lamin A/C gene mutations and how transcriptomic analysis of one model identified increased cardiac activity of the ERK1/2, JNK, and p38α MAP kinases. We describe methods used to measure the activity of these MAP kinases in mouse hearts and then discuss preclinical treatment protocols using pharmacological inhibitors to demonstrate their role in pathogenesis. Several of these kinase inhibitors are in clinical development and could potentially be used to treat human subjects with cardiomyopathy caused by lamin A/C gene mutations. PMID:26795484

  10. Human protein kinase C lota gene (PRKC1) is closely linked to the BTK gene in Xq21.3

    SciTech Connect

    Mazzarella, R.; Jones, C.; Schlessinger, D.

    1995-04-10

    The human X chromosome contains many disease loci, but only a small number of X-linked genes have been cloned and characterized. One approach to finding genes in genomic DNA uses partial sequencing of random cDNAs to develop {open_quotes}expressed sequence tags{close_quotes} (ESTs). Many authors have recently reported chromosomal localization of such ESTs using hybrid panels. Twenty ESTs specific for the X chromosome have been localized to defined regions with somatic cell hybrids, and 12 of them have been physically linked to markers that detect polymorphisms. One of these ESTs, EST02087, was physically linked in a 650-kb contig to the GLA ({alpha}-galactosidase) gene involved in Fabry disease. A comparison of this contig with a 7.5-Mb YAC contig indicated that this gene is also within 250 kb of the src-like protein-tyrosine kinase BTK (X-linked agammaglobulinemia protein-tyrosine kinase) gene in Xq21.3. 14 refs., 1 fig.

  11. Abl2/Arg controls dendritic spine and dendrite arbor stability via distinct cytoskeletal control pathways.

    PubMed

    Lin, Yu-Chih; Yeckel, Mark F; Koleske, Anthony J

    2013-01-30

    Rho family GTPases coordinate cytoskeletal rearrangements in neurons, and mutations in their regulators are associated with mental retardation and other neurodevelopmental disorders (Billuart et al., 1998; Kutsche et al., 2000; Newey et al., 2005; Benarroch, 2007). Chromosomal microdeletions encompassing p190RhoGAP or its upstream regulator, the Abl2/Arg tyrosine kinase, have been observed in cases of mental retardation associated with developmental defects (Scarbrough et al., 1988; James et al., 1996; Takano et al., 1997; Chaabouni et al., 2006; Leal et al., 2009). Genetic knock-out of Arg in mice leads to synapse, dendritic spine, and dendrite arbor loss accompanied by behavioral deficits (Moresco et al., 2005; Sfakianos et al., 2007). To elucidate the cell-autonomous mechanisms by which Arg regulates neuronal stability, we knocked down Arg in mouse hippocampal neuronal cultures. We find that Arg knockdown significantly destabilizes dendrite arbors and reduces dendritic spine density by compromising dendritic spine stability. Inhibiting RhoA prevents dendrite arbor loss following Arg knockdown in neurons, but does not block spine loss. Interestingly, Arg-deficient neurons exhibit increased miniature EPSC amplitudes, and their remaining spines exhibit larger heads deficient in the actin stabilizing protein cortactin. Spine destabilization in Arg knockdown neurons is prevented by blocking NMDA receptor-dependent relocalization of cortactin from spines, or by forcing cortactin into spines via fusion to an actin-binding region of Arg. Thus, Arg employs distinct mechanisms to selectively regulate spine and dendrite stability: Arg dampens activity-dependent disruption of cortactin localization to stabilize spines and attenuates Rho activity to stabilize dendrite arbors.

  12. Regulation of pyruvate dehydrogenase kinase isoform 4 (PDK4) gene expression by glucocorticoids and insulin.

    PubMed

    Connaughton, Sara; Chowdhury, Farhana; Attia, Ramy R; Song, Shulan; Zhang, Yi; Elam, Marshall B; Cook, George A; Park, Edwards A

    2010-02-05

    The pyruvate dehydrogenase complex (PDC) catalyzes the conversion of pyruvate to acetyl-CoA in mitochondria and is a key regulatory enzyme in the oxidation of glucose to acetyl-CoA. Phosphorylation of PDC by the pyruvate dehydrogenase kinases (PDK) inhibits its activity. The expression of the pyruvate dehydrogenase kinase 4 (PDK4) gene is increased in fasting and other conditions associated with the switch from the utilization of glucose to fatty acids as an energy source. Transcription of the PDK4 gene is elevated by glucocorticoids and inhibited by insulin. In this study, we have investigated the factors involved in the regulation of the PDK4 gene by these hormones. Glucocorticoids stimulate PDK4 through two glucocorticoid receptor (GR) binding sites located more than 6000 base pairs upstream of the transcriptional start site. Insulin inhibits the glucocorticoid induction in part by causing dissociation of the GR from the promoter. Previously, we found that the estrogen related receptor alpha (ERRalpha) stimulates the expression of PDK4. Here, we determined that one of the ERRalpha binding sites contributes to the insulin inhibition of PDK4. A binding site for the forkhead transcription factor (FoxO1) is adjacent to the ERRalpha binding sites. FoxO1 participates in the glucocorticoid induction of PDK4 and the regulation of this gene by insulin. Our data demonstrate that glucocorticoids and insulin each modulate PDK4 gene expression through complex hormone response units that contain multiple factors.

  13. Retinoblastoma cancer suppressor gene product is a substrate of the cell cycle regulator cdc2 kinase.

    PubMed Central

    Lin, B T; Gruenwald, S; Morla, A O; Lee, W H; Wang, J Y

    1991-01-01

    The retinoblastoma gene product (RB) is a nuclear protein which has been shown to function as a tumor suppressor. It is phosphorylated from S to M phase of the cell cycle and dephosphorylated in G1. This suggests that the function of RB is regulated by its phosphorylation in the cell cycle. Ten phosphotryptic peptides are found in human RB proteins. The pattern of RB phosphorylation does not change from S to M phases of the cell cycle. Hypophosphorylated RB prepared from insect cells infected with an RB-recombinant baculovirus is used as a substrate for in vitro phosphorylation reactions. Of several protein kinases tested, only cdc2 kinase phosphorylates RB efficiently and all 10 peptides can be phosphorylated by cdc2 in vitro. Removal of cdc2 from mitotic cell extracts by immunoprecipitation causes a concomitant depletion of RB kinase activity. These results indicate that cdc2 or a kinase with similar substrate specificity is involved in the cell cycle-dependent phosphorylation of the RB protein. Images PMID:2009861

  14. Arabidopsis Raf-Like Mitogen-Activated Protein Kinase Kinase Kinase Gene Raf43 Is Required for Tolerance to Multiple Abiotic Stresses

    PubMed Central

    Virk, Nasar; Li, Dayong; Tian, Limei; Huang, Lei; Hong, Yongbo; Li, Xiaohui; Zhang, Yafen; Liu, Bo; Zhang, Huijuan; Song, Fengming

    2015-01-01

    Mitogen-activated protein kinase (MAPK) cascades are critical signaling modules that mediate the transduction of extracellular stimuli into intracellular response. A relatively large number of MAPKKKs have been identified in a variety of plant genomes but only a few of them have been studied for their biological function. In the present study, we identified an Arabidopsis Raf-like MAPKKK gene Raf43 and studied its function in biotic and abiotic stress response using a T-DNA insertion mutant raf43-1 and two Raf43-overexpressing lines Raf43-OE#1 and Raf43-OE#13. Expression of Raf43 was induced by multiple abiotic and biotic stresses including treatments with drought, mannitol and oxidative stress or defense signaling molecule salicylic acid and infection with necrotrophic fungal pathogen Botrytis cinerea. Seed germination and seedling root growth of raf43-1 were significantly inhibited on MS medium containing mannitol, NaCl, H2O2 or methyl viologen (MV) while seed germination and seedling root growth of the Raf43-OE#1 and Raf43-OE#13 lines was similar to wild type Col-0 under the above stress conditions. Soil-grown raf43-1 plants exhibited reduced tolerance to MV, drought and salt stress. Abscisic acid inhibited significantly seed germination and seedling root growth of the raf43-1 line but had no effect on the two Raf43-overexpressing lines. Expression of stress-responsive RD17 and DREB2A genes was significantly down-regulated in raf43-1 plants. However, the raf43-1 and Raf43-overexpressing plants showed similar disease phenotype to the wild type plants after infection with B. cinerea or Pseudomonas syringae pv. tomato DC3000. Our results demonstrate that Raf43, encoding for a Raf-like MAPKKK, is required for tolerance to multiple abiotic stresses in Arabidopsis. PMID:26222830

  15. Discovery of rice essential genes by characterizing a CRISPR-edited mutation of closely related rice MAP kinase genes.

    PubMed

    Minkenberg, Bastian; Xie, Kabin; Yang, Yinong

    2017-02-01

    The clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated protein 9 nuclease (Cas9) system depends on a guide RNA (gRNA) to specify its target. By efficiently co-expressing multiple gRNAs that target different genomic sites, the polycistronic tRNA-gRNA gene (PTG) strategy enables multiplex gene editing in the family of closely related mitogen-activated protein kinase (MPK) genes in Oryza sativa (rice). In this study, we identified MPK1 and MPK6 (Arabidopsis AtMPK6 and AtMPK4 orthologs, respectively) as essential genes for rice development by finding the preservation of MPK functional alleles and normal phenotypes in CRISPR-edited mutants. The true knock-out mutants of MPK1 were severely dwarfed and sterile, and homozygous mpk1 seeds from heterozygous parents were defective in embryo development. By contrast, heterozygous mpk6 mutant plants completely failed to produce homozygous mpk6 seeds. In addition, the functional importance of specific MPK features could be evaluated by characterizing CRISPR-induced allelic variation in the conserved kinase domain of MPK6. By simultaneously targeting between two and eight genomic sites in the closely related MPK genes, we demonstrated 45-86% frequency of biallelic mutations and the successful creation of single, double and quadruple gene mutants. Indels and fragment deletion were both stably inherited to the next generations, and transgene-free mutants of rice MPK genes were readily obtained via genetic segregation, thereby eliminating any positional effects of transgene insertions. Taken together, our study reveals the essentiality of MPK1 and MPK6 in rice development, and enables the functional discovery of previously inaccessible genes or domains with phenotypes masked by lethality or redundancy.

  16. The doublecortin and doublecortin-like kinase 1 genes cooperate in murine hippocampal development.

    PubMed

    Tanaka, Teruyuki; Koizumi, Hiroyuki; Gleeson, Joseph G

    2006-07-01

    The doublecortin (Dcx) and doublecortin-like kinase 1 (Dclk) genes are developmentally expressed neuronal microtubule-associated proteins. Humans with DCX mutations show a severe defect in hippocampal development, but targeted deletion in mouse shows only a defect in pyramidal neuron lamination. There is significant sequence overlap between Dcx and Dclk, suggesting functional redundancy. Here we show that the two genes display overlapping expression patterns in developing mouse hippocampus. Targeted deletion of Dclk shows no appreciable developmental defect in the hippocampus, but removal of both genes shows severe hippocampal lamination defects involving the entire cornu ammonis and dentate gyrus fields that mimic the human phenotype. These results suggest these genes are partially functionally redundant in the formation of the murine hippocampus.

  17. Evidence of gene deletion of p21 (WAF1/CIP1), a cyclin-dependent protein kinase inhibitor, in thyroid carcinomas.

    PubMed Central

    Shi, Y.; Zou, M.; Farid, N. R.; al-Sedairy, S. T.

    1996-01-01

    Eukaryotic cell cycle progression is controlled by a host of cyclin/cyclin-dependent kinases (Cdks), that are themselves regulated by multiple factors, including a group of small cyclin-Cdk inhibitor proteins (p15, p16, p21 and p27). The involvement of Cdk inhibitors in carcinogenesis has been demonstrated by the studies of p16. p53 is frequently mutated in thyroid carcinomas and p21/Waf1 is a downstream effector of p53. It is conceivable that genetic defects of genes downstream in the p53 pathway could also be oncogenic. We, therefore, examined a series of 57 thyroid tumour specimens (eight follicular adenomas and 49 carcinomas) for deletion and point mutation of the p21/Waf1 gene. Three different kinds of deletions ranging from 349 to 450 bp were detected in five papillary carcinoma specimens by reverse transcription-polymerase chain reaction (RT-PCR). All the deletions were involved in the second exon of the p21/Waf1 gene. RT-PCR single strand conformational polymorphism (SSCP) analysis of remaining samples failed to reveal any point mutations in the coding region of the gene, except for a polymorphism at codon 31 (Ser to Arg). Genomic Southern blot analysis did not demonstrate any gene deletion or rearrangement in these samples, indicating abnormal RNA splicing may be involved. Analysis of intron-exon boundary and the coding region of the second exon did not reveal any mutation except for a point mutation (C to G) located 16 bp downstream from the splice donor site of the second intron in three out of five samples with p21/Waf1 deletions. Whether the mutation plays any role in aberrant RNA splicing remains to be determined. Among the five samples with p21/Waf1 gene deletions, none of them simultaneously carried a p53 or retinoblastoma (Rb) gene mutation. No p21/Waf1 abnormality was found in the benign adenomas. Thus, 12.5% (5/40) of thyroid papillary carcinoma specimens harboured p21/Waf1 gene deletions. Our data suggest that p21/Waf1 gene deletion is involved

  18. Construction and Expression of Sugar Kinase Transcriptional Gene Fusions by Using the Sinorhizobium meliloti ORFeome▿

    PubMed Central

    Humann, Jodi L.; Schroeder, Brenda K.; Mortimer, Michael W.; House, Brent L.; Yurgel, Svetlana N.; Maloney, Scott C.; Ward, Kristel L.; Fallquist, Heather M.; Ziemkiewicz, Hope T.; Kahn, Michael L.

    2008-01-01

    The Sinorhizobium meliloti ORFeome project cloned 6,314 open reading frames (ORFs) into a modified Gateway entry vector system from which the ORFs could be transferred to destination vectors in vivo via bacterial conjugation. In this work, a reporter gene destination vector, pMK2030, was constructed and used to generate ORF-specific transcriptional fusions to β-glucuronidase (gusA) and green fluorescent protein (gfp) reporter genes. A total of 6,290 ORFs were successfully transferred from the entry vector library into pMK2030. To demonstrate the utility of this system, reporter plasmids corresponding to 30 annotated sugar kinase genes were integrated into the S. meliloti SM1021 and/or SM8530 genome. Expression of these genes was measured using a high-throughput β-glucuronidase assay to track expression on nine different carbon sources. Six ORFs integrated into SM1021 and SM8530 had different basal levels of expression in the two strains. The annotated activities of three other sugar kinases were also confirmed. PMID:18791020

  19. Characterization of a protein kinase gene in allelic association with the spinal muscular atrophy locus

    SciTech Connect

    Wang, C.H.; Carter, T.A.; Kleyn, P.W.

    1994-09-01

    A protein kinase gene has been identified from a 400 Kb minimal genetic region which defines the spinal muscular atrophy (SMA) locus. A highly polymorphic microsatellite marker (D5S1414) isolated from a yeast artificial chromosome (YAC) clone within this interval detects linkage disequilibrium with the SMA locus in 32 Polish families (Yule`s coefficient: 0.92) and maps to an intron of the protein kinase gene. Exon amplification was used to isolate coding sequences from a YAC-derived phage subclone containing D5S1414. Five exons were identified and a GenBank search using the BLAST program showed complete homology of these exons with a protein kinase gene. The gene is expressed in all tissues checked to far. Full-length cDNAs have been identified from both normal and SMA brain libraries and by reverse-transcriptase (RT) PCR from RNA of various tissues. The cDNA sequences will be reported. The genomic sequences flanking each exon were determined by direct sequencing of the homologous phage. The marker D5S1414 was located within the intronic sequence between exons 6 and 7. To screen for disease mutations, PCR was performed across each exon including the flanking splice sites in normal controls and SMA samples shown to be homozygous across the region by haplotyping. Comparative sequence analysis of the products together with the RT-PCR from normal and SMA brain RNA has identified several candidate polymorphisms. To date, the most interesting lead is an intronic polymorphism possibly affecting exon splicing in a homozygous SMA patient. An updated mutation search will be reported.

  20. Promoter sequence of 3-phosphoglycerate kinase gene 1 of lactic acid-producing fungus rhizopus oryzae and a method of expressing a gene of interest in fungal species

    DOEpatents

    Gao, Johnway [Richland, WA; Skeen, Rodney S [Pendleton, OR

    2002-10-15

    The present invention provides the promoter clone discovery of phosphoglycerate kinase gene 1 of a lactic acid-producing filamentous fungal strain, Rhizopus oryzae. The isolated promoter can constitutively regulate gene expression under various carbohydrate conditions. In addition, the present invention also provides a design of an integration vector for the transformation of a foreign gene in Rhizopus oryzae.

  1. Promoter sequence of 3-phosphoglycerate kinase gene 2 of lactic acid-producing fungus rhizopus oryzae and a method of expressing a gene of interest in fungal species

    DOEpatents

    Gao, Johnway [Richland, WA; Skeen, Rodney S [Pendleton, OR

    2003-03-04

    The present invention provides the promoter clone discovery of phosphoglycerate kinase gene 2 of a lactic acid-producing filamentous fungal strain, Rhizopus oryzae. The isolated promoter can constitutively regulate gene expression under various carbohydrate conditions. In addition, the present invention also provides a design of an integration vector for the transformation of a foreign gene in Rhizopus oryzae.

  2. Kinase Gene Expression Profiling of Metastatic Clear Cell Renal Cell Carcinoma Tissue Identifies Potential New Therapeutic Targets

    PubMed Central

    Ramaker, Ryne C.; Cooper, Sara J.; Chen, Dongquan; Sudarshan, Sunil; Wei, Shi; Guru, Arjun S.; Zhao, Amy; Cooper, Tiffiny; Della Manna, Deborah L.; Naik, Gurudatta; Myers, Richard M.; Sonpavde, Guru

    2016-01-01

    Kinases are therapeutically actionable targets. Kinase inhibitors targeting vascular endothelial growth factor receptors (VEGFR) and mammalian target of rapamycin (mTOR) improve outcomes in metastatic clear cell renal cell carcinoma (ccRCC), but are not curative. Metastatic tumor tissue has not been comprehensively studied for kinase gene expression. Paired intra-patient kinase gene expression analysis in primary tumor (T), matched normal kidney (N) and metastatic tumor tissue (M) may assist in identifying drivers of metastasis and prioritizing therapeutic targets. We compared the expression of 519 kinase genes using NanoString in T, N and M in 35 patients to discover genes over-expressed in M compared to T and N tissue. RNA-seq data derived from ccRCC tumors in The Cancer Genome Atlas (TCGA) were used to demonstrate differential expression of genes in primary tumor tissue from patients that had metastasis at baseline (n = 79) compared to those that did not develop metastasis for at least 2 years (n = 187). Functional analysis was conducted to identify key signaling pathways by using Ingenuity Pathway Analysis. Of 10 kinase genes overexpressed in metastases compared to primary tumor in the discovery cohort, 9 genes were also differentially expressed in TCGA primary tumors with metastasis at baseline compared to primary tumors without metastasis for at least 2 years: EPHB2, AURKA, GSG2, IKBKE, MELK, CSK, CHEK2, CDC7 and MAP3K8; p<0.001). The top pathways overexpressed in M tissue were pyridoxal 5'-phosphate salvage, salvage pathways of pyrimidine ribonucleotides, NF-kB signaling, NGF signaling and cell cycle control of chromosomal replication. The 9 kinase genes validated to be over-expressed in metastatic ccRCC may represent currently unrecognized but potentially actionable therapeutic targets that warrant functional validation. PMID:27574806

  3. Kinase Gene Expression Profiling of Metastatic Clear Cell Renal Cell Carcinoma Tissue Identifies Potential New Therapeutic Targets.

    PubMed

    Ghatalia, Pooja; Yang, Eddy S; Lasseigne, Brittany N; Ramaker, Ryne C; Cooper, Sara J; Chen, Dongquan; Sudarshan, Sunil; Wei, Shi; Guru, Arjun S; Zhao, Amy; Cooper, Tiffiny; Della Manna, Deborah L; Naik, Gurudatta; Myers, Richard M; Sonpavde, Guru

    2016-01-01

    Kinases are therapeutically actionable targets. Kinase inhibitors targeting vascular endothelial growth factor receptors (VEGFR) and mammalian target of rapamycin (mTOR) improve outcomes in metastatic clear cell renal cell carcinoma (ccRCC), but are not curative. Metastatic tumor tissue has not been comprehensively studied for kinase gene expression. Paired intra-patient kinase gene expression analysis in primary tumor (T), matched normal kidney (N) and metastatic tumor tissue (M) may assist in identifying drivers of metastasis and prioritizing therapeutic targets. We compared the expression of 519 kinase genes using NanoString in T, N and M in 35 patients to discover genes over-expressed in M compared to T and N tissue. RNA-seq data derived from ccRCC tumors in The Cancer Genome Atlas (TCGA) were used to demonstrate differential expression of genes in primary tumor tissue from patients that had metastasis at baseline (n = 79) compared to those that did not develop metastasis for at least 2 years (n = 187). Functional analysis was conducted to identify key signaling pathways by using Ingenuity Pathway Analysis. Of 10 kinase genes overexpressed in metastases compared to primary tumor in the discovery cohort, 9 genes were also differentially expressed in TCGA primary tumors with metastasis at baseline compared to primary tumors without metastasis for at least 2 years: EPHB2, AURKA, GSG2, IKBKE, MELK, CSK, CHEK2, CDC7 and MAP3K8; p<0.001). The top pathways overexpressed in M tissue were pyridoxal 5'-phosphate salvage, salvage pathways of pyrimidine ribonucleotides, NF-kB signaling, NGF signaling and cell cycle control of chromosomal replication. The 9 kinase genes validated to be over-expressed in metastatic ccRCC may represent currently unrecognized but potentially actionable therapeutic targets that warrant functional validation.

  4. Modulation of Colorectal Cancer Risk by Polymorphisms in 51Gln/His, 64Ile/Val, and 148Asp/Glu of APEX Gene; 23Gly/Ala of XPA Gene; and 689Ser/Arg of ERCC4 Gene

    PubMed Central

    Dziki, L.; Dziki, A.; Mik, M.; Kabzinski, J.

    2017-01-01

    Polymorphisms in DNA repair genes may affect the activity of the BER (base excision repair) and NER (nucleotide excision repair) systems. Using DNA isolated from blood taken from patients (n = 312) and a control group (n = 320) with CRC, we have analyzed the polymorphisms of selected DNA repair genes and we have demonstrated that genotypes 51Gln/His and 148Asp/Glu of APEX gene and 23Gly/Ala of XPA gene may increase the risk of colorectal cancer. At the same time analyzing the gene-gene interactions, we suggest the thesis that the main factor to be considered when analyzing the impact of polymorphisms on the risk of malignant transformation should be intergenic interactions. Moreover, we are suggesting that some polymorphisms may have impact not only on the malignant transformation but also on the stage of the tumor. PMID:28386271

  5. Listeria monocytogenes 10403S Arginine Repressor ArgR Finely Tunes Arginine Metabolism Regulation under Acidic Conditions

    PubMed Central

    Cheng, Changyong; Dong, Zhimei; Han, Xiao; Sun, Jing; Wang, Hang; Jiang, Li; Yang, Yongchun; Ma, Tiantian; Chen, Zhongwei; Yu, Jing; Fang, Weihuan; Song, Houhui

    2017-01-01

    Listeria monocytogenes is able to colonize human and animal intestinal tracts and to subsequently cross the intestinal barrier, causing systemic infection. For successful establishment of infection, L. monocytogenes must survive the low pH environment of the stomach. L. monocytogenes encodes a functional ArgR, a transcriptional regulator belonging to the ArgR/AhrC arginine repressor family. We aimed at clarifying the specific functions of ArgR in arginine metabolism regulation, and more importantly, in acid tolerance of L. monocytogenes. We showed that ArgR in the presence of 10 mM arginine represses transcription and expression of the argGH and argCJBDF operons, indicating that L. monocytogenes ArgR plays the classical role of ArgR/AhrC family proteins in feedback inhibition of the arginine biosynthetic pathway. Notably, transcription and expression of arcA (encoding arginine deiminase) and sigB (encoding an alternative sigma factor B) were also markedly repressed by ArgR when bacteria were exposed to pH 5.5 in the absence of arginine. However, addition of arginine enabled ArgR to derepress the transcription and expression of these two genes. Electrophoretic mobility shift assays showed that ArgR binds to the putative ARG boxes in the promoter regions of argC, argG, arcA, and sigB. Reporter gene analysis with gfp under control of the argG promoter demonstrated that ArgR was able to activate the argG promoter. Unexpectedly, deletion of argR significantly increased bacterial survival in BHI medium adjusted to pH 3.5 with lactic acid. We conclude that this phenomenon is due to activation of arcA and sigB. Collectively, our results show that L. monocytogenes ArgR finely tunes arginine metabolism through negative transcriptional regulation of the arginine biosynthetic operons and of the catabolic arcA gene in an arginine-independent manner during lactic acid-induced acid stress. ArgR also appears to activate catabolism as well as sigB transcription by anti

  6. Identification of Phosphoglycerate Kinase 1 (PGK1) as a reference gene for quantitative gene expression measurements in human blood RNA

    PubMed Central

    2011-01-01

    Background Blood is a convenient sample and increasingly used for quantitative gene expression measurements with a variety of diseases including chronic fatigue syndrome (CFS). Quantitative gene expression measurements require normalization of target genes to reference genes that are stable and independent from variables being tested in the experiment. Because there are no genes that are useful for all situations, reference gene selection is an essential step to any quantitative reverse transcription-PCR protocol. Many publications have described appropriate genes for a wide variety of tissues and experimental conditions, however, reference genes that may be suitable for the analysis of CFS, or human blood RNA derived from whole blood as well as isolated peripheral blood mononuclear cells (PBMCs), have not been described. Findings Literature review and analyses of our unpublished microarray data were used to narrow down the pool of candidate reference genes to six. We assayed whole blood RNA from Tempus tubes and cell preparation tube (CPT)-collected PBMC RNA from 46 subjects, and used the geNorm and NormFinder algorithms to select the most stable reference genes. Phosphoglycerate kinase 1 (PGK1) was one of the optimal normalization genes for both whole blood and PBMC RNA, however, additional genes differed for the two sample types; Ribosomal protein large, P0 (RPLP0) for PBMC RNA and Peptidylprolyl isomerase B (PPIB) for whole blood RNA. We also show that the use of a single reference gene is sufficient for normalization when the most stable candidates are used. Conclusions We have identified PGK1 as a stable reference gene for use with whole blood RNA and RNA derived from PBMC. When stable genes are selected it is possible to use a single gene for normalization rather than two or three. Optimal normalization will improve the ability of results from PBMC RNA to be compared with those from whole blood RNA and potentially allows comparison of gene expression results

  7. Fission yeast Cdk7 controls gene expression through both its CAK and C-terminal domain kinase activities.

    PubMed

    Devos, Maxime; Mommaerts, Elise; Migeot, Valerie; van Bakel, Harm; Hermand, Damien

    2015-05-01

    Cyclin-dependent kinase (Cdk) activation and RNA polymerase II transcription are linked by the Cdk7 kinase, which phosphorylates Cdks as a trimeric Cdk-activating kinase (CAK) complex, and serine 5 within the polymerase II (Pol II) C-terminal domain (CTD) as transcription factor TFIIH-bound CAK. However, the physiological importance of integrating these processes is not understood. Besides the Cdk7 ortholog Mcs6, fission yeast possesses a second CAK, Csk1. The two enzymes have been proposed to act redundantly to activate Cdc2. Using an improved analogue-sensitive Mcs6-as kinase, we show that Csk1 is not a relevant CAK for Cdc2. Further analyses revealed that Csk1 lacks a 20-amino-acid sequence required for its budding yeast counterpart, Cak1, to bind Cdc2. Transcriptome profiling of the Mcs6-as mutant in the presence or absence of the budding yeast Cak1 kinase, in order to uncouple the CTD kinase and CAK activities of Mcs6, revealed an unanticipated role of the CAK branch in the transcriptional control of the cluster of genes implicated in ribosome biogenesis and cell growth. The analysis of a Cdc2 CAK site mutant confirmed these data. Our data show that the Cdk7 kinase modulates transcription through its well-described RNA Pol II CTD kinase activity and also through the Cdc2-activating kinase activity.

  8. Fission Yeast Cdk7 Controls Gene Expression through both Its CAK and C-Terminal Domain Kinase Activities

    PubMed Central

    Devos, Maxime; Mommaerts, Elise; Migeot, Valerie; van Bakel, Harm

    2015-01-01

    Cyclin-dependent kinase (Cdk) activation and RNA polymerase II transcription are linked by the Cdk7 kinase, which phosphorylates Cdks as a trimeric Cdk-activating kinase (CAK) complex, and serine 5 within the polymerase II (Pol II) C-terminal domain (CTD) as transcription factor TFIIH-bound CAK. However, the physiological importance of integrating these processes is not understood. Besides the Cdk7 ortholog Mcs6, fission yeast possesses a second CAK, Csk1. The two enzymes have been proposed to act redundantly to activate Cdc2. Using an improved analogue-sensitive Mcs6-as kinase, we show that Csk1 is not a relevant CAK for Cdc2. Further analyses revealed that Csk1 lacks a 20-amino-acid sequence required for its budding yeast counterpart, Cak1, to bind Cdc2. Transcriptome profiling of the Mcs6-as mutant in the presence or absence of the budding yeast Cak1 kinase, in order to uncouple the CTD kinase and CAK activities of Mcs6, revealed an unanticipated role of the CAK branch in the transcriptional control of the cluster of genes implicated in ribosome biogenesis and cell growth. The analysis of a Cdc2 CAK site mutant confirmed these data. Our data show that the Cdk7 kinase modulates transcription through its well-described RNA Pol II CTD kinase activity and also through the Cdc2-activating kinase activity. PMID:25691663

  9. Novel PANK2 gene mutations in two Chinese siblings with atypical pantothenate kinase-associated neurodegeneration.

    PubMed

    Shan, Jingli; Wen, Bing; Zhu, Jun; Lin, Pengfei; Zheng, Jinfan; Yan, Chuanzhu

    2013-04-01

    Pantothenate kinase-associated neurodegeneration (PKAN) is an autosomal-recessive disorder characterized by neurodegeneration and iron accumulation in the brain. Classic and atypical PKAN are distinguished on the basis of age at onset and disease progression. PANK2, localized on chromosome20p13, is confirmed as the responsible gene. We report two Chinese siblings with atypical PKAN, who had a 26- and 24-year disease course, respectively. Brain MRI scans of the two siblings showed the specific "eye of the tiger" sign. Genetic analysis identified novel compound heterozygous mutations (IVS1-2 A>T, c.T1130C) in PANK2 gene, which were confirmed to be deleterious. We verify the clinical heterogeneity even in siblings with identical genotype and expand the gene mutation pool for PKAN.

  10. XRCC1 Arg194Trp and Arg399Gln polymorphisms and arsenic methylation capacity are associated with urothelial carcinoma

    SciTech Connect

    Chiang, Chien-I; Huang, Ya-Li; Chen, Wei-Jen; Shiue, Horng-Sheng; Huang, Chao-Yuan; Pu, Yeong-Shiau; Lin, Ying-Chin; Hsueh, Yu-Mei

    2014-09-15

    The association between DNA repair gene polymorphisms and bladder cancer has been widely studied. However, few studies have examined the correlation between urothelial carcinoma (UC) and arsenic or its metabolites. The aim of this study was to examine the association between polymorphisms of the DNA repair genes, XRCC1 Arg194Trp, XRCC1 Arg399Gln, XRCC3 Thr241Met, and XPD Lys751Gln, with urinary arsenic profiles and UC. To this end, we conducted a hospital-based case–control study with 324 UC patients and 647 age- and gender-matched non-cancer controls. Genomic DNA was used to examine the genotype of XRCC1 Arg194Trp, XRCC1 Arg399Gln, XRCC3 Thr241Met, and XPD Lys751Gln by PCR-restriction fragment length polymorphism analysis (PCR-RFLP). Urinary arsenic profiles were measured by high performance liquid chromatography (HPLC) linked with hydride generator and atomic absorption spectrometry. The XRCC1 399 Gln/Gln and 194 Arg/Trp and Trp/Trp genotypes were significantly related to UC, and the odds ratio (OR) and 95% confidence interval (95%CI) were 1.68 (1.03–2.75) and 0.66 (0.48–0.90), respectively. Participants with higher total urinary arsenic levels, a higher percentage of inorganic arsenic (InAs%) and a lower percentage of dimethylarsinic acid (DMA%) had a higher OR of UC. Participants carrying XRCC1 risk diplotypes G-C/G-C, A-C/A-C, and A-T/G-T, and who had higher total arsenic levels, higher InAs%, or lower DMA% compared to those with other XRCC1 diplotypes had a higher OR of UC. Our results suggest that the XRCC1 399 Gln/Gln and 194 Arg/Arg DNA repair genes play an important role in poor arsenic methylation capacity, thereby increasing the risk of UC in non-obvious arsenic exposure areas. - Highlights: • The XRCC1 399Gln/Gln genotype was significantly associated with increased OR of UC. • The XRCC1 194 Arg/Trp and Trp/Trp genotype had a significantly decreased OR of UC. • Combined effect of the XRCC1 genotypes and poor arsenic methylation capacity on

  11. Radiation induction of the receptor tyrosine kinase gene Ptk-3 in normal rat astrocytes

    SciTech Connect

    Sakuma, S.; Hideyuki, S.; Akihiro, I.

    1995-07-01

    Radiation-induced gene expression was examined in rat astrocyte cultures using differential display of mRNA via reverse transcriptase-polymerase chain reaction. A 0.3-kb cDNA that was consistently observed in irradiated cultures but not in unirradiated cultures was cloned and sequenced. It was found to be identical to Ptk-3, a receptor tyrosine kinase gene identified recently. The protein encoded by Ptk-3 is a member of a novel class of receptor tyrosine kinases whose extracellular domain contains regions of homology with coagulation factors V and VIII and complement component C1. Northern blot analysis revealed that the expression of Ptk-3 was increased in rat astrocytes by 0.5 h after exposure to 10 Gy and remained at the same elevated level for at least 24 h. The maximum increase occurred after 5 Gy cloning studies indicated the presence of at least two Ptk-3 mRNA transcripts, which are probable the result of an alternative splicing mechanism. The short isoform lacks a 37 amino acid sequence in the glycine/proline-rich juxtamembrane region. The splicing pattern of the Ptk-3 gene was not altered by radiation. However, the ratios of the longer to the shorter mRNA transcripts differed between adult cortex, neonatal cortex and in vitro astrocyte cultures. 36 refs., 5 figs.

  12. An S receptor kinase gene in self-compatible Brassica napus has a 1-bp deletion.

    PubMed Central

    Goring, D R; Glavin, T L; Schafer, U; Rothstein, S J

    1993-01-01

    S locus glycoprotein (SLG) and S locus receptor kinase (SRK) cDNAs were isolated from an S allele present in a number of self-compatible Brassica napus lines. This A10 allele did not segregate with self-incompatibility in crosses involving other self-incompatible B. napus lines. The SLG-A10 cDNA was found to contain an intact open reading frame and was predicted to encode an SLG protein with sequence similarities to those previously associated with phenotypically strong self-incompatibility reactions. SLG-A10 transcripts were detected in the developing stigma at steady state levels even higher than those detected for SLG alleles linked with self-incompatibility. Analysis of the corresponding SRK-A10 cDNA showed that it was very similar to other S locus receptor kinase genes and was expressed predominantly in the stigma. However, a 1-bp deletion was detected in the SRK gene toward the 3' end of the SLG homology domain. This deletion would lead to premature termination of translation and the production of a truncated SRK protein. The A10 allele was determined to represent a B. oleracea S allele based on its segregation pattern with the B. oleracea S24 allele when both these alleles were present in the same B. napus background. These results suggest that a functional SRK gene is required for Brassica self-incompatibility. PMID:8518554

  13. The human protein kinase C gamma gene (PRKCG) as a susceptibility locus for behavioral disinhibition.

    PubMed

    Schlaepfer, Isabel R; Clegg, Hilary V; Corley, Robin P; Crowley, Thomas J; Hewitt, John K; Hopfer, Christian J; Krauter, Kenneth; Lessem, Jeffrey; Rhee, Soo Hyun; Stallings, Michael C; Wehner, Jeanne M; Young, Susan E; Ehringer, Marissa A

    2007-06-01

    This study explores the association between a highly heritable behavioral disinhibition phenotype and the protein kinase C gamma (PRKCG) gene in an ethnically diverse youth sample from Colorado, USA. The rationale for this study was based on the impulsive behavior and increased ethanol consumption observed in the protein kinase C gamma (PKC-gamma)-deficient mouse model. Two composite behavioral disinhibition phenotypes and their component behavioral scores [conduct disorder, attention-deficit hyperactivity disorder (ADHD), substance experimentation (SUB) and novelty-seeking] were examined for association with five independent PRKCG single nucleotide polymorphisms (SNPs). Association analysis for the five individual SNPs revealed modest genetic association of Exon 14 (rs2242244) and Upstream (rs307941) markers with the behavioral disinhibition composite variables in the combined, Hispanic and African-American samples. Additionally, haplotype-based association analysis for two SNPs located in Intron 3 (rs402691) and Exon 6 (rs3745406) indicated a significant overall association of the PRKCG locus with the ADHD-hyperactive subscale scores in the combined and Caucasian samples, supporting the relation between impulsive behaviors and the PRKCG gene. A significant haplotype association was also observed with SUB scores but only in the Hispanic ethnic group, highlighting the marker variability for each ethnic group. In conclusion, our results support the role of the PKC-gamma enzyme in behavioral impulsivity previously observed in mice. This study provides the first exploration of the PRKCG gene and its association with behavioral disinhibition and warrants further study in other larger population samples.

  14. Tyrosine kinase activation in breast carcinoma with correlation to HER-2/neu gene amplification and receptor overexpression.

    PubMed

    Bhargava, R; Naeem, R; Marconi, S; Luszcz, J; Garb, J; Gasparini, R; Otis, C N

    2001-12-01

    The HER-2/neu oncogene encodes a transmembrane receptor with intrinsic tyrosine kinase activity. A pilot study was performed to investigate downstream effects of HER-2/neu (or related growth factor receptor) activation by identifying phosphorylated tyrosine. Fifty-four breast carcinomas were evaluated for HER-2/neu overexpression by the HercepTest (Dako, Carpinteria, CA) and the monoclonal CB11 antibody (Ventana, Tucson, AZ). Phosphotyrosine (an indication of tyrosine kinase activity) was detected by an antiphosphotyrosine mouse monoclonal antibody (Upstate Biotechnology, Lake Placid, NY). The gene amplification status was evaluated in 50 of the 54 cases by fluorescence in situ hybridization (FISH) using the Ventana gene probe. The HER-2/neu oncogene amplification was detected in 28% (14 of 50) of cases. Of the 14 cases showing oncogene amplification, tyrosine kinase activity was detected in 9 (64.2%) cases. There was moderate agreement between HER-2/neu gene amplification and tyrosine kinase activity (kappa = 0.43). Immunohistochemical staining of 3+ (with both HercepTest and CB11) showed better agreement with HER-2/neu oncogene amplification and increased tyrosine kinase activity than 2+ immunohistochemical staining. Overall, oncogene amplification and overexpression correlated with increased tyrosine kinase activity, supporting the mechanism of tyrosine kinase activation by HER-2/neu amplification and overexpression. However, 7 cases showing increased tyrosine kinase activity did not show gene amplification or 3+ receptor expression (by either HercepTest or CB11), raising the possibility of other growth factor receptors operating via the tyrosine kinase pathway. There was no apparent correlation between tyrosine kinase activity and hormone receptor status (estrogen or progesterone). Increased tyrosine kinase activity is more commonly associated with higher-grade tumors and thus may correlate with aggressive biologic behavior in breast carcinoma. The results of

  15. Founder p.Arg 446* mutation in the PDHX gene explains over half of cases with congenital lactic acidosis in Roma children.

    PubMed

    Ivanov, Ivan S; Azmanov, Dimitar N; Ivanova, Mariya B; Chamova, Teodora; Pacheva, Ilyana H; Panova, Margarita V; Song, Sharon; Morar, Bharti; Yordanova, Ralitsa V; Galabova, Fani K; Sotkova, Iglika G; Linev, Alexandar J; Bitchev, Stoyan; Shearwood, Anne-Marie J; Kancheva, Dalia; Gabrikova, Dana; Karcagi, Veronika; Guergueltcheva, Velina; Geneva, Ina E; Bozhinova, Veneta; Stoyanova, Vili K; Kremensky, Ivo; Jordanova, Albena; Savov, Aleksey; Horvath, Rita; Brown, Matthew A; Tournev, Ivailo; Filipovska, Aleksandra; Kalaydjieva, Luba

    2014-01-01

    Investigation of 31 of Roma patients with congenital lactic acidosis (CLA) from Bulgaria identified homozygosity for the R446* mutation in the PDHX gene as the most common cause of the disorder in this ethnic group. It accounted for around 60% of patients in the study and over 25% of all CLA cases referred to the National Genetic Laboratory in Bulgaria. The detection of a homozygous patient from Hungary and carriers among population controls from Romania and Slovakia suggests a wide spread of the mutation in the European Roma population. The clinical phenotype of the twenty R446* homozygotes was relatively homogeneous, with lactic acidosis crisis in the first days or months of life as the most common initial presentation (15/20 patients) and delayed psychomotor development and/or seizures in infancy as the leading manifestations in a smaller group (5/20 patients). The subsequent clinical picture was dominated by impaired physical growth and a very consistent pattern of static cerebral palsy-like encephalopathy with spasticity and severe to profound mental retardation seen in over 80% of cases. Most patients had a positive family history. We propose testing for the R446* mutation in PDHX as a rapid first screening in Roma infants with metabolic acidosis. It will facilitate and accelerate diagnosis in a large proportion of cases, allow early rehabilitation to alleviate the chronic clinical course, and prevent further affected births in high-risk families.

  16. Two Different Transcripts of a LAMMER Kinase Gene Play Opposite Roles in Disease Resistance1[OPEN

    PubMed Central

    Xiao, Wenfei; Xia, Fan; Liu, Hongbo; Xiao, Jinghua

    2016-01-01

    Alternative splicing of genes can increase protein diversity and affect mRNA stability. Genome-wide transcriptome sequencing has demonstrated that alternative splicing occurs in a large number of intron-containing genes of different species. However, despite the phenomenon having been known for decades, it is largely unknown how the alternatively spliced transcripts function differently. Here, we report that two alternatively spliced transcripts of the rice (Oryza sativa) LAMMER kinase gene OsDR11, long OsDR11L and short OsDR11S, play opposite roles in rice resistance against Xanthomonas oryzae pv oryzae (Xoo), which causes the most damaging bacterial disease in rice worldwide. Overexpressing OsDR11S or suppressing OsDR11L in rice enhanced resistance to Xoo, which was accompanied by an accumulation of jasmonic acid (JA) and induced expression of JA signaling genes. In contrast, suppressing OsDR11S was associated with increased susceptibility to Xoo, along with decreased levels of JA and expression of JA signaling genes. The OsDR11S and OsDR11L proteins colocalized in the nucleus. OsDR11L showed autophosphorylation activity in vitro, while OsDR11S did not. In the presence of OsDR11S, autophosphorylation of OsDR11L was inhibited, and overexpression of OsDR11S suppressed OsDR11L expression. OsDR11 appeared to contribute to a minor quantitative trait locus against Xoo. These results suggest that OsDR11L is a negative regulator in rice disease resistance, which may be associated with suppression of JA signaling. The results also suggest that OsDR11S may inhibit the function of OsDR11L at both the transcription and protein kinase activity levels, leading to resistance against Xoo. PMID:27621422

  17. Cloning, sequencing and characterization of a gene encoding dihydroxyacetone kinase from Zygosaccharomyces rouxii NRRL2547.

    PubMed

    Wang, Zheng-Xiang; Kayingo, Gerald; Blomberg, Anders; Prior, Bernard A

    2002-12-01

    The dihydroxyacetone pathway, an alternative pathway for the dissimilation of glycerol via reduction by glycerol dehydrogenase and subsequent phosphorylation by dihydroxyacetone (DHA) kinase, is activated in the yeasts Saccharomyces cerevisiae and Zygosaccharomyces rouxii during osmotic stress. In experiments aimed at investigating the physiological function of the DHA pathway in Z. rouxii, a typical osmotolerant yeast, we cloned and characterized a DAK gene encoding dihydroxyacetone kinase from Z. rouxii NRRL 2547. Sequence analysis revealed a 1761 bp open reading frame, encoding a peptide composed of 587 deduced amino acids with the predicted molecular weight of 61 664 Da. As the amino acid sequence was most closely homologous (68% identity) to the S. cerevisiae Dak1p, we named the gene and protein ZrDAK1 and ZrDak1p, respectively. A putative ATP binding site was also found but no consensus element associated with osmoregulation was found in the upstream region of the ZrDAK1 gene. The ZrDAK1 gene complemented a S. cerevisiae W303-1A dak1delta dak2 delta strain by improving the growth of the mutant on 50 mmol/l dihydroxyacetone and by increasing the tolerance to dihydroxyacetone in a medium containing 5% sodium chloride, suggesting that it is a functional homologue of the S. cerevisiae DAK1. However, expression of the ZrDAK1 gene in the S. cerevisiae dak1delta dak2 delta strain had no significant effect on glycerol levels during osmotic stress. The ZrDAK1 sequence has been deposited in the public data bases under Accession No. AJ294719; regions upstream and downstream of ZrDAK1are deposited as Accession Nos AJ294739 and AJ294720, respectively.

  18. CARD14 gene polymorphism c.C2458T (p.Arg820Trp) is associated with clinical features of psoriasis vulgaris in a Chinese cohort.

    PubMed

    Feng, Chunsheng; Wang, Tingting; Li, Shi-Jie; Fan, Yi-Ming; Shi, Ge; Zhu, Kun-Ju

    2016-03-01

    Genome-wide association studies have found the single nucleotide polymorphism (SNP) c.C2458T, at the caspase recruitment domain family member 14 (CARD14) gene, to be associated with psoriasis. But little is known about the association of c.C2458T and clinical features of psoriasis vulgaris (PsV) in a Chinese cohort. This study was undertaken to further explore the relationship between c.C2458T and risk of psoriasis in southern Chinese subjects and to evaluate the SNP effect on the clinical features of psoriasis. A case-control study was performed involving 345 PsV patients and 206 controls. The variant of c.C2458T was typed using a SNaPshot assay. Statistical analysis was performed using SPSS version 13.0 software. In analysis of the basic situation of the sample, no difference was observed between cases and controls for age and sex. In the frequency distribution of genotypes and alleles in patients and controls, we found no association between the SNP and the risk of PsV. We performed a stratified analysis according to the age of onset, family history and Psoriasis Area and Severity Index (PASI) subphenotypes. We found that the CC genotype was associated significantly with an increased familial history of PsV. The main finding of our study was that the CC genotype was more common in familial cases than in sporadic cases. However, there were no significant differences found in other subphenotypes of age of onset or PASI between patients positive and those negative for a particular phenotype. In conclusion, the SNP c.C2458T may have significant effects on heritability of PsV in our Chinese population.

  19. Cloning and characterization of HbMT2a, a metallothionein gene from Hevea brasiliensis Muell. Arg differently responds to abiotic stress and heavy metals

    SciTech Connect

    Li, Yan; Chen, Yue Yi; Yang, Shu Guang; Tian, Wei Min

    2015-05-22

    Metallothioneins (MTs) are of low molecular mass, cysteine-rich proteins. They play an important role in the detoxification of heavy metals and homeostasis of intracellular metal ions, and protecting against intracellular oxidative damages. In this study a full-length cDNA of type 2 plant metallothioneins, HbMT2a, was isolated from 25 mM Polyethyleneglycol (PEG) stressed leaves of Hevea brasiliensis by RACE. The HbMT2a was 372 bp in length and had a 237 bp open reading frame (ORF) encoding for a protein of 78 amino acid residues with molecular mass of 7.772 kDa. The expression of HbMT2a in the detached leaves of rubber tree clone RY7-33-97 was up-regulated by Me-JA, ABA, PEG, H{sub 2}O{sub 2}, Cu{sup 2+} and Zn{sup 2+}, but down-regulated by water. The role of HbMT2a protein in protecting against metal toxicity was demonstrated in vitro. PET-28a-HbMT2-beared Escherichia coli. Differential expression of HbMT2a upon treatment with 10 °C was observed in the detached leaves of rubber tree clone 93-114 which is cold-resistant and Reken501 which is cold-sensitive. The expression patterns of HbMT2a in the two rubber tree clones may be ascribed to a change in the level of endogenous H{sub 2}O{sub 2}. - Highlights: • Cloning an HbMT2a gene from rubber tree. • Analyzing expression patterns of HbMT2a upon abiotic stress and heavy metal stress. • Finding different expression patterns of HbMT2a among two Hevea germplasm. • The expressed protein of HbMT2a enhances copper and zinc tolerance in Escherichia coli.

  20. An EGFR-Src-Arg-cortactin pathway mediates functional maturation of invadopodia and breast cancer cell invasion.

    PubMed

    Mader, Christopher C; Oser, Matthew; Magalhaes, Marco A O; Bravo-Cordero, Jose Javier; Condeelis, John; Koleske, Anthony J; Gil-Henn, Hava

    2011-03-01

    Invasive carcinoma cells use specialized actin polymerization-driven protrusions called invadopodia to degrade and possibly invade through the extracellular matrix (ECM) during metastasis. Phosphorylation of the invadopodium protein cortactin is a master switch that activates invadopodium maturation and function. Cortactin was originally identified as a hyperphosphorylated protein in v-Src-transformed cells, but the kinase or kinases that are directly responsible for cortactin phosphorylation in invadopodia remain unknown. In this study, we provide evidence that the Abl-related nonreceptor tyrosine kinase Arg mediates epidermal growth factor (EGF)-induced cortactin phosphorylation, triggering actin polymerization in invadopodia, ECM degradation, and matrix proteolysis-dependent tumor cell invasion. Both Src and Arg localize to invadopodia and are required for EGF-induced actin polymerization. Notably, Arg overexpression in Src knockdown cells can partially rescue actin polymerization in invadopodia while Src overexpression cannot compensate for loss of Arg, arguing that Src indirectly regulates invadopodium maturation through Arg activation. Our findings suggest a novel mechanism by which an EGFR-Src-Arg-cortactin pathway mediates functional maturation of invadopodia and breast cancer cell invasion. Furthermore, they identify Arg as a novel mediator of invadopodia function and a candidate therapeutic target to inhibit tumor invasion in vivo.

  1. Calcium-dependent protein kinase (CDPK) and CDPK-related kinase (CRK) gene families in tomato: genome-wide identification and functional analyses in disease resistance.

    PubMed

    Wang, Ji-Peng; Xu, You-Ping; Munyampundu, Jean-Pierre; Liu, Tian-Yu; Cai, Xin-Zhong

    2016-04-01

    Calcium-dependent protein kinases (CDPKs) and CDPK-related kinases (CRKs) play multiple roles in plant. Nevertheless, genome-wide identification of these two families is limited to several plant species, and role of CRKs in disease resistance remains unclear. In this study, we identified the CDPK and CRK gene families in genome of the economically important crop tomato (Solanum lycopersicum L.) and analyzed their function in resistance to various pathogens. Twenty-nine CDPK and six CRK genes were identified in tomato genome. Both SlCDPK and SlCRK proteins harbored an STKc_CAMK type protein kinase domain, while only SlCDPKs contained EF-hand type Ca(2+) binding domain(s). Phylogenetic analysis revealed that plant CRK family diverged early from CDPKs, and shared a common ancestor gene with subgroup IV CDPKs. Subgroup IV SlCDPK proteins were basic and their genes contained 11 introns, which were distinguished from other subgroups but similar to CRKs. Subgroup I SlCDPKs generally did not carry an N-terminal myristoylation motif while those of the remaining subgroups and SlCRKs universally did. SlCDPK and SlCRK genes were differently responsive to pathogenic stimuli. Furthermore, silencing analyses demonstrated that SlCDPK18 and SlCDPK10 positively regulated nonhost resistance to Xanthomonas oryzae pv. oryzae and host resistance to Pseudomonas syringae pv. tomato (Pst) DC3000, respectively, while SlCRK6 positively regulated resistance to both Pst DC3000 and Sclerotinia sclerotiorum in tomato. In conclusion, CRKs apparently evolved from CDPK lineage, SlCDPK and SlCRK genes regulate a wide range of resistance and SlCRK6 is the first CRK gene proved to function in plant disease resistance.

  2. Synthesis and cytotoxic activity of 4-N-carboxybutyl-5-fluorocytosyl-Arg-Gln-Trp-Arg-Arg-Trp-Trp-Gln-Arg-NH₂.

    PubMed

    Somlai, Csaba; Correche, Estela; Olivella, Monica; Tolosa, Laia; Lechon, Maria José Gomez; Dombi, György; Tóth, Gábor K; Penke, Botond; Enriz, Ricardo D

    2012-07-01

    The chemical synthesis of 4-N-carboxybutyl-5-fluorocytosine (II) in solution phase starting from 5-fluorocytosine and the solid phase synthesis of Arg-Gln-Trp-Arg-Arg-Trp-Trp-Gln-Arg-NH(2) attached to the 4-N-carboxybutyl-5-fluorocytosine residue at the N-terminus of the peptide (III) via peptide bond formation is reported. The target compound exhibited a significant cytotoxic activity against a culture of HepG2 cells. In addition our results demonstrated that this new compound affect cell viability, produce mitochondrial dysfunction as well as interfere with intracellular calcium homeostasis control; leading to cell malfunction and death.

  3. Tumor necrosis factor gene expression is mediated by protein kinase C following activation by ionizing radiation.

    SciTech Connect

    Hallahan, D. E.; Virudachalam, S.; Sherman, M. L.; Huberman, E.; Kufe, D. W.; Weichselbaum, R. R.; Univ. of Chicago; Dana-Farber Cancer Inst.; Univ. of Chicago

    1991-01-01

    Tumor necrosis factor (TNF) production following X-irradiation has been implicated in the biological response to ionizing radiation. Protein kinase C (PKC) is suggested to participate in TNF transcriptional induction and X-ray-mediated gene expression. We therefore studied radiation-mediated TNF expression in HL-60 cells with diminished PKC activity produced by either pretreatment with protein kinase inhibitors or prolonged 12-O-tetradecanoylphorbol-13-acetate treatment. Both treatments resulted in attenuation of radiation-mediated TNF induction. Consistent with these results, we found no detectable induction of TNF expression following X-irradiation in the HL-60 variant deficient in PKC-mediated signal transduction. The rapid activation of PKC following {gamma}-irradiation was established using an in vitro assay measuring phosphorylation of a PKC specific substrate. A 4.5-fold increase in PKC activity occurred 15 to 30 s following irradiation, which declined to baseline at 60 s. Two-dimensional gel electrophoresis of phosphoproteins extracted from irradiated cells demonstrated in vivo phosphorylation of the PKC specific substrate Mr 80,000 protein at 45 s following X-irradiation. These findings indicate that signal transduction via the PKC pathway is required for the induction of TNF gene expression by ionizing radiation.

  4. The protein kinase TOUSLED is required for maintenance of transcriptional gene silencing in Arabidopsis

    PubMed Central

    Wang, Yu; Liu, Jun; Xia, Ran; Wang, Junguo; Shen, Jie; Cao, Rui; Hong, Xuhui; Zhu, Jian-Kang; Gong, Zhizhong

    2007-01-01

    TOUSLED-like kinases (TLKs) are highly conserved in plants and animals, but direct evidence linking TLKs and transcriptional gene silencing is lacking. We isolated two new alleles of TOUSLED (TSL). Mutations of TSL in ros1 reactivate the transcriptionally silent 35S-NPTII transgene and the transcriptionally silent endogenous loci TSI (TRANSCRIPTIONAL SILENCING INFORMATION). Chromatin immunoprecipitation (ChIP) analysis shows that histone H3Lys9 dimethylation is decreased in the reactivated transgene and endogenous TSI loci in the tsl ros1 mutant. However, there is no change in DNA methylation in the affected loci. Western blot and ChIP assay suggest that TSL might not be responsible for histone H3Ser10 phosphorylation. The tsl seedlings were more sensitive to DNA damage reagent methyl methanesulphonate and UV-B light. Our results provide direct evidence for a crucial role of the TOUSLED protein kinase in the maintenance of transcriptional gene silencing in some genomic regions in a DNA-methylation-independent manner in Arabidopsis. PMID:17110953

  5. The protein kinase TOUSLED is required for maintenance of transcriptional gene silencing in Arabidopsis.

    PubMed

    Wang, Yu; Liu, Jun; Xia, Ran; Wang, Junguo; Shen, Jie; Cao, Rui; Hong, Xuhui; Zhu, Jian-Kang; Gong, Zhizhong

    2007-01-01

    TOUSLED-like kinases (TLKs) are highly conserved in plants and animals, but direct evidence linking TLKs and transcriptional gene silencing is lacking. We isolated two new alleles of TOUSLED (TSL). Mutations of TSL in ros1 reactivate the transcriptionally silent 35S-NPTII transgene and the transcriptionally silent endogenous loci TSI (TRANSCRIPTIONAL SILENCING INFORMATION). Chromatin immunoprecipitation (ChIP) analysis shows that histone H3Lys9 dimethylation is decreased in the reactivated transgene and endogenous TSI loci in the tsl ros1 mutant. However, there is no change in DNA methylation in the affected loci. Western blot and ChIP assay suggest that TSL might not be responsible for histone H3Ser10 phosphorylation. The tsl seedlings were more sensitive to DNA damage reagent methyl methanesulphonate and UV-B light. Our results provide direct evidence for a crucial role of the TOUSLED protein kinase in the maintenance of transcriptional gene silencing in some genomic regions in a DNA-methylation-independent manner in Arabidopsis.

  6. Cloning and Characterization of a Receptor-Like Protein Kinase Gene Associated with Senescence

    PubMed Central

    Hajouj, Taleb; Michelis, Regina; Gepstein, Shimon

    2000-01-01

    Senescence-associated genes are up-regulated during plant senescence and many have been implicated in encoding enzymes involved in the metabolism of senescing tissues. Using the differential display technique, we identified a SAG in bean (Phaseolus vulgaris) leaf that was exclusively expressed during senescence and was designated senescence-associated receptor-like kinase (SARK). The deduced SARK polypeptide consists of a signal peptide, a leucine-rich repeat in the extracellular region, a single membrane-spanning domain, and the characteristic serine/threonine protein kinase domain. The mRNA level for SARK increased prior to the loss of chlorophyll and the decrease of chlorophyll a/b-binding protein mRNA. Detached mature bean leaves, which senesce at an accelerated rate compared with leaves on intact plants, showed a similar temporal pattern of SARK message accumulation. Light and cytokinin, which delayed the initiation of leaf senescence, also delayed SARK gene expression; in contrast, darkness and ethylene, which accelerated senescence, advanced the initial appearance of the SARK transcript. SARK protein accumulation exhibited a temporal pattern similar to that of its mRNA. A possible role for SARK in the regulation of leaf senescence was considered. PMID:11080306

  7. Identification and characterization of a novel serine-threonine kinase gene from the Xp22 region.

    PubMed

    Montini, E; Andolfi, G; Caruso, A; Buchner, G; Walpole, S M; Mariani, M; Consalez, G; Trump, D; Ballabio, A; Franco, B

    1998-08-01

    Eukaryotic protein kinases are part of a large and expanding family of proteins. Through our transcriptional mapping effort in the Xp22 region, we have isolated and sequenced the full-length transcript of STK9, a novel cDNA highly homologous to serine-threonine kinases. A number of human genetic disorders have been mapped to the region where STK9 has been localized including Nance-Horan (NH) syndrome, oral-facial-digital syndrome type 1 (OFD1), and a novel locus for nonsyndromic sensorineural deafness (DFN6). To evaluate the possible involvement of STK9 in any of the above-mentioned disorders, a 2416-bp full-length cDNA was assembled. The entire genomic structure of the gene, which is composed of 20 coding exons, was determined. Northern analysis revealed a transcript larger than 9.5 kb in several tissues including brain, lung, and kidney. The mouse homologue (Stk9) was identified and mapped in the mouse in the region syntenic to human Xp. This location is compatible with the location of the Xcat mutant, which shows congenital cataracts very similar to those observed in NH patients. Sequence homologies, expression pattern, and mapping information in both human and mouse make STK9 a candidate gene for the above-mentioned disorders.

  8. Transcriptional regulation of pig GYS1 gene by glycogen synthase kinase 3β (GSK3β).

    PubMed

    Wang, Yilin; Wang, Yan; Zhong, Tao; Guo, Jiazhong; Li, Li; Zhang, Hongping; Wang, Linjie

    2017-01-01

    Glycogen synthase kinase 3β (GSK3β) is a ubiquitous serine/threonine kinase and has important roles in glycogen metabolism biosynthesis. Studies have revealed that GSK3β can directly regulate the glycogen synthase activity, yet little is known about the regulation of GSK3β on GYS1 gene transcription. Here, we show that overexpression of GSK3β decreased the mRNA expression level of GYS1. Then we cloned approximately 1.5 kb of pig GYS1 gene promoter region, generated sequential deletion constructs, and evaluated their activity. A gradual increase of the promoter activity was seen with increasing length of the promoter sequence, reaching its highest activity to the sequence corresponding to nt -350 to +224, and then decreased. However, the activities of constructed promoter fragments show different responses to GSK3β co-transfection. By analyzing a series of GYS1 promoter reporter constructs, we have defined two crucial regions (-1488 to -539, -350 to -147) that are responsible for GSK3β-induced transcriptional repression. Furthermore, the ChIP results revealed that only the first and second NF-κB sites of GYS1 promoter could bind to p65, and overexpression of GSK3β induced a significant decrease in p65 binding to the second NF-κB binding site, suggesting that GSK3β may regulate expression of GYS1 gene through binding to the second rather than the first NF-κB site. These data suggest that the NF-κB plays important roles in the transcriptional activity of pig GYS1 gene regulated by GSK3β.

  9. Herpes simplex virus thymidine kinase gene therapy for rat malignant brain tumors.

    PubMed

    Vincent, A J; Vogels, R; Someren, G V; Esandi, M C; Noteboom, J L; Avezaat, C J; Vecht, C; Bekkum, D W; Valerio, D; Bout, A; Hoogerbrugge, P M

    1996-01-20

    Transfer of a herpes simplex virus-derived thymidine kinase (HSV-tk) gene into brain tumor cells and subsequent ganciclovir (GCV) treatment has been shown by others to be an effective treatment in rats with intracerebrally inoculated 9L gliosarcomas. Mechanism of action and reproducibility are, however, still a matter of debate. We have used the same model to test the therapeutic effects of both retrovirus- and adenovirus-mediated transfer of the HSV-tk gene followed by GCV treatment. Survival time of rats with intracerebral 9L tumors was significantly prolonged after a single administration of adenovirus carrying a HSV-tk gene as compared to controls. Retrovirus-mediated gene transfer also resulted in significantly prolonged survival time when recombinant retrovirus-producing cells were transplanted. Direct injection of the recombinant retrovirus, HSV-tk-expressing cells, virus-producing cells without GCV administration and recombinant retrovirus-lacZ or interleukin-2 (IL-2)-producing cells did not result in tumor cell kill. In the present study, no significant difference in survival of 9L brain tumor carrying rats was found after treatment with adenovirus as compared to retrovirus-mediated HSV-tk-mediated gene transfer and subsequent GCV treatment.

  10. Expression of protein kinase C genes during ontogenic development of the central nervous system

    SciTech Connect

    Sposi, N.M.; Bottero, L.; Testa, U.; Peschle, C.; Russo, G.

    1989-05-01

    The authors have analyzed the RNA expression of three protein kinase C (PKC) genes in (/alpha/, /beta/, and /gamma/) in human and murine central nervous systems during embryonic-fetal, perinatal, and adult life. Analysis of human brain poly(A)/sup +/ RNA indicates that expression of PKC /alpha/ and /beta/ genes can be detected as early as 6 weeks postconception, undergoes a gradual increase until 9 weeks postconception, and reaches its highest level in the adult stage,and that the PKC /gamma/ gene, although not expressed during embryonic and early fetal development, is abundantly expressed in the adult period. Similar developmental patterns were observed in human spinal cord and medulla oblongata. A detailed analysis of PKC gene expression during mammalian ontogeny was performed on poly(A)/sup +/ RNA from the brain cells of murine embryos at different stages of development and the brain cells of neonatal and adult mice. The ontogenic patterns were similar to those observed for human brain. Furthermore, they observed that the expression of PKC /gamma/ is induced in the peri- and postnatal phases. These results suggest that expression of PKC /alpha/, /beta/, and /gamma/ genes possibly mediates the development of central neuronal functions, and expression of PKC /gamma/ in particular may be involved in the development of peri- and postnatal functions.

  11. The pkI gene encoding pyruvate kinase I links to the luxZ gene which enhances bioluminescence of the lux operon from Photobacterium leiognathi.

    PubMed

    Lin, J W; Lu, H C; Chen, H Y; Weng, S F

    1997-10-09

    Partial 3'-end nucleotide sequence of the pkI gene (GenBank accession No. AF019143) from Photobacterium leiognathi ATCC 25521 has been determined, and the encoded pyruvate kinase I is deduced. Pyruvate kinase I is the key enzyme of glycolysis, which converts phosphoenol pyruvate to pyruvate. Alignment and comparison of pyruvate kinase Is from P. leiognathi, E. coli and Salmonella typhimurium show that they are homologous. Nucleotide sequence reveals that the pkI gene is linked to the luxZ gene that enhances bioluminescence of the lux operon from P. leiognathi. The gene order of the pkI and luxZ genes is-pk1-ter-->-R&R"-luxZ-ter"-->, whereas ter is transcriptional terminator for the pkI and related genes, and R&R" is the regulatory region and ter" is transcriptional terminator for the luxZ gene. It clearly elicits that the pkI gene and luxZ gene are divided to two operons. Functional analysis confirms that the potential hairpin loop omega T is the transcriptional terminator for the pkI and related genes. It infers that the pkI and related genes are simply linked to the luxZ gene in P. leiognathi genome.

  12. Arg/Abl2 modulates the affinity and stoichiometry of binding of cortactin to F-actin.

    PubMed

    MacGrath, Stacey M; Koleske, Anthony J

    2012-08-21

    The Abl family nonreceptor tyrosine kinase Arg/Abl2 interacts with cortactin, an Arp2/3 complex activator, to promote actin-driven cell edge protrusion. Both Arg and cortactin bind directly to filamentous actin (F-actin). While protein-protein interactions between Arg and cortactin have well-characterized downstream effects on the actin cytoskeleton, it is unclear whether and how Arg and cortactin affect each other's actin binding properties. We employ actin cosedimentation assays to show that Arg increases the stoichiometry of binding of cortactin to F-actin at saturation. Using a series of Arg deletion mutants and fragments, we demonstrate that the Arg C-terminal calponin homology domain is necessary and sufficient to increase the stoichiometry of binding of cortactin to F-actin. We also show that interactions between Arg and cortactin are required for optimal affinity between cortactin and the actin filament. Our data suggest a mechanism for Arg-dependent stimulation of binding of cortactin to F-actin, which may facilitate the recruitment of cortactin to sites of local actin network assembly.

  13. Novel homozygous mutation, c.400C>T (p.Arg134*), in the PVRL1 gene underlies cleft lip/palate-ectodermal dysplasia syndrome in an Asian patient.

    PubMed

    Yoshida, Kazue; Hayashi, Ryota; Fujita, Hideki; Kubota, Masaya; Kondo, Mai; Shimomura, Yutaka; Niizeki, Hironori

    2015-07-01

    Cleft lip/palate-ectodermal dysplasia syndrome is a rare, autosomal recessive disorder caused by homozygous loss-of-function mutations of the poliovirus receptor-like 1 (PVRL1) gene encoding nectin-1. Nectin-1 is a cell-cell adhesion molecule that is important for the initial step in the formation of adherens junctions and tight junctions; it is expressed in keratinocytes, neurons, and the developing face and palate. Clinical manifestations comprise a unique facial appearance with cleft lip/palate, ectodermal dysplasia, cutaneous syndactyly of the fingers and/or toes, and in some cases, mental retardation. We present the first report, to our knowledge, of an Asian individual with cleft lip/palate-ectodermal dysplasia syndrome with a novel PVRL1 mutation. A 7-year-old Japanese boy, the first child of a consanguineous marriage, showed hypohidrotic ectodermal dysplasia with sparse, brittle, fine, dry hair and hypodontia, the unique facial appearance with cleft lip/palate, cutaneous syndactyly of the fingers and mild mental retardation. Scanning electron microscopic examination of the hair demonstrated pili torti and pili trianguli et canaliculi. Mutation analysis of exon 2 of PVRL1 revealed a novel homozygous nonsense mutation, c.400C>T (p.Arg134*). His parents were heterozygous for the mutant alleles. All four PVRL1 mutations identified in cleft lip/palate-ectodermal dysplasia syndrome to date, including this study, resulted in truncated proteins that lack the transmembrane domain and intracellular domain of nectin-1, which is necessary to initiate the cell-cell adhesion process.

  14. AMP-activated protein kinase counteracted the inhibitory effect of glucose on the phosphoenolpyruvate carboxykinase gene expression in rat hepatocytes.

    PubMed

    Hubert, A; Husson, A; Chédeville, A; Lavoinne, A

    2000-09-22

    The effect of AMP-activated protein kinase (AMPK) in the regulation of the phosphoenolpyruvate carboxykinase (PEPCK) gene expression was studied in isolated rat hepatocytes. Activation of AMPK by AICAR counteracted the inhibitory effect of glucose on the PEPCK gene expression, both at the mRNA and the transcriptional levels. It is proposed that a target for AMPK is involved in the inhibitory effect of glucose on PEPCK gene transcription.

  15. Characterization and expression analysis of somatic embryogenesis receptor-like kinase genes from Phalaenopsis.

    PubMed

    Huang, Y W; Tsai, Y J; Chen, F C

    2014-12-18

    Somatic embryogenesis receptor-like kinase (SERK) genes have been found to be involved in the somatic embryogenesis of several plant species. We identified and characterized 5 PhSERK genes in the Phalaenopsis orchid. The amino acid sequences of PhSERKs and other SERK proteins are highly conserved, with the highest homology observed in the leucine-rich repeat-receptor-like kinase domain. All 5 PhSERKs were expressed in all Phalaenopsis organs examined (root, leaf, shoot apical meristem, and flower), with the strongest expression, particularly for PhSERK1 and 3, in the shoot apical meristem of mature plants. Expression of all PhSERKs was downregulated during early floral bud development and was upregulated gradually until the semi-open flower stage was reached. All 5 PhSERKs were expressed during both seed germination and protocorm-like-body (PLB) development. In germinated seeds, quantitative real-time PCR revealed upregulation of all PhSERKs except PhSERK4 at 1 week and downregulation after 4 weeks. The 5 PhSERKs were differentially expressed in the early stage of PLB development and maintained substantial levels during PLB formation, with PhSERK1 and 5 upregulated 1 week after culture and PhSERK2, 3, and 4 downregulated over this period. Because physical wounding of PLB stimulates secondary PLB formation, the PhSERK5 expression peak at week 3 coincided with visible and fully developed secondary PLBs. PhSERK5 may be important in PLB induction and subsequent development. Our PhSERK expression analysis revealed that these genes have a broad role during orchid plant development.

  16. Novel Polymorphisms in the Myosin Light Chain Kinase Gene Confer Risk for Acute Lung Injury

    PubMed Central

    Gao, Li; Grant, Audrey; Halder, Indrani; Brower, Roy; Sevransky, Jonathan; Maloney, James P.; Moss, Marc; Shanholtz, Carl; Yates, Charles R.; Meduri, Gianfranco Umberto; Shriver, Mark D.; Ingersoll, Roxann; Scott, Alan F.; Beaty, Terri H.; Moitra, Jaideep; Ma, Shwu Fan; Ye, Shui Q.; Barnes, Kathleen C.; Garcia, Joe G. N.

    2006-01-01

    The genetic basis of acute lung injury (ALI) is poorly understood. The myosin light chain kinase (MYLK) gene encodes the nonmuscle myosin light chain kinase isoform, a multifunctional protein involved in the inflammatory response (apoptosis, vascular permeability, leukocyte diapedesis). To examine MYLK as a novel candidate gene in sepsis-associated ALI, we sequenced exons, exon–intron boundaries, and 2 kb of 5′ UTR of the MYLK, which revealed 51 single-nucleotide polymorphisms (SNPs). Potential association of 28 MYLK SNPs with sepsis-associated ALI were evaluated in a case-control sample of 288 European American subjects (EAs) with sepsis alone, subjects with sepsis-associated ALI, or healthy control subjects, and a sample population of 158 African American subjects (AAs) with sepsis and ALI. Significant single locus associations in EAs were observed between four MYLK SNPs and the sepsis phenotype (P < 0.001), with an additional SNP associated with the ALI phenotype (P = 0.03). A significant association of a single SNP (identical to the SNP identified in EAs) was observed in AAs with sepsis (P = 0.002) and with ALI (P = 0.01). Three sepsis risk-conferring haplotypes in EAs were defined downstream of start codon of smooth muscle MYLK isoform, a region containing putative regulatory elements (P < 0.001). In contrast, multiple haplotypic analyses revealed an ALI-specific, risk-conferring haplotype at 5′ of the MYLK gene in both European and African Americans and an additional 3′ region haplotype only in African Americans. These data strongly implicate MYLK genetic variants to confer increased risk of sepsis and sepsis-associated ALI. PMID:16399953

  17. SOS4, a pyridoxal kinase gene, is required for root hair development in Arabidopsis.

    PubMed

    Shi, Huazhong; Zhu, Jian-Kang

    2002-06-01

    Root hair development in plants is controlled by many genetic, hormonal, and environmental factors. A number of genes have been shown to be important for root hair formation. Arabidopsis salt overly sensitive 4 mutants were originally identified by screening for NaCl-hypersensitive growth. The SOS4 (Salt Overly Sensitive 4) gene was recently isolated by map-based cloning and shown to encode a pyridoxal (PL) kinase involved in the production of PL-5-phosphate, which is an important cofactor for various enzymes and a ligand for certain ion transporters. The root growth of sos4 mutants is slower than that of the wild type. Microscopic observations revealed that sos4 mutants do not have root hairs in the maturation zone. The sos4 mutations block the initiation of most root hairs, and impair the tip growth of those that are initiated. The root hairless phenotype of sos4 mutants was complemented by the wild-type SOS4 gene. SOS4 promoter-beta-glucuronidase analysis showed that SOS4 is expressed in the root hair and other hair-like structures. Consistent with SOS4 function as a PL kinase, in vitro application of pyridoxine and pyridoxamine, but not PL, partially rescued the root hair defect in sos4 mutants. 1-Aminocyclopropane-1-carboxylic acid and 2,4-dichlorophenoxyacetic acid treatments promoted root hair formation in both wild-type and sos4 plants, indicating that genetically SOS4 functions upstream of ethylene and auxin in root hair development. The possible role of SOS4 in ethylene and auxin biosynthesis is discussed.

  18. Liver kinase B1 inhibits the expression of inflammation-related genes postcontraction in skeletal muscle.

    PubMed

    Chen, Ting; Moore, Timothy M; Ebbert, Mark T W; McVey, Natalie L; Madsen, Steven R; Hallowell, David M; Harris, Alexander M; Char, Robin E; Mackay, Ryan P; Hancock, Chad R; Hansen, Jason M; Kauwe, John S; Thomson, David M

    2016-04-15

    Skeletal muscle-specific liver kinase B1 (LKB1) knockout mice (skmLKB1-KO) exhibit elevated mitogen-activated protein kinase (MAPK) signaling after treadmill running. MAPK activation is also associated with inflammation-related signaling in skeletal muscle. Since exercise can induce muscle damage, and inflammation is a response triggered by damaged tissue, we therefore hypothesized that LKB1 plays an important role in dampening the inflammatory response to muscle contraction, and that this may be due in part to increased susceptibility to muscle damage with contractions in LKB1-deficient muscle. Here we studied the inflammatory response and muscle damage with in situ muscle contraction or downhill running. After in situ muscle contractions, the phosphorylation of both NF-κB and STAT3 was increased more in skmLKB1-KO vs. wild-type (WT) muscles. Analysis of gene expression via microarray and RT-PCR shows that expression of many inflammation-related genes increased after contraction only in skmLKB1-KO muscles. This was associated with mild skeletal muscle fiber membrane damage in skmLKB1-KO muscles. Gene markers of oxidative stress were also elevated in skmLKB1-KO muscles after contraction. Using the downhill running model, we observed significantly more muscle damage after running in skmLKB1-KO mice, and this was associated with greater phosphorylation of both Jnk and STAT3 and increased expression of SOCS3 and Fos. In conclusion, we have shown that the lack of LKB1 in skeletal muscle leads to an increased inflammatory state in skeletal muscle that is exacerbated by muscle contraction. Increased susceptibility of the muscle to damage may underlie part of this response.

  19. The myotonic dystrophy kinase 3{prime}-untranslated region and its effect on gene expression

    SciTech Connect

    Ang, C.W.Y.; Sabourin, L.A.; Narang, M.A.

    1994-09-01

    Myotonic dystrophy (DM) is an autosomal dominant neuromuscular disease involving the expansion of an unstable CTG repeat in the 3{prime}-untranslated (3{prime}-UTR) region of the DM kinase (DMK) gene. Increased levels of mRNA in congenital compared to normal tissue have been shown, suggesting elevated DMK levels may be responsible for the disease phenotype. To study the effect of the DMK 3{prime}UTR on gene expression, a reporter gene system was constructed using the constitutive CMV promoter with the chloramphenicol acetyl transferase (CAT) open reading frame and the DMK 3{prime}UTR containing from 5 repeats up to 90 repeats. Transient transfection into a rhabdomyosarcoma cell line shows a three-fold increase in CAT activity from constructs containing a wildtype 3{prime}UTR (5 and 20 repeats) compared to a control construct containing only a poly(A) signal. Reporter constructs with repeats in the protomutation (50 repeats) and mutation (90 repeats) range show a greater than 10-fold increase over control CAT activity. These results suggest the presence of elements in the DMK 3{prime}UTR capable of conferring increased gene expression. We are currently investigating cell-specific activity of the constructs and conducting deletion mapping to identify regulatory elements in the 3{prime}-UTR.

  20. A system for assaying homologous recombination at the endogenous human thymidine kinase gene

    SciTech Connect

    Benjamin, M.B.; Little, J.B. ); Potter, H. ); Yandell, D.W. Massachusetts Eye and Ear Infirmary, Boston Harvard Medical School, Boston, MA )

    1991-08-01

    A system for assaying human interchromosomal recombination in vitro was developed, using a cell line containing two different mutant thymidine kinase genes (TK) on chromosomes 17. Heteroalleles were generated in the TK{sup +/+} parent B-lymphoblast cell line WIL-2 by repeated exposure to the alkylating nitrogen mustard ICR-191, which preferentially causes +1 or {minus}1 frameshifts. Resulting TK{sup {minus}/{minus}} mutants were selected in medium containing the toxic thymidine analog trifluorothymidine. In two lines, heterozygous frameshifts were located in exons 4 and 7 of the TK gene separated by {approx}8 kilobases. These lines undergo spontaneous reversion to TK{sup +} at a frequency of < 10{sup {minus}7}, and revertants can be selected in cytidine/hypoxanthine/aminopterin/thymidine medium. The nature and location of these heteroallelic mutations make large deletions, rearrangements, nondisjunction, and reduplication unlikely mechanisms for reversion to TK{sup +}. The mode of reversion to TK{sup +} was specifically assessed by DNA sequencing, use of single-strand conformation polymorphisms, and analysis of various restriction fragment length polymorphisms (RFLPs) linked to the TK gene on chromosome 17. The data suggest that a proportion of revertants has undergone recombination and gene conversion at the TK locus, with concomitant loss of frameshifts and allele loss at linked RFLPs. Models are presented for the origin of two recombinants.

  1. Identification, nomenclature, and evolutionary relationships of mitogen-activated protein kinase (MAPK) genes in soybean.

    PubMed

    Neupane, Achal; Nepal, Madhav P; Piya, Sarbottam; Subramanian, Senthil; Rohila, Jai S; Reese, R Neil; Benson, Benjamin V

    2013-01-01

    Mitogen-activated protein kinase (MAPK) genes in eukaryotes regulate various developmental and physiological processes including those associated with biotic and abiotic stresses. Although MAPKs in some plant species including Arabidopsis have been identified, they are yet to be identified in soybean. Major objectives of this study were to identify GmMAPKs, assess their evolutionary relationships, and analyze their functional divergence. We identified a total of 38 MAPKs, eleven MAPKKs, and 150 MAPKKKs in soybean. Within the GmMAPK family, we also identified a new clade of six genes: four genes with TEY and two genes with TQY motifs requiring further investigation into possible legume-specific functions. The results indicated the expansion of the GmMAPK families attributable to the ancestral polyploidy events followed by chromosomal rearrangements. The GmMAPK and GmMAPKKK families were substantially larger than those in other plant species. The duplicated GmMAPK members presented complex evolutionary relationships and functional divergence when compared to their counterparts in Arabidopsis. We also highlighted existing nomenclatural issues, stressing the need for nomenclatural consistency. GmMAPK identification is vital to soybean crop improvement, and novel insights into the evolutionary relationships will enhance our understanding about plant genome evolution.

  2. Identification, Nomenclature, and Evolutionary Relationships of Mitogen-Activated Protein Kinase (MAPK) Genes in Soybean

    PubMed Central

    Neupane, Achal; Nepal, Madhav P.; Piya, Sarbottam; Subramanian, Senthil; Rohila, Jai S.; Reese, R. Neil; Benson, Benjamin V.

    2013-01-01

    Mitogen-activated protein kinase (MAPK) genes in eukaryotes regulate various developmental and physiological processes including those associated with biotic and abiotic stresses. Although MAPKs in some plant species including Arabidopsis have been identified, they are yet to be identified in soybean. Major objectives of this study were to identify GmMAPKs, assess their evolutionary relationships, and analyze their functional divergence. We identified a total of 38 MAPKs, eleven MAPKKs, and 150 MAPKKKs in soybean. Within the GmMAPK family, we also identified a new clade of six genes: four genes with TEY and two genes with TQY motifs requiring further investigation into possible legume-specific functions. The results indicated the expansion of the GmMAPK families attributable to the ancestral polyploidy events followed by chromosomal rearrangements. The GmMAPK and GmMAPKKK families were substantially larger than those in other plant species. The duplicated GmMAPK members presented complex evolutionary relationships and functional divergence when compared to their counterparts in Arabidopsis. We also highlighted existing nomenclatural issues, stressing the need for nomenclatural consistency. GmMAPK identification is vital to soybean crop improvement, and novel insights into the evolutionary relationships will enhance our understanding about plant genome evolution. PMID:24137047

  3. Phosphatidylinositol phosphate 5-kinase genes respond to phosphate deficiency for root hair elongation in Arabidopsis thaliana.

    PubMed

    Wada, Yukika; Kusano, Hiroaki; Tsuge, Tomohiko; Aoyama, Takashi

    2015-02-01

    Plants drastically alter their root system architecture to adapt to different underground growth conditions. During phosphate (Pi) deficiency, most plants including Arabidopsis thaliana enhance the development of lateral roots and root hairs, resulting in bushy and hairy roots. To elucidate the signal pathway specific for the root hair elongation response to Pi deficiency, we investigated the expression of type-B phosphatidylinositol phosphate 5-kinase (PIP5K) genes, as a quantitative factor for root hair elongation in Arabidopsis. At young seedling stages, the PIP5K3 and PIP5K4 genes responded to Pi deficiency in steady-state transcript levels via PHR1-binding sequences (P1BSs) in their upstream regions. Both pip5k3 and pip5k4 single mutants, which exhibit short-root-hair phenotypes, remained responsive to Pi deficiency for root hair elongation; however the pip5k3pip5k4 double mutant exhibited shorter root hairs than the single mutants, and lost responsiveness to Pi deficiency at young seedling stages. In the tactical complementation line in which modified PIP5K3 and PIP5K4 genes with base substitutions in their P1BSs were co-introduced into the double mutant, root hairs of young seedlings had normal lengths under Pi-sufficient conditions, but were not responsive to Pi deficiency. From these results, we conclude that a Pi-deficiency signal is transferred to the pathway for root hair elongation via the PIP5K genes.

  4. Tyrosine kinase domain mutations of EGFR gene in head and neck squamous cell carcinoma

    PubMed Central

    Vatte, Chittibabu; Al Amri, Ali M; Cyrus, Cyril; Chathoth, Shahanas; Acharya, Sadananda; Hashim, Tariq Mohammad; Al Ali, Zhara; Alshreadah, Saleh Tawfeeq; Alsayyah, Ahmed; Al-Ali, Amein K

    2017-01-01

    Background Epidermal growth factor receptor (EGFR) is a commonly altered gene that is identified in various cancers, including head and neck squamous cell carcinoma (HNSCC). Therefore, EGFR is a promising molecular marker targeted by monoclonal antibodies and small molecule inhibitors targeting the tyrosine kinase (TK) domain. Objective The objective of this study was to investigate the spectrum of mutations in exons 18, 19, 20, and 21 of the EGFR gene in HNSCC patients. Materials and methods This retrospective study included 47 confirmed HNSCC cases. Mutations in the TK domain, exons 18, 19, 20, and 21 of the EGFR gene, were detected by Scorpion® chemistry and ARMS® technologies on Rotor-Gene Q real-time polymerase chain reaction. Results The tumors exhibited EGFR-TK domain mutations in 57% of cases. Four cases of T790M mutations were reported for the first time among HNSCC patients. Out of the total mutations, L861Q (exon 21), exon 20 insertions and deletions of exon 19 accounted for the majority of mutations (21%, 19%, and 17%, respectively). EGFR mutation status was correlated with the higher grade (P=0.026) and advanced stage (P=0.034) of HNSCC tumors. Conclusion Higher frequency of EGFR-TK domain mutations together with the presence of the T790M mutation suggests that identification of these mutations might streamline the therapy and provide a better prognosis in HNSCC cases. PMID:28352186

  5. Flatworms have lost the right open reading frame kinase 3 gene during evolution

    PubMed Central

    Breugelmans, Bert; Ansell, Brendan R. E.; Young, Neil D.; Amani, Parisa; Stroehlein, Andreas J.; Sternberg, Paul W.; Jex, Aaron R.; Boag, Peter R.; Hofmann, Andreas; Gasser, Robin B.

    2015-01-01

    All multicellular organisms studied to date have three right open reading frame kinase genes (designated riok-1, riok-2 and riok-3). Current evidence indicates that riok-1 and riok-2 have essential roles in ribosome biosynthesis, and that the riok-3 gene assists this process. In the present study, we conducted a detailed bioinformatic analysis of the riok gene family in 25 parasitic flatworms (platyhelminths) for which extensive genomic and transcriptomic data sets are available. We found that none of the flatworms studied have a riok-3 gene, which is unprecedented for multicellular organisms. We propose that, unlike in other eukaryotes, the loss of RIOK-3 from flatworms does not result in an evolutionary disadvantage due to the unique biology and physiology of this phylum. We show that the loss of RIOK-3 coincides with a loss of particular proteins associated with essential cellular pathways linked to cell growth and apoptosis. These findings indicate multiple, key regulatory functions of RIOK-3 in other metazoan species. Taking advantage of a known partial crystal structure of human RIOK-1, molecular modelling revealed variability in nucleotide binding sites between flatworm and human RIOK proteins. PMID:25976756

  6. The Protein Kinase KIS Impacts Gene Expression during Development and Fear Conditioning in Adult Mice

    PubMed Central

    Manceau, Valérie; Kremmer, Elisabeth; Nabel, Elizabeth G.; Maucuer, Alexandre

    2012-01-01

    The brain-enriched protein kinase KIS (product of the gene UHMK1) has been shown to phosphorylate the human splicing factor SF1 in vitro. This phosphorylation in turn favors the formation of a U2AF65-SF1-RNA complex which occurs at the 3′ end of introns at an early stage of spliceosome assembly. Here, we analyzed the effects of KIS knockout on mouse SF1 phosphorylation, physiology, adult behavior, and gene expression in the neonate brain. We found SF1 isoforms are differently expressed in KIS-ko mouse brains and fibroblasts. Re-expression of KIS in fibroblasts restores a wild type distribution of SF1 isoforms, confirming the link between KIS and SF1. Microarray analysis of transcripts in the neonate brain revealed a subtle down-regulation of brain specific genes including cys-loop ligand-gated ion channels and metabolic enzymes. Q-PCR analyses confirmed these defects and point to an increase of pre-mRNA over mRNA ratios, likely due to changes in splicing efficiency. While performing similarly in prepulse inhibition and most other behavioral tests, KIS-ko mice differ in spontaneous activity and contextual fear conditioning. This difference suggests that disregulation of gene expression due to KIS inactivation affects specific brain functions. PMID:22937132

  7. Functional expression of the Herpes simplex virus thymidine kinase gene in Escherichia coli K-12.

    PubMed

    Kit, S; Otsuka, H; Qavi, H; Kit, M

    1981-12-01

    The recombinant plasmid pAGO contains the Herpes simplex virus type 1 (HSV-1) thymidine kinase (TK) gene and consists of a 2-kb PvuII fragment of HSV-1 DNA inserted into the PvuII site of pBR322. A deletion mutant of pAGO, designated pMH110, has been isolated which removes the normal HSV-1 TK gene promoter but places the promoter of the pBR322 tetracycline-resistance (tetr) gene only about 400 bp from the translational start codon of the HSV-1 TK polypeptide. In contrast to pAGO, which transforms mouse LM(TK-) cells to TK+ but is only weakly expressed in TK- bacteria, pMH110 not only efficiently transforms LM(TK-) cells to TK+ but also enables TK- Escherichia coli K-12 cells to form colonies on selective plates containing 5-fluorodeoxyuridine (FdUrd) plus thymidine (dThd) and to exhibit fully restored ability to incorporate [3H]dThd into DNA. The levels of TK activity expressed by bacteria harboring pMH110 were about as high as those expressed by bacteria harboring plasmid pTK3, which contains the wild-type E. coli TK gene. The TK activity expressed in bacteria harboring pMH110 was partially purified and shown to be HSV-1-specific by serological and disc PAGE analyses and by experiments demonstrating that this enzyme phosphorylated [125I]deoxycytidine.

  8. Assignment of the protein kinase C delta polypeptide gene (PRKCD) to human chromosome 3 and mouse chromosome 14.

    PubMed

    Huppi, K; Siwarski, D; Goodnight, J; Mischak, H

    1994-01-01

    The protein kinase C (pkc) enzymes are a family of serine-threonine protein kinases, each encoded by a distinct and separate gene. The chromosomal locations of human PRKCA, PRKCB, and PRKCG have previously been established. We now report that PRKCD, a novel member of the pkc gene family, maps to human chromosome 3. The chromosomal location of Pkcd has also been determined in the mouse by analysis of recombination frequency in an interspecific panel of backcross mice. We find that the locus encoding pkcd resides proximal to nucleoside phosphorylase (Np-2) and Tcra on mouse chromosome 14 in a region syntenic with human 3p.

  9. EML4-ALK fusion gene and efficacy of an ALK kinase inhibitor in lung cancer

    PubMed Central

    Koivunen, Jussi P.; Mermel, Craig; Zejnullahu, Kreshnik; Murphy, Carly; Lifshits, Eugene; Holmes, Alison J.; Choi, Hwan Geun; Kim, Jhingook; Chiang, Derek; Thomas, Roman; Lee, Jinseon; Richards, William G.; Sugarbaker, David J.; Ducko, Christopher; Lindeman, Neal; Marcoux, J. Paul; Engelman, Jeffrey A.; Gray, Nathanael S.; Lee, Charles; Meyerson, Matthew; Jänne, Pasi A.

    2011-01-01

    Purpose The EML4-ALK fusion gene has been detected in ~7% of Japanese non-small cell lung cancers (NSCLC). We determined the frequency of EML4-ALK in Caucasian NSCLCs and in NSCLC cell lines. We also determined whether TAE684, a specific ALK kinase inhibitor, would inhibit the growth of EML4-ALK containing cell lines in vitro and in vivo. Experimental Design We screened 305 primary NSCLCs (both US (n=138) and Korean (n=167) patients) and 83 NSCLC cell lines using RT-PCR and by exon array analyses. We evaluated the efficacy of TAE684 against NSCLC cell lines in vitro and in vivo. Results We detected 4 different variants, including two novel variants, of EML4-ALK using RT-PCR in 8/305 tumors (3%) and in 3/83 (3.6%) NSCLC cell lines. All EML4-ALK containing tumors and cell lines were adenocarcinomas. EML4-ALK was detected more frequently in NSCLC patients who were never or light (< 10 pack years) cigarette smokers compared to current/former smokers (6% vs. 1%; p=0.049). TAE684 inhibited the growth of 1 of 3 (H3122) EML4-ALK containing cell lines in vitro and in vivo, inhibited Akt phosphorylation and caused apoptosis. In another EML4-ALK cell line, DFCI032, TAE684 was ineffective due to co-activation of EGFR and ERBB2. The combination of TAE684 and CL-387,785 (EGFR/ERBB2 kinase inhibitor), inhibited growth and Akt phosphorylation and led to apoptosis in the DFCI032 cell line. Conclusions EML4-ALK is found in the minority of NSCLCs. ALK kinase inhibitors alone or in combination may nevertheless be clinically effective treatments for NSCLC patients whose tumors contain EML4-ALK. PMID:18594010

  10. Two Cdc2 Kinase Genes with Distinct Functions in Vegetative and Infectious Hyphae in Fusarium graminearum

    PubMed Central

    Liu, Huiquan; Zhang, Shijie; Ma, Jiwen; Dai, Yafeng; Li, Chaohui; Lyu, Xueliang; Wang, Chenfang; Xu, Jin-Rong

    2015-01-01

    Eukaryotic cell cycle involves a number of protein kinases important for the onset and progression through mitosis, most of which are well characterized in the budding and fission yeasts and conserved in other fungi. However, unlike the model yeast and filamentous fungi that have a single Cdc2 essential for cell cycle progression, the wheat scab fungus Fusarium graminearum contains two CDC2 orthologs. The cdc2A and cdc2B mutants had no obvious defects in growth rate and conidiation but deletion of both of them is lethal, indicating that these two CDC2 orthologs have redundant functions during vegetative growth and asexual reproduction. However, whereas the cdc2B mutant was normal, the cdc2A mutant was significantly reduced in virulence and rarely produced ascospores. Although deletion of CDC2A had no obvious effect on the formation of penetration branches or hyphopodia, the cdc2A mutant was limited in the differentiation and growth of infectious growth in wheat tissues. Therefore, CDC2A plays stage-specific roles in cell cycle regulation during infectious growth and sexual reproduction. Both CDC2A and CDC2B are constitutively expressed but only CDC2A was up-regulated during plant infection and ascosporogenesis. Localization of Cdc2A- GFP to the nucleus but not Cdc2B-GFP was observed in vegetative hyphae, ascospores, and infectious hyphae. Complementation assays with chimeric fusion constructs showed that both the N- and C-terminal regions of Cdc2A are important for its functions in pathogenesis and ascosporogenesis but only the N-terminal region is important for its subcellular localization. Among the Sordariomycetes, only three Fusarium species closely related to F. graminearum have two CDC2 genes. Furthermore, F. graminearum uniquely has two Aurora kinase genes and one additional putative cyclin gene, and its orthologs of CAK1 and other four essential mitotic kinases in the budding yeast are dispensable for viability. Overall, our data indicate that cell cycle

  11. Molecular variation and evolution of the tyrosine kinase domains of insulin receptor IRa and IRb genes in Cyprinidae.

    PubMed

    Kong, XiangHui; Wang, XuZhen; He, ShunPing

    2011-07-01

    The insulin receptor (IR) gene plays an important role in regulating cell growth, differentiation and development. In the present study, DNA sequences of insulin receptor genes, IRa and IRb, were amplified and sequenced from 37 representative species of the Cyprinidae and from five outgroup species from non-cyprinid Cypriniformes. Based on coding sequences (CDS) of tyrosine kinase regions of IRa and IRb, molecular evolution and phylogenetic relationships were analyzed to better understand the characteristics of IR gene divergence in the family Cyprinidae. IRa and IRb were clustered into one lineage in the gene tree of the IR gene family, reconstructed using the unweighted pair group method with arithmetic mean (UPGMA). IRa and IRb have evolved into distinct genes after IR gene duplication in Cyprinidae. For each gene, molecular evolution analyses showed that there was no significant difference among different groups in the reconstructed maximum parsimony (MP) tree of Cyprinidae; IRa and IRb have been subjected to similar evolutionary pressure among different lineages. Although the amino acid sequences of IRa and IRb tyrosine kinase regions were highly conserved, our analyses showed that there were clear sequence variations between the tyrosine kinase regions of IRa and IRb proteins. This indicates that IRa and IRb proteins might play different roles in the insulin signaling pathway.

  12. Phosphoinositide 3-kinase δ gene mutation predisposes to respiratory infection and airway damage

    PubMed Central

    Angulo, Ivan; Vadas, Oscar; Garçon, Fabien; Banham-Hall, Edward; Plagnol, Vincent; Leahy, Timothy R.; Baxendale, Helen; Coulter, Tanya; Curtis, James; Wu, Changxin; Blake-Palmer, Katherine; Perisic, Olga; Smyth, Deborah; Maes, Mailis; Fiddler, Christine; Juss, Jatinder; Cilliers, Deirdre; Markelj, Gašper; Chandra, Anita; Farmer, George; Kielkowska, Anna; Clark, Jonathan; Kracker, Sven; Debré, Marianne; Picard, Capucine; Pellier, Isabelle; Jabado, Nada; Morris, James A.; Barcenas-Morales, Gabriela; Fischer, Alain; Stephens, Len; Hawkins, Phillip; Barrett, Jeffrey C.; Abinun, Mario; Clatworthy, Menna; Durandy, Anne; Doffinger, Rainer; Chilvers, Edwin; Cant, Andrew J.; Kumararatne, Dinakantha; Okkenhaug, Klaus; Williams, Roger L.; Condliffe, Alison; Nejentsev, Sergey

    2014-01-01

    Genetic mutations cause primary immunodeficiencies (PIDs), which predispose to infections. Here we describe Activated PI3K-δ Syndrome (APDS), a PID associated with a dominant gain-of-function mutation E1021K in the p110δ protein, the catalytic subunit of phosphoinositide 3-kinase δ (PI3Kδ), encoded by the PIK3CD gene. We found E1021K in 17 patients from seven unrelated families, but not among 3,346 healthy subjects. APDS was characterized by recurrent respiratory infections, progressive airway damage, lymphopenia, increased circulating transitional B cells, increased IgM and reduced IgG2 levels in serum and impaired vaccine responses. The E1021K mutation enhanced membrane association and kinase activity of p110δ. Patient-derived lymphocytes had increased levels of phosphatidylinositol 3,4,5-trisphosphate and phosphorylated AKT protein and were prone to activation-induced cell death. Selective p110δ inhibitors IC87114 and GS-1101 reduced the activity of the mutant enzyme in vitro, suggesting a therapeutic approach for patients with APDS. PMID:24136356

  13. GCN-2 dependent inhibition of protein synthesis activates osmosensitive gene transcription via WNK and Ste20 kinase signaling

    PubMed Central

    Lee, Elaine Choung-Hee

    2012-01-01

    Increased gpdh-1 transcription is required for accumulation of the organic osmolyte glycerol and survival of Caenorhabditis elegans during hypertonic stress. Our previous work has shown that regulators of gpdh-1 (rgpd) gene knockdown constitutively activates gpdh-1 expression. Fifty-five rgpd genes play essential roles in translation suggesting that inhibition of protein synthesis is an important signal for regulating osmoprotective gene transcription. We demonstrate here that translation is reduced dramatically by hypertonic stress or knockdown of rgpd genes encoding aminoacyl-tRNA synthetases and eukaryotic translation initiation factors (eIFs). Toxin-induced inhibition of translation also activates gpdh-1 expression. Hypertonicity-induced translation inhibition is mediated by general control nonderepressible (GCN)-2 kinase signaling and eIF-2α phosphoryation. Loss of gcn-1 or gcn-2 function prevents eIF-2α phosphorylation, completely blocks reductions in translation, and inhibits gpdh-1 transcription. gpdh-1 expression is regulated by the highly conserved with-no-lysine kinase (WNK) and Ste20 kinases WNK-1 and GCK-3, which function in the GCN-2 signaling pathway downstream from eIF-2α phosphorylation. Our previous work has shown that hypertonic stress causes rapid and dramatic protein damage in C. elegans and that inhibition of translation reduces this damage. The current studies demonstrate that reduced translation also serves as an essential signal for activation of WNK-1/GCK-3 kinase signaling and subsequent transcription of gpdh-1 and possibly other osmoprotective genes. PMID:23076791

  14. GCN-2 dependent inhibition of protein synthesis activates osmosensitive gene transcription via WNK and Ste20 kinase signaling.

    PubMed

    Lee, Elaine Choung-Hee; Strange, Kevin

    2012-12-15

    Increased gpdh-1 transcription is required for accumulation of the organic osmolyte glycerol and survival of Caenorhabditis elegans during hypertonic stress. Our previous work has shown that regulators of gpdh-1 (rgpd) gene knockdown constitutively activates gpdh-1 expression. Fifty-five rgpd genes play essential roles in translation suggesting that inhibition of protein synthesis is an important signal for regulating osmoprotective gene transcription. We demonstrate here that translation is reduced dramatically by hypertonic stress or knockdown of rgpd genes encoding aminoacyl-tRNA synthetases and eukaryotic translation initiation factors (eIFs). Toxin-induced inhibition of translation also activates gpdh-1 expression. Hypertonicity-induced translation inhibition is mediated by general control nonderepressible (GCN)-2 kinase signaling and eIF-2α phosphoryation. Loss of gcn-1 or gcn-2 function prevents eIF-2α phosphorylation, completely blocks reductions in translation, and inhibits gpdh-1 transcription. gpdh-1 expression is regulated by the highly conserved with-no-lysine kinase (WNK) and Ste20 kinases WNK-1 and GCK-3, which function in the GCN-2 signaling pathway downstream from eIF-2α phosphorylation. Our previous work has shown that hypertonic stress causes rapid and dramatic protein damage in C. elegans and that inhibition of translation reduces this damage. The current studies demonstrate that reduced translation also serves as an essential signal for activation of WNK-1/GCK-3 kinase signaling and subsequent transcription of gpdh-1 and possibly other osmoprotective genes.

  15. A novel pantothenate kinase gene (PANK2) is defective in Hallervorden-Spatz syndrome.

    PubMed

    Zhou, B; Westaway, S K; Levinson, B; Johnson, M A; Gitschier, J; Hayflick, S J

    2001-08-01

    Hallervorden-Spatz syndrome (HSS) is an autosomal recessive neurodegenerative disorder associated with iron accumulation in the brain. Clinical features include extrapyramidal dysfunction, onset in childhood, and a relentlessly progressive course. Histologic study reveals iron deposits in the basal ganglia. In this respect, HSS may serve as a model for complex neurodegenerative diseases, such as Parkinson disease, Alzheimer disease, Huntington disease and human immunodeficiency virus (HIV) encephalopathy, in which pathologic accumulation of iron in the brain is also observed. Thus, understanding the biochemical defect in HSS may provide key insights into the regulation of iron metabolism and its perturbation in this and other neurodegenerative diseases. Here we show that HSS is caused by a defect in a novel pantothenate kinase gene and propose a mechanism for oxidative stress in the pathophysiology of the disease.

  16. Modulation of human c-mpl gene expression by thrombopoietin through protein kinase C.

    PubMed

    Sunohara, M; Morikawa, S; Sato, T; Sato, I; Sato, T; Fuse, A

    2003-01-01

    The c-Mpl, thrombopoietin (TPO) receptor specificially controls megakaryocytic growth and differentiation. TPO increased the c-mpl promoter activity determined by a transient expression system using a vector containing the luciferase gene as a reporter in the human megakaryoblastic cell line CMK. The maximal promoter activity of c-mpl was obtained 24 hr after pretreatment with TPO for 3 hr and then declined with time. This increase was completely abolished by protein kinase C (PKC) inhibitors (GF109203, calphostin C and H7). Phorbol 12-myristate 13-acetate (PMA) treatment led to an increase in c-mpl promoter activity. These results demonstrate that the promoter activity of c-mpl is modulated by transcription through a PKC-dependent pathway.

  17. Overexpression of polyphosphate kinase gene (ppk) increases bioinsecticide production by Bacillus thuringiensis.

    PubMed

    Doruk, Tugrul; Avican, Ummehan; Camci, Irem Yalim; Gedik, Sedef Tunca

    2013-05-06

    Polyphosphate (polyP), synthesized by polyP kinase (PPK) using the terminal phosphate of ATP as substrate, performs important functions in every living cell. The present work reports on the relationship between polyP metabolism and bioinsecticide production in Bacillus thuringiensis subsp. israelensis (Bti). The ppk gene of Bti was cloned into vector pHT315 and the effect of its overexpression on endotoxin production was determined. Endotoxin production by the recombinant strain was found to be consistently higher than that by the wild type strain and the strain that carried the empty plasmid. The toxicity of the recombinant mutant strain (LC50 5.8±0.6ngml(-1)) against late 2nd instar Culex quinquefasciatus was about 7.7 times higher than that of Bti (LC50 44.9±7ngml(-1)). To our knowledge this is the first reported study which relates polyP metabolism with bioinsecticide biosynthesis.

  18. Amino acid substitutions in the thymidine kinase gene of induced acyclovir-resistant herpes simplex virus type 1

    NASA Astrophysics Data System (ADS)

    Hussin, Ainulkhir; Nor, Norefrina Shafinaz Md; Ibrahim, Nazlina

    2013-11-01

    Acyclovir (ACV) is an antiviral drug of choice in healthcare setting to treat infections caused by herpes viruses, including, but not limited to genital herpes, cold sores, shingles and chicken pox. Acyclovir resistance has emerged significantly due to extensive use and misuse of this antiviral in human, especially in immunocompromised patients. However, it remains unclear about the amino acid substitutions in thymidine (TK) gene, which specifically confer the resistance-associated mutation in herpes simplex virus. Hence, acyclovir-resistant HSV-1 was selected at high concentration (2.0 - 4.5 μg/mL), and the TK-gene was subjected to sequencing and genotypic characterization. Genotypic sequences comparison was done using HSV-1 17 (GenBank Accesion no. X14112) for resistance-associated mutation determination whereas HSV-1 KOS, HSV-1 473/08 and HSV clinical isolates sequences were used for polymorphism-associated mutation. The result showed that amino acid substitutions at the non-conserved region (UKM-1: Gln34Lys, UKM-2: Arg32Ser & UKM-5: Arg32Cys) and ATP-binding site (UKM-3: Tyr53End & UKM-4: Ile54Leu) of the TK-gene. These discoveries play an important role to extend another dimension to the evolution of acyclovir-resistant HSV-1 and suggest that selection at high ACV concentration induced ACV-resistant HSV-1 evolution. These findings also expand the knowledge on the type of mutations among acyclovir-resistant HSV-1. In conclusion, HSV-1 showed multiple strategies to exhibit acyclovir resistance, including amino acid substitutions in the TK gene.

  19. Regulation of floral patterning and organ identity by Arabidopsis ERECTA-family receptor kinase genes.

    PubMed

    Bemis, Shannon M; Lee, Jin Suk; Shpak, Elena D; Torii, Keiko U

    2013-12-01

    Due to the lack of cell migration, plant organogenesis relies on coordinated cell proliferation, cell growth, and differentiation. A flower possesses a complex structure, with sepals and petals constituting the perianth, and stamens and pistils where male and female gametophytes differentiate. While advances have been made in our understanding of gene regulatory networks controlling flower development, relatively little is known of how cell-cell coordination influences floral organ specification. The Arabidopsis ERECTA (ER)-family receptor kinases, ER, ER-LIKE1 (ERL1), and ERL2, regulate inflorescence architecture, organ shape, and epidermal stomatal patterning. Here it is reported that ER-family genes together regulate floral meristem organization and floral organ identity. The stem cell marker CLAVATA3 exhibits misplaced expression in the floral meristems of the er erl1 erl2 mutant. Strikingly, homeotic conversion of sepals to carpels was observed in er erl1 erl2 flowers. Consistently, ectopic expression of AGAMOUS, which determines carpel identity, was detected in er erl1 erl2 flower primordia. Among the known downstream components of ER-family receptor kinases in stomatal patterning, YODA (YDA) is also required for proper floral patterning. YDA and the ER-family show complex, synergistic genetic interactions: er erl1 erl2 yda quadruple mutant plants become extremely small, callus-like masses. While a constitutively active YDA fully rescues stomatal clustering in er erl1 erl2, it only partially rescues er erl1 erl2 flower defects. The study suggests that ER-family signalling is crucial for ensuring proper expression domains of floral meristem and floral organ identity determinants, and further implies the existence of a non-canonical downstream pathway.

  20. The primary structure of the Saccharomyces cerevisiae gene for 3-phosphoglycerate kinase.

    PubMed Central

    Hitzeman, R A; Hagie, F E; Hayflick, J S; Chen, C Y; Seeburg, P H; Derynck, R

    1982-01-01

    The DNA sequence of the gene for the yeast glycolytic enzyme, 3-phosphoglycerate kinase (PGK), has been obtained by sequencing part of a 3.1 kbp HindIII fragment obtained from the yeast genome. The structural gene sequence corresponds to a reading frame of 1251 bp coding for 416 amino acids with no intervening DNA sequences. The amino acid sequence is approximately 65 percent homologous with human and horse PGK protein sequences and is in general agreement with the published protein sequence for yeast PGK. As for other highly expressed structural genes in yeast, the coding sequence is highly codon biased with 95 percent of the amino acids coded for by a select 25 codons (out of 61 possible). Besides structural DNA sequence, 291 bp of 5'-flanking sequence and 286 bp of 3'-flanking sequence were determined. Transcription starts 36 nucleotides upstream from the translational start and stops 86-93 nucleotides downstream from the translational stop. These results suggest a non-polyadenylated mRNA length of 1373 to 1380 nucleotides, which is consistent with the observed length of 1500 nucleotides for polyadenylated PGK mRNA. A sequence TATATATAAA is found at 145 nucleotides upstream from the translational start. This sequence resembles the TATAAA box that is possibly associated with RNA polymerase II binding. Images PMID:6296791

  1. The mouse muscle creatine kinase promoter faithfully drives reporter gene expression in transgenic Xenopus laevis.

    PubMed

    Lim, Wayland; Neff, Eric S; Furlow, J David

    2004-06-17

    Developing Xenopus laevis experience two periods of muscle differentiation, once during embryogenesis and again at metamorphosis. During metamorphosis, thyroid hormone induces both muscle growth in the limbs and muscle death in the tail. In mammals, the muscle creatine kinase (MCK) gene is activated during the differentiation from myoblasts to myocytes and has served as both a marker for muscle development and to drive transgene expression in transgenic mice. Transcriptional control elements are generally highly conserved throughout evolution, potentially allowing mouse promoter use in transgenic X. laevis. This paper compares endogenous X. laevis MCK gene expression and the mouse MCK (mMCK) promoter driving a green fluorescent protein reporter in transgenic X. laevis. The mMCK promoter demonstrated strong skeletal muscle-specific transgene expression in both the juvenile tadpole and adult frog. Therefore, our results clearly demonstrate the functional conservation of regulatory sequences in vertebrate muscle gene promoters and illustrate the utility of using X. laevis transgenesis for detailed comparative study of mammalian promoter activity in vivo.

  2. Dynamic Regulation of the Adenosine Kinase Gene during Early Postnatal Brain Development and Maturation

    PubMed Central

    Kiese, Katharina; Jablonski, Janos; Boison, Detlev; Kobow, Katja

    2016-01-01

    The ubiquitous metabolic intermediary and nucleoside adenosine is a “master regulator” in all living systems. Under baseline conditions adenosine kinase (ADK) is the primary enzyme for the metabolic clearance of adenosine. By regulating the availability of adenosine, ADK is a critical upstream regulator of complex homeostatic and metabolic networks. Not surprisingly, ADK dysfunction is involved in several pathologies, including diabetes, epilepsy, and cancer. ADK protein exists in the two isoforms nuclear ADK-L, and cytoplasmic ADK-S, which are subject to dynamic expression changes during brain development and in response to brain injury; however, gene expression changes of the Adk gene as well as regulatory mechanisms that direct the cell-type and isoform specific expression of ADK have never been investigated. Here we analyzed potential gene regulatory mechanisms that may influence Adk expression including DNA promoter methylation, histone modifications and transcription factor binding. Our data suggest binding of transcription factor SP1 to the Adk promoter influences the regulation of Adk expression. PMID:27812320

  3. VIP1 response elements mediate mitogen-activated protein kinase 3-induced stress gene expression

    PubMed Central

    Pitzschke, Andrea; Djamei, Armin; Teige, Markus; Hirt, Heribert

    2009-01-01

    The plant pathogen Agrobacterium tumefaciens transforms plant cells by delivering its T-DNA into the plant cell nucleus where it integrates into the plant genome and causes tumor formation. A key role of VirE2-interacting protein 1 (VIP1) in the nuclear import of T-DNA during Agrobacterium-mediated plant transformation has been unravelled and VIP1 was shown to undergo nuclear localization upon phosphorylation by the mitogen-activated protein kinase MPK3. Here, we provide evidence that VIP1 encodes a functional bZIP transcription factor that stimulates stress-dependent gene expression by binding to VIP1 response elements (VREs), a DNA hexamer motif. VREs are overrepresented in promoters responding to activation of the MPK3 pathway such as Trxh8 and MYB44. Accordingly, plants overexpressing VIP1 accumulate high levels of Trxh8 and MYB44 transcripts, whereas stress-induced expression of these genes is impaired in mpk3 mutants. Trxh8 and MYB44 promoters are activated by VIP1 in a VRE-dependent manner. VIP1 strongly enhances expression from a synthetic promoter harboring multiple VRE copies and directly interacts with VREs in vitro and in vivo. Chromatin immunoprecipitation assays of the MYB44 promoter confirm that VIP1 binding to VREs is enhanced under conditions of MPK3 pathway stimulation. These results provide molecular insight into the cellular mechanism of target gene regulation by the MPK3 pathway. PMID:19820165

  4. Activity, Expression and Function of a Second Drosophila Protein Kinase a Catalytic Subunit Gene

    PubMed Central

    Melendez, A.; Li, W.; Kalderon, D.

    1995-01-01

    The DC2 gene was isolated previously on the basis of sequence similarity to DCO, the major Drosophila protein kinase A (PKA) catalytic subunit gene. We show here that the 67-kD Drosophila DC2 protein behaves as a PKA catalytic subunit in vitro. DC2 is transcribed in mesodermal anlagen of early embryos. This expression depends on dorsal but on neither twist nor snail activity. DC2 transcriptional fusions mimic this embryonic expression and are also expressed in subsets of cells in the optic lamina, wing disc and leg discs of third instar larvae. A saturation screen of a small deficiency interval containing DC2 for recessive lethal mutations yielded no DC2 alleles. We therefore isolated new deficiencies to generate deficiency trans-heterozygotes that lacked DC2 activity. These animals were viable and fertile. The absence of DC2 did not affect the viability or phenotype of imaginal disc cells lacking DC0 activity or embryonic hatching of animals with reduced DC0 activity. Furthermore, transgenes expressing DC2 from a DC0 promoter did not efficiently rescue a variety of DC0 mutant phenotypes. These observations indicate that DC2 is not an essential gene and is unlikely to be functionally redundant with DC0, which has multiple unique functions during development. PMID:8601490

  5. Role of the promoter in the regulation of the thymidine kinase gene

    SciTech Connect

    Travali, S.; Lipson, K.E.; Jaskulski, D.; Lauret, E.; Baserga, R.

    1988-04-01

    To identify the regulatory elements of the human thymidine kinase (TK) gene, the authors established stable cell lines carrying different chimeric constructs of the TK gene. Their results can be summarized as follows. (i) When the TK coding sequence is under the control of the calcyclin promoter (a promoter that is activated when G/sub o/ cells are stimulated by growth factors), TK mRNA levels are higher in G/sub 1/-arrested cells than in proliferating cells: (ii) when the TK coding sequence is under the control of the promoter of heat shock protein HSP70, steady-state levels of TK mRNA are highest after heat shock, regardless of the position of the cells in the cell cycle; (iii) the bacterial CAT gene under the control of the human TK promoter is maximally expressed in the S phase; (iv) the TK cDNA driven by the simian virus 40 promoter is also maximally expressed in the S phase; and (v) TK enzyme activity is always at a maximum in the S phase, even when the levels of TK mRNA are highest in nonproliferating cells. The authors conclude that although the TK coding sequence may also play some role, the TK promoter has an important role in the cell cycle regulation of TK mRNA levels.

  6. T Cell Receptor-Independent Basal Signaling via Erk and Abl Kinases Suppresses RAG Gene Expression

    PubMed Central

    Roose, Jeroen P; Diehn, Maximilian; Tomlinson, Michael G; Lin, Joseph; Alizadeh, Ash A; Botstein, David; Brown, Patrick O

    2003-01-01

    Signal transduction pathways guided by cellular receptors commonly exhibit low-level constitutive signaling in a continuous, ligand-independent manner. The dynamic equilibrium of positive and negative regulators establishes such a tonic signal. Ligand-independent signaling by the precursors of mature antigen receptors regulates development of B and T lymphocytes. Here we describe a basal signal that controls gene expression profiles in the Jurkat T cell line and mouse thymocytes. Using DNA microarrays and Northern blots to analyze unstimulated cells, we demonstrate that expression of a cluster of genes, including RAG-1 and RAG-2, is repressed by constitutive signals requiring the adapter molecules LAT and SLP-76. This TCR-like pathway results in constitutive low-level activity of Erk and Abl kinases. Inhibition of Abl by the drug STI-571 or inhibition of signaling events upstream of Erk increases RAG-1 expression. Our data suggest that physiologic gene expression programs depend upon tonic activity of signaling pathways independent of receptor ligation. PMID:14624253

  7. Inhibition by interferon of biochemical transformation induced by cloned herpesvirus thymidine kinase genes.

    PubMed

    Otsuka, H; Qavi, H; Kit, S

    1982-10-01

    To learn whether interferon could prevent the biochemical transformations induced by cloned herpesvirus thymidine kinase (TK) genes, LM(TK-) mouse fibroblast cultures were pretreated for 24 h with 2.4-40 international units (I.U.)/ml mouse alpha + beta interferon, and subsequently transformed to the TK+ phenotype with recombinant plasmids containing the herpes simplex virus type 1 (HSV-1) TK gene (pAGO and pMH110) and the marmoset herpesvirus (MarHV) TK gene (pMAR035). Mouse alpha + beta interferon inhibited transformation and the inhibition was interferon dose-dependent. Transformation was also inhibited when LM(TK-) cells were pretreated for 2-5 h with 40 I.U./ml interferon. Maximal inhibitions of TK+ colony formation were observed following a 9-20 h pretreatment period with interferon. In contrast, 40 I.U./ml interferon treatment for 20 h did not reduce the rate or extent of LM(TK-) cell growth. Experiments in which cultures were first treated with plasmid pAGO and only afterwards treated with interferon also showed that, as the interferon concentration used, interferon did not inhibit the outgrowth of transformated colonies. Enzyme assays showed that pretreatment with interferon inhibited the induction of TK activity in cells that had been transfected with pAGO DNA.

  8. Chromatin structure is required to block transcription of the methylated herpes simplex virus thymidine kinase gene

    SciTech Connect

    Buschhausen, G.; Wittig, B.; Graessmann, M.; Graessmann, A.

    1987-03-01

    Inhibition of herpes simplex virus (HSV) thymidine kinase (TK) gene transcription (pHSV-106, pML-BPV-TK4) by DNA methylation is an indirect effect, which occurs with a latency period of approx. 8 hr microinjection of the DNA into TK/sup -/ rat 2 and mouse LTK/sup -/ cells. The authors have strong evidence that chromatin formation is critical for the transition of the injected DNA from methylation insensitivity to methylation sensitivity. Chromatin was reconstituted in vitro by using methylated and mock-methylated HSV TK DNA and purified chicken histone octamers. After microinjection, the methylated chromatin was always biologically inactive, as tested by autoradiography of the cells after incubation with (/sup 3/H)thymidine and by RNA dot blot analysis. However, in transformed cell lines, reactivation of the methylated chromatic occurred after treatment with 5-azacytidine. Furthermore, integration of the TK chromatin into the host genome is not required to block expression of the methylated TK gene. Mouse cells that contained the pML-BPV-TK4 chromatin permanently in an episomal state also did not support TK gene expression as long as the TK DNA remained methylated.

  9. c-Abl and Arg induce cathepsin-mediated lysosomal degradation of the NM23-H1 metastasis suppressor in invasive cancer

    PubMed Central

    Sledziona, James; Cibull, Michael L.; Wang, Chi; Richards, Dana L.; Neltner, Janna M.; Beach, Carol; McCorkle, Joseph R.; Kaetzel, David M.; Plattner, Rina

    2014-01-01

    Metastasis suppressors comprise a growing class of genes whose downregulation triggers metastatic progression. In contrast to tumor suppressors, metastasis suppressors are rarely mutated or deleted, and little is known regarding the mechanisms by which their expression is downregulated. Here, we demonstrate that the metastasis suppressor, NM23-H1, is degraded by lysosomal cysteine cathepsins (L,B), which directly cleave NM23-H1. In addition, activation of c-Abl and Arg oncoproteins induces NM23-H1 degradation in invasive cancer cells by increasing cysteine cathepsin transcription and activation. Moreover, c-Abl activates cathepsins by promoting endosome maturation, which facilitates trafficking of NM23-H1 to the lysosome where it is degraded. Importantly, the invasion- and metastasis-promoting activity of c-Abl/Arg is dependent on their ability to induce NM23-H1 degradation, and the pathway is clinically relevant as c-Abl/Arg activity and NM23-H1 expression are inversely correlated in primary breast cancers and melanomas. Thus, we demonstrate a novel mechanism by which cathepsin expression is upregulated in cancer cells (via Abl kinases). We also identify a novel role for intracellular cathepsins in invasion and metastasis (degradation of a metastasis suppressor). Finally, we identify novel crosstalk between oncogenic and metastasis suppressor pathways, thereby providing mechanistic insight into the process of NM23-H1 loss, which may pave the way for new strategies to restore NM23-H1 expression and block metastatic progression. PMID:24096484

  10. c-Abl and Arg induce cathepsin-mediated lysosomal degradation of the NM23-H1 metastasis suppressor in invasive cancer.

    PubMed

    Fiore, L S; Ganguly, S S; Sledziona, J; Cibull, M L; Wang, C; Richards, D L; Neltner, J M; Beach, C; McCorkle, J R; Kaetzel, D M; Plattner, R

    2014-09-04

    Metastasis suppressors comprise a growing class of genes whose downregulation triggers metastatic progression. In contrast to tumor suppressors, metastasis suppressors are rarely mutated or deleted, and little is known regarding the mechanisms by which their expression is downregulated. Here, we demonstrate that the metastasis suppressor, NM23-H1, is degraded by lysosomal cysteine cathepsins (L,B), which directly cleave NM23-H1. In addition, activation of c-Abl and Arg oncoproteins induces NM23-H1 degradation in invasive cancer cells by increasing cysteine cathepsin transcription and activation. Moreover, c-Abl activates cathepsins by promoting endosome maturation, which facilitates trafficking of NM23-H1 to the lysosome where it is degraded. Importantly, the invasion- and metastasis-promoting activity of c-Abl/Arg is dependent on their ability to induce NM23-H1 degradation, and the pathway is clinically relevant as c-Abl/Arg activity and NM23-H1 expression are inversely correlated in primary breast cancers and melanomas. Thus, we demonstrate a novel mechanism by which cathepsin expression is upregulated in cancer cells (via Abl kinases). We also identify a novel role for intracellular cathepsins in invasion and metastasis (degradation of a metastasis suppressor). Finally, we identify novel crosstalk between oncogenic and metastasis suppressor pathways, thereby providing mechanistic insight into the process of NM23-H1 loss, which may pave the way for new strategies to restore NM23-H1 expression and block metastatic progression.

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

    SciTech Connect

    Hazra, Saugata; Sabini, Eliszbetta; Ort, Stephan; Konrad, Manfred; Lavie, Arnon

    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.

  12. Phosphagen kinase in Schistosoma japonicum: characterization of its enzymatic properties and determination of its gene structure.

    PubMed

    Tokuhiro, Shinji; Uda, Kouji; Yano, Hiroko; Nagataki, Mitsuru; Jarilla, Blanca R; Suzuki, Tomohiko; Agatsuma, Takeshi

    2013-04-01

    Phosphagen kinases (PKs) play a major role in the regulation of energy metabolism in animals. Creatine kinase (CK) is the sole PK in vertebrates, whereas several PKs are present in invertebrates. Here, we report the enzymatic properties and gene structure of PK in the trematode Schistosoma japonicum (Sj). SjPK has a unique contiguous dimeric structure comprising domain 1 (D1) and domain 2 (D2). The three states of the recombinant SjPK (D1, D2, and D1D2) show a specific activity for the substrate taurocyamine. The comparison of the two domains of SjPK revealed that D1 had a high turnover rate (kcat=52.91) and D2 exhibited a high affinity for taurocyamine (Km(Tauro) =0.53±0.06). The full-length protein exhibited higher affinity for taurocyamine (Km(Tauro) =0.47±0.03) than the truncated domains (D1=1.30±0.10, D2=0.53±0.06). D1D2 also exhibited higher catalytic efficiency (kcat/Km(Tauro) =82.98) than D1 (40.70) and D2 (29.04). These results demonstrated that both domains of SjTKD1D2 interacted efficiently and remained functional. The three-dimensional structure of SjPKD1 was constructed by the homology modeling based on the transition state analog complex state of Limulus AK. This protein model of SjPKD1 suggests that the overall structure is almost conserve between SjPKD1 and Limulus AK except for the flexible loops, that is, particularly guanidino-specificity (GS) region, which is associated with the recognition of the corresponding guanidino substrate. The constructed NJ tree and the comparison of exon/intron organization suggest that SjTK has evolved from an arginine kinase (AK) gene. SjTK has potential as a novel antihelminthic drug target as it is absent in mammals and its strong activity may imply a significant role for this protein in the energy metabolism of the parasite.

  13. Overexpression of the MAP kinase gene OsMAPK33 enhances sensitivity to salt stress in rice (Oryza sativa L.)

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Mitogen-activated protein kinases (MAPK) signaling cascades are activated by extracellular stimuli such as environmental stresses and pathogens in higher eukaryotic plants. To know more about MAPK signaling in plants, a MAPK cDNA clone, OsMAPK33 was isolated from rice. The gene is mainly induced by ...

  14. THE GRK4 SUBFAMILY OF G PROTEIN-COUPLED RECEPTOR KINASES: ALTERNATIVE SPLICING, GENE ORGANIZATION, AND SEQUENCE CONSERVATION

    EPA Science Inventory

    The GRK4 subfamily of G protein-coupled receptor kinases. Alternative splicing, gene organization, and sequence conservation.

    Premont RT, Macrae AD, Aparicio SA, Kendall HE, Welch JE, Lefkowitz RJ.

    Department of Medicine, Howard Hughes Medical Institute, Duke Univer...

  15. Diversification of Lrk/Tak kinase gene clusters is associated with subfunctionalization and cultivar-specific transcript accumulation in barley.

    PubMed

    Hu, Pingsha; Wise, Roger P

    2008-08-01

    Lrk (Lr10 receptor-like kinase) and Tak (Triticum aestivum kinase) belong to the receptor-like kinase (RLK) supergene family in higher plants. Three Lrk/Tak gene regions spanning greater than 600 kb were identified via a genome-wide survey of barley gene-rich BAC clones. Two Lrk/Tak gene clusters are positioned on barley chromosome 3 (3H) and another is localized on chromosome 5 (1H), with each Lrk and Tak open reading frame physically positioned in a back-to-back orientation. Thirteen new Lrk/Tak-like fragments were cloned from the two clusters on 3H and the single cluster on 1H, respectively, and compared phylogenetically with other grass Lrk/Tak-like genes, including a 280-kb Lrk/Tak cluster on rice chromosome 1S. Physically clustered Lrk/Tak-like genes always form monophyletic groups; this suggests that the primary mechanism of expansion of the Lrk/Tak RLK super family was by tandem duplication, of which most members were duplicated after speciation of the Poaceae. Cultivar-dependent transcript accumulation of some Lrk/Tak family members on 3H, as revealed via Barley1 GeneChip microarray analysis, is consistent with the hypothesis of subfunctionalization of Lrk/Tak members following tandem duplication.

  16. Polymorphism of FGFR4 Gly388Arg does not confer an increased risk to breast cancer development.

    PubMed

    Naidu, R; Har, Y C; Taib, N A

    2009-01-01

    The genotype analysis of the Gly and Arg allele at codon 388 of fibroblast growth factor receptor-4 (FGFR4) gene was evaluated using polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) method in a hospital-based Malaysian population. Peripheral blood samples were collected from 387 breast cancer patients and 252 normal and healthy women who had no history of any malignancy. The aim of the present study was to evaluate the association between the FGFR4 Gly388Arg polymorphism and breast cancer risk as well as clinicopathological parameters of the patients. The Gly/Gly, Gly/Arg, Arg/Arg, and Arg allele genotypes were detected in 46.3%, 44.4%, 9.3%, and 53.7% of breast cancer cases, respectively. The distribution of genotype (p = 0.204) and allele (p = 0.086) frequencies of FGFR4 polymorphism were not significantly different between the breast cancer cases and normal individuals. Women who were Arg/ Arg homozygotes (OR = 1.714, 95% CI 0.896-3.278), Gly/Arg heterozygotes (OR = 1.205, 95% CI 0.863-1.683), carriers of Arg allele genotype (OR = 1.269, 95% CI 0.921-1.750), or Arg allele (OR = 1.246, 95% CI 0.970-1.602) were not associated with breast cancer risk. The Arg allele genotype was significantly associated with lymph node metastases (p = 0.001) but not with other clinicopathological parameters. Our findings suggest that the polymorphic variant at codon 388 of FGFR4 gene does not confer increased risk to breast cancer development but it may be a potential genetic marker for tumor prognosis.

  17. p38 Mitogen-Activated Protein Kinase Pathway Regulates Genes during Proliferation and Differentiation in Oligodendrocytes

    PubMed Central

    Haines, Jeffery D.; Fulton, Debra L.; Richard, Stephane; Almazan, Guillermina

    2015-01-01

    We have previously shown that p38 mitogen-activated protein kinase (p38 MAPK) is important for oligodendrocyte (OLG) differentiation and myelination. However, the precise cellular mechanisms by which p38 regulates OLG differentiation remain largely unknown. To determine whether p38 functions in part through transcriptional events in regulating OLG identity, we performed microarray analysis on differentiating oligodendrocyte progenitors (OLPs) treated with a p38 inhibitor. Consistent with a role in OLG differentiation, pharmacological inhibition of p38 down-regulated the transcription of genes that are involved in myelin biogenesis, transcriptional control and cell cycle. Proliferation assays showed that OLPs treated with the p38 inhibitor retained a proliferative capacity which could be induced upon application of mitogens demonstrating that after two days of p38-inhibition OLGs remained poised to continue mitosis. Together, our results suggest that the p38 pathway regulates gene transcription which can coordinate OLG differentiation. Our microarray dataset will provide a useful resource for future studies investigating the molecular mechanisms by which p38 regulates oligodendrocyte differentiation and myelination. PMID:26714323

  18. Five histidine kinases perceive osmotic stress and regulate distinct sets of genes in Synechocystis.

    PubMed

    Paithoonrangsarid, Kalyanee; Shoumskaya, Maria A; Kanesaki, Yu; Satoh, Syusei; Tabata, Satoshi; Los, Dmitry A; Zinchenko, Vladislav V; Hayashi, Hidenori; Tanticharoen, Morakot; Suzuki, Iwane; Murata, Norio

    2004-12-17

    Microorganisms respond to hyperosmotic stress via changes in the levels of expression of large numbers of genes. Such responses are essential for acclimation to a new osmotic environment. To identify factors involved in the perception and transduction of signals caused by hyperosmotic stress, we examined the response of Synechocystis sp. PCC 6803, which has proven to be a particularly useful microorganism in similar analyses. We screened knockout libraries of histidine kinases (Hiks) and response regulators (Rres) in Synechocystis by DNA microarray and slot-blot hybridization analyses, and we identified several two-component systems, which we designated Hik-Rre systems, namely, Hik33-Rre31, Hik34-Rre1, and Hik10-Rre3, as well as Hik16-Hik41-Rre17, as the transducers of hyperosmotic stress. We also identified Hik2-Rre1 as a putative additional two-component system. Each individual two-component system regulated the transcription of a specific group of genes that were responsive to hyperosmotic stress.

  19. Structure and chromosomal localization of the genomic locus encoding the Kiz1 LIM-kinase gene

    SciTech Connect

    Bernard, O.; Burkitt, V.; Webb, G.C.

    1996-08-01

    We have cloned and characterized the mouse gene encoding Kiz1/Limk1, a new member of the zinc-finger LIM family that also has a kinase domain. The gene encompasses 25 kb of the mouse genome, and the organization of its 16 exons does not correlate with its functional domains. The promoter region of Kiz1/Limk1 was identified by cloning a 1.06-kb genomic fragment upstream from the first ATG in a promotorless CAT vector. This construct was demonstrated to drive CAT expression in Jurkat cells. The promoter sequence lacks conventional TATA and CAAT motifs but contains consensus binding sequences for several transcriptional regulators implicated in control of transcription in many different cell types, including Sp1, Ets, and E2A. Analysis of the chromosomal localization of KIZ1/LIMK1 indicates that it lies on human chromosome 17 in the region 17q25 and on mouse Chromosome 5, band G2. 15 refs., 3 figs., 1 tab.

  20. Adenosine kinase, glutamine synthetase and EAAT2 as gene therapy targets for temporal lobe epilepsy.

    PubMed

    Young, D; Fong, D M; Lawlor, P A; Wu, A; Mouravlev, A; McRae, M; Glass, M; Dragunow, M; During, M J

    2014-12-01

    Astrocytes are an attractive cell target for gene therapy, but the validation of new therapeutic candidates is needed. We determined whether adeno-associated viral (AAV) vector-mediated overexpression of glutamine synthetase (GS) or excitatory amino-acid transporter 2 (EAAT2), or expression of microRNA targeting adenosine kinase (miR-ADK) in hippocampal astrocytes in the rat brain could modulate susceptibility to kainate-induced seizures and neuronal cell loss. Transgene expression was found predominantly in astrocytes following direct injection of glial-targeting AAV9 vectors by 3 weeks postinjection. ADK expression in miR-ADK vector-injected rats was reduced by 94-96% and was associated with an ~50% reduction in the duration of kainate-induced seizures and greater protection of dentate hilar neurons but not CA3 neurons compared with miR-control vector-injected rats. In contrast, infusion of AAV-GS and EAAT2 vectors did not afford any protection against seizures or neuronal damage as the level of transcriptional activity of the glial fibrillary acidic promoter was too low to drive any significant increase in transgenic GS or EAAT2 relative to the high endogenous levels of these proteins. Our findings support ADK as a prime therapeutic target for gene therapy of temporal lobe epilepsy and suggest that alternative approaches including the use of stronger glial promoters are needed to increase transgenic GS and EAAT2 expression to levels that may be required to affect seizure induction and propagation.

  1. Adaptation of a retrovirus as a eucaryotic vector transmitting the herpes simplex virus thymidine kinase gene

    SciTech Connect

    Tabin, C.J.; Hoffman, J.W.; Goff, S.P.; Weinberg, R.A.

    1982-04-01

    The authors investigated the feasibility of using retroviruses as vectors for transferring DNA sequences into animal cells. The thymidine kinase (tk) gene of herpes simplex virus was chosen as a convenient model. The internal BamHI fragments of a DNA clone of Moloney leukemia virus (MLV) were replaced with a purified BamHI DNA segment containing the tk gene. Chimeric genomes were created carrying the tk insert on both orientations relative to the MLV sequence. Each was transfected into TK/sup -/ cells along with MLV helper virus, and TK/sup +/ colonies were obtained by selection in the presence of hypoxanthine, aminopterin, and thymidine (HAT). Virus collected from TK/sup +/-transformed, MLV producer cells passed the TK/sup +/ phenotype to TK/sup -/ cells. Nonproducer cells were isolated, and TK/sup +/ transducing virus was subsequently rescued from them. The chimeric virus showed single-hit kinetics in infections. Virion and cellular RNA and cellular DNA from infected cells were all shown to contain sequences which hybridized to both MLV- and tk-specific probes. The sizes of these sequences were consistent with those predicted for the chimeric virus. In all respects studied, the chimeric MLV-tk virus behaved like known replication-defective retroviruses. These experiments suggest great general applicability of retroviruses as eucaryotic vectors.

  2. Activity, expression and function of a second Drosophila protein kinase a catalytic subunit gene

    SciTech Connect

    Melendez, A.; Li, W.; Kalderon, D.

    1995-12-01

    The DC2 was isolated previously on the basis of sequence similarity to DC0, the major Drosophila protein kinase A (PKA) catalytic subunit gene. We show here that the 67-kD Drosophila DC2 protein behaves as a PKA catalytic subunit in vitro. DC2 is transcribed in mesodermal anlagen of early embryos. This expression depends on dorsal but on neither twist nor snail activity. DC2 transcriptional fusions mimic this embryonic expression and are also expressed in subsets of cells in the optic lamina, wing disc and leg discs of third instar larvae. A saturation screen of a small deficiency interval containing DC2 for recessive lethal mutations yielded no DC2 alleles. We therefore isolated new deficiencies to generate deficiency trans-heterozygotes that lacked DC2 activity. These animals were viable and fertile. The absence of DC2 promoter did not efficiently rescue a variety of DC0 mutant phenotypes. These observations indicate that DC2 is not an essential gene and is unlikely to be functionally redundant with DC0, which has multiple unique functions during development. 62 refs., 10 figs., 2 tabs.

  3. Genome of Acanthamoeba castellanii highlights extensive lateral gene transfer and early evolution of tyrosine kinase signaling

    PubMed Central

    2013-01-01

    Background The Amoebozoa constitute one of the primary divisions of eukaryotes, encompassing taxa of both biomedical and evolutionary importance, yet its genomic diversity remains largely unsampled. Here we present an analysis of a whole genome assembly of Acanthamoeba castellanii (Ac) the first representative from a solitary free-living amoebozoan. Results Ac encodes 15,455 compact intron-rich genes, a significant number of which are predicted to have arisen through inter-kingdom lateral gene transfer (LGT). A majority of the LGT candidates have undergone a substantial degree of intronization and Ac appears to have incorporated them into established transcriptional programs. Ac manifests a complex signaling and cell communication repertoire, including a complete tyrosine kinase signaling toolkit and a comparable diversity of predicted extracellular receptors to that found in the facultatively multicellular dictyostelids. An important environmental host of a diverse range of bacteria and viruses, Ac utilizes a diverse repertoire of predicted pattern recognition receptors, many with predicted orthologous functions in the innate immune systems of higher organisms. Conclusions Our analysis highlights the important role of LGT in the biology of Ac and in the diversification of microbial eukaryotes. The early evolution of a key signaling facility implicated in the evolution of metazoan multicellularity strongly argues for its emergence early in the Unikont lineage. Overall, the availability of an Ac genome should aid in deciphering the biology of the Amoebozoa and facilitate functional genomic studies in this important model organism and environmental host. PMID:23375108

  4. Novel mutation in the PANK2 gene leads to pantothenate kinase-associated neurodegeneration in a Pakistani family.

    PubMed

    Saleheen, Danish; Ali, Tuba; Aly, Zarmeneh; Khealani, Bhojo; Frossard, Philippe M

    2007-10-01

    Pantothenate kinase-associated neurodegeneration is an autosomal-recessive disorder associated with the accumulation of iron in the basal ganglia. The disease presents with dystonia, rigidity, and gait impairment, leading to restriction of activities and loss of ambulation. The disorder is caused by defective iron metabolism associated with mutations in the PANK2 gene, which codes for the pantothenate kinase enzyme. We report on a mutation screen conducted in two siblings to establish a molecular diagnosis of the disease and a genetic test for the family.

  5. nArgBP2 as a hub molecule in the etiology of various neuropsychiatric disorders

    PubMed Central

    Lee, Sang-Eun; Chang, Sunghoe

    2016-01-01

    Recent studies have strongly implicated postsynaptic scaffolding proteins such as SAPAP3 or Shank3 in the pathogenesis of various mood disorders, including autism spectrum disorder, bipolar disorder (BD), and obsessive-compulsive disorders. Neural Abelson-related gene-binding protein 2 (nArgBP2) was originally identified as a protein that interacts with SAPAP3 and Shank3. Recent study shows that the genetic deletion of nArgBP2 in mice leads to manic/bipolar-like behavior resembling symptoms of BD. However, the function of nArgBP2 at synapse, or its connection with the synaptic dysfunctions, is completely unknown. This study provides compelling evidence that nArgBP2 regulates the spine morphogenesis through the activation of Rac1/WAVE/PAK/cofilin pathway, and that its ablation causes a robust and selective inhibition of excitatory synapse formation, by controlling actin dynamics. Our results revealed the underlying mechanism for the synaptic dysfunction caused by nArgBP2 downregulation that associates with analogous human BD. Moreover, since nArgBP2 interacts with key proteins involved in various neuropsychiatric disorders, our finding implies that nArgBP2 could function as a hub linking various etiological factors of different mood disorders. [BMB Reports 2016; 49(9): 457-458] PMID:27530683

  6. Developmental regulation of the gene for chimeric calcium/calmodulin-dependent protein kinase in anthers

    NASA Technical Reports Server (NTRS)

    Poovaiah, B. W.; Xia, M.; Liu, Z.; Wang, W.; Yang, T.; Sathyanarayanan, P. V.; Franceschi, V. R.

    1999-01-01

    Chimeric Ca(2+)/calmodulin-dependent protein kinase (CCaMK) was cloned from developing anthers of lily (Lilium longiflorum Thumb. cv. Nellie White) and tobacco (Nicotiana tabacum L. cv. Xanthi). Previous biochemical characterization and structure/function studies had revealed that CCaMK has dual modes of regulation by Ca(2+) and Ca(2+)/calmodulin. The unique structural features of CCaMK include a catalytic domain, a calmodulin-binding domain, and a neural visinin-like Ca(2+)-binding domain. The existence of these three features in a single polypeptide distinguishes it from other kinases. Western analysis revealed that CCaMK is expressed in a stage-specific manner in developing anthers. Expression of CCaMK was first detected in pollen mother cells and continued to increase, reaching a peak around the tetrad stage of meiosis. Following microsporogenesis, CCaMK expression rapidly decreased and at later stages of microspore development, no expression was detected. A tobacco genomic clone of CCaMK was isolated and transgenic tobacco plants were produced carrying the CCaMK promoter fused to the beta-glucuronidase reporter gene. Both CCaMK mRNA and protein were detected in the pollen sac and their localizations were restricted to the pollen mother cells and tapetal cells. Consistent results showing a stage-specific expression pattern were obtained by beta-glucuronidase analysis, in-situ hybridization and immunolocalization. The stage- and tissue-specific appearance of CCaMK in anthers suggests that it could play a role in sensing transient changes in free Ca(2+) concentration in target cells, thereby controlling developmental events in the anther.

  7. Gene control of tyrosine kinase TIE2 and vascular manifestations of infections

    PubMed Central

    Ghosh, Chandra C.; David, Sascha; Zhang, Ruyang; Berghelli, Anthony; Milam, Katelyn; Higgins, Sarah J.; Hunter, Jon; Mukherjee, Aditi; Wei, Yongyue; Tran, Mei; Suber, Freeman; Kobzik, Lester; Kain, Kevin C.; Lu, Shulin; Santel, Ansgar; Yano, Kiichiro; Guha, Prajna; Dumont, Daniel J.; Christiani, David C.; Parikh, Samir M.

    2016-01-01

    Ligands of the endothelial-enriched tunica interna endothelial cell kinase 2 (Tie2) are markedly imbalanced in severe infections associated with vascular leakage, yet regulation of the receptor itself has been understudied in this context. Here, we show that TIE2 gene expression may constitute a novel vascular barrier control mechanism in diverse infections. Tie2 expression declined rapidly in wide-ranging models of leak-associated infections, including anthrax, influenza, malaria, and sepsis. Forced Tie2 suppression sufficed to attenuate barrier function and sensitize endothelium to permeability mediators. Rapid reduction of pulmonary Tie2 in otherwise healthy animals attenuated downstream kinase signaling to the barrier effector vascular endothelial (VE)-cadherin and induced vascular leakage. Compared with wild-type littermates, mice possessing one allele of Tie2 suffered more severe vascular leakage and higher mortality in two different sepsis models. Common genetic variants that influence TIE2 expression were then sought in the HapMap3 cohort. Remarkably, each of the three strongest predicted cis-acting SNPs in HapMap3 was also associated with the risk of acute respiratory distress syndrome (ARDS) in an intensive care unit cohort of 1,614 subjects. The haplotype associated with the highest TIE2 expression conferred a 28% reduction in the risk of ARDS independent of other major clinical variables, including disease severity. In contrast, the most common haplotype was associated with both the lowest TIE2 expression and 31% higher ARDS risk. Together, the results implicate common genetic variation at the TIE2 locus as a determinant of vascular leak-related clinical outcomes from common infections, suggesting new tools to identify individuals at unusual risk for deleterious complications of infection. PMID:26884170

  8. 5' flanking sequence and structure of a gene encoding rat 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase.

    PubMed Central

    Darville, M I; Crepin, K M; Hue, L; Rousseau, G G

    1989-01-01

    The synthesis and degradation of fructose 2,6-bisphosphate, a ubiquitous stimulator of glycolysis, are catalyzed by 6-phosphofructo-2-kinase (EC 2.7.1.105) and fructose-2,6-bisphosphatase (EC 3.1.3.46), respectively. In liver, these two activities belong to separate domains of the same 470-residue polypeptide. Various mRNAs have been described for this bifunctional enzyme, which is controlled by hormonal and metabolic signals. To understand the origin and regulation of these mRNAs, we have characterized rat genomic clones encoding the liver isozyme, which is regulated by cAMP-dependent protein kinase, and the muscle isozyme, which is not. We describe here a 55-kilobase gene that encodes these isozymes by alternative splicing from two promoters. Each of the putative promoters was sequenced over about 3 kilobases and found to include nucleotide motifs for binding regulatory factors. The two isozymes share the same 13 exons and differ only by the first exon that, in the liver but not in the muscle isozyme, contains the serine phosphorylated by cAMP-dependent protein kinase. The gene was assigned to the X chromosome. An analysis of the exon limits of 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase in relation to its functional domains and to its similarity with other proteins plus its G + C content at the third codon position suggests that this gene originates from several fusion events. Images PMID:2549541

  9. Evaluation of Reference Genes for Quantitative Real-Time PCR Analysis of the Gene Expression in Laticifers on the Basis of Latex Flow in Rubber Tree (Hevea brasiliensis Muell. Arg.).

    PubMed

    Chao, Jinquan; Yang, Shuguang; Chen, Yueyi; Tian, Wei-Min

    2016-01-01

    Latex exploitation-caused latex flow is effective in enhancing latex regeneration in laticifer cells of rubber tree. It should be suitable for screening appropriate reference gene for analysis of the expression of latex regeneration-related genes by quantitative real-time PCR (qRT-PCR). In the present study, the expression stability of 23 candidate reference genes was evaluated on the basis of latex flow by using geNorm and NormFinder algorithms. Ubiquitin-protein ligase 2a (UBC2a) and ubiquitin-protein ligase 2b (UBC2b) were the two most stable genes among the selected candidate references in rubber tree clones with differential duration of latex flow. The two genes were also high-ranked in previous reference gene screening across different tissues and experimental conditions. By contrast, the transcripts of latex regeneration-related genes fluctuated significantly during latex flow. The results suggest that screening reference gene during latex flow should be an efficient and effective clue for selection of reference genes in qRT-PCR.

  10. Evaluation of Reference Genes for Quantitative Real-Time PCR Analysis of the Gene Expression in Laticifers on the Basis of Latex Flow in Rubber Tree (Hevea brasiliensis Muell. Arg.)

    PubMed Central

    Chao, Jinquan; Yang, Shuguang; Chen, Yueyi; Tian, Wei-Min

    2016-01-01

    Latex exploitation-caused latex flow is effective in enhancing latex regeneration in laticifer cells of rubber tree. It should be suitable for screening appropriate reference gene for analysis of the expression of latex regeneration-related genes by quantitative real-time PCR (qRT-PCR). In the present study, the expression stability of 23 candidate reference genes was evaluated on the basis of latex flow by using geNorm and NormFinder algorithms. Ubiquitin-protein ligase 2a (UBC2a) and ubiquitin-protein ligase 2b (UBC2b) were the two most stable genes among the selected candidate references in rubber tree clones with differential duration of latex flow. The two genes were also high-ranked in previous reference gene screening across different tissues and experimental conditions. By contrast, the transcripts of latex regeneration-related genes fluctuated significantly during latex flow. The results suggest that screening reference gene during latex flow should be an efficient and effective clue for selection of reference genes in qRT-PCR. PMID:27524995

  11. Sequences contained within the promoter of the human thymidine kinase gene can direct cell-cycle regulation of heterologous fusion genes.

    PubMed Central

    Kim, Y K; Wells, S; Lau, Y F; Lee, A S

    1988-01-01

    Recent evidence on the transcriptional regulation of the human thymidine kinase (TK) gene raises the possibility that cell-cycle regulatory sequences may be localized within its promoter. A hybrid gene that combines the TK 5' flanking sequence and the coding region of the bacterial neomycin-resistance gene (neo) has been constructed. Upon transfection into a hamster fibroblast cell line K12, the hybrid gene exhibits cell-cycle-dependent expression. Deletion analysis reveals that the region important for cell-cycle regulation is within -441 to -63 nucleotides from the transcriptional initiation site. This region (-441 to -63) also confers cell-cycle regulation to the herpes simplex virus thymidine kinase (HSVtk) promoter, which is not expressed in a cell-cycle manner. We conclude that the -441 to -63 sequence within the human TK promoter is important for cell-cycle-dependent expression. Images PMID:3413063

  12. Sequences contained within the promoter of the human thymidine kinase gene can direct cell-cycle regulation of heterologous fusion genes

    SciTech Connect

    Kim, Yongkyu; Wells, S.; Lau, Yunfai Chris; Lee, A.S. )

    1988-08-01

    Recent evidence on the transcriptional regulation of the human thymidine kinase (TK) gene raises the possibility that cell-cycle regulatory sequences may be localized within its promoter. A hybrid gene that combines the TK 5{prime} flanking sequence and the coding region of the bacterial neomycin-resistance gene (neo) has been constructed. Upon transfection into a hamster fibroblast cell line K12, the hybrid gene exhibits cell-cycle-dependent expression. Deletion analysis reveals that the region important for cell-cycle regulation is within {minus}441 to {minus}63 nucleotides from the transcriptional initiation site. This region ({minus}441 to {minus}63) also confers cell-cycle regulation to the herpes simplex virus thymidine kinase (HSVtk) promoter, which is not expressed in a cell-cycle manner. The authors conclude that the {minus}441 to {minus}63 sequence within the human TK promoter is important for cell-cycle-dependent expression.

  13. Mutations in the Kinase Domain of the HER2/ERBB2 Gene Identified in a Wide Variety of Human Cancers.

    PubMed

    Wen, Wenhsiang; Chen, Wangjuh Sting; Xiao, Nick; Bender, Ryan; Ghazalpour, Anatole; Tan, Zheng; Swensen, Jeffrey; Millis, Sherri Z; Basu, Gargi; Gatalica, Zoran; Press, Michael F

    2015-09-01

    The HER2 (official name ERBB2) gene encodes a membrane receptor in the epidermal growth factor receptor family amplified and overexpressed in adenocarcinoma. Activating mutations also occur in several cancers. We report mutation analyses of the HER2 kinase domain in 7497 histologically diverse cancers. Forty-five genes, including the kinase domain of HER2 with HER2 IHC and dual in situ hybridization, were analyzed in tumors from 7497 patients with cancer, including 850 breast, 770 colorectal, 910 non-small cell lung, 823 uterine or cervical, 1372 ovarian, and 297 pancreatic cancers, as well as 323 melanomas and 2152 other solid tumors. Sixty-nine HER2 kinase domain mutations were identified in tumors from 68 patients (approximately 1% of all cases, ranging from absent in sarcomas to 4% in urothelial cancers), which included previously published activating mutations and 13 novel mutations. Fourteen cases with coexisting HER2 mutation and amplification and/or overexpression were identified. Fifty-two of 68 patients had additional mutations in other analyzed genes, whereas 16 patients (23%) had HER2 mutations identified as the sole driver mutation. HER2 mutations coexisted with HER2 gene amplification and overexpression and with mutations in other functionally important genes. HER2 mutations were identified as the only driver mutation in a significant proportion of solid cancers. Evaluation of anti-HER2 therapies in nonamplified, HER2-mutated cancers is warranted.

  14. Genome-Wide Identification and Expression Analysis of the Mitogen-Activated Protein Kinase Gene Family in Cassava

    PubMed Central

    Yan, Yan; Wang, Lianzhe; Ding, Zehong; Tie, Weiwei; Ding, Xupo; Zeng, Changying; Wei, Yunxie; Zhao, Hongliang; Peng, Ming; Hu, Wei

    2016-01-01

    Mitogen-activated protein kinases (MAPKs) play central roles in plant developmental processes, hormone signaling transduction, and responses to abiotic stress. However, no data are currently available about the MAPK family in cassava, an important tropical crop. Herein, 21 MeMAPK genes were identified from cassava. Phylogenetic analysis indicated that MeMAPKs could be classified into four subfamilies. Gene structure analysis demonstrated that the number of introns in MeMAPK genes ranged from 1 to 10, suggesting large variation among cassava MAPK genes. Conserved motif analysis indicated that all MeMAPKs had typical protein kinase domains. Transcriptomic analysis suggested that MeMAPK genes showed differential expression patterns in distinct tissues and in response to drought stress between wild subspecies and cultivated varieties. Interaction networks and co-expression analyses revealed that crucial pathways controlled by MeMAPK networks may be involved in the differential response to drought stress in different accessions of cassava. Expression of nine selected MAPK genes showed that these genes could comprehensively respond to osmotic, salt, cold, oxidative stressors, and abscisic acid (ABA) signaling. These findings yield new insights into the transcriptional control of MAPK gene expression, provide an improved understanding of abiotic stress responses and signaling transduction in cassava, and lead to potential applications in the genetic improvement of cassava cultivars. PMID:27625666

  15. Nitric oxide regulation of gene transcription via soluble guanylate cyclase and type I cGMP-dependent protein kinase.

    PubMed

    Idriss, S D; Gudi, T; Casteel, D E; Kharitonov, V G; Pilz, R B; Boss, G R

    1999-04-02

    Nitric oxide (NO) regulates the expression of multiple genes but in most cases its precise mechanism of action is unclear. We used baby hamster kidney (BHK) cells, which have very low soluble guanylate cyclase and cGMP-dependent protein kinase (G-kinase) activity, and CS-54 arterial smooth muscle cells, which express these two enzymes, to study NO regulation of the human fos promoter. The NO-releasing agent Deta-NONOate (ethanamine-2,2'-(hydroxynitrosohydrazone)bis-) had no effect on a chloramphenicol acetyltransferase (CAT) reporter gene under control of the fos promoter in BHK cells transfected with an empty vector or in cells transfected with a G-kinase Ibeta expression vector. In BHK cells transfected with expression vectors for guanylate cyclase, Deta-NONOate markedly increased the intracellular cGMP concentration and caused a small (2-fold) increase in CAT activity; the increased CAT activity appeared to be from cGMP activation of cAMP-dependent protein kinase. In BHK cells co-transfected with guanylate cyclase and G-kinase expression vectors, CAT activity was increased 5-fold in the absence of Deta-NONOate and 7-fold in the presence of Deta-NONOate. Stimulation of CAT activity in the absence of Deta-NONOate appeared to be largely from endogenous NO since we found that: (i) BHK cells produced high amounts of NO; (ii) CAT activity was partially inhibited by a NO synthase inhibitor; and (iii) the inhibition by the NO synthase inhibitor was reversed by exogenous NO. In CS-54 cells, we found that NO increased fos promoter activity and that the increase was prevented by a guanylate cyclase inhibitor. In summary, we found that NO activates the fos promoter by a guanylate cyclase- and G-kinase-dependent mechanism.

  16. Evaluation of ARG protein expression in mature B cell lymphomas compared to non-neoplastic reactive lymph node.

    PubMed

    Kabiri, Zahra; Salehi, Mansoor; Mokarian, Fariborz; Mohajeri, Mohammad Reza; Mahmoodi, Farzaneh; Keyhanian, Kianoosh; Doostan, Iman; Ataollahi, Mohammad Reza; Modarressi, Mohammad Hossein

    2009-01-01

    The participation of Abl-Related Gene (ARG) is demonstrated in pathogenesis of different human malignancies. However there is no conclusive evidence on ARG expression level in mature B cell lymphomas. In this study we evaluated ARG protein expression in Follicular Lymphoma (FL), Burkitt's Lymphoma (BL) and Diffused Large B Cell Lymphoma (DLBCL) in comparison with non-neoplastic lymph nodes. Semi-quantitative fluorescent ImmunoHistoChemistry was applied on 14, 7 and 4 patients with DLBCL, FL and BL respectively, adding to 4 normal and 4 reactive lymph nodes. The mean ratio of ARG/GAPDH expression was significantly different (p<0.00) between lymphomas and control samples, with DLBCL having the highest ARG expression amongst all. Over expression of ARG was seen in FL and BL, with FL expressing statistically more ARG than BL. Moreover, the ARG/GAPDH expression ratio increased from DLBCL stage I towards stage VI, all showing significantly more ARG expression than FL and BL (in all cases p<0.00).

  17. Genome-wide identification, characterisation and expression profiles of calcium-dependent protein kinase genes in barley (Hordeum vulgare L.).

    PubMed

    Fedorowicz-Strońska, Olga; Koczyk, Grzegorz; Kaczmarek, Małgorzata; Krajewski, Paweł; Sadowski, Jan

    2017-02-01

    In plant cells, calcium-dependent protein kinases (CDPKs) are important sensors of Ca(2+) flux resulting from various environmental stresses like cold, drought or salt stress. Previous genome sequence analysis and comparative studies in Arabidopsis (Arabidopsis thaliana L.) and rice (Oryza sativa L.) defined a multi-gene family of CDPKs. Here, we identified and characterised the CDPK gene complement of the model plant, barley (Hordeum vulgare L.). Comparative analysis encompassed phylogeny reconstruction based on newly available barley genome sequence, as well as established model genomes (e.g. O. sativa, A. thaliana, Brachypodium distachyon). Functional gene copies possessed characteristic CDPK domain architecture, including a serine/threonine kinase domain and four regulatory EF-hand motifs. In silico verification was followed by measurements of transcript abundance via real-time polymerase chain reaction (PCR). The relative expression of CDPK genes was determined in the vegetative growth stage under intensifying drought stress conditions. The majority of barley CDPK genes showed distinct changes in patterns of expression during exposure to stress. Our study constitutes evidence for involvement of the barley CDPK gene complement in signal transduction pathways relating to adaptation to drought. Our bioinformatics and transcriptomic analyses will provide an important foundation for further functional dissection of the barley CDPK gene family.

  18. Gammaherpesvirus gene expression and DNA synthesis are facilitated by viral protein kinase and histone variant H2AX.

    PubMed

    Mounce, Bryan C; Tsan, Fei Chin; Droit, Lindsay; Kohler, Sarah; Reitsma, Justin M; Cirillo, Lisa A; Tarakanova, Vera L

    2011-11-25

    Gammaherpesvirus protein kinases are an attractive therapeutic target as they support lytic replication and latency. Via an unknown mechanism these kinases enhance expression of select viral genes and DNA synthesis. Importantly, the kinase phenotypes have not been examined in primary cell types. Mouse gammaherpesvirus-68 (MHV68) protein kinase orf36 activates the DNA damage response (DDR) and facilitates lytic replication in primary macrophages. Significantly, H2AX, a DDR component and putative orf36 substrate, enhances MHV68 replication. Here we report that orf36 facilitated expression of RTA, an immediate early MHV68 gene, and DNA synthesis during de novo infection of primary macrophages. H2AX expression supported efficient RTA transcription and phosphorylated H2AX associated with RTA promoter. Furthermore, viral DNA synthesis was attenuated in H2AX-deficient macrophages, suggesting that the DDR system was exploited throughout the replication cycle. The interactions between a cancer-associated gammaherpesvirus and host tumor suppressor system have important implications for the pathogenesis of gammaherpesvirus infection.

  19. The HPr(Ser) Kinase of Streptococcus salivarius: Purification, Properties, and Cloning of the hprK Gene

    PubMed Central

    Brochu, Denis; Vadeboncoeur, Christian

    1999-01-01

    In gram-positive bacteria, HPr, a protein of the phosphoenolpyruvate:sugar phosphotransferase system, is phosphorylated on a serine residue at position 46 by an ATP-dependent protein kinase. The HPr(Ser) kinase of Streptococcus salivarius ATCC 25975 was purified, and the encoding gene (hprK) was cloned by using a nucleotide probe designed from the N-terminal amino acid sequence. The predicted amino acid sequence of the S. salivarius enzyme showed 45% identity with the Bacillus subtilis enzyme, the conserved residues being located mainly in the C-terminal half of the protein. The predicted hprK gene product has a molecular mass of 34,440 Da and a pI of 5.6. These values agree well with those found experimentally by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate, molecular sieve chromatography in the presence of guanidine hydrochloride, and chromatofocusing using the purified protein. The native protein migrates on a Superdex 200 HR column as a 330,000-Da protein, suggesting that the HPr(Ser) kinase is a decamer. The enzyme requires Mg2+ for activity and functions optimally at pH 7.5. Unlike the enzyme from other gram-positive bacteria, the HPr(Ser) kinase from S. salivarius is not stimulated by FDP or other glycolytic intermediates. The enzyme is inhibited by inorganic phosphate, and its Kms for HPr and ATP are 31 μM and 1 mM, respectively. PMID:9922231

  20. Cell-cycle-specific interaction of nuclear DNA-binding proteins with a CCAAT sequence from the human thymidine kinase gene.

    PubMed Central

    Knight, G B; Gudas, J M; Pardee, A B

    1987-01-01

    Induction of thymidine kinase parallels the onset of DNA synthesis. To investigate the transcriptional regulation of the thymidine kinase gene, we have examined whether specific nuclear factors interact in a cell-cycle-dependent manner with sequences upstream of this gene. Two inverted CCAAT boxes near the transcriptional initiation sites were observed to form complexes with nuclear DNA-binding proteins. The nature of the complexes changes dramatically as the cells approach DNA synthesis and correlates well with the previously reported transcriptional increase of the thymidine kinase gene. Images PMID:3479796

  1. Zonal induction of mixed lineage kinase ZPK/DLK/MUK gene expression in regenerating mouse liver.

    PubMed

    Douziech, M; Grondin, G; Loranger, A; Marceau, N; Blouin, R

    1998-08-28

    ZPK/DLK/MUK is a serine/theronine kinase believed to be involved in the regulation of cell growth and differentiation. To further explore the suggested participation of ZPK/DLK/MUK in this process, we examined the expression and cellular localization of ZPK/DLK/MUK mRNA in regenerating mouse liver following partial hepatectomy by ribonuclease protection assay and in situ hybridization. The steady-state level of APK/DLKMUK mRNA was very low in normal and sham-operated mouse livers, whereas a marked and transient increase was observed in the regenerating liver. While ZPK/DLK/MUK mRNAs were rarely detected in hepatocytes from all zones of the normal liver, hepatocytes of regenerating liver exhibit a gradient of expression ranging from low in the periportal zone, to intermediate in the mid-zone, to high in the pericentral zone. These findings demonstrate a transient stimulation of ZPK/DLK/MUK gene expression that correlates with the growth response of hepatocyte subpopulations in regenerating liver.

  2. Response gene to complement 32 protein promotes macrophage phagocytosis via activation of protein kinase C pathway.

    PubMed

    Tang, Rui; Zhang, Gui; Chen, Shi-You

    2014-08-15

    Macrophage phagocytosis plays an important role in host defense. The molecular mechanism, especially factors regulating the phagocytosis, however, is not completely understood. In the present study, we found that response gene to complement 32 (RGC-32) is an important regulator of phagocytosis. Although RGC-32 is induced and abundantly expressed in macrophage during monocyte-macrophage differentiation, RGC-32 appears not to be important for this process because RGC-32-deficient bone marrow progenitor can normally differentiate to macrophage. However, both peritoneal macrophages and bone marrow-derived macrophages with RGC-32 deficiency exhibit significant defects in phagocytosis, whereas RGC-32-overexpressed macrophages show increased phagocytosis. Mechanistically, RGC-32 is recruited to macrophage membrane where it promotes F-actin assembly and the formation of phagocytic cups. RGC-32 knock-out impairs F-actin assembly. RGC-32 appears to interact with PKC to regulate PKC-induced phosphorylation of F-actin cross-linking protein myristoylated alanine-rich protein kinase C substrate. Taken together, our results demonstrate for the first time that RGC-32 is a novel membrane regulator for macrophage phagocytosis.

  3. Extracellular-signal regulated kinase (Erk1/2), mitogen-activated protein kinase-activated protein kinase 2 (MK2) and tristetraprolin (TTP) comprehensively regulate injury-induced immediate early gene (IEG) response in in vitro liver organ culture.

    PubMed

    Tran, Doan Duy Hai; Koch, Alexandra; Saran, Shashank; Armbrecht, Marcel; Ewald, Florian; Koch, Martina; Wahlicht, Tom; Wirth, Dagmar; Braun, Armin; Nashan, Björn; Gaestel, Matthias; Tamura, Teruko

    2016-05-01

    Differentiated hepatocytes are long-lived and normally do not undergo cell division, however they have the unique capacity to autonomously decide their replication fate after liver injury. In this context, the key players of liver regeneration immediately after injury have not been adequately studied. Using an in vitro liver culture system, we show that after liver injury, p38 mitogen-activated protein kinase (p38MAPK), mitogen-activated protein kinase-activated protein kinase 2 (MK2) and extracellular-signal regulated kinase (Erk)1/2 were activated within 15 min and continued to be phosphorylated for more than 2h. Both p38MAPK and Erk1/2 were activated at the edge of the cut as well as on the liver surface where the mesothelial cell sheet expresses several cytokines. Notably, in human liver Erk1/2 was also activated under the mesothelial cell sheet shortly after liver resections. Furthermore, in in vitro liver slice culture immediate early genes (IEGs) were upregulated within 1-2 h and the S phase marker proliferation-cell-nuclear-antigen (PCNA) appeared 24 h after injury. Although Erk1/2 was activated after injury, in MK2 depleted liver a set of IEGs, such as Dusp1, Cox2, or c-Myc and proliferation marker gene Ki67 were not induced. In addition, in immortalized hepatocyte cells, THLE-2, the same subset of genes was upregulated upon stimulation with lipopolysaccharide (LPS), but not in the presence of MK2 inhibitor. The protein level of tristetraprolin (TTP), a substrate for MK2 that plays a role in mRNA degradation, was increased in the presence of MK2 inhibitor. In this context, the depletion of TTP gene rescued Dusp1, Cox2, or c-Myc upregulation in the presence of MK2 inhibitor. These data imply that MK2 pathway is positively involved in Erk1/2 induced IEG response after liver injury. These data also suggest that in vitro liver culture may be a useful tool for measuring the proliferation potential of hepatocytes in individual liver.

  4. Salt-inducible kinase 3, SIK3, is a new gene associated with hearing

    PubMed Central

    Wolber, Lisa E.; Girotto, Giorgia; Buniello, Annalisa; Vuckovic, Dragana; Pirastu, Nicola; Lorente-Cánovas, Beatriz; Rudan, Igor; Hayward, Caroline; Polasek, Ozren; Ciullo, Marina; Mangino, Massimo; Steves, Claire; Concas, Maria Pina; Cocca, Massilimiliano; Spector, Tim D.; Gasparini, Paolo; Steel, Karen P.; Williams, Frances M.K.

    2014-01-01

    Hearing function is known to be heritable, but few significant and reproducible associations of genetic variants have been identified to date in the adult population. In this study, genome-wide association results of hearing function from the G-EAR consortium and TwinsUK were used for meta-analysis. Hearing ability in eight population samples of Northern and Southern European ancestry (n = 4591) and the Silk Road (n = 348) was measured using pure-tone audiometry and summarized using principal component (PC) analysis. Genome-wide association analyses for PC1–3 were conducted separately in each sample assuming an additive model adjusted for age, sex and relatedness of subjects. Meta-analysis was performed using 2.3 million single-nucleotide polymorphisms (SNPs) tested against each of the three PCs of hearing ability in 4939 individuals. A single SNP lying in intron 6 of the salt-inducible kinase 3 (SIK3) gene was found to be associated with hearing PC2 (P = 3.7×10−8) and further supported by whole-genome sequence in a subset. To determine the relevance of this gene in the ear, expression of the Sik3 protein was studied in mouse cochlea of different ages. Sik3 was expressed in murine hair cells during early development and in cells of the spiral ganglion during early development and adulthood. Our results suggest a developmental role of Sik3 in hearing and may be required for the maintenance of adult auditory function. PMID:25060954

  5. Mitogen-activated protein kinase signaling controls basal and oncostatin M-mediated JUNB gene expression.

    PubMed

    Hicks, Mellissa J; Hu, Qiuping; Macrae, Erin; DeWille, James

    2015-05-01

    The mitogen-activated protein kinase (MAPK) pathway is aberrantly activated in many human cancers, including breast cancer. Activation of MAPK signaling is associated with the increased expression of a wide range of genes that promote cell survival, proliferation, and migration. This report investigated the influence of MAPK signaling on the regulation and expression of JUNB in human breast cancer cell lines. JUNB has been associated with tumor suppressor and oncogenic functions, with most reports describing JUNB as an oncogene in breast cancer. Our results indicated that JUNB expression is elevated in MCF10A(met), SKBR3, and MDA-MB-231 human breast cancer cell lines compared to nontransformed MCF10A mammary epithelial cells. Increased RAS/MAPK signaling in MCF10A(met) cells correlates with the increased association of RNA polymerase II (Pol II) phosphorylated on serine 5 (Pol IIser5p) with the JUNB proximal promoter. Pol IIser5p is the "transcription initiating" form of Pol II. Treatment with U0126, a MAPK pathway inhibitor, reduces Pol IIser5p association with the JUNB proximal promoter and reduces JUNB expression. Oncostatin M (OSM) enhances MAPK and STAT3 signaling and significantly induces JUNB expression. U0126 treatment reduces OSM-induced Pol IIser5p binding to the JUNB proximal promoter and JUNB expression, but does not reduce pSTAT3 levels or the association of pSTAT3 with the JUNB proximal promoter. These results demonstrate that the MAPK pathway plays a primary role in the control of JUNB gene expression by promoting the association of Pol IIser5p with the JUNB proximal promoter.

  6. Functional analysis of duplicated Symbiosis Receptor Kinase (SymRK) genes during nodulation and mycorrhizal infection in soybean (Glycine max).

    PubMed

    Indrasumunar, Arief; Wilde, Julia; Hayashi, Satomi; Li, Dongxue; Gresshoff, Peter M

    2015-03-15

    Association between legumes and rhizobia results in the formation of root nodules, where symbiotic nitrogen fixation occurs. The early stages of this association involve a complex of signalling events between the host and microsymbiont. Several genes dealing with early signal transduction have been cloned, and one of them encodes the leucine-rich repeat (LRR) receptor kinase (SymRK; also termed NORK). The Symbiosis Receptor Kinase gene is required by legumes to establish a root endosymbiosis with Rhizobium bacteria as well as mycorrhizal fungi. Using degenerate primer and BAC sequencing, we cloned duplicated SymRK homeologues in soybean called GmSymRKα and GmSymRKβ. These duplicated genes have high similarity of nucleotide (96%) and amino acid sequence (95%). Sequence analysis predicted a malectin-like domain within the extracellular domain of both genes. Several putative cis-acting elements were found in promoter regions of GmSymRKα and GmSymRKβ, suggesting a participation in lateral root development, cell division and peribacteroid membrane formation. The mutant of SymRK genes is not available in soybean; therefore, to know the functions of these genes, RNA interference (RNAi) of these duplicated genes was performed. For this purpose, RNAi construct of each gene was generated and introduced into the soybean genome by Agrobacterium rhizogenes-mediated hairy root transformation. RNAi of GmSymRKβ gene resulted in an increased reduction of nodulation and mycorrhizal infection than RNAi of GmSymRKα, suggesting it has the major activity of the duplicated gene pair. The results from the important crop legume soybean confirm the joint phenotypic action of GmSymRK genes in both mycorrhizal and rhizobial infection seen in model legumes.

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

    SciTech Connect

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

    1991-10-01

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

  8. Novel neurotrophic tyrosine kinase receptor type 1 gene mutation associated with congenital insensitivity to pain with anhidrosis.

    PubMed

    Lin, Yi-Pei; Su, Yi-Ning; Weng, Wen-Chin; Lee, Wang-Tso

    2010-12-01

    Congenital insensitivity to pain with anhidrosis (hereditary sensory and autonomic neuropathy type IV) is a rare autosomal recessive disorder caused by a defect in neurotrophic tyrosine kinase receptor and nerve growth factor, as reported in previous studies. This report is of a 6-month-old male infant with typical symptoms and signs of congenital insensitivity to pain with anhidrosis. He had a homozygous insertion mutation with c.2086_2087 ins C of neurotrophic tyrosine kinase receptor type 1 (NTRK1) gene with both parents as heterozygous carriers. This mutation may have a strong relation to hereditary sensory and autonomic neuropathy type IV Taiwanese patients. This is the youngest reported patient in Taiwan and first reported with congenital insensitivity to pain with mutation of NTRK1 gene inherited from the parents. Early diagnosis may provide appropriate medical care and education for these children and their families for better prognosis.

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

    PubMed

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

    1997-02-03

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

  10. [Cytotoxicity of cytosine deaminase and herpes simplex virus thymidine kinase genes in melanoma cells is independent on promoter strength].

    PubMed

    Alekseenko, I V; Kuz'min, D V; Pleshkan, V V; Zinov'eva, M V; Sverdlov, E D

    2013-01-01

    In preparation of the therapeutic genetic constructs aimed to the gene-programmed enzymatic transformation of the non-toxic prodrug into toxin within cancer cells the right choice of regulatory elements (promoters and enhancers) is essential. This is widely accepted that the efficiency of the gene therapy constructions is dependent, in particular, on the strength of promoters driving the expression of the therapeutic genes. In this work we demonstrated, using the melanoma-specific promoters and enhancers of human melanoma inhibitory activity and mouse tyrosinase gene, that for the development of cytotoxic effect the promoter strength is not of primary importance. In the case of HSVtk, coding for the herpes simplex virus thymidine kinase, and FCU1, coding for cytosine deaminase/uracil phosphoribosyltransferase hybrid protein genes, their cytotoxic activity was determined by the quantity of the added prodrug.

  11. Somatic mutations and germline sequence variants in the expressed tyrosine kinase genes of patients with de novo acute myeloid leukemia

    PubMed Central

    Xiang, Zhifu; Walgren, Richard; Zhao, Yu; Kasai, Yumi; Miner, Tracie; Ries, Rhonda E.; Lubman, Olga; Fremont, Daved H.; McLellan, Michael D.; Payton, Jacqueline E.; Westervelt, Peter; DiPersio, John F.; Link, Daniel C.; Walter, Matthew J.; Graubert, Timothy A.; Watson, Mark; Baty, Jack; Heath, Sharon; Shannon, William D.; Nagarajan, Rakesh; Bloomfield, Clara D.; Mardis, Elaine R.; Wilson, Richard K.; Ley, Timothy J.

    2008-01-01

    Activating mutations in tyrosine kinase (TK) genes (eg, FLT3 and KIT) are found in more than 30% of patients with de novo acute myeloid leukemia (AML); many groups have speculated that mutations in other TK genes may be present in the remaining 70%. We performed high-throughput resequencing of the kinase domains of 26 TK genes (11 receptor TK; 15 cytoplasmic TK) expressed in most AML patients using genomic DNA from the bone marrow (tumor) and matched skin biopsy samples (“germline”) from 94 patients with de novo AML; sequence variants were validated in an additional 94 AML tumor samples (14.3 million base pairs of sequence were obtained and analyzed). We identified known somatic mutations in FLT3, KIT, and JAK2 TK genes at the expected frequencies and found 4 novel somatic mutations, JAK1V623A, JAK1T478S, DDR1A803V, and NTRK1S677N, once each in 4 respective patients of 188 tested. We also identified novel germline sequence changes encoding amino acid substitutions (ie, nonsynonymous changes) in 14 TK genes, including TYK2, which had the largest number of nonsynonymous sequence variants (11 total detected). Additional studies will be required to define the roles that these somatic and germline TK gene variants play in AML pathogenesis. PMID:18270328

  12. Expression of the benign HEXA mutations, Arg247Trp and Arg249Trp, associated with beta-hexosaminidase A pseudodeficiency

    SciTech Connect

    Cao, Z.; Petroulakis, E.; Salo, T.

    1994-09-01

    {beta}-Hexosaminidase (Hex A) is a heterodimer of {alpha} and {beta} subunits encoded by the HEXA and HEXB genes, respectively. Mutations in the HEXA gene typically cause Tay-Sachs disease or less severe forms of G{sub M2} gangliosidosis. However, two benign mutations (Arg247Trp and Arg249Trp) in the {alpha}-subunit of Hex A account for Hex A deficiency in {approximately}36% of non-Jewish enzyme-defined Tay-Sachs disease carriers. These mutations do not result in any apparent clinical phenotype in individuals who are genetic compounds with a second disease-causing mutation. We expressed the {alpha}-subunit harboring each of the benign mutations separately to study activity toward the synthetic substrate, 4-MUGS, for comparison to activity from enzymes containing mutations associated with other forms of G{sub M2} gangliosidosis. The C739T (Arg247Trp;benign), C745T (Arg 249Trp; benign), G805A (Gly269Ser; adult-onset), G749A (Gly250Asp; juvenile), and C508T (Arg170Trp; infantile) mutations were introduced into the {alpha}-subunit cDNA. These were transfected alone, or with the {beta}-subunit cDNA, to generate Hex S ({alpha}{alpha}) or Hex A ({alpha}{beta}), respectively. The activities were monitored using 4-MUGS, and the levels of {alpha}-subunit protein were assessed by Western blotting. Repeated experiments show that the benign mutations produce approximately 35% of normal Hex S and 40% of normal Hex A activity. This level is much higher than that of Hex A harbouring the Gly169Ser adult-onset mutation (12%). A sequential decrease in expressed Hex A activity is observed as mutations associated with more severe phenotypes are expressed. The benign mutations also result in lower levels of mature {alpha}-subunit protein compared to normal, and slightly reduced levels of {alpha}-subunit precursor protein. The Hex A deficiency resulting from benign mutations is not as great as that associated with disease-causing mutations.

  13. Chromatinized Protein Kinase C-θ Directly Regulates Inducible Genes in Epithelial to Mesenchymal Transition and Breast Cancer Stem Cells

    PubMed Central

    Zafar, Anjum; Wu, Fan; Hardy, Kristine; Li, Jasmine; Tu, Wen Juan; McCuaig, Robert; Harris, Janelle; Khanna, Kum Kum; Attema, Joanne; Gregory, Philip A.; Goodall, Gregory J.; Harrington, Kirsti; Dahlstrom, Jane E.; Boulding, Tara; Madden, Rebecca; Tan, Abel; Milburn, Peter J.

    2014-01-01

    Epithelial to mesenchymal transition (EMT) is activated during cancer invasion and metastasis, enriches for cancer stem cells (CSCs), and contributes to therapeutic resistance and disease recurrence. Signal transduction kinases play a pivotal role as chromatin-anchored proteins in eukaryotes. Here we report for the first time that protein kinase C-theta (PKC-θ) promotes EMT by acting as a critical chromatin-anchored switch for inducible genes via transforming growth factor β (TGF-β) and the key inflammatory regulatory protein NF-κB. Chromatinized PKC-θ exists as an active transcription complex and is required to establish a permissive chromatin state at signature EMT genes. Genome-wide analysis identifies a unique cohort of inducible PKC-θ-sensitive genes that are directly tethered to PKC-θ in the mesenchymal state. Collectively, we show that cross talk between signaling kinases and chromatin is critical for eliciting inducible transcriptional programs that drive mesenchymal differentiation and CSC formation, providing novel mechanisms to target using epigenetic therapy in breast cancer. PMID:24891615

  14. Expansion of the Receptor-Like Kinase/Pelle Gene Family and Receptor-Like Proteins in Arabidopsis1[w

    PubMed Central

    Shin-Han, Shiu; Bleecker, Anthony B.

    2003-01-01

    Receptor-like kinases (RLKs) are a family of transmembrane proteins with versatile N-terminal extracellular domains and C-terminal intracellular kinases. They control a wide range of physiological responses in plants and belong to one of the largest gene families in the Arabidopsis genome with more than 600 members. Interestingly, this gene family constitutes 60% of all kinases in Arabidopsis and accounts for nearly all transmembrane kinases in Arabidopsis. Analysis of four fungal, six metazoan, and two Plasmodium sp. genomes indicates that the family was represented in all but fungal genomes, indicating an ancient origin for the family with a more recent expansion only in the plant lineages. The RLK/Pelle family can be divided into several subfamilies based on three independent criteria: the phylogeny based on kinase domain sequences, the extracellular domain identities, and intron locations and phases. A large number of receptor-like proteins (RLPs) resembling the extracellular domains of RLKs are also found in the Arabidopsis genome. However, not all RLK subfamilies have corresponding RLPs. Several RLK/Pelle subfamilies have undergone differential expansions. More than 33% of the RLK/Pelle members are found in tandem clusters, substantially higher than the genome average. In addition, 470 of the RLK/Pelle family members are located within the segmentally duplicated regions in the Arabidopsis genome and 268 of them have a close relative in the corresponding regions. Therefore, tandem duplications and segmental/whole-genome duplications represent two of the major mechanisms for the expansion of the RLK/Pelle family in Arabidopsis. PMID:12805585

  15. The gene for creatine kinase, mitochondrial 2 (sarcomeric; CKMT2), maps to chromosome 5q13. 3

    SciTech Connect

    Richard, I.; Devaud, C. ); Cherif, D.; Cohen, D.; Beckmann, J.S. )

    1993-10-01

    YAC clones for the creatine kinase, mitochrondial 2 (sarcomeric; CKMT2), gene were isolated. One of these YACs was localized on chromosome 5q13.3 by fluorescence in situ hybridization. A polymorphic dinucleotide repeat (heterozygosity 0.77) was identified within the seventh intron of the CKMT2 gene. Genotyping of CEPH families allowed positioning of CKMT2 on the multipoint map of chromosome 5 between D5S424 and D5S428, distal to spinal muscular atrophy (SMA) (5q12-q14). 8 refs., 1 fig., 2 tabs.

  16. Analysis of the Mitogen-activated protein kinase kinase 4 (MAP2K4) tumor suppressor gene in ovarian cancer

    PubMed Central

    2011-01-01

    Background MAP2K4 is a putative tumor and metastasis suppressor gene frequently found to be deleted in various cancer types. We aimed to conduct a comprehensive analysis of this gene to assess its involvement in ovarian cancer. Methods We screened for mutations in MAP2K4 using High Resolution Melt analysis of 149 primary ovarian tumors and methylation at the promoter using Methylation-Specific Single-Stranded Conformation Polymorphism analysis of 39 tumors. We also considered the clinical impact of changes in MAP2K4 using publicly available expression and copy number array data. Finally, we used siRNA to measure the effect of reducing MAP2K4 expression in cell lines. Results In addition to 4 previously detected homozygous deletions, we identified a homozygous 16 bp truncating deletion and a heterozygous 4 bp deletion, each in one ovarian tumor. No promoter methylation was detected. The frequency of MAP2K4 homozygous inactivation was 5.6% overall, and 9.8% in high-grade serous cases. Hemizygous deletion of MAP2K4 was observed in 38% of samples. There were significant correlations of copy number and expression in three microarray data sets. There was a significant correlation between MAP2K4 expression and overall survival in one expression array data set, but this was not confirmed in an independent set. Treatment of JAM and HOSE6.3 cell lines with MAP2K4 siRNA showed some reduction in proliferation. Conclusions MAP2K4 is targeted by genetic inactivation in ovarian cancer and restricted to high grade serous and endometrioid carcinomas in our cohort. PMID:21575258

  17. Myeloid Cell Arg1 Inhibits Control of Arthritogenic Alphavirus Infection by Suppressing Antiviral T Cells

    PubMed Central

    Burrack, Kristina S.; Tan, Jeslin J. L.; McCarthy, Mary K.; Her, Zhisheng; Berger, Jennifer N.; Ng, Lisa F. P.; Morrison, Thomas E.

    2015-01-01

    Arthritogenic alphaviruses, including Ross River virus (RRV) and chikungunya virus (CHIKV), are responsible for explosive epidemics involving millions of cases. These mosquito-transmitted viruses cause inflammation and injury in skeletal muscle and joint tissues that results in debilitating pain. We previously showed that arginase 1 (Arg1) was highly expressed in myeloid cells in the infected and inflamed musculoskeletal tissues of RRV- and CHIKV-infected mice, and specific deletion of Arg1 from myeloid cells resulted in enhanced viral control. Here, we show that Arg1, along with other genes associated with suppressive myeloid cells, is induced in PBMCs isolated from CHIKV-infected patients during the acute phase as well as the chronic phase, and that high Arg1 expression levels were associated with high viral loads and disease severity. Depletion of both CD4 and CD8 T cells from RRV-infected Arg1-deficient mice restored viral loads to levels detected in T cell-depleted wild-type mice. Moreover, Arg1-expressing myeloid cells inhibited virus-specific T cells in the inflamed and infected musculoskeletal tissues, but not lymphoid tissues, following RRV infection in mice, including suppression of interferon-γ and CD69 expression. Collectively, these data enhance our understanding of the immune response following arthritogenic alphavirus infection and suggest that immunosuppressive myeloid cells may contribute to the duration or severity of these debilitating infections. PMID:26436766

  18. Leptin receptor expression and Gln223Arg polymorphism as prognostic markers in oral and oropharyngeal cancer.

    PubMed

    Rodrigues, P R S; Maia, L L; Santos, M; Peterle, G T; Alves, L U; Takamori, J T; Souza, R P; Barbosa, W M; Mercante, A M C; Nunes, F D; Carvalho, M B; Tajara, E H; Louro, I D; Silva-Conforti, A M A

    2015-11-25

    The leptin gene product is released into the blood stream, passes through the blood-brain barrier, and finds the leptin receptor (LEPR) in the central nervous system. This hormone regulates food intake, hematopoiesis, inflammation, immunity, differentiation, and cell proliferation. The LEPR Gln223Arg polymorphism has been reported to alter receptor function and expression, both of which have been related with prognostics in several tumor types. Furthermore, several studies have shown a relationship between the Gln223Arg polymorphism and tumor development, and its role in oral and oropharyngeal squamous cell carcinoma is now well understood. In this study, 315 DNA samples were used for LEPR Gln223Arg genotyping and 87 primary oral and oropharyngeal squamous cell carcinomas were used for immunohistochemical expression analysis, such that a relationship between these and tumor development and prognosis could be established. Homozygous LEPR Arg223 was found to be associated with a 2-fold reduction in oral and oropharyngeal cancer risk. In contrast, the presence of the Arg223 allele in tumors was associated with worse disease-free and disease-specific survival. Low LEPR expression was found to be an independent risk factor, increasing the risk for lymph node metastasis 4-fold. In conclusion, the Gln223Arg polymorphism and LEPR expression might be valuable markers for oral and oropharyngeal cancer, suggesting that LEPR might serve as a potential target for future therapies.

  19. Protein kinase A activation of the surfactant protein B gene is mediated by phosphorylation of thyroid transcription factor 1.

    PubMed

    Yan, C; Whitsett, J A

    1997-07-11

    Thyroid transcription factor 1 (TTF-1) is a homeodomain-containing nuclear transcription factor expressed in epithelial cells of the lung and thyroid. TTF-1 binds to and activates the transcription of genes expressed selectively in the respiratory epithelium including pulmonary surfactant A, B, C and Clara cell secretory protein. Transfection with a plasmid encoding the cyclic AMP-dependent protein kinase (protein kinase A; PKA) catalytic subunit, Cat-beta, stimulated the phosphorylation of a TTF-1-flag fusion protein 6-7-fold in H441 pulmonary adenocarcinoma cells. Recombinant TTF-1 was phosphorylated by purified PKA catalytic subunit in the presence of [gamma-32P]ATP. PKA catalytic subunit family members, Cat-alpha and Cat-beta, markedly enhanced the transcriptional activation of surfactant B gene promoters by TTF-1 in vitro. Peptide mapping was used to identify a PKA phosphorylation site at the NH2 terminus of TTF-1. A 17-amino acid synthetic peptide comprising this site completely inhibited the PKA-dependent phosphorylation of TTF-1 in vitro. A substitution mutation of TTF-1 (Thr9 two head right arrow Ala) abolished phosphorylation by PKA and reduced transactivation of the surfactant B gene promoter. Transfection with a plasmid encoding the cAMP regulatory element binding factor inhibited transcriptional activity of the surfactant protein B gene promoter. Phosphorylation of TTF-1 mediates PKA-dependent activation of surfactant protein B gene transcription.

  20. Cloning and characterization of the major promoter of the human protein kinase C beta gene. Regulation by phorbol esters.

    PubMed

    Obeid, L M; Blobe, G C; Karolak, L A; Hannun, Y A

    1992-10-15

    The expression of the beta isoenzyme for protein kinase C is regulated developmentally and in response to inducers of cell differentiation (such as phorbol esters and 1 alpha,25-dihydroxyvitamin D3). The 5' segment of the gene for protein kinase C beta was cloned from a human leukocyte genomic library in EMBL3 bacteriophage. This segment of the gene (greater than 54 kilobases in length) encompassed the coding sequence for the amino-terminal regulatory domain of the enzyme, the 5'-untranslated region, and the 5'-flanking region. Initiation of transcription was identified by S1 nuclease analysis and confirmed by RNase protection analysis at 197 base pairs 5' of the initiator ATG. Sequence analysis of the 5'-flanking region revealed it to be extremely G+C-rich (> 80%) with many features of a CpG island. Comparison of sequence with known cis-regulatory motifs disclosed a number of potential regulatory elements including an octamer binding motif at -76, Sp1-binding sites at -94 and -63, E boxes at -110, -26, and +18, an AP-1 site at -442, and an AP-2 site at -330. To demonstrate promoter activity, a 630-base pair fragment extending from -587 to +43 was subcloned in front of a promoterless luciferase gene. This fragment was able to drive the expression of luciferase in transient transfections of human hematopoietic cells. Deletion analysis demonstrated that a fragment -111 to +43 was necessary and sufficient for promoter activity; this fragment did not contain TATA or CAAT motifs. The promoter was stimulated 8-20-fold by phorbol esters accounting for the previously observed transcriptional activation of protein kinase C beta. This phorbol ester responsiveness was conferred by the basal promoter (-111 to +43) and was independent of the AP-1 site. These results define a novel mechanism of protein kinase C autoregulation at a transcriptional level.

  1. Dioxin-dependent activation of murine Cyp1a-1 gene transcription requires protein kinase C-dependent phosphorylation.

    PubMed Central

    Carrier, F; Owens, R A; Nebert, D W; Puga, A

    1992-01-01

    Transcriptional activation of the murine Cyp1a-1 (cytochrome P(1)450) gene by inducers such as 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) (dioxin) requires the aromatic hydrocarbon (Ah) receptor and the interaction of an inducer-receptor complex with one or more of the Ah-responsive elements (AhREs) located about 1 kb upstream from the transcriptional initiation site. We find that treatment of mouse hepatoma Hepa-1 cells with 2-aminopurine, an inhibitor of protein kinase activity, inhibits CYP1A1 mRNA induction by TCDD as well as the concomitant increase in CYP1A1 enzyme activity. Formation of DNA-protein complexes between the Ah receptor and its AhRE target is also inhibited by 2-aminopurine, as determined by gel mobility shift assays. Phosphorylation is required for the formation of Ah receptor-specific complexes, since in vitro dephosphorylation of nuclear extracts from TCDD-treated Hepa-1 cells abolishes the capacity of the Ah receptor to form specific complexes with its cognate AhRE sequences. To determine whether any one of several known protein kinases was involved in the transcriptional regulation of the Cyp1a-1 gene, we treated Hepa-1 cells with nine other protein kinase inhibitors prior to induction with TCDD; nuclear extracts from these cells were analyzed for their capacity to form specific DNA-protein complexes. Only extracts from cells treated with staurosporine, a protein kinase C inhibitor, were unable to form these complexes. In addition, staurosporine completely inhibited CYP1A1 mRNA induction by TCDD. Depletion of protein kinase C by prolonged treatment with phorbol ester led to the complete suppression of CYP1A1 mRNA induction by TCDD. We conclude that (i) phosphorylation is necessary for the formation of a transcriptional complex and for transcriptional activation of the Cyp1a-1 gene; (ii) the phosphorylation site(s) exists on at least one of the proteins constituting the transcriptional complex, possibly the Ah receptor itself; and (iii) the

  2. Analysis of the murine Dtk gene identifies conservation of genomic structure within a new receptor tyrosine kinase subfamily

    SciTech Connect

    Lewis, P.M.; Crosier, K.E.; Crosier, P.S.

    1996-01-01

    The receptor tyrosine kinase Dtk/Tyro 3/Sky/rse/brt/tif is a member of a new subfamily of receptors that also includes Axl/Ufo/Ark and Eyk/Mer. These receptors are characterized by the presence of two immunoglobulin-like loops and two fibronectin type III repeats in their extracellular domains. The structure of the murine Dtk gene has been determined. The gene consists of 21 exons that are distributed over 21 kb of genomic DNA. An isoform of Dtk is generated by differential splicing of exons from the 5{prime} region of the gene. The overall genomic structure of Dtk is virtually identical to that determined for the human UFO gene. This particular genomic organization is likely to have been duplicated and closely maintained throughout evolution. 38 refs., 3 figs., 1 tab.

  3. Myosin Light Chain Kinase (MLCK) Gene Influences Exercise Induced Muscle Damage during a Competitive Marathon

    PubMed Central

    Valero, Marjorie; Lara, Beatriz; Salinero, Juan José; Gallo-Salazar, César; Areces, Francisco

    2016-01-01

    Myosin light chain kinase (MLCK) phosphorylates the regulatory light chain (RLC) of myosin producing increases in force development during skeletal muscle contraction. It has been suggested that MLCK gene polymorphisms might alter RLC phosphorylation thereby decreasing the ability to produce force and to resist strain during voluntary muscle contractions. Thus, the genetic variations in the MLCK gene might predispose some individuals to higher values of muscle damage during exercise, especially during endurance competitions. The aim of this investigation was to determine the influence of MLCK genetic variants on exercise-induced muscle damage produced during a marathon. Sixty-seven experienced runners competed in a marathon race. The MLCK genotype (C37885A) of these marathoners was determined. Before and after the race, a sample of venous blood was obtained to assess changes in serum myoglobin concentrations and leg muscle power changes were measured during a countermovement jump. Self-reported leg muscle pain and fatigue were determined by questionnaires. A total of 59 marathoners (88.1%) were CC homozygotes and 8 marathoners (11.9%) were CA heterozygotes. The two groups of participants completed the race with a similar time (228 ± 33 vs 234 ± 39 min; P = 0.30) and similar self-reported values for fatigue (15 ± 2 vs 16 ± 2 A.U.; P = 0.21) and lower-limb muscle pain (6.2 ± 1.7 vs 6.6 ± 1.8 cm; P = 0.29). However, CC marathoners presented higher serum myoglobin concentrations (739 ± 792 vs 348 ± 144 μg·mL-1; P = 0.03) and greater pre-to-post- race leg muscle power reduction (-32.7 ± 15.7 vs -21.2 ± 21.6%; P = 0.05) than CA marathoners. CA heterozygotes for MLCK C37885A might present higher exercise-induced muscle damage after a marathon competition than CC counterparts. PMID:27483374

  4. Chloroplast phosphoglycerate kinase from Euglena gracilis: endosymbiotic gene replacement going against the tide.

    PubMed

    Nowitzki, Ulrich; Gelius-Dietrich, Gabriel; Schwieger, Maike; Henze, Katrin; Martin, William

    2004-10-01

    Two chloroplast phosphoglycerate kinase isoforms from the photosynthetic flagellate Euglena gracilis were purified to homogeneity, partially sequenced, and subsequently cDNAs encoding phosphoglycerate kinase isoenzymes from both the chloroplast and cytosol of E. gracilis were cloned and sequenced. Chloroplast phosphoglycerate kinase, a monomeric enzyme, was encoded as a polyprotein precursor of at least four mature subunits that were separated by conserved tetrapeptides. In a Neighbor-Net analysis of sequence similarity with homologues from numerous prokaryotes and eukaryotes, cytosolic phosphoglycerate kinase of E. gracilis showed the highest similarity to cytosolic and glycosomal homologues from the Kinetoplastida. The chloroplast isoenzyme of E. gracilis did not show a close relationship to sequences from other photosynthetic organisms but was most closely related to cytosolic homologues from animals and fungi.

  5. Statistical Analysis of DWPF ARG-1 Data

    SciTech Connect

    Harris, S.P.

    2001-03-02

    A statistical analysis of analytical results for ARG-1, an Analytical Reference Glass, blanks, and the associated calibration and bench standards has been completed. These statistics provide a means for DWPF to review the performance of their laboratory as well as identify areas of improvement.

  6. ARG portable neutron radiography. Final report

    SciTech Connect

    Barton, J.P.

    1995-04-01

    In this report all available neutron radiographic data, including results of tests run at LANL, McClellan AFB, and University of Virginia, will be combined to outline specific transportable neutron radiography systems that could achieve the desired results as a complement to x-radiography capabilities for the Accident Response Group (ARG).

  7. The upstream muscle-specific enhancer of the rat muscle creatine kinase gene is composed of multiple elements.

    PubMed Central

    Horlick, R A; Benfield, P A

    1989-01-01

    A series of constructs that links the rat muscle creatine kinase promoter to the bacterial chloramphenicol acetyltransferase gene was generated. These constructs were introduced into differentiating mouse C2C12 myogenic cells to localize sequences that are important for up-regulation of the creatine kinase gene during myogenic differentiation. A muscle-specific enhancer element responsible for induction of chloramphenicol acetyltransferase expression during myogenesis was localized to a 159-base-pair region from 1,031 to 1,190 base pairs upstream of the transcription start site. Analysis of transient expression experiments using promoters mutated by deletion indicated the presence of multiple functional domains within this muscle-specific regulatory element. A DNA fragment spanning this region was used in DNase I protection experiments. Nuclear extracts derived from C2 myotubes protected three regions (designated E1, E2, and E3) on this fragment from digestion, which indicated there may be three or more trans-acting factors that interact with the creatine kinase muscle enhancer. Gel retardation assays revealed that factors able to bind specifically to E1, E2, and E3 are present in a wide variety of tissues and cell types. Transient expression assays demonstrated that elements in regions E1 and E3, but not necessarily E2, are required for full enhancer activity. Images PMID:2761536

  8. Mitogen-activated protein kinase kinase 1/2 inhibition and angiotensin II converting inhibition in mice with cardiomyopathy caused by lamin A/C gene mutation

    SciTech Connect

    Muchir, Antoine; Wu, Wei; Sera, Fusako; Homma, Shunichi; Worman, Howard J.

    2014-10-03

    Highlights: • Both ACE and MEK1/2 inhibition are beneficial on cardiac function in Lmna cardiomyopathy. • MEK1/2 inhibitor has beneficial effects beyond ACE inhibition for Lmna cardiomyopathy. • These results provide further preclinical rationale for a clinical trial of a MEK1/2 inhibitor. - Abstract: Background: Mutations in the LMNA gene encoding A-type nuclear lamins can cause dilated cardiomyopathy with or without skeletal muscular dystrophy. Previous studies have shown abnormally increased extracellular signal-regulated kinase 1/2 activity in hearts of Lmna{sup H222P/H222P} mice, a small animal model. Inhibition of this abnormal signaling activity with a mitogen-activated protein kinase kinase 1/2 (MEK1/2) inhibitor has beneficial effects on heart function and survival in these mice. However, such treatment has not been examined relative to any standard of care intervention for dilated cardiomyopathy or heart failure. We therefore examined the effects of an angiotensin II converting enzyme (ACE) inhibitor on left ventricular function in Lmna{sup H222P/H222P} mice and assessed if adding a MEK1/2 inhibitor would provide added benefit. Methods: Male Lmna{sup H222P/H222P} mice were treated with the ACE inhibitor benazepril, the MEK1/2 inhibitor selumetinib or both. Transthoracic echocardiography was used to measure left ventricular diameters and fractional shortening was calculated. Results: Treatment of Lmna{sup H222P/H222P} mice with either benazepril or selumetinib started at 8 weeks of age, before the onset of detectable left ventricular dysfunction, lead to statistically significantly increased fractional shortening compared to placebo at 16 weeks of age. There was a trend towards a great value for fractional shortening in the selumetinib-treated mice. When treatment was started at 16 weeks of age, after the onset of left ventricular dysfunction, the addition of selumetinib treatment to benazepril lead to a statistically significant increase in left

  9. Calcium/calmodulin-dependent protein kinase type IV is a target gene of the Wnt/beta-catenin signaling pathway.

    PubMed

    Arrázola, Macarena S; Varela-Nallar, Lorena; Colombres, Marcela; Toledo, Enrique M; Cruzat, Fernando; Pavez, Leonardo; Assar, Rodrigo; Aravena, Andrés; González, Mauricio; Montecino, Martín; Maass, Alejandro; Martínez, Servet; Inestrosa, Nibaldo C

    2009-12-01

    Calcium/calmodulin-dependent protein kinase IV (CaMKIV) plays a key role in the regulation of calcium-dependent gene expression. The expression of CaMKIV and the activation of CREB regulated genes are involved in memory and neuronal survival. We report here that: (a) a bioinformatic analysis of 15,476 promoters of the human genome predicted several Wnt target genes, being CaMKIV a very interesting candidate; (b) CaMKIV promoter contains TCF/LEF transcription motifs similar to those present in Wnt target genes; (c) biochemical studies indicate that lithium and the canonical ligand Wnt-3a induce CaMKIV mRNA and protein expression levels in rat hippocampal neurons as well as CaMKIV promoter activity; (d) treatment of hippocampal neurons with Wnt-3a increases the binding of beta-catenin to the CaMKIV promoter: (e) In vivo activation of the Wnt signaling improve spatial memory impairment and restores the expression of CaMKIV in a mice double transgenic model for Alzheimer's disease which shows decreased levels of the kinase. We conclude that CaMKIV is regulated by the Wnt signaling pathway and that its expression could play a role in the neuroprotective function of the Wnt signaling against the Alzheimer's amyloid peptide.

  10. Adoptive immunotherapy for leukemia: donor lymphocytes transduced with the herpes simplex thymidine kinase gene for remission induction. HGTRI 0103.

    PubMed

    Link, C J; Burt, R K; Traynor, A E; Drobyski, W R; Seregina, T; Levy, J P; Gordon, L; Rosen, S T; Burns, W H; Camitta, B; Casper, J; Horowitz, M; Juckett, M; Lawton, C; Margolis, D; Pietryga, D; Rowlings, P; Taylor, C; Furtado, M; Stefka, J; Gupta-Burt, S; Kaiser, H; Vesole, D H

    1998-01-01

    This study will evaluate the safety and efficacy of allogenic donor lymphocyte infusions in patients who have relapsed hematologic malignancies after allogeneic bone marrow transplantation (BMT). Donor lymphocyte transfusions have resulted in the cure of some patients with relapsed leukemia or lymphoproliferative disorder after allogeneic BMT, but has been complicated by the development of graft versus host disease (GvHD). We hypothesize that a retroviral vector containing the Herpes simplex thymidine kinase (HStk) gene will allow for retention of the anti-leukemia response of transfused donor lymphocytes while allowing for the adverse effects of GVHD to be mitigated. Patients with relapsed hematologic malignancies after allogeneic BMT will be infused with ex vivo gene modified donor lymphocytes. The Herpes Simplex thymidine kinase (HStk) gene will be transduced into the cells ex vivo using LTKOSN. 1 vector supernate. Insertion of the HStk gene into lymphocytes confers a sensitivity to the anti-herpes drug ganciclovir (GCV). This selective destruction of donor lymphocytes in situ will be used to abrogate the effect of graft versus host disease, if it develops.

  11. Novel compound heterozygous mutations in the PANK2 gene in a Chinese patient with atypical pantothenate kinase-associated neurodegeneration.

    PubMed

    Zhang, Yu-hu; Tang, Bei-sha; Zhao, Ai-ling; Xia, Kun; Long, Zhi-gao; Guo, Ji-feng; Westaway, Shawn K; Hayflick, Susan J

    2005-07-01

    We investigated the presence of mutations in the pantothenate kinase (PANK2) gene in a 27-year-old male Chinese patient with atypical pantothenate kinase-associated neurodegeneration (PKAN), formerly Hallervorden-Spatz syndrome. Automated DNA sequence analyses revealed compound heterozygous mutations in the exon 3 and 5. This patient had a 10-year history of PKAN characterized by a slight tremor of the right hand when writing at onset and a slow progressive rigidity of the neck and the right arm and resting tremor in upper extremities. Dysarthria, dysphagia, and dystonic-athetoid movements of the face and right fingers were marked. Magnetic resonance showed the typical "eye-of-the-tiger" sign.

  12. Tank-Binding Kinase 1 (TBK1) Gene and Open-Angle Glaucomas (An American Ophthalmological Society Thesis)

    PubMed Central

    Fingert, John H.; Robin, Alan L.; Scheetz, Todd E.; Kwon, Young H.; Liebmann, Jeffrey M.; Ritch, Robert; Alward, Wallace L.M.

    2016-01-01

    Purpose To investigate the role of TANK-binding kinase 1 (TBK1) gene copy-number variations (ie, gene duplications and triplications) in the pathophysiology of various open-angle glaucomas. Methods In previous studies, we discovered that copy-number variations in the TBK1 gene are associated with normal-tension glaucoma. Here, we investigated the prevalence of copy-number variations in cohorts of patients with other open-angle glaucomas—juvenile-onset open-angle glaucoma (n=30), pigmentary glaucoma (n=209), exfoliation glaucoma (n=225), and steroid-induced glaucoma (n=79)—using a quantitative polymerase chain reaction assay. Results No TBK1 gene copy-number variations were detected in patients with juvenile-onset open-angle glaucoma, pigmentary glaucoma, or steroid-induced glaucoma. A TBK1 gene duplication was detected in one (0.44%) of the 225 exfoliation glaucoma patients. Conclusions TBK1 gene copy-number variations (gene duplications and triplications) have been previously associated with normal-tension glaucoma. An exploration of other open-angle glaucomas detected a TBK1 copy-number variation in a patient with exfoliation glaucoma, which is the first example of a TBK1 mutation in a glaucoma patient with a diagnosis other than normal-tension glaucoma. A broader phenotypic range may be associated with TBK1 copy-number variations, although mutations in this gene are most often detected in patients with normal-tension glaucoma. PMID:27881886

  13. Chimeric Plant Calcium/Calmodulin-Dependent Protein Kinase Gene with a Neural Visinin-Like Calcium-Binding Domain

    NASA Technical Reports Server (NTRS)

    Patil, Shameekumar; Takezawa, D.; Poovaiah, B. W.

    1995-01-01

    Calcium, a universal second messenger, regulates diverse cellular processes in eukaryotes. Ca-2(+) and Ca-2(+)/calmodulin-regulated protein phosphorylation play a pivotal role in amplifying and diversifying the action of Ca-2(+)- mediated signals. A chimeric Ca-2(+)/calmodulin-dependent protein kinase (CCaMK) gene with a visinin-like Ca-2(+)- binding domain was cloned and characterized from lily. The cDNA clone contains an open reading frame coding for a protein of 520 amino acids. The predicted structure of CCaMK contains a catalytic domain followed by two regulatory domains, a calmodulin-binding domain and a visinin-like Ca-2(+)-binding domain. The amino-terminal region of CCaMK contains all 11 conserved subdomains characteristic of serine/threonine protein kinases. The calmodulin-binding region of CCaMK has high homology (79%) to alpha subunit of mammalian Ca-2(+)/calmodulin-dependent protein kinase. The calmodulin-binding region is fused to a neural visinin-like domain that contains three Ca-2(+)-binding EF-hand motifs and a biotin-binding site. The Escherichia coli-expressed protein (approx. 56 kDa) binds calmodulin in a Ca-2(+)-dependent manner. Furthermore, Ca-45-binding assays revealed that CCaMK directly binds Ca-2(+). The CCaMK gene is preferentially expressed in developing anthers. Southern blot analysis revealed that CCaMK is encoded by a single gene. The structural features of the gene suggest that it has multiple regulatory controls and could play a unique role in Ca-2(+) signaling in plants.

  14. Genome-Wide Analysis and Evolution of the Pto-Like Protein Kinase (PLPK) Gene Family in Pepper

    PubMed Central

    Venkatesh, Jelli; Jahn, Molly; Kang, Byoung-Cheorl

    2016-01-01

    The tomato Pto gene, which encodes a serine/threonine kinase (STK) domain-containing protein, confers resistance to bacterial speck disease caused by Pseudomonas syringae pv. tomato (Pst). In this study, in vivo recognition assays using PVX constructs showed that AvrPto was specifically recognized in the pepper genotypes. This AvrPto recognition caused a nonhost hypersensitive response (HR) and localization of the PVX::AvrPto fusion protein to inoculated pepper leaf tissues, which indicates the presence of a similar Pto recognition mechanism in pepper as in tomato. However, genome-wide analysis in pepper revealed no Pto clade corresponding to that in tomato, suggesting an alternative system for Pto recognition in pepper. Nevertheless, 25 Pto-like protein kinases (PLPKs) with a highly conserved STK domain have been identified in the pepper genome. For the majority of the amino acid sites in the STK domain of Ptos and PLPKs, nonsynonymous (dN) to synonymous (dS) nucleotide substitution ratios (ω) were less than one, suggesting that purifying selection played a predominant role in the evolutionary process. However, some amino acid sites were found to be subjected to episodic positive selection in the course of evolution of Pto homologs, and, thus, different evolutionary processes might have shaped the Pto gene family in plants. Based on RNA-seq data, PLPK genes and other Pto pathway genes, such as Prf, Pti1, Pti5, and Pti6 were expressed in all tested pepper genotypes. Therefore, the nonhost HR against Pst in pepper may be due to the recognition of the AvrPto effector by a PLPK homolog, and subsequent action of downstream components of the Pto signaling pathway. However, the possibility remains that the recognition of AvrPto in pepper plants may involve activities of other receptor like kinases (RLKs). The identification of the PLPKs in this study will serve as a foundation for further efforts to understand the roles of PLPKs in nonhost resistance. PMID:27536870

  15. Arg972 insulin receptor substrate-1 enhances tumor necrosis factor-α-induced apoptosis in osteoblasts.

    PubMed

    You, Yunhui; Liu, Shiqing; Peng, Lijuan; Long, Mei; Deng, Hongxiang; Zhao, Hongjun

    2015-07-01

    The presence of Arg972 insulin receptor substrate-1 (IRS-1) is associated with impaired insulin/IRS-1 signaling to activate phosphatidylinositol-3 kinase (PI3K). Tumor necrosis factor-α (TNF-α), an inflammatory cytokine with a central role in the pathogenesis of rheumatoid arthritis (RA), induces apoptosis in osteoblasts, which are the principal cell type responsible for bone loss in RA. In our previous study, an association between Arg972 IRS-1 and a high risk and severity of RA was identified. In the present study, the effects of Arg972 IRS-1 and IRS-1 on TNF-α-induced apoptosis in human osteoblasts were examined. Normal and RA osteoblasts were stably transfected with Arg972 IRS-1 and IRS-1. In addition, cells were stably transduced with IRS-1-shRNA to knock down IRS1. Following stimulation with 10 nM insulin for 30 min, the stable overexpression of Arg972 IRS-1 and knock down of IRS-1 significantly decreased IRS-1-associated PI3K activity and Akt activation/phosphorylation at serine 473 (ser473) and enhanced TNF-α-induced apoptosis in normal and in RA osteoblasts. By contrast, the stable overexpression of IRS-1 significantly increased the levels of IRS-1-associated PI3K activity and Akt phosphorylation (ser473) and inhibited TNF-α-induced apoptosis, which was eliminated by pretreatment with 50 µn BJM120, a selective PI3K inhibitor, for 30 min. In conclusion, the present study provided the first evidence, to the best of our knowledge, that insulin stimulation of Arg972 IRS-1 and IRS-1 enhanced and inhibited TNF-α-induced apoptosis, respectively in normal and RA osteoblasts by a PI3K‑dependent mechanism. These findings suggest that insulin/IRS-1 signaling is important in the pathogenesis of RA.

  16. c-Abl and Arg are activated in human primary melanomas, promote melanoma cell invasion via distinct pathways, and drive metastatic progression.

    PubMed

    Ganguly, S S; Fiore, L S; Sims, J T; Friend, J W; Srinivasan, D; Thacker, M A; Cibull, M L; Wang, C; Novak, M; Kaetzel, D M; Plattner, R

    2012-04-05

    Despite 35 years of clinical trials, there is little improvement in 1-year survival rates for patients with metastatic melanoma, and the disease is essentially untreatable if not cured surgically. The paucity of chemotherapeutic agents that are effective for treating metastatic melanoma indicates a dire need to develop new therapies. Here, we found a previously unrecognized role for c-Abl and Arg in melanoma progression. We demonstrate that the kinase activities of c-Abl and Arg are elevated in primary melanomas (60%), in a subset of benign nevi (33%) and in some human melanoma cell lines. Using siRNA and pharmacological approaches, we show that c-Abl/Arg activation is functionally relevant because it is requiredfor melanoma cell proliferation, survival and invasion. Significantly, we identify the mechanism by which activated c-Abl promotes melanoma invasion by showing that it transcriptionally upregulates matrix metalloproteinase-1 (MMP-1), and using rescue approaches we demonstrate that c-Abl promotes invasion through a STAT3 → MMP-1 pathway. Additionally, we show that c-Abl and Arg are not merely redundant, as active Arg drives invasion in a STAT3-independent manner, and upregulates MMP-3 and MT1-MMP, in addition to MMP-1. Most importantly, c-Abl and Arg not only promote in vitro processes important for melanoma progression, but also promote metastasis in vivo, as inhibition of c-Abl/Arg kinase activity with the c-Abl/Arg inhibitor, nilotinib, dramatically inhibits metastasis in a mouse model. Taken together, these data identify c-Abl and Arg as critical, novel, drug targets in metastatic melanoma, and indicate that nilotinib may be useful in preventing metastasis in patients with melanomas harboring active c-Abl and Arg.

  17. Construction of Poxviruses as Cloning Vectors: Insertion of the Thymidine Kinase Gene from Herpes Simplex Virus into the DNA of Infectious Vaccinia Virus

    NASA Astrophysics Data System (ADS)

    Panicali, Dennis; Paoletti, Enzo

    1982-08-01

    We have constructed recombinant vaccinia viruses containing the thymidine kinase gene from herpes simplex virus. The gene was inserted into the genome of a variant of vaccinia virus that had undergone spontaneous deletion as well as into the 120-megadalton genome of the large prototypic vaccinia variant. This was accomplished via in vivo recombination by contransfection of eukaryotic tissue culture cells with cloned BamHI-digested thymidine kinase gene from herpes simplex virus containing flanking vaccinia virus DNA sequences and infectious rescuing vaccinia virus. Pure populations of the recombinant viruses were obtained by replica filter techniques or by growth of the recombinant virus in biochemically selective medium. The herpes simplex virus thymidine kinase gene, as an insert in vaccinia virus, is transcribed in vivo and in vitro, and the fidelity of in vivo transcription into a functional gene product was detected by the phosphorylation of 5-[125I]iodo-2'-deoxycytidine.

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

    SciTech Connect

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

    1996-09-01

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

  19. The dual role of DksA protein in the regulation of Escherichia coli pArgX promoter

    PubMed Central

    Łyżeń, Robert; Maitra, Amarnath; Milewska, Klaudia; Kochanowska-Łyżeń, Maja; Hernandez, V. James; Szalewska-Pałasz, Agnieszka

    2016-01-01

    Gene expression regulation by the stringent response effector, ppGpp, is facilitated by DksA protein; however DksA and ppGpp can play independent roles in transcription. In Escherichia coli, the pArgX promoter which initiates the transcription of four tRNA genes was shown to be inhibited by ppGpp. Our studies on the role of DksA in pArgX regulation revealed that it can stimulate transcription by increasing the binding of RNA polymerase to the promoter and the productive transcription complex formation. However, when DksA is present together with ppGpp a severe down-regulation of promoter activity is observed. Our results indicate that DksA facilitates the effects of ppGpp to drive formation of inactive dead-end complexes formed by RNA polymerase at the ArgX promoter. In vivo, ppGpp-mediated regulation of pArgX transcription is dependent on DksA activity. The potential mechanisms of opposing pArgX regulation by ppGpp and DksA are discussed. pArgX is the first reported example of the promoter stimulated by DksA and inhibited by ppGpp in vitro when an overall inhibition occurs in the presence of both regulators. A dual role is thus proposed for DksA in the regulation of the pArgX promoter activity. PMID:27915292

  20. Assignment of the human deoxycytidine kinase (DCK) gene to chromosome 4 band q13. 3-q21. 1

    SciTech Connect

    Stegmann, A.P.A.; Honders, M.W.; Bolk, M.W.J.; Willemze, R.; Landegent, J.E.; Wessels, J. )

    1993-08-01

    The enzyme deoxycytidine kinase (DCK) is the key enzyme of the salvage pathway for pyrimidine synthesis. It is responsible for the phosphorylation of deoxycytidine and several deoxycytidine analogues that are used as antimetabolites in the treatment of human cancers. For instance, the cytotoxic activity of 1-[beta]-D-arabinofuranosylcytosine (AraC), used in the chemotherapy of acute myeloid leukemia (AML), is dependent on its phosphorylation by DCK. The occurrence of clinical AraC resistance, which is usually marked by functional DCK deficiency, is one of the major obstacles in the successful treatment of AML. The cDNA sequence of the DCK gene was published and, more recently, mutational inactivation of the DCK gene has been described as a possible cause of DCK deficiency. In this study the authors report on the chromosomal localization of the DCK gene by means of fluorescence in situ hybridization. 6 refs., 2 figs.

  1. Complete genomic organization of the human erythroid p55 gene (MPP1), a membrane-associated guanylate kinase homologue

    SciTech Connect

    Kim, A.C.; Metzenberg, A.B.; Sahr, K.E.

    1996-01-15

    Human p55 is an abundantly palmitoylated phosphoprotein of the erythroid membrane. It is the prototype of a newly discovered family of membrane-associated proteins termed MAGUKs (membrane-associated guanylate kinase homologues). The MAGUKs interact with the cytoskeleton and regulate cell proliferation, signaling pathways, and intercellular junctions. Here, we report the complete intron-exon map of the human erythroid p55 gene (HGMW-approved symbol MPP1). The structure of the p55 gene was determined from cosmid clones isolated from a cosmid library specific for the human X chromosome. There is a single copy of the p55 gene, composed of 12 exons and spanning approximately 28 kb in the q28 region of the human X chromosome. The exon sizes range from 69 (exon 5) to 203 bp (intron 2) to {approximately}14 kb (intron 1). The intron-exon boundaries conform to the donor/acceptor consensus sequence, GT-AG, for splice junctions. Several of the exon boundaries correspond to the boundaries of functional domains in the p55 protein. These domains include a SH3 motif and a region that binds to cytoskeletal protein 4.1. In addition, a comparison of the genomic and the primary structures of p55 reveals a highly conserved phosphotyrosine domain located between the protein 4.1 binding domain and the guanylate kinase domain. Finally, promoter activity measurements of the region immediately upstream of the p55 gene, which contains several cis-elements commonly found in housekeeping genes, suggest that a CpG island may be associated with the p55 gene expression in vivo. 42 refs., 5 figs., 1 tab.

  2. Liposomal delivery of the herpes simplex virus thymidine kinase gene in glioma: improvement of cell sensitization to ganciclovir.

    PubMed

    Zerrouqi, A; Rixe, O; Ghoumari, A M; Yarovoi, S V; Mouawad, R; Khayat, D; Soubrane, C

    1996-01-01

    In this study, we investigated whether the regulation and the copy number of the herpes simplex virus thymidine kinase (HSVtk) gene increased the sensitization to ganciclovir (GCV) of glioma cell lines (Rat C6 and human U118-MG) using liposome-mediated gene transfer. Three recombinant plasmids carrying the HSVtk gene driven by the thymidine kinase promoter in single (pAGo) and double copy (pYED) or by the human cytomegalovirus promoter (pCMVtk) were used for the transfection. The DNA delivery was optimized by screening a panel of cationic liposomes using Lac-Z and luciferase as reporter genes. The efficiency of transfection reached 33% to 36% in vitro but only 18.6% in vivo after an intratumoral injection of DNA-liposome complexes. Moreover, after transfection of the three plasmids, the cell-killing effect of GCV was evaluated. A significant enhancement (four- to fivefold) of the cell sensitivity to GCV was shown in pCMVtk and pYED as compared with pAGo-transfected cells in both cell lines. According to the plasmid, the effect of the HSVtk/GCV system was confirmed by in vivo experiments and was objectified by a higher tumor weight reduction with pCMVtk (49%) than pAGo (27%). From these results, we conclude that (1) the gene transfer can be achieved by cationic liposomes both in vitro and in vivo and that (2) using this type of vector, the antitumor effect of the HSVtk/GCV system could be potentiated by the up-regulation of HSVtk gene duplication.

  3. Multiplex image-based autophagy RNAi screening identifies SMCR8 as ULK1 kinase activity and gene expression regulator

    PubMed Central

    Jung, Jennifer; Nayak, Arnab; Schaeffer, Véronique; Starzetz, Tatjana; Kirsch, Achim K; Müller, Stefan; Dikic, Ivan; Mittelbronn, Michel; Behrends, Christian

    2017-01-01

    Autophagy is an intracellular recycling and degradation pathway that depends on membrane trafficking. Rab GTPases are central for autophagy but their regulation especially through the activity of Rab GEFs remains largely elusive. We employed a RNAi screen simultaneously monitoring different populations of autophagosomes and identified 34 out of 186 Rab GTPase, GAP and GEF family members as potential autophagy regulators, amongst them SMCR8. SMCR8 uses overlapping binding regions to associate with C9ORF72 or with a C9ORF72-ULK1 kinase complex holo-assembly, which function in maturation and formation of autophagosomes, respectively. While focusing on the role of SMCR8 during autophagy initiation, we found that kinase activity and gene expression of ULK1 are increased upon SMCR8 depletion. The latter phenotype involved association of SMCR8 with the ULK1 gene locus. Global mRNA expression analysis revealed that SMCR8 regulates transcription of several other autophagy genes including WIPI2. Collectively, we established SMCR8 as multifaceted negative autophagy regulator. DOI: http://dx.doi.org/10.7554/eLife.23063.001 PMID:28195531

  4. Cloning of a phosphatidylinositol 4-kinase gene based on fiber strength transcriptome QTL mapping in the cotton species Gossypium barbadense.

    PubMed

    Liu, H W; Shi, R F; Wang, X F; Pan, Y X; Zang, G Y; Ma, Z Y

    2012-09-25

    Sea Island cotton (Gossypium barbadense) is highly valued for its superior fiber qualities, especially fiber strength. Based on a transcript-derived fragment originated from transcriptome QTL mapping, a fiber strength related candidate gene of phosphatidylinositol 4-kinase cDNA, designated as GbPI4K, was first cloned, and its expression was characterized in the secondary cell wall thickening stage of G. barbadense fibers. The ORF of GbPI4K was found to be 1926 bp in length and encoded a predicted protein of 641 amino acid residues. The putative protein contained a clear PI3/4K kinase catalytic domain and fell into the plant type II PI4K cluster in phylogenetic analysis. In this study, the expression of cotton PI4K protein was also induced in Escherichia coli BL21 (DE3) as a fused protein. Semi-quantitative RT-PCR analysis showed that the gene expressed in the root, hypocotyl and leaf of the cotton plants. Real-time RT-PCR indicated that this gene in Sea Island cotton fibers expressed 10 days longer than that in Upland cotton fibers, and the main expression difference of PI4K between Sea Island cotton and Upland cotton in fibers was located in the secondary cell wall thickening stage of the fiber. Further analysis indicated that PI4K is a crucial factor in the ability of Rac proteins to regulate phospholipid signaling pathways.

  5. Convergent evidence identifying MAP/microtubule affinity-regulating kinase 1 (MARK1) as a susceptibility gene for autism.

    PubMed

    Maussion, Gilles; Carayol, Jérôme; Lepagnol-Bestel, Aude-Marie; Tores, Frédéric; Loe-Mie, Yann; Milbreta, Ulla; Rousseau, Francis; Fontaine, Karine; Renaud, Julie; Moalic, Jean-Marie; Philippi, Anne; Chedotal, Alain; Gorwood, Philip; Ramoz, Nicolas; Hager, Jörg; Simonneau, Michel

    2008-08-15

    Autism spectrum disorders (ASDs) are common, heritable, but genetically heterogeneous neurodevelopmental conditions. We recently defined a susceptibility locus for ASDs on chromosome 1q41-q42. High-resolution single-nucleotide polymorphisms (126 SNPs) genotyping across the chromosome 1q41-q42 region, followed by a MARK1 (microtubule affinity-regulating kinase 1)-tagged-SNP association study in 276 families with autism from the Autism Genetic Research Exchange, showed that several SNPs within the MARK1 gene were significantly associated with ASDs by transmission disequilibrium tests. Haplotype rs12740310*C-rs3737296*G-rs12410279*A was overtransmitted (P(corrected)= 0.0016), with a relative risk for autism of 1.8 in homozygous carriers. Furthermore, ASD-associated SNP rs12410279 modulates the level of transcription of MARK1. We found that MARK1 was overexpressed in the prefrontal cortex (BA46) but not in cerebellar granule cells, on postmortem brain tissues from patients. MARK1 displayed an accelerated evolution along the lineage leading to humans, suggesting possible involvement of this gene in cognition. MARK1 encodes a kinase-regulating microtubule-dependent transport in axons and dendrites. Both overexpression and silencing of MARK1 resulted in significantly shorter dendrite length in mouse neocortical neurons and modified dendritic transport speed. As expected for a gene encoding a key polarity determinant Par-1 protein kinase, MARK1 is involved in axon-dendrite specification. Thus, MARK1 overexpression in humans may be responsible for subtle changes in dendritic functioning.

  6. Characterization and expression of the gene encoding En-MAPK1, an intestinal cell kinase (ICK)-like kinase activated by the autocrine pheromone-signaling loop in the Polar Ciliate, Euplotes nobilii.

    PubMed

    Candelori, Annalisa; Luporini, Pierangelo; Alimenti, Claudio; Vallesi, Adriana

    2013-04-03

    In the protozoan ciliate Euplotes, a transduction pathway resulting in a mitogenic cell growth response is activated by autocrine receptor binding of cell type-specific, water-borne signaling protein pheromones. In Euplotes raikovi, a marine species of temperate waters, this transduction pathway was previously shown to involve the phosphorylation of a nuclear protein kinase structurally similar to the intestinal-cell and male germ cell-associated kinases described in mammals. In E. nobilii, which is phylogenetically closely related to E. raikovi but inhabits Antarctic and Arctic waters, we have now characterized a gene encoding a structurally homologous kinase. The expression of this gene requires +1 translational frameshifting and a process of intron splicing for the production of the active protein, designated En-MAPK1, which contains amino acid substitutions of potential significance for cold-adaptation.

  7. Relationship between bacterial virulence and nucleotide metabolism: a mutation in the adenylate kinase gene renders Yersinia pestis avirulent.

    PubMed Central

    Munier-Lehmann, Hélène; Chenal-Francisque, Viviane; Ionescu, Mihaela; Chrisova, Petya; Foulon, Jeannine; Carniel, Elisabeth; Bârzu, Octavian

    2003-01-01

    Nucleoside monophosphate kinases (NMPKs) are essential catalysts for bacterial growth and multiplication. These enzymes display high primary sequence identities among members of the family Enterobacteriaceae. Yersinia pestis, the causative agent of plague, belongs to this family. However, it was previously shown that its thymidylate kinase (TMPKyp) exhibits biochemical properties significantly different from those of its Escherichia coli counterpart [Chenal-Francisque, Tourneux, Carniel, Christova, Li de la Sierra, Barzu and Gilles (1999) Eur. J. Biochem. 265, 112-119]. In this work, the adenylate kinase (AK) of Y. pestis (AKyp) was characterized. As with TMPKyp, AKyp displayed a lower thermodynamic stability than other studied AKs. Two mutations in AK (Ser129Phe and Pro87Ser), previously shown to induce a thermosensitive growth defect in E. coli, were introduced into AKyp. The recombinant variants had a lower stability than wild-type AKyp and a higher susceptibility to proteolytic digestion. When the Pro87Ser substitution was introduced into the chromosomal adk gene of Y. pestis, growth of the mutant strain was altered at the non-permissive temperature of 37 degree C. In virulence testings, less than 50 colony forming units (CFU) of wild-type Y. pestis killed 100% of the mice upon subcutaneous infection, whereas bacterial loads as high as 1.5 x 10(4) CFU of the adk mutant were unable to kill any animals. PMID:12879903

  8. The stabilization effect of dielectric constant and acidic amino acids on arginine-arginine (Arg-Arg) pairings: database survey and computational studies.

    PubMed

    Zhang, Zhengyan; Xu, Zhijian; Yang, Zhuo; Liu, Yingtao; Wang, Jin'an; Shao, Qiang; Li, Shujin; Lu, Yunxiang; Zhu, Weiliang

    2013-05-02

    Database survey in this study revealed that about one-third of the protein structures deposited in the Protein Data Bank (PDB) contain arginine-arginine (Arg-Arg) pairing with a carbon···carbon (CZ···CZ) interaction distance less than 5 Å. All the Arg-Arg pairings were found to bury in a polar environment composed of acidic residues, water molecules, and strong polarizable or negatively charged moieties from binding site or bound ligand. Most of the Arg-Arg pairings are solvent exposed and 68.3% Arg-Arg pairings are stabilized by acidic residues, forming Arg-Arg-Asp/Glu clusters. Density functional theory (DFT) was then employed to study the effect of environment on the pairing structures. It was revealed that Arg-Arg pairings become thermodynamically stable (about -1 kcal/mol) as the dielectric constant increases to 46.8 (DMSO), in good agreement with the results of the PDB survey. DFT calculations also demonstrated that perpendicular Arg-Arg pairing structures are favorable in low dielectric constant environment, while in high dielectric constant environment parallel structures are favorable. Additionally, the acidic residues can stabilize the Arg-Arg pairing structures to a large degree. Energy decomposition analysis of Arg-Arg pairings and Arg-Arg-Asp/Glu clusters showed that both solvation and electrostatic energies contribute significantly to their stability. The results reported herein should be very helpful for understanding Arg-Arg pairing and its application in drug design.

  9. A cotton fiber associated cyclin-dependent kinase A gene: Characterization and chromosomal location

    Technology Transfer Automated Retrieval System (TEKTRAN)

    A cotton fiber cell normally originates and elongates as a single ovular epidermal cell. The cessation of fiber cell division and ensuing elongation imply that the cell cycle is differentially regulated in fiber cells. Cyclin-dependent kinases (CDKs) play a central role in the regulation of cell cy...

  10. Assignment of the rat genes coding for phenylalanine hydroxylase (PAH), tyrosine aminotransferase (TAT), and pyruvate kinase (PKL) to chromosomes 7, 19, 2, respectively.

    PubMed

    Fulchignoni-Lataud, M C; Weiss, M C; Szpirer, C; Levan, G

    1990-01-01

    A panel of hybrid clones segregating rat chromosomes in a mouse background was used to determine the chromosomal localization of three genes specifically expressed in hepatocytes. The phenylalanine hydroxylase, tyrosine aminotransferase, and pyruvate kinase genes were assigned to rat chromosomes 7, 19, and 2, respectively.

  11. Basal expression of the cystic fibrosis transmembrane conductance regulator gene is dependent on protein kinase A activity.

    PubMed Central

    McDonald, R A; Matthews, R P; Idzerda, R L; McKnight, G S

    1995-01-01

    The cystic fibrosis transmembrane conductance regulator (CFTR) functions as a Cl- channel that becomes activated after phosphorylation by cAMP-dependent protein kinase (PKA). We demonstrate that PKA also plays a crucial role in maintaining basal expression of the CFTR gene in the human colon carcinoma cell line T84. Inhibition of PKA activity by expression of a dominant-negative regulatory subunit or treatment with the PKA-selective inhibitor N-[2-(p-bromocinnamylamino)ethyl]-5-isoquinolinesulfonamide (H-89) caused a complete suppression of CFTR gene expression without affecting other constitutively active genes. Basal expression of a 2.2-kb region of the CFTR promoter linked to a luciferase reporter gene (CFTR-luc) exhibited the same dependence on PKA. The ability of cAMP to induce CFTR over basal levels is cell-type specific. In T84 cells, both the endogenous CFTR gene and CFTR-luc exhibited only a modest inducibility (approximately 2-fold), whereas in the human choriocarcinoma cell line JEG-3, CFTR-luc could be induced at least 4-fold. A variant cAMP-response element is present at position -48 to -41 in the CFTR promoter, and mutation of this sequence blocks basal expression. We conclude that cAMP, acting through PKA, is an essential regulator of basal CFTR gene expression and may mediate an induction of CFTR in responsive cell types. Images Fig. 1 Fig. 3 PMID:7543684

  12. Abscisic Acid and Gibberellin Differentially Regulate Expression of Genes of the SNF1-Related Kinase Complex in Tomato Seeds1

    PubMed Central

    Bradford, Kent J.; Downie, A. Bruce; Gee, Oliver H.; Alvarado, Veria; Yang, Hong; Dahal, Peetambar

    2003-01-01

    The SNF1/AMP-activated protein kinase subfamily plays central roles in metabolic and transcriptional responses to nutritional or environmental stresses. In yeast (Saccharomyces cerevisiae) and mammals, activating and anchoring subunits associate with and regulate the activity, substrate specificity, and cellular localization of the kinase subunit in response to changing nutrient sources or energy demands, and homologous SNF1-related kinase (SnRK1) proteins are present in plants. We isolated cDNAs corresponding to the kinase (LeSNF1), regulatory (LeSNF4), and localization (LeSIP1 and LeGAL83) subunits of the SnRK1 complex from tomato (Lycopersicon esculentum Mill.). LeSNF1 and LeSNF4 complemented yeast snf1 and snf4 mutants and physically interacted with each other and with LeSIP1 in a glucose-dependent manner in yeast two-hybrid assays. LeSNF4 mRNA became abundant at maximum dry weight accumulation during seed development and remained high when radicle protrusion was blocked by abscisic acid (ABA), water stress, far-red light, or dormancy, but was low or undetected in seeds that had completed germination or in gibberellin (GA)-deficient seeds stimulated to germinate by GA. In leaves, LeSNF4 was induced in response to ABA or dehydration. In contrast, LeSNF1 and LeGAL83 genes were essentially constitutively expressed in both seeds and leaves regardless of the developmental, hormonal, or environmental conditions. Regulation of LeSNF4 expression by ABA and GA provides a potential link between hormonal and sugar-sensing pathways controlling seed development, dormancy, and germination. PMID:12857836

  13. Effect of the Arg389Gly β₁-adrenoceptor polymorphism on plasma renin activity and heart rate, and the genotype-dependent response to metoprolol treatment.

    PubMed

    Petersen, Morten; Andersen, Jon T; Jimenez-Solem, Espen; Broedbaek, Kasper; Hjelvang, Brian R; Henriksen, Trine; Frandsen, Erik; Forman, Julie L; Torp-Pedersen, Christian; Køber, Lars; Poulsen, Henrik E

    2012-09-01

    1. A gene-drug interaction has been indicated between β₁-adrenoceptor-selective beta-blockers and the Arg389Gly polymorphism (rs1801253) in the adrenergic beta-1 receptor gene (ADRB1). In the present study, we investigated the effect of the ADRB1 Arg389Gly polymorphism on plasma renin activity (PRA) and heart rate (HR), as well as genotype-dependent responses to metoprolol and exercise. 2. Twenty-nine healthy male subjects participated in two treatment periods (placebo and 200 mg/day metoprolol). A 15 min submaximal exercise test was performed after each treatment period and PRA and HR were measured before and after exercise. 3. Before exercise, median PRA was lower in Gly/Gly subjects than in Arg/Arg subjects after both placebo (P = 0.030) and metoprolol (P = 0.020) treatment. After placebo, the exercise-induced increase in PRA was greater in Gly/Gly than Arg/Gly and Arg/Arg subjects (P = 0.033). The linear association between log(PRA) and log(metoprolol concentration) varied significantly between genotypes (P = 0.024). In Gly/Gly subjects, PRA decreased significantly with metoprolol concentration before (P = 0.025) and after exercise (P < 0.001), whereas in Arg/Gly and Arg/Arg subjects metoprolol concentration had no effect on PRA. The effect of metoprolol concentration on PRA in Gly/Gly subjects was enhanced by exercise (P = 0.044). No significant differences in HR were seen between genotype groups. 4. Resting PRA was lower in Gly/Gly than Arg/Arg subjects and the effect of exercise and metoprolol concentration on PRA was stronger in Gly/Gly subjects than with the other two genotypes. Thus, Gly/Gly heart failure patients may require lower doses of metoprolol than other patients to block neurohumoral hyperactivity.

  14. Origin, structure, and regulation of argK, encoding the phaseolotoxin-resistant ornithine carbamoyltransferase in Pseudomonas syringae pv. phaseolicola, and functional expression of argK in transgenic tobacco.

    PubMed Central

    Hatziloukas, E; Panopoulos, N J

    1992-01-01

    Pseudomonas syringae pv. phaseolicola produces the tripeptide N delta(N'-sulfo-diaminophosphinyl)-ornithylalanyl-homoarginin e (phaseolotoxin), which functions as a chlorosis-inducing toxin in the bean halo blight disease by inhibiting ornithine carbamoyltransferase (OCT). The bacterium possesses duplicate OCT genes, one of which, argK, encodes a toxin-resistant enzyme (ROCT) and imparts resistance to phaseolotoxin. We sequenced the argK gene from strain NPS3121, defined its promoter region, analyzed its regulation, and characterized its transcripts. The gene probably originated from another organism, since it is very distantly related to the argF gene encoding the housekeeping toxin-sensitive OCT and has low G+C content compared with the bacterial genome as a whole and with other protein-coding genes from P. syringae pv. phaseolicola. Optimized alignments of 13 OCT sequences allowed us to define key residues that may be responsible for toxin resistance and to identify a distinct prokaryotic amino acid signature, in ROCT, which argues for a prokaryotic origin of argK. An in-frame fusion of the argK coding region with the chloroplast transit peptide segment of the pea rbcS gene was introduced in Nicotiana tabacum by Agrobacterium-mediated transformation. The presence of an ROCT activity in transgenic plants was demonstrated by in vitro and in vivo assays. Some plants were toxin resistant, suggesting that pathogen-derived resistance to the toxin should be feasible in the pathogen's host. Images PMID:1522066

  15. Novel mutations in cyclin-dependent kinase-like 5 (CDKL5) gene in Indian cases of Rett syndrome.

    PubMed

    Das, Dhanjit Kumar; Mehta, Bhakti; Menon, Shyla R; Raha, Sarbani; Udani, Vrajesh

    2013-03-01

    Rett syndrome is a severe neurodevelopmental disorder, almost exclusively affecting females and characterized by a wide spectrum of clinical manifestations. Both the classic and atypical forms of Rett syndrome are primarily due to mutations in the methyl-CpG-binding protein 2 (MECP2) gene. Mutations in the X-linked cyclin-dependent kinase-like 5 (CDKL5) gene have been identified in patients with atypical Rett syndrome, X-linked infantile spasms sharing common features of generally early-onset seizures and mental retardation. CDKL5 is known as serine/threonine protein kinase 9 (STK9) and is mapped to the Xp22 region. It has a conserved serine/threonine kinase domain within its amino terminus and a large C-terminal region. Disease-causing mutations are distributed in both the amino terminal domain and in the large C-terminal domain. We have screened the CDKL5 gene in 44 patients with atypical Rett syndrome who had tested negative for MECP2 gene mutations and have identified 6 sequence variants, out of which three were novel and three known mutations. Two of these novel mutations p.V966I and p.A1011V were missense and p.H589H a silent mutation. Other known mutations identified were p.V999M, p.Q791P and p.T734A. Sequence homology for all the mutations revealed that the two mutations (p.Q791P and p.T734A) were conserved across species. This indicated the importance of these residues in structure and function of the protein. The damaging effects of these mutations were analysed in silico using PolyPhen-2 online software. The PolyPhen-2 scores of p.Q791P and p.T734A were 0.998 and 0.48, revealing that these mutations could be deleterious and might have potential functional effect. All other mutations had a low score suggesting that they might not alter the activity of CDKL5. We have also analysed the position of the mutations in the CDKL5 protein and found that all the mutations were present in the C-terminal domain of the protein. The C-terminal domain is required for

  16. Environmental analysis of typical antibiotic-resistant bacteria and ARGs in farmland soil chronically fertilized with chicken manure.

    PubMed

    Zhao, Xiang; Wang, Jinhua; Zhu, Lusheng; Ge, Weili; Wang, Jun

    2017-03-21

    Antibiotics and the corresponding resistant bacteria and resistance genes (ARGs) are generally considered emerging pollutants. To assess the impacts of tetracycline (TC) and sulfonamide (SA) antibiotics that are eliminated with fecaluria as drug prototypes, farmland soil used to research long-term fertilization with chicken manure was collected at four sites in Shandong Province. In this study, the rates of bacterial drug resistance to the same antibiotic decreased with an increase in the concentration of that antibiotic, and the resistance rates to TCs were lower than those to SAs. PCR of ARGs revealed that the ARGs detected at the highest frequency were the TC resistance genes tetW and tetO and the SA resistance genes sul1 and sul2. Real-time qPCR showed that the quantities of ARGs in farmland soil fertilized with chicken manure were significantly greater compared with the control soil. Moreover, significant correlations (R(2)=0.9525, p<0.05) between the number of sul ARGs and the total SA concentration were observed in all of the soil samples. In summary, this study showed that SAs can induce the appearance of ARGs and pollute the soil environment.

  17. A Novel Nonsense Mutation in PANK2 Gene in Two Patients with Pantothenate Kinase-Associated Neurodegeneration

    PubMed Central

    Ghafouri-Fard, Soudeh; Yassaee, Vahid Reza; Rezayi, Alireza; Hashemi-Gorji, Feyzollah; Alipour, Nasrin; Miryounesi, Mohammad

    2016-01-01

    Pantothenate kinase- associated neurodegeneration (PKAN) syndrome is a rare autosomal recessive disorder characterized by progressive extrapyramidal dysfunction and iron accumulation in the brain and axonal spheroids in the central nervous system. It has been shown that the disorder is caused by mutations in PANK2 gene which codes for a mitochondrial enzyme participating in coenzyme A biosynthesis. Here we report two cases of classic PKAN syndrome with early onset of neurodegenerative disorder. Mutational analysis has revealed that both are homozygous for a novel nonsense mutation in PANK2 gene (c.T936A (p.C312X)). The high prevalence of consanguineous marriages in Iran raises the likelihood of occurrence of autosomal recessive disorders such as PKAN and necessitates proper premarital genetic counseling. Further research is needed to provide the data on the prevalence of PKAN and identification of common PANK2 mutations in Iranian population. PMID:28357202

  18. Functional identification of the promoter for the gene encoding the alpha subunit of calcium/calmodulin-dependent protein kinase II.

    PubMed Central

    Olson, N J; Massé, T; Suzuki, T; Chen, J; Alam, D; Kelly, P T

    1995-01-01

    To examine the expression of the alpha subunit of calcium/calmodulin-dependent protein kinase II, various 5' flanking genomic sequences were inserted into a chloramphenicol acetyltransferase (CAT) reporter plasmid and CAT enzyme activities were analyzed in transfected NB2a neuroblastoma cells and mRNA transcription was analyzed by nuclease protection assays. A core promoter was identified which contained an essential TATA element located 162 nt 5' to the transcription start site. Sequences 3' to the transcription start site, as well as 5' to the TATA element, increased levels of CAT activity in transfected cells. The alpha-subunit gene promoter displayed higher CAT activities, relative to a simian virus 40 promoter, in transfected neuronal cell lines than in nonneuronal cell lines. Results also suggested that sequence surrounding the natural alpha-gene transcription initiation site may be important for targeting transcription initiation 162 nt downstream of its TATA element. Images Fig. 1 Fig. 3 PMID:7878035

  19. Fine mapping of the sunflower resistance locus Pl(ARG) introduced from the wild species Helianthus argophyllus.

    PubMed

    Wieckhorst, S; Bachlava, E; Dussle, C M; Tang, S; Gao, W; Saski, C; Knapp, S J; Schön, C-C; Hahn, V; Bauer, E

    2010-11-01

    Downy mildew, caused by Plasmopara halstedii, is one of the most destructive diseases in cultivated sunflower (Helianthus annuus L.). The dominant resistance locus Pl(ARG) originates from silverleaf sunflower (H. argophyllus Torrey and Gray) and confers resistance to all known races of P. halstedii. We mapped Pl(ARG) on linkage group (LG) 1 of (cms)HA342 × ARG1575-2, a population consisting of 2,145 F(2) individuals. Further, we identified resistance gene candidates (RGCs) that cosegregated with Pl(ARG) as well as closely linked flanking markers. Markers from the target region were mapped with higher resolution in NDBLOS(sel) × KWS04, a population consisting of 2,780 F(2) individuals that does not segregate for Pl(ARG). A large-insert sunflower bacterial artificial chromosome (BAC) library was screened with overgo probes designed for markers RGC52 and RGC151, which cosegregated with Pl(ARG). Two RGC-containing BAC contigs were anchored to the Pl(ARG) region on LG 1.

  20. Characterization of a mitogen-activated protein kinase gene from cucumber required for trichoderma-conferred plant resistance.

    PubMed

    Shoresh, Michal; Gal-On, Amit; Leibman, Diana; Chet, Ilan

    2006-11-01

    The fungal biocontrol agent Trichoderma asperellum has been recently shown to induce systemic resistance in plants through a mechanism that employs jasmonic acid and ethylene signal transduction pathways. Mitogen-activated protein kinase (MAPK) proteins have been implicated in the signal transduction of a wide variety of plant stress responses. Here we report the identification and characterization of a Trichoderma-induced MAPK (TIPK) gene function in cucumber (Cucumis sativus). Similar to its homologs, wound-induced protein kinase, MPK3, and MPK3a, TIPK is also induced by wounding. Normally, preinoculation of roots with Trichoderma activates plant defense mechanisms, which result in resistance to the leaf pathogen Pseudomonas syringae pv lachrymans. We used a unique attenuated virus vector, Zucchini yellow mosaic virus (ZYMV-AGII), to overexpress TIPK protein and antisense (AS) RNA. Plants overexpressing TIPK were more resistant to pathogenic bacterial attack than control plants, even in the absence of Trichoderma preinoculation. On the other hand, plants expressing TIPK-AS revealed increased sensitivity to pathogen attack. Moreover, Trichoderma preinoculation could not protect these AS plants against subsequent pathogen attack. We therefore demonstrate that Trichoderma exerts its protective effect on plants through activation of the TIPK gene, a MAPK that is involved in signal transduction pathways of defense responses.

  1. Genome-wide identification, characterization and expression analysis of populus leucine-rich repeat receptor-like protein kinase genes

    PubMed Central

    2013-01-01

    Background Leucine-rich repeat receptor-like kinases (LRR-RLKs) comprise the largest group within the receptor-like kinase (RLK) superfamily in plants. This gene family plays critical and diverse roles in plant growth, development and stress response. Although the LRR-RLK families in Arabidopsis and rice have been previously analyzed, no comprehensive studies have been performed on this gene family in tree species. Results In this work, 379 LRR-RLK genes were retrieved from the Populus trichocarpa genome and further grouped into 14 subfamilies based on their structural and sequence similarities. Approximately 82% (312 out of 379) of the PtLRR-RLK genes are located in segmental duplication blocks indicating the role of duplication process in the expansion of this gene family. The conservation and variation in motif composition and intron/exon arrangement among PtLRR-RLK subfamilies were analyzed to provide additional support for their phylogenetic relationship and more importantly to indicate the potential divergence in their functions. Expression profiling of PtLRR-RLKs showed that they were differentially expressed in different organs and tissues and some PtLRR-RLKs were specifically expressed in meristem tissues, which indicated their potential involvement in tissue development and differentiation. For most AtLRR-RLKs with defined functions, Populus homologues exhibiting similar expression patterns could be identified, which might indicate the functional conservation during evolution. Among 12 types of environmental cues analyzed by the genome-wide microarray data, PtLRR-RLKs showed specific responses to shoot organogenesis, wounding, low ammonium feeding, hypoxia and seasonal dormancy, but not to drought, re-watering after drought, flooding, AlCl3 treatment and bacteria or fungi treatments. Conclusions This study provides the first comprehensive genomic analysis of the Populus LRR-RLK gene family. Segmental duplication contributes significantly to the expansion

  2. Toward Understanding the Functional Role of Ss-riok-1, a RIO Protein Kinase-Encoding Gene of Strongyloides stercoralis

    PubMed Central

    Yuan, Wang; Lok, James B.; Stoltzfus, Jonathan D.; Gasser, Robin B.; Fang, Fang; Lei, Wei-Qiang; Fang, Rui; Zhou, Yan-Qin; Zhao, Jun-Long; Hu, Min

    2014-01-01

    Background Some studies of Saccharomyces cerevisiae and mammals have shown that RIO protein kinases (RIOKs) are involved in ribosome biogenesis, cell cycle progression and development. However, there is a paucity of information on their functions in parasitic nematodes. We aimed to investigate the function of RIOK-1 encoding gene from Strongyloides stercoralis, a nematode parasitizing humans and dogs. Methodology/Principal Findings The RIOK-1 protein-encoding gene Ss-riok-1 was characterized from S. stercoralis. The full-length cDNA, gDNA and putative promoter region of Ss-riok-1 were isolated and sequenced. The cDNA comprises 1,828 bp, including a 377 bp 5′-UTR, a 17 bp 3′-UTR and a 1,434 bp ORF encoding a protein of 477 amino acids containing a RIOK-1 signature motif. The genomic sequence of the Ss-riok-1 coding region is 1,636 bp in length and has three exons and two introns. The putative promoter region comprises 4,280 bp and contains conserved promoter elements, including four CAAT boxes, 12 GATA boxes, eight E-boxes (CANNTG) and 38 TATA boxes. The Ss-riok-1 gene is transcribed throughout all developmental stages with the highest transcript abundance in the infective third-stage larva (iL3). Recombinant Ss-RIOK-1 is an active kinase, capable of both phosphorylation and auto-phosphorylation. Patterns of transcriptional reporter expression in transgenic S. stercoralis larvae indicated that Ss-RIOK-1 is expressed in neurons of the head, body and tail as well as in pharynx and hypodermis. Conclusions/Significance The characterization of the molecular and the temporal and spatial expression patterns of the encoding gene provide first clues as to functions of RIOKs in the biological processes of parasitic nematodes. PMID:25101874

  3. Transcriptional Regulation of the SMK1 Mitogen-Activated Protein Kinase Gene during Meiotic Development in Saccharomyces cerevisiae

    PubMed Central

    Pierce, Michael; Wagner, Marisa; Xie, Jianxin; Gailus-Durner, Valérie; Six, John; Vershon, Andrew K.; Winter, Edward

    1998-01-01

    Meiotic development (sporulation) in Saccharomyces cerevisiae is characterized by an ordered pattern of gene expression, with sporulation-specific genes classified as early, middle, mid-late, or late depending on when they are expressed. SMK1 encodes a mitogen-activated protein kinase required for spore morphogenesis that is expressed as a middle sporulation-specific gene. Here, we identify the cis-acting DNA elements that regulate SMK1 transcription and characterize the phenotypes of mutants with altered expression patterns. The SMK1 promoter contains an upstream activating sequence (UASS) that specifically interacts with the transcriptional activator Abf1p. The Abf1p-binding sites from the early HOP1 and the middle SMK1 promoters are functionally interchangeable, demonstrating that these elements do not play a direct role in their differential transcriptional timing. Timing of SMK1 expression is determined by another cis-acting DNA sequence termed MSE (for middle sporulation element). The MSE is required not only for activation of SMK1 transcription during middle sporulation but also for its repression during vegetative growth and early meiosis. In addition, the SMK1 MSE can repress vegetative expression in the context of the HOP1 promoter and convert HOP1 from an early to a middle gene. SMK1 function is not contingent on its tight transcriptional regulation as a middle sporulation-specific gene. However, promoter mutants with different quantitative defects in SMK1 transcript levels during middle sporulation show distinct sporulation phenotypes. PMID:9742114

  4. Identification of cis-acting regulatory elements in the promoter region of the rat brain creatine kinase gene.

    PubMed Central

    Hobson, G M; Molloy, G R; Benfield, P A

    1990-01-01

    The functional organization of the rat brain creatine kinase (ckb) promoter was analyzed by deletion, linker scanning, and substitution mutagenesis. Mutations were introduced into the ckb promoter of hybrid ckb/neo (neomycin resistance gene) genes, and the mutant genes were expressed transiently in HeLa cells. Expression was assayed by primer extension analysis of neo RNA, which allowed the transcription start sites and the amount of transcription to be determined. Transfections and primer extension reactions were internally controlled by simultaneous analysis of transcription from the adenovirus VA gene located on the same plasmid as the hybrid ckb/neo gene. We demonstrate that 195 bp of the ckb promoter is sufficient for efficient in vivo expression in HeLa cells. A nonconsensus TTAA element at -28 bp appears to provide the TATA box function for the ckb promoter in vivo. Two CCAAT elements, one at -84 bp and the other at -54 bp, and a TATAAA TA element (a consensus TATA box sequence) at -66 bp are required for efficient transcription from the TTAA element. In addition, we present evidence that the consensus beta-globin TATA box responds to the TATAAATA element in the same way as the ckb nonconsensus TTAA element. Images PMID:2247071

  5. Ultrasound Microbubble-Mediated Delivery of Integrin-Linked Kinase Gene Improves Endothelial Progenitor Cells Dysfunction in Pre-Eclampsia

    PubMed Central

    Cui, Kai; Yan, Ting; Luo, Qingqing; Zheng, Yanfang; Liu, Xiaoxia; Huang, Xiaoyu

    2014-01-01

    Pre-eclampsia (PE) is a specific vascular complication in pregnancy whose precise mechanism is still unclear. We hypothesized that endothelial progenitor cells (EPCs), the precursor of endothelial cells, might be impaired in patients with PE and hold a great promise for the treatment of PE. In the present study, we analyzed the EPCs number and expression of integrin-linked kinase (ILK) in PE patients. We confirmed that both EPCs number and ILK expression were diminished in PE patients. Next, we transfected EPCs with ILK gene using ultrasonic microbubble technique (UMT) for the first time, as UMT is a novel type of gene transfer technology showing promising applications in stem cells apart from EPCs. To further investigate the transfection efficiency of UMT, RT-PCR analysis and western blot were used to examine the messenger RNA (mRNA) and protein level of ILK. After transfection of the ILK gene, EPCs function was tested to illustrate the role of ILK in cell proliferation, apoptosis, migration, and secretion. The results of the in vitro study suggested that UMT, a novel gene delivery system, could be considered a potent physical method for EPCs transfection. Moreover, the growth and angiogenetic properties of EPCs are enhanced by introducing ILK. This study may afford a new trend for EPCs transfection and gene therapy in PE. PMID:24564279

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

    PubMed

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

    2014-08-01

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

  7. Nuclear localization of lymphocyte-specific protein tyrosine kinase (Lck) and its role in regulating LIM domain only 2 (Lmo2) gene

    SciTech Connect

    Venkitachalam, Srividya; Chueh, Fu-Yu; Yu, Chao-Lan

    2012-01-20

    Highlights: Black-Right-Pointing-Pointer Lmo2 expression is elevated in Lck-transformed cells. Black-Right-Pointing-Pointer Both endogenous and exogenous Lck localize in the nucleus. Black-Right-Pointing-Pointer Nuclear Lck is active in Lck-transformed cells. Black-Right-Pointing-Pointer Lck binds to the promoter region of Lmo2 gene in vivo. Black-Right-Pointing-Pointer In contrast to JAK2, Lck does not increase histone H3 phosphorylation on Tyr 41. -- Abstract: LIM domain only protein 2 (Lmo2) is a transcription factor that plays a critical role in the development of T-acute lymphoblastic leukemia (T-ALL). A previous report established a link between Lmo2 expression and the nuclear presence of oncogenic Janus kinase 2 (JAK2), a non-receptor protein tyrosine kinase. The oncogenic JAK2 kinase phosphorylates histone H3 on Tyr 41 that leads to the relief of Lmo2 promoter repression and subsequent gene expression. Similar to JAK2, constitutive activation of lymphocyte-specific protein tyrosine kinase (Lck) has been implicated in lymphoid malignancies. However, it is not known whether oncogenic Lck regulates Lmo2 expression through a similar mechanism. We show here that Lmo2 expression is significantly elevated in T cell leukemia LSTRA overexpressing active Lck kinase and in HEK 293 cells expressing oncogenic Y505FLck kinase. Nuclear localization of active Lck kinase was confirmed in both Lck-transformed cells by subcellular fractionation and immunofluorescence microscopy. More importantly, in contrast to oncogenic JAK2, oncogenic Lck kinase does not result in significant increase in histone H3 phosphorylation on Tyr 41. Instead, chromatin immunoprecipitation experiment shows that oncogenic Y505FLck kinase binds to the Lmo2 promoter in vivo. This result raises the possibility that oncogenic Lck may activate Lmo2 promoter through direct interaction.

  8. Gene and protein kinase expression profiling of reactive oxygen species-associated lipotoxicity in the pancreatic beta-cell line MIN6.

    PubMed

    Wang, Xiaolin; Li, Hui; De Leo, Domenica; Guo, Wanbei; Koshkin, Vasilij; Fantus, I George; Giacca, Adria; Chan, Catherine B; Der, Sandy; Wheeler, Michael B

    2004-01-01

    Oligonucleotide microarrays were used to define oleic acid (OA)-regulated gene expression and proteomic technology to screen protein kinases in MIN6 insulinoma cells. The effects of oxidative stress caused by OA and potential protective effects of N-acetyl-L-cysteine (NAC), a scavenger of reactive oxygen species (ROS), on global gene expression and beta-cell function were investigated. Long-term exposure of MIN6 cells to OA led to a threefold increase in basal insulin secretion, a 50% decrease in insulin content, an inhibition of glucose-stimulated insulin secretion (GSIS), and a twofold increase in the level of ROS. The addition of NAC normalized both the OA-induced insulin content and ROS elevation, but it failed to restore GSIS. Microarray studies and subsequent quantitative PCR analysis showed that OA consistently regulated the expression of 45 genes involved in metabolism, cell growth, signal transduction, transcription, and protein processing. The addition of NAC largely normalized the expression of the OA-regulated genes involved in cell growth and differentiation but not other functions. A protein kinase screen showed that OA regulated the expression and/or phosphorylation levels of kinases involved in stress-response mitogen-activated protein kinase, phosphatidylinositol 3-kinase, and cell cycle control pathways. Importantly, these findings indicate that chronic OA exposure can impair beta-cell function through ROS-dependent and -independent mechanisms.

  9. A ras-dependent pathway abolishes activity of a muscle-specific enhancer upstream from the muscle creatine kinase gene.

    PubMed Central

    Sternberg, E A; Spizz, G; Perry, M E; Olson, E N

    1989-01-01

    Differentiation of skeletal myoblasts is accompanied by induction of a series of tissue-specific genes whose products are required for the specialized functions of the mature muscle fiber. The program for myogenic differentiation is subject to negative control by several peptide growth factors and by the products of mutationally activated ras oncogenes, which persistently activate intracellular cascades normally triggered by specific growth factors. Previously, we reported that induction of the muscle creatine kinase (mck) gene during myogenesis was dependent on a distal upstream enhancer that cooperated with a proximal promoter to direct high levels of expression in developing muscle cells (E. A. Sternberg, G. Spizz, W. M. Perry, D. Vizard, T. Weil, and E. N. Olson, Mol. Cell. Biol. 8:2896-2909). To investigate the mechanisms whereby ras blocks the induction of muscle-specific genes, we have examined the ability of mck 5' regulatory elements to direct expression of the linked reporter gene for chloramphenicol acetyltransferase (cat) in C2 myoblasts bearing mutant N-ras and H-ras oncogenes. In this paper we report that expression of activated ras alleles abolishes activity of the mck upstream enhancer but does not affect the activity of the mck promoter. The ability of ras to repress the expression of mck-cat fusion genes that have been transfected either transiently or stably into myoblasts suggests that ras may exert its effects on muscle-specific genes through mechanisms independent of chromatin configurations or DNA methylation. These results also suggest that ras blocks establishment of the myogenic phenotype by preventing the accumulation of regulatory factors required for transcriptional induction of muscle-specific genes. Images PMID:2651901

  10. The activity-regulated cytoskeletal-associated protein (Arc/Arg3.1) is required for memory consolidation of pavlovian fear conditioning in the lateral amygdala.

    PubMed

    Ploski, Jonathan E; Pierre, Vicki J; Smucny, Jason; Park, Kevin; Monsey, Melissa S; Overeem, Kathie A; Schafe, Glenn E

    2008-11-19

    The activity-regulated cytoskeletal-associated protein (Arc/Arg3.1) is an immediate early gene that has been widely implicated in hippocampal-dependent learning and memory and is believed to play an integral role in synapse-specific plasticity. Here, we examined the role of Arc/Arg3.1 in amygdala-dependent Pavlovian fear conditioning. We first examined the regulation of Arc/Arg3.1 mRNA and protein after fear conditioning and LTP-inducing stimulation of thalamic inputs to the lateral amygdala (LA). Quantitative real-time PCR, in situ hybridization, Western blotting and immunohistochemistry revealed a significant upregulation of Arc/Arg3.1 mRNA and protein in the LA relative to controls. In behavioral experiments, intra-LA infusion of an Arc/Arg3.1 antisense oligodeoxynucleotide (ODN) was observed to be anatomically restricted to the LA, taken up by LA cells, and to promote significant knockdown of Arc/Arg3.1 protein. Rats given intra-LA infusions of multiple doses of the Arc/Arg3.1 ODN showed an impairment of LTM (tested approximately 24 later), but no deficit in STM (tested 3 h later) relative to controls infused with scrambled ODN. Finally, to determine whether upregulation of Arc/Arg3.1 occurs downstream of ERK/MAPK activation, we examined Arc/Arg3.1 expression in rats given intra-LA infusion of the MEK inhibitor U0126. Relative to vehicle controls, infusion of U0126 impaired training-induced increases in Arc/Arg3.1 expression. These findings suggest that Arc/Arg3.1 expression in the amygdala is required for fear memory consolidation, and further suggest that Arc/Arg3.1 regulation in the LA is downstream of the ERK/MAPK signaling pathway.

  11. Src subfamily kinases regulate nuclear export and degradation of transcription factor Nrf2 to switch off Nrf2-mediated antioxidant activation of cytoprotective gene expression.

    PubMed

    Niture, Suryakant K; Jain, Abhinav K; Shelton, Phillip M; Jaiswal, Anil K

    2011-08-19

    Nrf2 (NF-E2-related factor 2) is a nuclear transcription factor that in response to chemical and radiation stress regulates coordinated induction of a battery of cytoprotective gene expressions leading to cellular protection. In this study, we investigated the role of Src kinases in the regulation of Nrf2 and downstream signaling. siRNA-mediated inhibition of Fyn, Src, Yes, and Fgr, but not Lyn, in mouse hepatoma Hepa-1 cells, led to nuclear accumulation of Nrf2 and up-regulation of Nrf2 downstream gene expression. Mouse embryonic fibroblasts with combined deficiency of Fyn/Src/Yes/Fgr supported results from siRNA. In addition, steady-state overexpression of Fyn, Src, and Yes phosphorylated Nrf2Tyr568 that triggered nuclear export and degradation of Nrf2 and down-regulation of Nrf2 downstream gene expression. Exposure of cells to antioxidant, oxidant, or UV radiation increased nuclear import of Fyn, Src, and Yes kinases, which phosphorylated Nrf2Tyr568 resulting in nuclear export and degradation of Nrf2. Further analysis revealed that stress-activated GSK3β acted upstream to the Src kinases and phosphorylated the Src kinases, leading to their nuclear localization and Nrf2 phosphorylation. The overexpression of Src kinases in Hepa-1 cells led to decreased Nrf2, increased apoptosis, and decreased cell survival. Mouse embryonic fibroblasts deficient in Src kinases showed nuclear accumulation of Nrf2, induction of Nrf2 and downstream gene expression, reduced apoptosis, and increased cell survival. The studies together demonstrate that Src kinases play a critical role in nuclear export and degradation of Nrf2, thereby providing a negative feedback mechanism to switch off Nrf2 activation and restore normal cellular homeostasis.

  12. Oncoprotein kinase

    DOEpatents

    Karin, Michael; Hibi, Masahiko; Lin, Anning

    2001-02-27

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

  13. Gene-Environment Interactions Target Mitogen-activated Protein 3 Kinase 1 (MAP3K1) Signaling in Eyelid Morphogenesis*

    PubMed Central

    Mongan, Maureen; Meng, Qinghang; Wang, Jingjing; Kao, Winston W.-Y.; Puga, Alvaro; Xia, Ying

    2015-01-01

    Gene-environment interactions determine the biological outcomes through mechanisms that are poorly understood. Mouse embryonic eyelid closure is a well defined model to study the genetic control of developmental programs. Using this model, we investigated how exposure to dioxin-like environmental pollutants modifies the genetic risk of developmental abnormalities. Our studies reveal that mitogen-activated protein 3 kinase 1 (MAP3K1) signaling is a focal point of gene-environment cross-talk. Dioxin exposure, acting through the aryl hydrocarbon receptor (AHR), blocked eyelid closure in genetic mutants in which MAP3K1 signaling was attenuated but did not disturb this developmental program in either wild type or mutant mice with attenuated epidermal growth factor receptor or WNT signaling. Exposure also markedly inhibited c-Jun phosphorylation in Map3k1+/− embryonic eyelid epithelium, suggesting that dioxin-induced AHR pathways can synergize with gene mutations to inhibit MAP3K1 signaling. Our studies uncover a novel mechanism through which the dioxin-AHR axis interacts with the MAP3K1 signaling pathways during fetal development and provide strong empirical evidence that specific gene alterations can increase the risk of developmental abnormalities driven by environmental pollutant exposure. PMID:26109068

  14. Overexpression of GbRLK, a putative receptor-like kinase gene, improved cotton tolerance to Verticillium wilt.

    PubMed

    Jun, Zhao; Zhang, Zhiyuan; Gao, Yulong; Zhou, Lei; Fang, Lei; Chen, Xiangdong; Ning, Zhiyuan; Chen, Tianzi; Guo, Wangzhen; Zhang, Tianzhen

    2015-10-08

    Verticillium dahliae is a causative fungal pathogen and only a few genes have been identified that exhibit critical roles in disease resistance and few has shown positive effects on the resistance to Verticillium wilt in transgenic cotton. We cloned a receptor-like kinase gene (GbRLK) induced by Verticillium dahliae (VD) in the disease-resistant cotton Gossypium barbadense cv. Hai7124. Northern blotting revealed that the GbRLK was induced by VD at 96 h after inoculation. The functional GbRLK is from D subgenome since a single base deletion results in a frameshift or dysfunctional homologue in the A subgenome in tetraploid cotton. To verify the function of GbRLK, we developed the overexpression transgenic GbRLK cotton and Arabidopsis lines, and found that they all showed the higher resistance to Verticillium in the greenhouse and field trial. The results of the expression profile using transgenic and non-transgenic Arabidopsis thaliana revealed that the GbRLK regulated expressions of a series genes associated with biotic and abiotic stresses. Therefore, we propose that the increased resistance to Verticillium dahliae infection in transgnic plants could result from reduction in the damage of water loss and regulation of defense gene expression.

  15. Pyrroloquinoline quinone and a quinoprotein kinase support γ-radiation resistance in Deinococcus radiodurans and regulate gene expression.

    PubMed

    Rajpurohit, Yogendra Singh; Desai, Shruti Sumeet; Misra, Hari Sharan

    2013-06-01

    Deinococcus radiodurans is known for its extraordinary resistance to various DNA damaging agents including γ-radiation and desiccation. The pqqE:cat and Δdr2518 mutants making these cells devoid of pyrroloquinoline quinone (PQQ) and a PQQ inducible Ser/Thr protein kinase, respectively, became sensitive to γ-radiation. Transcriptome analysis of these mutants showed differential expression of the genes including those play roles in oxidative stress tolerance and (DSB) repair in D. radiodurans and in genome maintenance and stress response in other bacteria. Escherichia coli cells expressing DR2518 and PQQ showed improved resistance to γ-radiation, which increased further when both DR2518 and PQQ were present together. Although, profiles of genes getting affected in these mutants were different, there were still a few common genes showing similar expression trends in both the mutants and some others as reported earlier in oxyR and pprI mutant of this bacterium. These results suggested that PQQ and DR2518 have independent roles in γ-radiation resistance of D. radiodurans but their co-existence improves radioresistance further, possibly by regulating differential expression of the genes important for bacterial response to oxidative stress and DNA damage.

  16. Extracellular simian virus 40 induces an ERK/MAP kinase-independent signalling pathway that activates primary response genes and promotes virus entry.

    PubMed

    Dangoria, N S; Breau, W C; Anderson, H A; Cishek, D M; Norkin, L C

    1996-09-01

    Simian virus 40 (SV40) binding to growth-arrested cells activated an intracellular signalling pathway that induced the up-regulation of the primary response genes c-myc, c-jun and c-sis within 30 min and of JE within 90 min. The up-regulation of the primary response genes occurred in the presence of cycloheximide and when UV-inactivated SV40 was adsorbed to cells. SV40 binding did not activate Raf or mitogen-activated protein kinase (MAP/ERK1), or mobilize intracellular Ca2+. The SV40-induced up-regulation of c-myc and c-jun was blocked by the tyrosine kinase inhibitor, genistein, and by the protein kinase C (PKC) inhibitor, calphostin C, but not by expression of the MAP kinase-specific phosphatase, MKP-1. These results suggest that the SV40-induced signalling pathway includes the activities of a tyrosine kinase and a Ca(2+)-independent isoform of PKC, but not of Raf or MAP kinase. Finally, SV40 infectious entry into cells was specifically and reversibly blocked by genistein.

  17. Rearranged Anaplastic Lymphoma Kinase (ALK) Gene in Adult-Onset Papillary Thyroid Cancer Amongst Atomic Bomb Survivors

    PubMed Central

    Mukai, Mayumi; Takahashi, Keiko; Hayashi, Yuzo; Nakachi, Kei; Kusunoki, Yoichiro

    2012-01-01

    Background We previously noted that among atomic bomb survivors (ABS), the relative frequency of cases of adult papillary thyroid cancer (PTC) with chromosomal rearrangements (mainly RET/PTC) was significantly greater in those with relatively higher radiation exposure than those with lower radiation exposure. In contrast, the frequency of PTC cases with point mutations (mainly BRAFV600E) was significantly lower in patients with relatively higher radiation exposure than those with lower radiation exposure. We also found that among ABS, the frequency of PTC cases with no detectable gene alterations in RET, neurotrophic tyrosine kinase receptor 1 (NTRK1), BRAF, or RAS was significantly higher in patients with relatively higher radiation exposure than those with lower radiation exposure. However, in ABS with PTC, the relationship between the presence of the anaplastic lymphoma kinase (ALK) gene fused with other gene partners and radiation exposure has received little study. In this study, we tested the hypothesis that the relative frequency of rearranged ALK in ABS with PTC, and with no detectable gene alterations in RET, NTRK1, BRAF, or RAS, would be greater in those having relatively higher radiation exposures. Methods The 105 subjects in the study were drawn from the Life Span Study cohort of ABS of Hiroshima and Nagasaki who were diagnosed with PTC between 1956 and 1993. Seventy-nine were exposed (>0 mGy), and 26 were not exposed to A-bomb radiation. In the 25 ABS with PTC, and with no detectable gene alterations in RET, NTRK1, BRAF, or RAS, we examined archival, formalin-fixed, paraffin-embedded PTC specimens for rearrangement of ALK using reverse transcription–polymerase chain reaction and 5′ rapid amplification of cDNA ends (5′ RACE). Results We found rearranged ALK in 10 of 19 radiation-exposed PTC cases, but none among 6 patients with PTC with no radiation exposure. In addition, solid/trabecular-like architecture in PTC was closely associated with ALK

  18. Liver X receptor α is involved in the transcriptional regulation of the 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase gene.

    PubMed

    Zhao, Li-Feng; Iwasaki, Yasumasa; Nishiyama, Mitsuru; Taguchi, Takafumi; Tsugita, Makoto; Okazaki, Mizuho; Nakayama, Shuichi; Kambayashi, Machiko; Fujimoto, Shimpei; Hashimoto, Koshi; Murao, Koji; Terada, Yoshio

    2012-05-01

    The activity of 6-phosphofructo-1-kinase is strictly controlled by fructose-2,6-bisphosphate, the level of which is regulated by another enzyme, 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase (PFK2/FBP2). PFK2/FBP2 is a bifunctional enzyme, having kinase and phosphatase activities, and regulates both glycolysis and gluconeogenesis. Here, we examined the hormonal regulation of the PFK2/FBP2 gene in vitro using the reporter assay, the electromobility shift assay (EMSA), and the chromatin immunoprecipitation (ChIP) assay in HuH7 cells and also using the mouse liver in vivo. We found that the transcriptional activity of the PFK2/FBP2 gene was stimulated by insulin and inhibited by cAMP and glucocorticoid. Liver X receptor (LXR) α showed a potent and specific stimulatory effect on PFK2/FBP2 gene transcription. Deletion and mutagenesis analyses identified the LXR response element (LXRE) in the 5'-promoter region of the PFK2/FBP2 gene. Binding of LXRα was confirmed by the EMSA and ChIP assay. Endogenous PFK2/FBP2 mRNA in the mouse liver was increased in the fasting/refeeding state compared with the fasting state. Altogether, PFK2/FBP2 gene transcription is found to be regulated in a way that is more similar to other glycolytic enzyme genes than to gluconeogenic genes. Furthermore, our data strongly suggest that LXRα is one of the key regulators of PFK2/FBP2 gene transcription.

  19. GENETIC VARIATION IN THE BETA-3-ADRENORECEPTOR GENE (TRP64ARG POLYMORPHISM) AND THEIR INFLUENCE ON ANTHROPOMETRIC PARAMETERS AND INSULIN RESISTANCE AFTER A HIGH PROTEIN/LOW CARBOHYDRATE VERSUS A STANDARD HYPOCALORIC DIET.

    PubMed

    de Luis, Daniel Antonio; Aller, Rocío; Izaola, Olatz; de la Fuente, Beatriz; Romero, Enrique

    2015-08-01

    Introducción: la variante Trp64Arg del receptor Beta ha sido relacionada con un aumento del peso corporal y resistencia a la insulina. Objetivo: el objetivo de nuestro estudio fue investigar la influencia del polimorfismo (rs 4994) del gen del receptor adrenérgico-Beta-3 en la respuesta metabólica y la pérdida de peso en un estudio de intervención a medio plazo con una dieta con alto contenido en proteínas/baja en carbohidratos vs una dieta hipocalórica estándar (1.000 kcal / día). Material y métodos: se evaluó una muestra de 284 sujetos obesos con un diseño de ensayo aleatorio. Se realizó una evaluación nutricional al inicio y al final de un período de 9 meses en el que los sujetos recibieron una de las dos dietas (dieta HP: alta en proteínas/baja en carbohidratos vs dieta S: dieta estándar). Resultados: no hubo diferencias significativas entre los efectos positivos (sobre el peso, el índice de masa corporal, la circunferencia de la cintura, la masa grasa, la presión arterial sistólica y los niveles de leptina) en los dos genotipos con ambas dietas. Con ambas dietas y solo en el genotipo salvaje (dieta HP vs dieta S), colesterol total (-10,1 ± 3,9 mg / dl vs -10,1 ± 2,2 mg / dl; p> 0,05), colesterol LDL (-9,5 ± 2,1 mg / dl vs -8,5 ± 2,3 mg / dl; p> 0,05) y los triglicéridos (-19,1 ± 2,1 mg / dl vs -14,3 ± 2,1 mg / dl; p> 0,05) disminuyeron. La mejoría de estos parámetros fue similar en sujetos con dieta HP vs dieta HS. Con la dieta HP y solo en el genotipo salvaje, los niveles de insulina (-3,7 ± 1,9 UI / L; p.

  20. A genomewide overexpression screen identifies genes involved in the phosphatidylinositol 3-kinase pathway in the human protozoan parasite Entamoeba histolytica.

    PubMed

    Koushik, Amrita B; Welter, Brenda H; Rock, Michelle L; Temesvari, Lesly A

    2014-03-01

    Entamoeba histolytica is a protozoan parasite that causes amoebic dysentery and liver abscess. E. histolytica relies on motility, phagocytosis, host cell adhesion, and proteolysis of extracellular matrix for virulence. In eukaryotic cells, these processes are mediated in part by phosphatidylinositol 3-kinase (PI3K) signaling. Thus, PI3K may be critical for virulence. We utilized a functional genomics approach to identify genes whose products may operate in the PI3K pathway in E. histolytica. We treated a population of trophozoites that were overexpressing genes from a cDNA library with a near-lethal dose of the PI3K inhibitor wortmannin. This screen was based on the rationale that survivors would be overexpressing gene products that directly or indirectly function in the PI3K pathway. We sequenced the overexpressed genes in survivors and identified a cDNA encoding a Rap GTPase, a protein previously shown to participate in the PI3K pathway. This supports the validity of our approach. Genes encoding a coactosin-like protein, EhCoactosin, and a serine-rich E. histolytica protein (SREHP) were also identified. Cells overexpressing EhCoactosin or SREHP were also less sensitive to a second PI3K inhibitor, LY294002. This corroborates the link between these proteins and PI3K. Finally, a mutant cell line with an increased level of phosphatidylinositol (3,4,5)-triphosphate, the product of PI3K activity, exhibited increased expression of SREHP and EhCoactosin. This further supports the functional connection between these proteins and PI3K in E. histolytica. To our knowledge, this is the first forward-genetics screen adapted to reveal genes participating in a signal transduction pathway in this pathogen.

  1. Genome of Epinotia aporema granulovirus (EpapGV), a polyorganotropic fast killing betabaculovirus with a novel thymidylate kinase gene

    PubMed Central

    2012-01-01

    Background Epinotia aporema (Lepidoptera: Tortricidae) is an important pest of legume crops in South America. Epinotia aporema granulovirus (EpapGV) is a baculovirus that causes a polyorganotropic infection in the host larva. Its high pathogenicity and host specificity make EpapGV an excellent candidate to be used as a biological control agent. Results The genome of Epinotia aporema granulovirus (EpapGV) was sequenced and analyzed. Its circular double-stranded DNA genome is 119,082 bp in length and codes for 133 putative genes. It contains the 31 baculovirus core genes and a set of 19 genes that are GV exclusive. Seventeen ORFs were unique to EpapGV in comparison with other baculoviruses. Of these, 16 found no homologues in GenBank, and one encoded a thymidylate kinase. Analysis of nucleotide sequence repeats revealed the presence of 16 homologous regions (hrs) interspersed throughout the genome. Each hr was characterized by the presence of 1 to 3 clustered imperfect palindromes which are similar to previously described palindromes of tortricid-specific GVs. Also, one of the hrs (hr4) has flanking sequences suggestive of a putative non-hr ori. Interestingly, two more complex hrs were found in opposite loci, dividing the circular dsDNA genome in two halves. Gene synteny maps showed the great colinearity of sequenced GVs, being EpapGV the most dissimilar as it has a 20 kb-long gene block inversion. Phylogenetic study performed with 31 core genes of 58 baculoviral genomes suggests that EpapGV is the baculovirus isolate closest to the putative common ancestor of tortricid specific betabaculoviruses. Conclusions This study, along with previous characterization of EpapGV infection, is useful for the better understanding of the pathology caused by this virus and its potential utilization as a bioinsecticide. PMID:23051685

  2. Gene Expression Profile of Calcium/Calmodulin-Dependent Protein Kinase IIα in Rat's Hippocampus during Morphine Withdrawal

    PubMed Central

    Ahmadi, Shamseddin; Amiri, Shahin; Rafieenia, Fatemeh; Rostamzadeh, Jalal

    2013-01-01

    Introduction Calcium/calmodulin-dependent protein kinase II (CaMKII) which is highly expressed in the hippocampus is known to play a pivotal role in reward-related memories and morphine dependence. Methods In the present study, repeated morphine injections once daily for 7 days was done to induce morphine tolerance in male Wistar rats, after which gene expression profile of α-isoform of CaMKII (CaMKIIα) in the hippocampus was evaluated upon discontinuation of morphine injection over 21 days of morphine withdrawal. Control groups received saline for 7 consecutive days. For gene expression study, rats’ brains were removed and the hippocampus was dissected in separate groups on days 1, 3, 7, 14, and 21 since discontinuation of of morphine injection. A semi-quantitative RT-PCR method was used to evaluate the gene expression profile. Results Tolerance to morphine was verified by a significant decrease in morphine analgesia in a hotplate test on day 8 (one day after the final repeated morphine injections). Results showed that gene expression of CaMKIIα at mRNA level on day 1, 3, 7, 14 and 21 of morphine withdrawal was significantly altered as compared to the saline control group. Post hoc Tukey's test revealed a significantly enhanced CaMKIIα gene expression on day 14. Discussion It can be concluded that CaMKIIα gene expression during repeated injections of morphine is increased and this increase continues up to 14 days of withdrawal then settles at a new set point. Therefore, the strong morphine reward-related memory in morphine abstinent animals may, at least partly be attributed to, the up-regulation of CaMKIIα in the hippocampus over 14 days of morphine withdrawal. PMID:25337341

  3. Unifying Vertical and Nonvertical Evolution: A Stochastic ARG-based Framework

    PubMed Central

    Bloomquist, Erik W.; Suchard, Marc A.

    2010-01-01

    Evolutionary biologists have introduced numerous statistical approaches to explore nonvertical evolution, such as horizontal gene transfer, recombination, and genomic reassortment, through collections of Markov-dependent gene trees. These tree collections allow for inference of nonvertical evolution, but only indirectly, making findings difficult to interpret and models difficult to generalize. An alternative approach to explore nonvertical evolution relies on phylogenetic networks. These networks provide a framework to model nonvertical evolution but leave unanswered questions such as the statistical significance of specific nonvertical events. In this paper, we begin to correct the shortcomings of both approaches by introducing the “stochastic model for reassortment and transfer events” (SMARTIE) drawing upon ancestral recombination graphs (ARGs). ARGs are directed graphs that allow for formal probabilistic inference on vertical speciation events and nonvertical evolutionary events. We apply SMARTIE to phylogenetic data. Because of this, we can typically infer a single most probable ARG, avoiding coarse population dynamic summary statistics. In addition, a focus on phylogenetic data suggests novel probability distributions on ARGs. To make inference with our model, we develop a reversible jump Markov chain Monte Carlo sampler to approximate the posterior distribution of SMARTIE. Using the BEAST phylogenetic software as a foundation, the sampler employs a parallel computing approach that allows for inference on large-scale data sets. To demonstrate SMARTIE, we explore 2 separate phylogenetic applications, one involving pathogenic Leptospirochete and the other Saccharomyces. PMID:20525618

  4. A polymorphism in the protein kinase C gene PRKCB is associated with α2-adrenoceptor-mediated vasoconstriction

    PubMed Central

    Ruohonen, Saku; Valve, Laura; Muszkat, Mordechai; Sofowora, Gbenga G.; Kurnik, Daniel; Stein, C. Michael; Perola, Markus; Scheinin, Mika; Snapir, Amir

    2013-01-01

    Objectives α2-Adrenoceptors (α2-AR) mediate both constriction and dilatation of blood vessels. There is substantial inter-individual variability in dorsal hand vein (DHV) constriction responses to α2-AR agonist activation. Genetic factors appear to contribute significantly to this variation. The present study was designed to identify genetic factors contributing to the inter-individual variability in α2-AR-mediated vascular constriction induced by the selective α2-AR agonist dexmedetomidine. Methods DHV constriction responses to local infusion of dexmedetomidine were assessed by measuring changes in vein diameter with a linear variable differential transformer. The outcome variable was log-transformed dexmedetomidine ED50 for constriction. A genome-wide association study (GWAS) of 433,378 single nucleotide polymorphisms (SNPs) was performed for the sensitivity of DHV responses in 64 healthy Finnish subjects. 20 SNPs were selected based on the GWAS results and their associations with the ED50 of dexmedetomidine were tested in an independent North American study population of 68 healthy individuals. Results In both study populations (GWAS and replication samples), the SNP rs9922316 in the gene for protein kinase C type β was consistently associated with dexmedetomidine ED50 for dorsal hand vein constriction (unadjusted p = 0.00016 for the combined population). Conclusions Genetic variation in protein kinase C type β may contribute to the inter-individual variation in dorsal hand vein constriction responses to α2-AR activation by the agonist dexmedetomidine. PMID:23337848

  5. Escherichia coli dihydroxyacetone kinase controls gene expression by binding to transcription factor DhaR.

    PubMed

    Bächler, Christoph; Schneider, Philipp; Bähler, Priska; Lustig, Ariel; Erni, Bernhard

    2005-01-26

    Dihydroxyacetone (Dha) kinases are a sequence-conserved family of enzymes, which utilize either ATP (in animals, plants, bacteria) or the bacterial phosphoenolpyruvate carbohydrate phosphotransferase system (PTS) as a source of high-energy phosphate. The PTS-dependent kinase of Escherichia coli consists of three subunits: DhaK contains the Dha binding site, DhaL contains ADP as cofactor for the double displacement of phosphate from DhaM to Dha, and DhaM provides a phospho-histidine relay between the PTS and DhaL::ADP. DhaR is a transcription activator belonging to the AAA+ family of enhancer binding proteins. It stimulates transcription of the dhaKLM operon from a sigma70 promoter and autorepresses dhaR transcription. Genetic and biochemical studies indicate that the enzyme subunits DhaL and DhaK act antagonistically as coactivator and corepressor of the transcription activator by mutually exclusive binding to the sensing domain of DhaR. In the presence of Dha, DhaL is dephosphorylated and DhaL::ADP displaces DhaK and stimulates DhaR activity. In the absence of Dha, DhaL::ADP is converted by the PTS to DhaL::ATP, which does not bind to DhaR.

  6. Living with high putrescine: expression of ornithine and arginine biosynthetic pathway genes in high and low putrescine producing poplar cells.

    PubMed

    Page, Andrew F; Minocha, Rakesh; Minocha, Subhash C

    2012-01-01

    Arginine (Arg) and ornithine (Orn), both derived from glutamate (Glu), are the primary substrates for polyamine (PA) biosynthesis, and also play important roles as substrates and intermediates of overall N metabolism in plants. Their cellular homeostasis is subject to multiple levels of regulation. Using reverse transcription quantitative PCR (RT-qPCR), we studied changes in the expression of all genes of the Orn/Arg biosynthetic pathway in response to up-regulation [via transgenic expression of mouse Orn decarboxylase (mODC)] of PA biosynthesis in poplar (Populus nigra × maximowiczii) cells grown in culture. Cloning and sequencing of poplar genes involved in the Orn/Arg biosynthetic pathway showed that they have high homology with similar genes in other plants. The expression of the genes of Orn, Arg and PA biosynthetic pathway fell into two hierarchical clusters; expression of one did not change in response to high putrescine, while members of the other cluster showed a shift in expression pattern during the 7-day culture cycle. Gene expression of branch point enzymes (N-acetyl-Glu synthase, Orn aminotransferase, Arg decarboxylase, and spermidine synthase) in the sub-pathways, constituted a separate cluster from those involved in intermediary reactions of the pathway (N-acetyl-Glu kinase, N-acetyl-Glu-5-P reductase, N-acetyl-Orn aminotransferase, N (2)-acetylOrn:N-acetyl-Glu acetyltransferase, N (2)-acetyl-Orn deacetylase, Orn transcarbamylase, argininosuccinate synthase, carbamoylphosphate synthetase, argininosuccinate lyase, S-adenosylmethionine decarboxylase, spermine synthase). We postulate that expression of all genes of the Glu-Orn-Arg pathway is constitutively coordinated and is not influenced by the increase in flux rate through this pathway in response to increased utilization of Orn by mODC; thus the pathway involves mostly biochemical regulation rather than changes in gene expression. We further suggest that Orn itself plays a major role in the

  7. Mutational analysis of the Saccharomyces cerevisiae SNF1 protein kinase and evidence for functional interaction with the SNF4 protein.

    PubMed Central

    Celenza, J L; Carlson, M

    1989-01-01

    The SNF1 gene of Saccharomyces cerevisiae encodes a protein-serine/threonine kinase that is required for derepression of gene expression in response to glucose limitation. We present evidence that the protein kinase activity is essential for SNF1 function: substitution of Arg for Lys in the putative ATP-binding site results in a mutant phenotype. A polyhistidine tract near the N terminus was found to be dispensable. Deletion of the large region C terminal to the kinase domain only partially impaired SNF1 function, causing expression of invertase to be somewhat reduced but still glucose repressible. The function of the SNF4 gene, another component of the regulatory system, was required for maximal in vitro activity of the SNF1 protein kinase. Increased SNF1 gene dosage partially alleviated the requirement for SNF4. C-terminal deletions of SNF1 also reduced dependence on SNF4. Our findings suggest that SNF4 acts as a positive effector of the kinase but does not serve a regulatory function in signaling glucose availability. Images PMID:2557546

  8. A novel gene mutation in PANK2 in a patient with an atypical form of pantothenate kinase-associated neurodegeneration.

    PubMed

    Pérez-González, E A; Chacón-Camacho, O F; Arteaga-Vázquez, J; Zenteno, J C; Mutchinick, O M

    2013-11-01

    Pantothenate kinase-associated neurodegeneration (PKAN) disease is an autosomal recessive neurodegenerative disorder with iron storage in the brain due to PANK2 gene mutations. Brain magnetic resonance imaging (MRI) shows the typical "eye-of-the-tiger" sign. The aim of the present study was to describe clinical, MRI and molecular findings in a 26-year-old male with atypical PKAN disease in whom, brain MRI scans showed bilateral pallidal T2-hypointensity with a small central region of T2-hyperintensity, resembling the "eye-of-the-tiger" typical image. Genetic analysis identified two mutations in PANK2: c.1561G>A and c.1663G>A, being the latter never described before. Due to limited phenotype-genotype correlation among patients with movement disorders, if "eye-of-the-tiger" brain MRI is present, PANK2 mutations investigation are needed to confirm PKAN disease.

  9. The mammalian AMP-activated protein kinase complex mediates glucose regulation of gene expression in the yeast Saccharomyces cerevisiae.

    PubMed

    Ye, Tian; Bendrioua, Loubna; Carmena, David; García-Salcedo, Raúl; Dahl, Peter; Carling, David; Hohmann, Stefan

    2014-06-05

    The AMP-activated protein kinase (AMPK) controls energy homeostasis in eukaryotic cells. Here we expressed hetero-trimeric mammalian AMPK complexes in a Saccharomyces cerevisiae mutant lacking all five genes encoding yeast AMPK/SNF1 components. Certain mammalian complexes complemented the growth defect of the yeast mutant on non-fermentable carbon sources. Phosphorylation of the AMPK α1-subunit was glucose-regulated, albeit not by the Glc7-Reg1/2 phosphatase, which performs this function on yeast AMPK/SNF1. AMPK could take over SNF1 function in glucose derepression. While indirectly acting anti-diabetic drugs had no effect on AMPK in yeast, compound 991 stimulated α1-subunit phosphorylation. Our results demonstrate a remarkable functional conservation of AMPK and that glucose regulation of AMPK may not be mediated by regulatory features of a specific phosphatase.

  10. Mitogen-activated protein kinase kinase 5 (MKK5)-mediated signalling cascade regulates expression of iron superoxide dismutase gene in Arabidopsis under salinity stress.

    PubMed

    Xing, Yu; Chen, Wei-hua; Jia, Wensuo; Zhang, Jianhua

    2015-09-01

    Superoxide dismutases (SODs) are involved in plant adaptive responses to biotic and abiotic stresses but the upstream signalling process that modulates their expression is not clear. Expression of two iron SODs, FSD2 and FSD3, was significantly increased in Arabidopsis in response to NaCl treatment but blocked in transgenic MKK5-RNAi plant, mkk5. Using an assay system for transient expression in protoplasts, it was found that mitogen-activated protein kinase kinase 5 (MKK5) was also activated in response to salt stress. Overexpression of MKK5 in wild-type plants enhanced their tolerance to salt treatments, while mkk5 mutant exhibited hypersensitivity to salt stress in germination on salt-containing media. Moreover, another kinase, MPK6, was also involved in the MKK5-mediated iron superoxide dismutase (FSD) signalling pathway in salt stress. The kinase activity of MPK6 was totally turned off in mkk5, whereas the activity of MPK3 was only partially blocked. MKK5 interacted with the MEKK1 protein that was also involved in the salt-induced FSD signalling pathway. These data suggest that salt-induced FSD2 and FSD3 expressions are influenced by MEKK1 via MKK5-MPK6-coupled signalling. This MAP kinase cascade (MEKK1, MKK5, and MPK6) mediates the salt-induced expression of iron superoxide dismutases.

  11. The muscle creatine kinase gene is regulated by multiple upstream elements, including a muscle-specific enhancer

    SciTech Connect

    Jaynes, J.B.; Johnson, J.E.; Buskin, J.N.; Gartside, C.L.; Hauschka, S.D.

    1988-01-01

    Muscle creatine kinase (MCK) is induced to high levels during skeletal muscle differentiation. The authors examined the upstream regulatory elements of the mouse MCK gene which specify its activation during myogenesis in culture. Fusion genes containing up to 3,300 nucleotides (nt) of MCK 5' flanking DNA in various positions and orientations relative to the bacterial chloramphenicol acetyltransferase (CAT) structural gene were transfected into cultured cells. Transient expression of CAT was compared between proliferating and differentiated MM14 mouse myoblasts and with nonmyogenic mouse L cells. The major effector of high-level expression was found to have the properties of a transcriptional enhancer. This element, located between 1,050 and 1,256 nt upstream of the transcription start site, was also found to have a major influence on the tissue and differentiation specificity of MCK expression; it activated either the MCK promoter or heterologous promoters only in differentiated muscle cells. Comparisons of viral and cellular enhancer sequences with the MCK enhancer revealed some similarities to essential regions of the simian virus 40 enhancer as well as to a region of the immunoglobulin heavy-chain enhancer, which has been implicated in tissue-specific protein binding. Even in the absence of the enhancer, low-level expression from a 776-nt MCK promoter retained differentiation specificity. In addition to positive regulatory elements, our data provide some evidence for negative regulatory elements with activity in myoblasts. These may contribute to the cell type and differentiation specificity of MCK expression.

  12. Expression of muscle-gene-specific isozymes of phosphorylase and creatine kinase in innervated cultured human muscle

    PubMed Central

    1986-01-01

    Isozymes of creatine kinase and glycogen phosphorylase are excellent markers of skeletal muscle maturation. In adult innervated muscle only the muscle-gene-specific isozymes are present, whereas aneurally cultured human muscle has predominantly the fetal pattern of isozymes. We have studied the isozyme pattern of human muscle cultured in monolayer and innervated by rat embryo spinal cord explants for 20-42 d. In this culture system, large groups of innervated muscle fibers close to the ventral part of the spinal cord explant continuously contracted. The contractions were reversibly blocked by 1 mM d- tubocurarine. In those innervated fibers, the total activity and the muscle-gene-specific isozymes of both enzymes increased significantly. The amount of muscle-gene-specific isozymes directly correlated with the duration of innervation. Control noninnervated muscle fibers from the same dishes as the innervated fibers remained biochemically immature. This study demonstrated that de novo innervation of human muscle cultured in monolayer exerts a time-related maturational influence that is not mediated by a diffusable neural factor. PMID:3771644

  13. Genetic Characterization of Plasmodium Putative Pantothenate Kinase Genes Reveals Their Essential Role in Malaria Parasite Transmission to the Mosquito.

    PubMed

    Hart, Robert J; Cornillot, Emmanuel; Abraham, Amanah; Molina, Emily; Nation, Catherine S; Ben Mamoun, Choukri; Aly, Ahmed S I

    2016-09-20

    The metabolic machinery for the biosynthesis of Coenzyme A (CoA) from exogenous pantothenic acid (Vitamin B5) has long been considered as an excellent target for the development of selective antimicrobials. Earlier studies in the human malaria parasite Plasmodium falciparum have shown that pantothenate analogs interfere with pantothenate phosphorylation and block asexual blood stage development. Although two eukaryotic-type putative pantothenate kinase genes (PanK1 and PanK2) have been identified in all malaria parasite species, their role in the development of Plasmodium life cycle stages remains unknown. Here we report on the genetic characterization of PanK1 and PanK2 in P. yoelii. We show that P. yoelii parasites lacking either PanK1 or PanK2 undergo normal asexual stages development and sexual stages differentiation, however they are severely deficient in ookinete, oocyst and sporozoite formation inside the mosquito vector. Quantitative transcriptional analyses in wild-type and knockout parasites demonstrate an important role for these genes in the regulation of expression of other CoA biosynthesis genes. Together, our data provide the first genetic evidence for the importance of the early steps of pantothenate utilization in the regulation of CoA biosynthesis and malaria parasite transmission to Anopheles mosquitoes.

  14. Intron sequences of arginine kinase in an intertidal snail suggest an ecotype-specific selective sweep and a gene duplication

    PubMed Central

    Kemppainen, P; Lindskog, T; Butlin, R; Johannesson, K

    2011-01-01

    Many species with restricted gene flow repeatedly respond similarly to local selection pressures. To fully understand the genetic mechanisms behind this process, the phylogeographic history of the species (inferred from neutral markers) as well as the loci under selection need to be known. Here we sequenced an intron in the arginine kinase gene (Ark), which shows strong clinal variation between two locally adapted ecotypes of the flat periwinkle, Littorina fabalis. The ‘small-sheltered' ecotype was almost fixed for one haplotype, H1, in populations on both sides of the North Sea, unlike the ‘large-moderately exposed ecotype', which segregated for ten different haplotypes. This contrasts with neutral markers, where the two ecotypes are equally variable. H1 could have been driven to high frequency in an ancestral population and then repeatedly spread to sheltered habitats due to local selection pressures with the colonization of both sides of the North Sea, after the last glacial maximum (∼18 000 years ago). An alternative explanation is that a positively selected mutation, in or linked to Ark, arose after the range expansion and secondarily spread through sheltered populations throughout the distribution range, causing this ecotype to evolve in a concerted fashion. Also, we were able to sequence up to four haplotypes consistently from some individuals, suggesting a gene duplication in Ark. PMID:20877396

  15. Deletion of the thymidine kinase gene induces complete attenuation of the Georgia isolate of African swine fever virus.

    PubMed

    Sanford, B; Holinka, L G; O'Donnell, V; Krug, P W; Carlson, J; Alfano, M; Carrillo, C; Wu, Ping; Lowe, Andre; Risatti, G R; Gladue, D P; Borca, M V

    2016-02-02

    African swine fever virus (ASFV) is the etiological agent of a contagious and often lethal viral disease of domestic pigs. There are no vaccines to control Africa swine fever (ASF). Experimental vaccines have been developed using genetically modified live attenuated ASFVs obtained by specifically deleting virus genes involved in virulence, including the thymidine kinase (TK) gene. TK has been shown to be involved in the virulence of several viruses, including ASFV. Here we report the construction of a recombinant virus (ASFV-G/V-ΔTK) obtained by deleting the TK gene in a virulent strain of ASFV Georgia adapted to replicate in Vero cells (ASFV-G/VP30). ASFV-G/P-ΔTK demonstrated decreased replication both in primary swine macrophage cell cultures and in Vero cells compared with ASFV-G/VP30. In vivo, intramuscular administration of up to 10(6) TCID50 of ASFV-G/V-ΔTK does not result in ASF disease. However, these animals are not protected when challenged with the virulent parental Georgia strain.

  16. Genetic Characterization of Plasmodium Putative Pantothenate Kinase Genes Reveals Their Essential Role in Malaria Parasite Transmission to the Mosquito

    PubMed Central

    Hart, Robert J.; Cornillot, Emmanuel; Abraham, Amanah; Molina, Emily; Nation, Catherine S.; Ben Mamoun, Choukri; Aly, Ahmed S. I.

    2016-01-01

    The metabolic machinery for the biosynthesis of Coenzyme A (CoA) from exogenous pantothenic acid (Vitamin B5) has long been considered as an excellent target for the development of selective antimicrobials. Earlier studies in the human malaria parasite Plasmodium falciparum have shown that pantothenate analogs interfere with pantothenate phosphorylation and block asexual blood stage development. Although two eukaryotic-type putative pantothenate kinase genes (PanK1 and PanK2) have been identified in all malaria parasite species, their role in the development of Plasmodium life cycle stages remains unknown. Here we report on the genetic characterization of PanK1 and PanK2 in P. yoelii. We show that P. yoelii parasites lacking either PanK1 or PanK2 undergo normal asexual stages development and sexual stages differentiation, however they are severely deficient in ookinete, oocyst and sporozoite formation inside the mosquito vector. Quantitative transcriptional analyses in wild-type and knockout parasites demonstrate an important role for these genes in the regulation of expression of other CoA biosynthesis genes. Together, our data provide the first genetic evidence for the importance of the early steps of pantothenate utilization in the regulation of CoA biosynthesis and malaria parasite transmission to Anopheles mosquitoes. PMID:27644319

  17. Nucleotide sequence of the Rhodobacter capsulatus fruK gene, which encodes fructose-1-phosphate kinase: evidence for a kinase superfamily including both phosphofructokinases of Escherichia coli.

    PubMed Central

    Wu, L F; Reizer, A; Reizer, J; Cai, B; Tomich, J M; Saier, M H

    1991-01-01

    The fruK gene encoding fructose-1-phosphate kinase (FruK), located within the fructose (fru)-catabolic operon of Rhodobacter capsulatus, was sequenced. FruK of R. capsulatus (316 amino acids; molecular weight = 31,232) is the same size as and is homologous to FruK of Escherichia coli, phosphofructokinase B (PfkB) of E. coli, phosphotagatokinase of Staphylococcus aureus, and ribokinase of E. coli. These proteins therefore make up a family of homologous proteins, termed the PfkB family. A phylogenetic tree for this new family was constructed. Sequence comparisons plus chemical inactivation studies suggested the lack of involvement of specific residues in catalysis. Although the Rhodobacter FruK differed markedly from the other enzymes within the PfkB family with respect to amino acid composition, these enzymes exhibited similar predicted secondary structural features. A large internal segment of the Rhodobacter FruK was found to be similar in sequence to the domain bearing the sugar bisphosphate-binding region of the large subunit of ribulose 1,5-bisphosphate carboxylase/oxygenase of plants and bacteria. Proteins of the PfkB family did not exhibit statistically significant sequence identity with PfkA of E. coli. PfkA, however, is homologous to other prokaryotic and eukaryotic ATP- and PPi-dependent Pfks (the PfkA family). These eukaryotic, ATP-dependent enzymes each consist of a homotetramer (mammalian) or a heterooctamer (yeasts), with each subunit containing an internal duplication of the size of the entire PfkA protein of E. coli. In some of these enzymes, additional domains are present. A phylogenetic tree was constructed for the PfkA family and revealed that the bacterial enzymes closely resemble the N-terminal domains of the eukaryotic enzyme subunits whereas the C-terminal domains have diverged more extensively. The PPi-dependent Pfk of potato is only distantly related to the ATP-dependent enzymes. On the basis of their similar functions, sizes, predicted

  18. RNA interference suppression of the receptor tyrosine kinase Torso gene impaired pupation and adult emergence in Leptinotarsa decemlineata.

    PubMed

    Zhu, Tao-Tao; Meng, Qing-Wei; Guo, Wen-Chao; Li, Guo-Qing

    2015-12-01

    In Drosophila melanogaster prothoracic gland (PG) cells, Torso mediates prothoracicotropic hormone (PTTH)-triggered mitogen activated protein kinase (MAPK) pathway (consisting of four core components Ras, Raf, MEK and ERK) to stimulate ecdysteroidogenesis. In this study, LdTorso, LdRas, LdRaf and LdERK were cloned in Leptinotarsa decemlineata. The four genes were highly or moderately expressed in the larval prothoracic glands. At the first- to third-instar stages, their expression levels were higher just before and right after the molt, and were lower in the mid instars. At the fourth-instar stage, their transcript levels were higher before prepupal stage. RNA interference-mediated knockdown of LdTorso delayed larval development, increased pupal weight, and impaired pupation and adult emergence. Moreover, knockdown of LdTorso decreased the mRNA levels of LdRas, LdRaf and LdERK, repressed the transcription of two ecdysteroidogenesis genes (LdPHM and LdDIB), lowered 20E titer, and downregulated the expression of several 20E-response genes (LdEcR, LdUSP, LdHR3 and LdFTZ-F1). Furthermore, silencing of LdTorso induced the expression of a JH biosynthesis gene LdJHAMT, increased JH titer, and activated the transcription of a JH early-inducible gene LdKr-h1. Thus, our results suggest that Torso transduces PTTH-triggered MAPK signal to regulate ecdysteroidogenesis in the PGs in a non-drosophiline insect.

  19. Decreased in vivo virulence and altered gene expression by a Brucella melitensis light-sensing histidine kinase mutant.

    PubMed

    Gourley, Christopher R; Petersen, Erik; Harms, Jerome; Splitter, Gary

    2015-03-01

    Brucella species utilize diverse virulence factors. Previously, Brucella abortus light-sensing histidine kinase was identified as important for cellular infection. Here, we demonstrate that a Brucella melitensis LOV-HK (BM-LOV-HK) mutant strain has strikingly different gene expression than wild type. General stress response genes including the alternative sigma factor rpoE1 and its anti-anti-sigma factor phyR were downregulated, while flagellar, quorum sensing (QS), and type IV secretion system genes were upregulated in the ΔBM-LOV-HK strain vs. wild type. Contextually, expression results agree with other studies of transcriptional regulators involving ΔrpoE1, ΔphyR, ΔvjbR, and ΔblxR (ΔbabR) Brucella strains. Additionally, deletion of BM-LOV-HK decreases virulence in mice. During C57BL/6 mouse infection, the ΔBM-LOV-HK strain had 2 logs less CFUs in the spleen 3 days postinfection, but similar levels 6 days post infection compared to wild type. Infection of IRF-1(-/-) mice more specifically define ΔBM-LOV-HK strain attenuation with fewer bacteria in spleens and significantly increased survival of mutant vs. wild-type infected IRF-1(-/-) mice. Upregulation of flagella, QS, and VirB genes, along with downregulation of rpoE1 and related sigma factor, rpoH2 (BMEI0280) suggest that BM-LOV-HK modulates both QS and general stress response regulatory components to control Brucella gene expression on a global level.

  20. Contribution of retinoblastoma LOH and the p53 Arg/Pro polymorphism to cervical cancer.

    PubMed

    Eltahir, Huda A; Elhassan, Ahmed M; Ibrahim, Muntaser E

    2012-09-01

    Epidemiological studies indicate that infections by certain types of human papillomaviruses (HPVs) are causally linked to the development of cervical cancer. It is also known that HPV infections alone do not cause progression to cervical cancer, as additional genetic changes such as loss of distinct chromosomal regions, inactivation of tumor-suppressor genes and activation of oncogenes must also occur in order for malignant transformation to take place. In the present study, 78 patients diagnosed with cervical cancer and 36 cervical cancer-free cases (control) were analyzed for high-risk HPV genotypes (16 and 18) by polymerase chain reaction (PCR). Loss of heterozygosity (LOH) of the retinoblastoma gene (Rb) at two polymorphic intronic sites (intron 1 and 17) and the p53 polymorphism in codon 72 were detected by RFLP and allele-specific PCR, respectively. HPV 16 and 18 were found at frequencies of 93.6 and 8.3% in the cervical cancer and control samples, respectively. LOH was detected in 63% of patients in intron 1 and/or intron 17. p53 allele frequency for Arg/Arg was 43.6% (34/78), for Arg/Pro 37.2% (29/78) and for Pro/Pro 19.2% (15/78). The relative risk (RR) of LOH and Arg/Arg alone was 1.7 and 1.1, respectively, while the combined RR for Rb LOH and p53 Arg/Arg was 2.5. The present study showed a significant association of the chromosomal allelic loss of Rb in Sudanese cervical cancer patients, while no such association was observed with other parameters, such as clinical stage and degree of differentiation; hence, it cannot be a determinant of tumor behavior in cervical carcinoma. Although the p53 arginine allele is itself an important risk factor for cervical cancer, the combined risk with LOH of Rb, which appears to be greater, might indicate a possible epistatic effect of the two genes/polymorphisms.

  1. Minimum requirements for inhibition of smooth-muscle myosin light-chain kinase by synthetic peptides.

    PubMed Central

    Hunt, J T; Floyd, D M; Lee, V G; Little, D K; Moreland, S

    1989-01-01

    Although the amino acid residues that are important for peptide substrates of myosin light-chain kinase have been reported, those that are important for peptide inhibitors of this enzyme have not previously been investigated. Synthetic peptides based on the sequence Lys11-Lys12-Arg13-Ala-Ala-Arg16-Ala-Thr-Ser19 -Asn-Val21-Phe22-Ala of the chicken gizzard myosin light chain were tested as inhibitors of pig carotid-artery myosin light-chain kinase. The basic amino acid residues of the known myosin light-chain kinase inhibitor Lys-Lys-Arg-Ala-Ala-Arg-Ala-Thr-Ser-NH2 (IC50 = 14 microM) [Pearson, Misconi & Kemp (1986) J. Biol. Chem. 261, 25-27] were shown to be the important residues that contribute to inhibitor potency, as evidence by the finding that the hexapeptide Lys-Lys-Arg-Ala-Ala-Arg-NH2 had an IC50 value of 22 microM. This indicates that binding of the phosphorylatable serine residue to myosin light-chain kinase, which is of obvious importance for a substrate, does not enhance the potency of an inhibitor. With the aim of preparing more potent inhibitors, peptides Lys-Lys-Arg-Ala-Ala-Arg-Ala-Ala-Xaa-NH2 were prepared with a variety of amino acids substituted for the phosphorylatable serine residue. None of these peptides was a more potent inhibitor than the serine peptide. PMID:2920029

  2. Regulation of argA operon expression in Escherichia coli K-12: cell-free synthesis of beta-galactosidase under argA control.

    PubMed Central

    Kelker, N; Eckhardt, T

    1977-01-01

    Regulation of argA operon expression in Escherichia coli K-12 was studied in a cell-free, deoxyribonucleic acid-dependent, enzyme-synthesizing system. lambdaAZ-7 deoxyribonucleic acid, which carries a fusion of the lacZ structural gene to the argA operon so that beta-galactosidase synthesis is under argA regulation, was used as the template. To eliminate extraneous readthrough from lambda promoters, lambda repressor was introduced into the synthesis mixtures by preparing the S-30 component from a strain (514X5a-12-29) that carries a multicopy hybrid plasmid (pKB252) containing the lambdacI gene. Under these conditions beta-galactosidase synthesis was repressed 90% by the arginine repressor when a sufficient concentration of L-arginine was present. This repression could be overcome by escape synthesis when the lambdaAZ-7 deoxyribonucleic acid concentration in the synthesis mixtures was increased. Guanosine 3'-diphosphate-5'-diphosphate stimulated beta-galactosidase synthesis from this template. PMID:410786

  3. MAP kinase pathway gene copy alterations in NRAS/BRAF wild-type advanced melanoma.

    PubMed

    Orouji, Elias; Orouji, Azadeh; Gaiser, Timo; Larribère, Lionel; Gebhardt, Christoffer; Utikal, Jochen

    2016-05-01

    Recent therapeutic advances have improved melanoma patientś clinical outcome. Novel therapeutics targeting BRAF, NRAS and cKit mutant melanomas are widely used in clinical practice. However therapeutic options in NRAS(wild-type) /BRAF(wild-type) /cKit(wild-type) melanoma patients are limited. Our study shows that gene copy numbers of members of the MAPK signaling pathway vary in different melanoma subgroups. NRAS(wild-type) /BRAF(wild-type) melanoma metastases are characterized by significant gains of MAP2K1 (MEK1) and MAPK3 (ERK1) gene loci. These additional gene copies could lead to an activation of the MAPK signaling pathway via a gene-dosage effect. Our results suggest that downstream analyses of the pMEK and pERK expression status in NRAS(wild-type) /BRAF(wild-type) melanoma patients identify patients that could benefit from targeted therapies with MEK and ERK inhibitors.

  4. BRO1, a novel gene that interacts with components of the Pkc1p-mitogen-activated protein kinase pathway in Saccharomyces cerevisiae.

    PubMed Central

    Nickas, M E; Yaffe, M P

    1996-01-01

    Yeast cells with mutations in BRO1 display phenotypes similar to those caused by deletion of BCK1, a gene encoding a MEK kinase that functions in a mitogen-activated protein kinase pathway mediating maintenance of cell integrity. bro1 cells exhibit a temperature-sensitive growth defect that is suppressed by the addition of osmotic stabilizers or Ca2+ to the growth medium or by additional copies of the BCK1 gene. At permissive temperatures, bro1 mutants are sensitive to caffeine and respond abnormally to nutrient limitation. A null mutation in BRO1 is synthetically lethal with null mutations in BCK1, MPK1, which encodes a mitogen-activated protein kinase that functions downstream of Bck1p, or PKC1, a gene encoding a protein kinase C homolog that activates Bck1p. Analysis of the isolated BRO1 gene revealed that it encodes a novel, 97-kDa polypeptide which contains a putative SH3 domain-binding motif and is homologous to a protein of unknown function in Caenorhabditis elegans. PMID:8649366

  5. Epigenetic Activation of μ-Opioid Receptor Gene via Increased Expression and Function of Mitogen- and Stress-Activated Protein Kinase 1.

    PubMed

    Wagley, Yadav; Law, Ping-Yee; Wei, Li-Na; Loh, Horace H

    2017-04-01

    Since the discovery of μ-opioid receptor (MOR) gene two decades ago, various regulatory factors have been shown to interact with the MOR promoter and modulate transcript levels. However, the majority of early transcriptional studies on MOR gene have not addressed how intracellular signaling pathways mediate extracellular modulators. In this study, we demonstrate that MOR epigenetic regulation requires multiple coordinated signals converging at the MOR promoter, involving mitogen-activated protein kinase (MAPK) activation and mitogen- and stress-activated protein kinase 1 (MSK1)-ranges of intracellular signaling pathways similar to those activated by opioid agonists. Inhibiting p38 MAPK or extracellular signal-regulated kinase (ERK) 1/2 MAPK (upstream activators of MSK1) reduced MOR expression levels; accordingly, the functional role of MSK1, but not MSK2, was demonstrated using genetic approaches. However, for maximal MSK1 effect, an open chromatin configuration was required, because in vitro CpG methylation of the MOR promoter abolished MSK1 activity. Finally, endogenous MSK1 levels concomitantly increased to regulate MOR gene expression during neuronal differentiation of P19 cells, suggesting a conserved role of this kinase in the epigenic activation of MOR in neurons. Taken together, our findings indicate that the expression of MOR gene requires the activity of intracellular signaling pathways that have been implicated in the behavioral outcomes of opioid drugs, which suggests that an autoregulatory mechanism may function in opioid systems.

  6. Involvement of protein kinases and calcium in the NO-signalling cascade for defence-gene induction in ozonated tobacco plants.

    PubMed

    Pasqualini, S; Reale, L; Calderini, O; Pagiotti, R; Ederli, L

    2012-07-01

    This study analyses the signalling pathways triggered by nitric oxide (NO) in response to ozone (O(3)) fumigation of tobacco plants, with particular attention to protein kinase cascades and free cytosolic Ca(2+) in defence-gene activation. NO was visualized with the NO probe DAF-FM. Using a pharmacological approach, the effects of different inhibitors on the expression profiles of NO-dependent defence genes were monitored using RT-PCR. The assay of the kinase activity of the immunoprecipitates complexes shows that O(3) stimulates a 48 kDa salicylic acid (SA)-induced protein kinase (SIPK) in an NO-dependent manner. The O(3)-induced alternative oxidase 1a (AOX1a) and phenylalanine ammonia lyase a (PALa) genes are modulated by phosphorylation by protein kinases, and SIPK might have a role in this up-regulation. By contrast, protein dephosphorylation mediates pathogenesis-related protein 1a (PR1a) expression in O(3)-treated tobacco plants. Ca(2+) is essential, but not sufficient, to promote NO accumulation in ozonated tobacco plants. Intracellular Ca(2+) transients are also essential for PALa up-regulation and cGMP-induced PR1a expression. Partial dependence on intracellular Ca(2+) suggests two different pathways of SA accumulation and PR1a induction. A model summarizing the signalling networks involving NO, SA, and the cellular messengers in this O(3)-induced defence gene activation is proposed.

  7. Partial hypogonadotropic hypogonadism associated with the Leu266Arg and Gln106Arg mutation of the gonadotropin-releasing hormone receptor.

    PubMed

    Quintos, J B; Krotz, Stephan; Vogiatzi, Maria G; Kralickova, Milena; New, Maria I

    2009-02-01

    We describe a patient with partial hypogonadotropic hypogonadism caused by a compound heterozygous GnRH-R mutation. She is a 20-year-old tall, eunuchoid female referred for evaluation of primary amenorrhea. Spontaneous thelarche occurred at the age of 15 years. Breast and pubic hair were at Tanner stages 3 and 4, respectively. Evaluation revealed low plasma estradiol level and absence of withdrawal bleeding after progestin challenge. Pelvic ultrasonography showed a small uterus and ovaries. Bone age was delayed at 14.5 years. Bone mineral density showed osteopenia. Endogenous LH secretory pattern was abnormal with low amplitude and frequency, but responded to pulsatile GnRH administration. The coding exons of the GnRH-R gene were amplified and the PCR products were sequenced bidirectionally. Two different mutations were identified: one in exon 1 (Gln106Arg) and the other in exon 3 (Leu266Arg).

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

    PubMed

    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.

  9. Crystallization and preliminary X-ray diffraction analysis of the arginine repressor ArgR from Bacillus halodurans

    PubMed Central

    Kang, Jina; Park, Young Woo; Yeo, Hyun Ku; Lee, Jae Young

    2015-01-01

    The arginine repressor (ArgR) is a transcriptional regulator which regulates genes encoding proteins involved in arginine biosynthesis and the arginine catabolic pathway. ArgR from the alkaliphilic bacterium Bacillus halodurans was cloned and overexpressed in Escherichia coli. ArgR (Bh2777) from B. halodurans is composed of 149 amino-acid residues with a molecular mass of 16 836 Da. ArgR was crystallized at 296 K using 1,2-propanediol as a precipitant. Crystals of N-terminally His-tagged ArgR were obtained by the sitting-drop vapour-diffusion method. Dehydrated crystals showed a dramatic improvement in diffraction quality and diffracted to 2.35 Å resolution. The crystals belonged to the cubic space group I23, with unit-cell parameters a = b = c = 104.68 Å. The asymmetric unit contained one monomer of ArgR, which generates a trimer by the threefold axis of the space group, giving a crystal volume per mass (V M) of 2.98 Å3 Da−1 and a solvent content of 56.8%. PMID:25760703

  10. A differentially regulated AP2/ERF transcription factor gene cluster acts downstream of a MAP kinase cascade to modulate terpenoid indole alkaloid biosynthesis in Catharanthus roseus.

    PubMed

    Paul, Priyanka; Singh, Sanjay K; Patra, Barunava; Sui, Xueyi; Pattanaik, Sitakanta; Yuan, Ling

    2017-02-01

    Catharanthus roseus produces bioactive terpenoid indole alkaloids (TIAs), including the chemotherapeutics, vincristine and vinblastine. Transcriptional regulation of TIA biosynthesis is not fully understood. The jasmonic acid (JA)-responsive AP2/ERF transcription factor (TF), ORCA3, and its regulator, CrMYC2, play key roles in TIA biosynthesis. ORCA3 forms a physical cluster with two uncharacterized AP2/ERFs, ORCA4 and 5. Here, we report that (1) the ORCA gene cluster is differentially regulated; (2) ORCA4, while overlapping functionally with ORCA3, modulates an additional set of TIA genes. Unlike ORCA3, ORCA4 overexpression resulted in dramatic increase of TIA accumulation in C. roseus hairy roots. In addition, CrMYC2 is capable of activating ORCA3 and co-regulating TIA pathway genes concomitantly with ORCA3. The ORCA gene cluster and CrMYC2 act downstream of a MAP kinase cascade that includes a previously uncharacterized MAP kinase kinase, CrMAPKK1. Overexpression of CrMAPKK1 in C. roseus hairy roots upregulated TIA pathways genes and increased TIA accumulation. This work provides detailed characterization of a TF gene cluster and advances our understanding of the transcriptional and post-translational regulatory mechanisms that govern TIA biosynthesis in C. roseus.

  11. Effect of the selective pressure of sub-lethal level of heavy metals on the fate and distribution of ARGs in the catchment scale.

    PubMed

    Xu, Yan; Xu, Jian; Mao, Daqing; Luo, Yi

    2017-01-01

    Our previous study demonstrated that high levels of antibiotic resistance genes (ARGs) in the Haihe River were directly attributed to the excessive use of antibiotics in animal agriculture. The antibiotic residues of the Xiangjiang River determined in this study were much lower than those of the Haihe River, but the relative abundance of 16 detected ARGs (sul1, sul2 and sul3, qepA, qnrA, qnrB, qnrD and qnrS, tetA, tetB, tetW, tetM, tetQ and tetO, ermB and ermC), were as high as the Haihe River particularly in the downstream of the Xiangjiang River which is close to the extensive metal mining. The ARGs discharged from the pharmaceutical wastewater treatment plant (PWWTP) are a major source of ARGs in the upstream of the Xiangjiang River. In the downstream, selective stress of heavy metals rather than source release had a significant influence on the distinct distribution pattern of ARGs. Some heavy metals showed a positive correlation with certain ARG subtypes. Additionally, there is a positive correlation between individual ARG subtypes and heavy metal resistance genes, suggesting that heavy metals may co select the ARGs on the same plasmid of antibiotic resistant bacteria. The co-selection mechanism between specific metal and antibiotic resistance was further confirmed by these isolations encoding the resistance genotypes to antibiotics and metals. To our knowledge, this is the first study on the fate and distribution of ARGs under the selective pressure exerted by heavy metals in the catchment scale. These results are beneficial to understand the fate, and to discern the contributors of ARGs from either the source release or the selective pressure by sub-lethal levels of environmental stressors during their transport on a river catchment scale.

  12. Identification of calcium-dependent protein kinase (CDPK): A multi-functional gene family in Rafflesia cantleyi

    NASA Astrophysics Data System (ADS)

    Amini, Safoora; Goh, Hoe-Han; Wan, Kiew-Lian

    2016-11-01

    Rafflesia, a parasitic plant that belongs to the Rafflesiaceae family, is notable for producing the largest flowers in the world. This study focused on identification of Calcium-dependent protein kinases (CDPKs) due to their vital roles in plant growth and development, biotic and abiotic stress responses, and hormone signaling. RNA-seq data generated from three bud stages of Rafflesia cantleyi ie BS1, BS2, and BS3 and were assembled. Based on the BLAST searches of Rafflesia unique transcripts (UTs) to Arabidopsis TAIR database, a total of 14 unique transcripts (UTs) were identified as CDPK1 to CDPK5, CDPK7 to CDPK11, CDPK16, CDPK18, CDPK19, and CDPK28. These genes are expressed at all three bud stages of R. cantleyi with up-regulation pattern at BS1 vs. BS2 and BS2 vs. BS3. This result shows that the expression of CDPK gene family increases by developmental progress in Rafflesia in order to regulate biochemical and molecular changes at the cellular level in response to exposure to environmental changes. However, CDPKs functions in plants growth and defense process still need more experimental evidence to deeply understand their biological roles in R. cantleyi.

  13. The MRE4 gene encodes a novel protein kinase homologue required for meiotic recombination in Saccharomyces cerevisiae.

    PubMed Central

    Leem, S H; Ogawa, H

    1992-01-01

    The MRE4 gene was cloned by complementation of the defects of meiotic recombination and haploidization in an mre4-1 mutant. Disruption of MRE4 resulted in reduced meiotic recombination and spore inviability. The mre4 spore lethality can be suppressed by spo13, a mutation that causes cells to bypass the reductional division. Analysis of meiotic DNA extracted from the mre4 mutant cells revealed that double-strand breaks occurred at the two sites of the HIS4-LEU2 recombination hot spot, but at a frequency of about 10-20% of the wild type. Northern blot analysis indicated that the MRE4 gene produces four transcripts of 1.63, 3.2, 4.0 and 6.2 kb. All of these transcripts are absent from mitotic cells and are meiotically induced. The DNA sequence of the MRE4 open reading frame predicts a 497-amino acids protein with a molecular mass of 56.8 kDa. The Mre4 protein contains highly conserved amino acid sequences found specifically in serine-threonine protein kinases. These results suggest that protein phosphorylation is required directly or indirectly for meiotic recombination. Images PMID:1741279

  14. The FvMK1 mitogen-activated protein kinase gene regulates conidiation, pathogenesis, and fumonisin production in Fusarium verticillioides.

    PubMed

    Zhang, Yueping; Choi, Yoon-E; Zou, Xuexiao; Xu, Jin-Rong

    2011-02-01

    Fusarium verticillioides is one of the most important fungal pathogens to cause destructive diseases of maize worldwide. Fumonisins produced by the fungus are harmful to human and animal health. To date, our understanding of the molecular mechanisms associated with pathogenicity and fumonisin biosynthesis in F. verticillioides is limited. Because MAP kinase pathways have been implicated in regulating diverse processes important for plant infection in phytopathogenic fungi, in this study we identified and functionally characterized the FvMK1 gene in F. verticillioides. FvMK1 is orthologous to FMK1 in F. oxysporum and GPMK1 in F. graminearum. The Fvmk1 deletion mutant was reduced in vegetative growth and production of microconidia. However, it was normal in sexual reproduction and increased in the production of macroconidia. In infection assays with developing corn kernels, the Fvmk1 mutant was non-pathogenic and failed to colonize through wounding sites. It also failed to cause stalk rot symptoms beyond the inoculation sites on corn stalks, indicating that FvMK1 is essential for plant infection. Furthermore, the Fvmk1 mutant was significantly reduced in fumonisin production and expression levels of FUM1 and FUM8, two genes involved in fumonisin biosynthesis. The defects of the Fvmk1 mutant were fully complemented by re-introducing the wild type FvMK1 allele. These results demonstrate that FvMK1 plays critical roles in the regulation of vegetative growth, asexual reproduction, fumonisin biosynthesis, and pathogenicity.

  15. xopAC-triggered Immunity against Xanthomonas Depends on Arabidopsis Receptor-Like Cytoplasmic Kinase Genes PBL2 and RIPK

    PubMed Central

    Guy, Endrick; Lautier, Martine; Chabannes, Matthieu; Roux, Brice; Lauber, Emmanuelle; Arlat, Matthieu; Noël, Laurent D.

    2013-01-01

    Xanthomonas campestris pv. campestris (Xcc) colonizes the vascular system of Brassicaceae and ultimately causes black rot. In susceptible Arabidopsis plants, XopAC type III effector inhibits by uridylylation positive regulators of the PAMP-triggered immunity such as the receptor-like cytoplasmic kinases (RLCK) BIK1 and PBL1. In the resistant ecotype Col-0, xopAC is a major avirulence gene of Xcc. In this study, we show that both the RLCK interaction domain and the uridylyl transferase domain of XopAC are required for avirulence. Furthermore, xopAC can also confer avirulence to both the vascular pathogen Ralstonia solanacearum and the mesophyll-colonizing pathogen Pseudomonas syringae indicating that xopAC-specified effector-triggered immunity is not specific to the vascular system. In planta, XopAC-YFP fusions are localized at the plasma membrane suggesting that XopAC might interact with membrane-localized proteins. Eight RLCK of subfamily VII predicted to be localized at the plasma membrane and interacting with XopAC in yeast two-hybrid assays have been isolated. Within this subfamily, PBL2 and RIPK RLCK genes but not BIK1 are important for xopAC-specified effector-triggered immunity and Arabidopsis resistance to Xcc. PMID:23951354

  16. Thymidine kinase/ganciclovir and cytosine deaminase/5-fluorocytosine suicide gene therapy-induced cell apoptosis in breast cancer cells.

    PubMed

    Kong, H; Tao, L; Qi, K; Wang, Y; Li, Q; Du, J; Huang, Z

    2013-09-01

    The present study was conducted to explore the efficacy of suicide gene therapy with thymidine kinase (TK) in combination with cytosine deaminase (CD) for breast cancer. The expression of CD/TK was detected in the infected cells by RT-PCR. The killing effect on MCF-7 cells following treatment was analyzed by MTT assay. The morphological characteristics of the cells were observed by electron microscopy, and the distribution of the cell cycle was analyzed by flow cytometry. Caspase‑3 and -8 activities were detected by absorption spectrometry. Cytotoxic assays showed that cells transfected with CD/TK became more sensitive to the prodrugs. Morphological features characteristic of apoptosis were noted in the MCF‑7 cells via electron microscopy. The experimental data showed that the proportion of MCF-7 cells during the different phases of the cell cycle varied significantly following treatment with the prodrugs. The activity of caspase‑3 gradually increased following treatment with increasing concentrations of the prodrugs. We conclude that the TK/ganciclovir and CD/5-fluorocytosine suicide gene system used here induces apoptosis in breast cancer cells, and provides a promising treatment modality for breast cancer.

  17. P-TEFb Kinase Complex Phosphorylates Histone H1 to Regulate Expression of Cellular and HIV-1 Genes*

    PubMed Central

    O'Brien, Siobhan K.; Cao, Hong; Nathans, Robin; Ali, Akbar; Rana, Tariq M.

    2010-01-01

    Transcription of HIV-1 genes depends on the RNA polymerase II kinase and elongation factor positive transcription elongation factor b (P-TEFb), the complex of cyclin T1 and CDK9. Recent evidence suggests that regulation of transcription by P-TEFb involves chromatin binding and modifying factors. To determine how P-TEFb may connect chromatin remodeling to transcription, we investigated the relationship between P-TEFb and histone H1. We identify histone H1 as a substrate for P-TEFb involved in cellular and HIV-1 transcription. We show that P-TEFb interacts with H1 and that P-TEFb inhibition by RNAi, flavopiridol, or dominant negative CDK9 expression correlates with loss of phosphorylation and mobility of H1 in vivo. Importantly, P-TEFb directs H1 phosphorylation in response to wild-type HIV-1 infection, but not Tat-mutant HIV-1 infection. Our results show that P-TEFb phosphorylates histone H1 at a specific C-terminal phosphorylation site. Expression of a mutant H1.1 that cannot be phosphorylated by P-TEFb also disrupts Tat transactivation in an HIV reporter cell line as well as transcription of the c-fos and hsp70 genes in HeLa cells. We identify histone H1 as a novel P-TEFb substrate, and our results suggest new roles for P-TEFb in both cellular and HIV-1 transcription. PMID:20551309

  18. A specific requirement of Arc/Arg3.1 for visual experience-induced homeostatic synaptic plasticity in mouse primary visual cortex.

    PubMed

    Gao, Ming; Sossa, Kenneth; Song, Lihua; Errington, Lauren; Cummings, Laurel; Hwang, Hongik; Kuhl, Dietmar; Worley, Paul; Lee, Hey-Kyoung

    2010-05-26

    Visual experience scales down excitatory synapses in the superficial layers of visual cortex in a process that provides an in vivo paradigm of homeostatic synaptic scaling. Experience-induced increases in neural activity rapidly upregulates mRNAs of immediate early genes involved in synaptic plasticity, one of which is Arc (activity-regulated cytoskeleton protein or Arg3.1). Cell biological studies indicate that Arc/Arg3.1 protein functions to recruit endocytic machinery for AMPA receptor internalization, and this action, together with its activity-dependent expression, rationalizes a role for Arc/Arg3.1 in homeostatic synaptic scaling. Here, we investigated the role of Arc/Arg3.1 in homeostatic scaling in vivo by examining experience-dependent development of layer 2/3 neurons in the visual cortex of Arc/Arg3.1 knock-out (KO) mice. Arc/Arg3.1 KOs show minimal changes in basal and developmental regulation of excitatory synaptic strengths but display a profound deficit in homeostatic regulation of excitatory synapses by visual experience. As additional evidence of specificity, we found that the visual experience-induced regulation of inhibitory synapses is normal, although the basal inhibitory synaptic strength is increased in the Arc/Arg3.1 KOs. Our results demonstrate that Arc/Arg3.1 plays a selective role in regulating visual experience-dependent homeostatic plasticity of excitatory synaptic transmission in vivo.

  19. Detection of anaplastic lymphoma kinase (ALK) gene rearrangement in non-small cell lung cancer and related issues in ALK inhibitor therapy: a literature review.

    PubMed

    Yi, Eunhee S; Chung, Jin-Haeng; Kulig, Kimary; Kerr, Keith M

    2012-06-01

    Anaplastic lymphoma kinase (ALK) encodes a receptor tyrosine kinase, and ALK gene rearrangement (ALK+) is implicated in the oncogenesis of non-small cell lung carcinomas (NSCLCs), especially adenocarcinomas. The ALK inhibitor crizotinib was approved in August 2011 by the US Food and Drug Administration (FDA) for treating late-stage NSCLCs that are ALK+, with a companion fluorescent in situ hybridization (FISH) test using the Vysis ALK Break Apart FISH Probe Kit. This review covers pertinent issues in ALK testing, including approaches to select target patients for the test, pros and cons of different detection methods, and mechanisms as well as monitoring of acquired crizotinib resistance in ALK+ NSCLCs.

  20. Activation of ATP binding for the autophosphorylation of DosS, a Mycobacterium tuberculosis histidine kinase lacking an ATP lid motif.

    PubMed

    Cho, Ha Yeon; Lee, Young-Hoon; Bae, Young-Seuk; Kim, Eungbin; Kang, Beom Sik

    2013-05-03

    The sensor histidine kinases of Mycobacterium tuberculosis, DosS and DosT, are responsible for sensing hypoxic conditions and consist of sensor and kinase cores responsible for accepting signals and phosphorylation activity, respectively. The kinase core contains a dimerization and histidine phosphate-accepting (DHp) domain and an ATP binding domain (ABD). The 13 histidine kinase genes of M. tuberculosis can be grouped based on the presence or absence of the ATP lid motif and F box (elements known to play roles in ATP binding) in their ABDs; DosS and DosT have ABDs lacking both these elements, and the crystal structures of their ABDs indicated that they were unsuitable for ATP binding, as a short loop covers the putative ATP binding site. Although the ABD alone cannot bind ATP, the kinase core is functional in autophosphorylation. Appropriate spatial arrangement of the ABD and DHp domain within the kinase core is required for both autophosphorylation and ATP binding. An ionic interaction between Arg(440) in the DHp domain and Glu(537) in the short loop of the ABD is available and may open the ATP binding site, by repositioning the short loop away from the site. Mutations at Arg(440) and Glu(537) reduce autophosphorylation activity. Unlike other histidine kinases containing an ATP lid, which protects bound ATP, DosS is unable to accept ATP until the ABD is properly positioned relative to the histidine; this may prevent unexpected ATP reactions. ATP binding can, therefore, function as a control mechanism for histidine kinase activity.

  1. Deletion of the gene Pip4k2c, a novel phosphatidylinositol kinase, results in hyperactivation of the immune system

    PubMed Central

    Shim, Hyeseok; Wu, Chuan; Ramsamooj, Shivan; Bosch, Kaitlyn N.; Chen, Zuojia; Emerling, Brooke M.; Yun, Jihye; Liu, Hui; Choo-Wing, Rayman; Yang, Zhiwei; Wulf, Gerburg M.; Kuchroo, Vijay Kumar; Cantley, Lewis C.

    2016-01-01

    Type 2 phosphatidylinositol-5-phosphate 4-kinase (PI5P4K) converts phosphatidylinositol-5-phosphate to phosphatidylinositol-4,5-bisphosphate. Mammals have three enzymes PI5P4Kα, PI5P4Kβ, and PI5P4Kγ, and these enzymes have been implicated in metabolic control, growth control, and a variety of stress responses. Here, we show that mice with germline deletion of type 2 phosphatidylinositol-5-phosphate 4-kinase gamma (Pip4k2c), the gene encoding PI5P4Kγ, appear normal in regard to growth and viability but have increased inflammation and T-cell activation as they age. Immune cell infiltrates increased in Pip4k2c−/− mouse tissues. Also, there was an increase in proinflammatory cytokines, including IFNγ, interleukin 12, and interleukin 2 in plasma of Pip4k2c−/− mice. Pip4k2c−/− mice had an increase in T-helper-cell populations and a decrease in regulatory T-cell populations with increased proliferation of T cells. Interestingly, mammalian target of rapamycin complex 1 (mTORC1) signaling was hyperactivated in several tissues from Pip4k2c−/− mice and treating Pip4k2c−/− mice with rapamycin reduced the inflammatory phenotype, resulting in a decrease in mTORC1 signaling in tissues and a decrease in proinflammatory cytokines in plasma. These results indicate that PI5P4Kγ plays a role in the regulation of the immune system via mTORC1 signaling. PMID:27313209

  2. MAPKAP kinase-2; a novel protein kinase activated by mitogen-activated protein kinase.

    PubMed Central

    Stokoe, D; Campbell, D G; Nakielny, S; Hidaka, H; Leevers, S J; Marshall, C; Cohen, P

    1992-01-01

    A novel protein kinase, which was only active when phosphorylated by the mitogen-activated protein kinase (MAP kinase), has been purified 85,000-fold to homogeneity from rabbit skeletal muscle. This MAP kinase activated protein kinase, termed MAPKAP kinase-2, was distinguished from S6 kinase-II (MAPKAP kinase-1) by its response to inhibitors, lack of phosphorylation of S6 peptides and amino acid sequence. MAPKAP kinase-2 phosphorylated glycogen synthase at Ser7 and the equivalent serine (*) in the peptide KKPLNRTLS*VASLPGLamide whose sequence is similar to the N terminus of glycogen synthase. MAPKAP kinase-2 was resolved into two monomeric species of apparent molecular mass 60 and 53 kDa that had similar specific activities and substrate specificities. Peptide sequences of the 60 and 53 kDa species were identical, indicating that they are either closely related isoforms or derived from the same gene. MAP kinase activated the 60 and 53 kDa forms of MAPKAP kinase-2 by phosphorylating the first threonine residue in the sequence VPQTPLHTSR. Furthermore, Mono Q chromatography of extracts from rat phaeochromocytoma and skeletal muscle demonstrated that two MAP kinase isoforms (p42mapk and p44mapk) were the only enzymes in these cells that were capable of reactivating MAPKAP kinase-2. These results indicate that MAP kinase activates at least two distinct protein kinases, suggesting that it represents a point at which the growth factor-stimulated protein kinase cascade bifurcates. Images PMID:1327754

  3. Molecular analysis of the SNF4 gene of Saccharomyces cerevisiae: evidence for physical association of the SNF4 protein with the SNF1 protein kinase.

    PubMed Central

    Celenza, J L; Eng, F J; Carlson, M

    1989-01-01

    The SNF4 gene is required for expression of glucose-repressible genes in response to glucose deprivation in Saccharomyces cerevisiae. Previous evidence suggested that SNF4 is functionally related to SNF1, another essential gene in this global regulatory system that encodes a protein kinase. Increased SNF1 gene dosage partially compensates for a mutation in SNF4, and the SNF4 function is required for maximal SNF1 protein kinase activity in vitro. We have cloned SNF4 and identified its 1.2-kilobase RNA, which is not regulated by glucose repression. A 36-kilodalton SNF4 protein is predicted from the nucleotide sequence. Disruption of the chromosomal SNF4 locus revealed that the requirement for SNF4 function is less stringent at low temperature (23 degrees C). A bifunctional SNF4-lacZ gene fusion that includes almost the entire SNF4 coding sequence was constructed. The fusion protein was shown by immunofluorescence microscopy to be distributed throughout the cell, with partial localization to the nucleus. The SNF4-beta-galactosidase protein coimmunoprecipitated with the SNF1 protein kinase, thus providing evidence for the physical association of the two proteins. Images PMID:2481228

  4. cAMP and cAMP-dependent protein kinase regulate the human heat shock protein 70 gene promoter activity.

    PubMed

    Choi, H S; Li, B; Lin, Z; Huang, E; Liu, A Y

    1991-06-25

    The theme of this study is an evaluation of the involvement of cAMP and cAMP-dependent protein kinase (PKA) in the regulation of the human heat shock protein (hsp) 70 gene promoter. Expression of a highly specific protein inhibitor of PKA (pRSVPKI) inhibited the basal as well as heat- and cadmium-induced expression of the cotransfected pHBCAT, a human hsp 70 promoter-driven reporter gene; this inhibition was dependent on the amount of pRSVPKI used. The effect of an expression vector of the RI regulatory subunit of PKA, pMTREV, was similar to that of pRSVPKI; pMTREV inhibited both the basal as well as the heat-induced expression of pHBCAT. The specificity of effects of these expression vectors was demonstrated by the lack of effect of a mutant PKI gene and by the unaffected expression of a reference gene (pRSV beta gal) under these conditions. Analysis of the effects of dibutyryl cAMP (1 mM), forskolin (10 microM), and 8-Br-cAMP (1 mM) on the transient expression of pHBCAT showed that these cAMP-elevating agents stimulated the hsp 70 promoter activity, whereas cAMP (1 mM) was without effect. Chloramphenicol acetyltransferase gene constructs with truncated or mutated hsp 70 promoter were used to define the cis-acting DNA element(s) that confer this cAMP stimulation; the heat induced (42 degrees C) expression was used as a control. Mutation of the adenovirus transcription factor element (pLSN-40/-26) greatly reduced the basal level of expression; forskolin had little or no effect on this adenovirus transcription factor-minus promoter, although the promoter activity was very heat inducible. The absence of a functional heat shock consensus element (HSE) in the construct pLSPNWT rendered the promoter heat insensitive; this construct was forskolin responsive although the magnitude of this stimulation was reduced when compared with that of a control construct with HSE. These results were corroborated by studies using consensus sequence of ATF (ATFE) and HSE as competitors

  5. Tyrosine kinase inhibitors for epidermal growth factor receptor gene mutation-positive non-small cell lung cancers: an update for recent advances in therapeutics.

    PubMed

    Chung, Clement

    2016-06-01

    The presence of activating gene mutations in the epidermal growth factor receptor of non-small cell lung cancer patients is predictive (improved progression-free survival and improved response rate) when treated with small molecule tyrosine kinase inhibitors such as gefitinib, erlotinib and afatinib. The two most common mutations that account for greater than 85% of all EGFR gene mutations are in-frame deletions in exon 19 (LREA deletions) and substitution in exon 21 (L858R). Exon 18 mutations occur much less frequently at about 4% of all EGFR gene mutations. Together, exon 19 deletion and exon 21 L858R gene substitution are present in about 10% of Caucasian patients and 20-40% of Asian patients with non-small cell lung cancer. T790M gene mutation at exon 20 is associated with acquired resistance to epidermal growth factor receptor tyrosine kinase inhibitors. Early studies showed that activating EGFR gene mutations are most common in patients with adenocarcinoma histology, women, never smokers and those of Asian ethnicity. A recent multi-center phase III trial suggested that frontline epidermal growth factor receptor tyrosine kinase inhibitor therapy with afatinib is associated with improved progression-free survival compared to chemotherapy regardless of race. Moreover, guidelines now suggest EGFR gene mutation testing should be conducted in all patients with lung adenocarcinoma or mixed lung cancers with an adenocarcinoma component, regardless of characteristics such as smoking status, gender or race. The success of targeted therapies in non-small cell lung cancer patients has changed the treatment paradigm in metastatic non-small cell lung cancer. However, despite a durable response of greater than a year, resistance to epidermal growth factor receptor tyrosine kinase inhibitors inevitably occurs. This mini-review describes the clinically relevant EGFR gene mutations and the efficacy/toxicity of small molecule epidermal growth factor receptor tyrosine kinase

  6. Characterization and mapping of the human rhodopsin kinase gene and screening of the gene for mutations in patients with retinitis pigmentosa

    SciTech Connect

    Khani, S.C.; Lin, D.; Magovcevic, I.

    1994-09-01

    Rhodopsin kinase (RK) is a cytosolic enzyme in rod photoreceptors that initiates the deactivation of the phototransductions cascade by phosphorylating photoactivated rhodopsin. Although the cDNA sequence of bovine RK has been determined previously, no human cDNA or genomic sequence has thus far been available for genetic studies. In order to investigate the possible role of this candidate gene in retinitis pigmentosa (RP) and allied diseases, we have isolated and characterized human cDNA and genomic clones derived from the RK locus. The coding sequence of the human gene is 1692 nucleotides in length and is split into seven exons. The human and the bovine sequence show 84% identity at the nucleotide level and 92% identity at the amino acid level. Thus far, the intronic sequences flanking each exon except for one have been determined. We have also mapped the human RK gene to chromosome 13q34 using fluorescence in situ hybridization. To our knowledge, no RP gene has as yet been linked to this region. However, since the substrate for RK (rhodopsin) and other members of the phototransduction cascade have been implicated in the pathogenesis of RP, it is conceivable that defects in RK can also cause some forms of this disease. We are evaluating this possibility by screening DNA from 173 patients with autosomal recessive RP and 190 patients with autosomal dominant RP. So far, we have found 11 patients with variant bands. In one patient with autosomal dominant RP we discovered the missense change Ser536Leu. Cosegregation studies and further sequencing of the variant bands are currently underway.

  7. Serine/threonine kinase gene Stpk-V, a key member of powdery mildew resistance gene Pm21, confers powdery mildew resistance in wheat.

    PubMed

    Cao, Aizhong; Xing, Liping; Wang, Xiaoyun; Yang, Xueming; Wang, Wei; Sun, Yulei; Qian, Chen; Ni, Jinlong; Chen, Yaping; Liu, Dajun; Wang, Xiue; Chen, Peidu

    2011-05-10

    Powdery mildew resistance gene Pm21, located on the chromosome 6V short arm of Haynaldia villosa and transferred to wheat as a 6VS·6AL translocation (T6VS·6AL), confers durable and broad-spectrum resistance to wheat powdery mildew. Pm21 has become a key gene resource for powdery mildew resistance breeding all over the world. In China, 12 wheat varieties containing Pm21 have been planted on more than 3.4 million hectares since 2002. Pm21 has been intractable to molecular genetic mapping because the 6VS does not pair and recombine with the 6AS. Moreover, all known accessions of H. villosa are immune to powdery mildew fungus. Pm21 is still defined by cytogenetics as a locus. In the present study, a putative serine and threonine protein kinase gene Stpk-V was cloned and characterized with an integrative strategy of molecular and cytogenetic techniques. Stpk-V is located on the Pm21 locus. The results of a single cell transient expression assay showed that Stpk-V could decrease the haustorium index dramatically. After the Stpk-V was transformed into a susceptible wheat variety Yangmai158, the characterized transgenic plants showed high and broad-spectrum powdery mildew resistance similar to T6VS·6AL. Silencing of the Stpk-V by virus-induced gene silencing in both T6VS·6AL and H. villosa resulted in their increased susceptibility. Stpk-V could be induced by Bgt and exogenous H(2)O(2), but it also mediated the increase of endogenous H(2)O(2), leading to cell death and plant resistance when the plant was attacked by Bgt.

  8. Identification of upstream and intragenic regulatory elements that confer cell-type-restricted and differentiation-specific expression on the muscle creatine kinase gene

    SciTech Connect

    Sternberg, E.A.; Spizz, G.; Perry, W.M.; Vizard, D.; Weil, T.; Olson, E.N.

    1988-07-01

    Terminal differentiation of skeletal myobalsts is accompanied by induction of a series of tissue-specific gene products, which includes the muscle isoenzymte of creatine kinase (MCK). To begin to define the sequences and signals involved in MCK regulation in developing muscle cells, the mouse MCK gene has been isolated. Sequence analysis of 4,147 bases of DNA surrounding the transcription initiation site revealed several interesting structural features, some of which are common to other muscle-specific genes and to cellular and viral enhancers.

  9. Functional consequences of a gene duplication and fusion event in an arginine kinase.

    PubMed

    Compaan, Deanne M; Ellington, W Ross

    2003-05-01

    Arginine kinase (AK) from the foot of the razor clam Ensis directus consists of two full-length AK domains, denoted D1 and D2, fused in a single polypeptide chain. The full-length cDNA for Ensis AK was obtained and its deduced amino acid sequence was analyzed in the context of the X-ray crystal structure of a typical, monomeric AK. Both domains of Ensis AK contain most of the residues currently thought to be critical in catalysis, suggesting that both AK domains are catalytically competent. The full-length Ensis AK, a D2-NusA-His-tag fusion protein and a D2-truncated AK (enterokinase cleavage product of the fusion protein) were expressed in Escherichia coli and purified. All recombinant AK constructs displayed high enzyme activity. Attempts at expressing active D1 alone, D2 alone or a D1-NusA-His-tag fusion protein were unsuccessful. The catalytic properties of the active proteins were compared with the corresponding properties of recombinant AK from the horseshoe crab Limulus polyphemus, which is a typical monomeric AK. In contrast to expectations, the kinetic results strongly suggest that Ensis AK has only one active domain, namely D2. The K(cat) values for all Ensis constructs were roughly twice that of typical AKs, indicating higher overall catalytic throughput at the competent active site. Furthermore, both the full-length and truncated D2 Ensis AKs showed no synergism of substrate binding unlike typical AKs. The D2-NusA-His-tag fusion construct actually displayed negative synergism of substrate binding, which means that, in effect, the first substrate bound acts as a competitive inhibitor of the second. The conservation of the structure of the apparently inactive D1 may be related to constraints imposed by structural changes that could potentially impact substrate binding in D2 and/or possibly influence the proper folding of the enzyme during synthesis. Overall, the results from the present study indicate that the AK contiguous dimer from Ensis directus

  10. Elongation factor 2 and fragile X mental retardation protein control the dynamic translation of Arc/Arg3.1 essential for mGluR-LTD.

    PubMed

    Park, Sungjin; Park, Joo Min; Kim, Sangmok; Kim, Jin-Ah; Shepherd, Jason D; Smith-Hicks, Constance L; Chowdhury, Shoaib; Kaufmann, Walter; Kuhl, Dietmar; Ryazanov, Alexey G; Huganir, Richard L; Linden, David J; Worley, Paul F

    2008-07-10

    Group I metabotropic glutamate receptors (mGluR) induce long-term depression (LTD) that requires protein synthesis. Here, we demonstrate that Arc/Arg3.1 is translationally induced within 5 min of mGluR activation, and this response is essential for mGluR-dependent LTD. The increase in Arc/Arg3.1 translation requires eEF2K, a Ca(2+)/calmodulin-dependent kinase that binds mGluR and dissociates upon mGluR activation, whereupon it phosphorylates eEF2. Phospho-eEF2 acts to slow the elongation step of translation and inhibits general protein synthesis but simultaneously increases Arc/Arg3.1 translation. Genetic deletion of eEF2K results in a selective deficit of rapid mGluR-dependent Arc/Arg3.1 translation and mGluR-LTD. This rapid translational mechanism is disrupted in the fragile X disease mouse (Fmr1 KO) in which mGluR-LTD does not require de novo protein synthesis but does require Arc/Arg3.1. We propose a model in which eEF2K-eEF2 and FMRP coordinately control the dynamic translation of Arc/Arg3.1 mRNA in dendrites that is critical for synapse-specific LTD.

  11. NcoI RFLP at the creatine kinase-muscle type gene locus (CKMM, chromosome 19)

    SciTech Connect

    Coerwinkel-Driessen, M.; Schepens, J.; van Zandvoort, P.; van Oost, B.; Mariman, E.; Wieringa, B. )

    1988-09-12

    A 3.2 kbp human genomic DNA fragment (BamHI-Sau3A) of the 3{prime} untranslated and 3{prime} flanking region of the CKMM gene was isolated and subcloned into the BamHI site of vector pSP64. The CKMM 3{prime}-probe identifies a 2-allele polymorphism with bands at 2.3 and 1.0 kbp (allele A) and 3.3 kbp (allele B). In addition a weak constant 4.2 kbp band is observed. This probe also detects a 2-allele TaqI RFLP reported previously, as either a 4.3 kbp (A) or a 4.2 kbp (B) band. The CKMM locus previously has been assigned to 19q13.2-q13.3. By Southern blot analysis of human-rodent somatic cell hybrids containing unique subregional fragments of chromosome 19 of man the authors have assigned the gene to 19q13.2. Co-dominant segregation was observed in 8 families with 3 generations.

  12. Generation of a High Number of Healthy Erythroid Cells from Gene-Edited Pyruvate Kinase Deficiency Patient-Specific Induced Pluripotent Stem Cells

    PubMed Central

    Garate, Zita; Quintana-Bustamante, Oscar; Crane, Ana M.; Olivier, Emmanuel; Poirot, Laurent; Galetto, Roman; Kosinski, Penelope; Hill, Collin; Kung, Charles; Agirre, Xabi; Orman, Israel; Cerrato, Laura; Alberquilla, Omaira; Rodriguez-Fornes, Fatima; Fusaki, Noemi; Garcia-Sanchez, Felix; Maia, Tabita M.; Ribeiro, Maria L.; Sevilla, Julian; Prosper, Felipe; Jin, Shengfang; Mountford, Joanne; Guenechea, Guillermo; Gouble, Agnes; Bueren, Juan A.; Davis, Brian R.; Segovia, Jose C.

    2015-01-01

    Summary Pyruvate kinase deficiency (PKD) is a rare erythroid metabolic disease caused by mutations in the PKLR gene. Erythrocytes from PKD patients show an energetic imbalance causing chronic non-spherocytic hemolytic anemia, as pyruvate kinase defects impair ATP production in erythrocytes. We generated PKD induced pluripotent stem cells (PKDiPSCs) from peripheral blood mononuclear cells (PB-MNCs) of PKD patients by non-integrative Sendai viral vectors. PKDiPSCs were gene edited to integrate a partial codon-optimized R-type pyruvate kinase cDNA in the second intron of the PKLR gene by TALEN-mediated homologous recombination (HR). Notably, we found allele specificity of HR led by the presence of a single-nucleotide polymorphism. High numbers of erythroid cells derived from gene-edited PKDiPSCs showed correction of the energetic imbalance, providing an approach to correct metabolic erythroid diseases and demonstrating the practicality of this approach to generate the large cell numbers required for comprehensive biochemical and metabolic erythroid analyses. PMID:26549847

  13. Chromosome mapping of the human arrestin (SAG), {beta}-arrestin 2 (ARRB2), and {beta}-adrenergic receptor kinase 2 (ADRBK2) genes

    SciTech Connect

    Calabrese, G.; Sallese, M.; Stornaiuolo, A.

    1994-09-01

    Two types of proteins play a major role in determining homologous desensitization of G-coupled receptors: {beta}-adrenergic receptor kinase ({beta}ARK), which phosphorylates the agonist-occupied receptor and its functional cofactor, {beta}-arrestin. Both {beta}ARK and {beta}-arrestin are members of multigene families. The family of G-protein-coupled receptor kinases includes rhodopsin kinase, {beta}ARK1, {beta}ARK2, IT11-A (GRK4), GRK5, and GRK6. The arrestin/{beta}-arrestin gene family includes arrestin (also known as S-antigen), {beta}-arrestin 1, and {beta}-arrestin 2. Here we report the chromosome mapping of the human genes for arrestin (SAG), {beta}arrestin 2 (ARRB2), and {beta}ARK2 (ADRBK2) by fluorescence in situ hybridization (FISH). FISH results confirmed the assignment of the gene coding for arrestin (SAG) to chromosome 2 and allowed us to refine its localization to band q37. The gene coding for {beta}-arrestin 2 (ARRB2) was mapped to chromosome 17p13 and that coding for {beta}ARK2 (ADRBK2) to chromosome 22q11. 17 refs., 1 fig.

  14. Association of p53 codon72 Arg>Pro polymorphism with susceptibility to nasopharyngeal carcinoma: evidence from a case-control study and meta-analysis.

    PubMed

    Sahu, S K; Chakrabarti, S; Roy, S D; Baishya, N; Reddy, R R; Suklabaidya, S; Kumar, A; Mohanty, S; Maji, S; Suryanwanshi, A; Rajasubramaniam, S; Asthana, M; Panda, A K; Singh, S P; Ganguly, S; Shaw, O P; Bichhwalia, A K; Sahoo, P K; Chattopadhyay, N R; Chatterjee, K; Kundu, C N; Das, A K; Kannan, R; Zorenpuii; Zomawia, E; Sema, S A; Singh, Y I; Ghosh, S K; Sharma, K; Das, B S; Choudhuri, T

    2016-05-09

    Tumor suppressor p53 is a critical player in the fight against cancer as it controls the cell cycle check point, apoptotic pathways and genomic stability. It is known to be the most frequently mutated gene in a wide variety of human cancers. Single-nucleotide polymorphism of p53 at codon72 leading to substitution of proline (Pro) in place of arginine (Arg) has been identified as a risk factor for development of many cancers, including nasopharyngeal carcinoma (NPC). However, the association of this polymorphism with NPC across the published literature has shown conflicting results. We aimed to conduct a case-control study for a possible relation of p53 codon72 Arg>Pro polymorphism with NPC risk in underdeveloped states of India, combine the result with previously available records from different databases and perform a meta-analysis to draw a more definitive conclusion. A total of 70 NPC patients and 70 healthy controls were enrolled from different hospitals of north-eastern India. The p53 codon72 Arg>Pro polymorphism was typed by polymerase chain reaction, which showed an association with NPC risk. In the meta-analysis consisting of 1842 cases and 2330 controls, it was found that individuals carrying the Pro allele and the ProPro genotype were at a significantly higher risk for NPC as compared with those with the Arg allele and the ArgArg genotype, respectively. Individuals with a ProPro genotype and a combined Pro genotype (ProPro+ArgPro) also showed a significantly higher risk for NPC over a wild homozygote ArgArg genotype. Additionally, the strength of each study was tested by power analysis and genotype distribution by Hardy-Weinberg equilibrium. The outcome of the study indicated that both allele frequency and genotype distribution of p53 codon72 Arg>Pro polymorphism were significantly associated with NPC risk. Stratified analyses based on ethnicity and source of samples supported the above result.

  15. The Pto kinase conferring resistance to tomato bacterial speck disease interacts with proteins that bind a cis-element of pathogenesis-related genes.

    PubMed Central

    Zhou, J; Tang, X; Martin, G B

    1997-01-01

    In tomato, the Pto kinase confers resistance to bacterial speck disease by recognizing the expression of a corresponding avirulence gene, avrPto, in the pathogen Pseudomonas syringae pv. tomato. Using the yeast two-hybrid system, we have identified three genes, Pti4, Pti5 and Pti6, that encode proteins that physically interact with the Pto kinase. Pti4/5/6 each encode a protein with characteristics that are typical of transcription factors and are similar to the tobacco ethylene-responsive element-binding proteins (EREBPs). Using a gel mobility-shift assay, we demonstrate that, similarly to EREBPs, Pti4/5/6 specifically recognize and bind to a DNA sequence that is present in the promoter region of a large number of genes encoding 'pathogenesis-related' (PR) proteins. Expression of several PR genes and a tobacco EREBP gene is specifically enhanced upon Pto-avrPto recognition in tobacco. These observations establish a direct connection between a disease resistance gene and the specific activation of plant defense genes. PMID:9214637

  16. Protein Kinases and Addiction

    PubMed Central

    Lee, Anna M.; Messing, Robert O.

    2011-01-01

    Although drugs of abuse have different chemical structures and interact with different protein targets, all appear to usurp common neuronal systems that regulate reward and motivation. Addiction is a complex disease that is thought to involve drug-induced changes in synaptic plasticity due to alterations in cell signaling, gene transcription, and protein synthesis. Recent evidence suggests that drugs of abuse interact with and change a common network of signaling pathways that include a subset of specific protein kinases. The best studied of these kinases are reviewed here and include extracellular signal-regulated kinase, cAMP-dependent protein kinase, cyclin-dependent protein kinase 5, protein kinase C, calcium/calmodulin-dependent protein kinase II, and Fyn tyrosine kinase. These kinases have been implicated in various aspects of drug addiction including acute drug effects, drug self-administration, withdrawal, reinforcement, sensitization, and tolerance. Identifying protein kinase substrates and signaling pathways that contribute to the addicted state may provide novel approaches for new pharma-cotherapies to treat drug addiction. PMID:18991950

  17. [Effect of the Gly972Arg, SNP43 and Prol2Ala polymorphisms of the genes IRS1, CAPN10 and PPARG2 on secondary failure to sulphonylurea and metformin in patients with type 2 diabetes in Yucatán, México].

    PubMed

    García-Escalante, María Guadalupe; Suárez-Solís, Víctor Manuel; López-Avila, María Teresa de Jesús; Pinto-Escalante, Doris del Carmen; Laviada-Molina, Hugo

    2009-03-01

    In Yucatán, 52% of patients with type 2 diabetes (DT2) present secondary failure to treatment associated with sulphonylurea and metformin. A possible explanation may be due to polymorphisms in the genes IRS1, CAPN10, PPARG2, which are involved in pancreatic beta cell dysfunction and a poor response to the action of insulin. The association of the polymorphisms Gly972Arg, SNP43, and Pro12Ala, of the genes IRS1, CAPN10, PPARG2, with the risk of failure to sulphonylurea and metformin therapies was determinated in patients with DT2 in Yucatán, México. One hundred and thirty and two subjects with DT2 were classified in groups of responders (HbA1c < 8%) and non-responders (HbA1c > 8%) to the treatment, according to the control of hyperglucemia with sulphonylurea and metformin. Demographic, anthropometric and metabolic data were obtained from each subject. The polymorphisms were identified by means of DNA analysis by PCR/RFLP and PCR/OAL. Genotypic and allelic frequencies and the Hardy-Weinberg equilibrium were determined. Statistical analyses consisted of X2 and multiple logistic regression tests (Epi-Info 2000 and SPSS version 12). Obese subjects carrying the genotype AA SNP43 showed 4.69 times more risk of failure to respond to treatment (p = 0.027), when compared with subjects sharing GA genotype: X2 (OR = 4.69, IC: 1.15-20.59) and multiple logistic regression, p = 0.048, (OR = 3.72, IC: 1.009-13.718). The interaction between genotype AA and the BMI > 27 showed also a significant difference (p = 0.009). The findings suggest the fact that polymorphism SNP43 may influence the response to treatment with sulphonylurea and metformin, the expression being dependent on obesity.

  18. Lack of neighborhood effects from a transcriptionally active phosphoglycerate kinase-neo cassette located between the murine beta-major and beta-minor globin genes.

    PubMed

    Kaufman, R M; Lu, Z H; Behl, R; Holt, J M; Ackers, G K; Ley, T J

    2001-07-01

    For the treatment of beta-globin gene defects, a homologous recombination-mediated gene correction approach would provide advantages over random integration-based gene therapy strategies. However, "neighborhood effects" from retained selectable marker genes in the targeted locus are among the key issues that must be taken into consideration for any attempt to use this strategy for gene correction. An Ala-to-Ile mutation was created in the beta6 position of the mouse beta-major globin gene (beta(6I)) as a step toward the development of a murine model system that could serve as a platform for therapeutic gene correction studies. The marked beta-major gene can be tracked at the level of DNA, RNA, and protein, allowing investigation of the impact of a retained phosphoglycerate kinase (PGK)-neo cassette located between the mutant beta-major and beta-minor globin genes on expression of these 2 neighboring genes. Although the PGK-neo cassette was expressed at high levels in adult erythroid cells, the abundance of the beta(6I) mRNA was indistinguishable from that of the wild-type counterpart in bone marrow cells. Similarly, the output from the beta-minor globin gene was also normal. Therefore, in this specific location, the retained, transcriptionally active PGK-neo cassette does not disrupt the regulated expression of the adult beta-globin genes. (Blood. 2001;98:65-73)

  19. ERECTA-family receptor kinase genes redundantly prevent premature progression of secondary growth in the Arabidopsis hypocotyl.

    PubMed

    Ikematsu, Shuka; Tasaka, Masao; Torii, Keiko U; Uchida, Naoyuki

    2017-03-01

    Secondary growth is driven by continuous cell proliferation and differentiation of the cambium that acts as vascular stem cells, producing xylem and phloem to expand vascular tissues laterally. During secondary growth of hypocotyls in Arabidopsis thaliana, the xylem undergoes a drastic phase transition from a parenchyma-producing phase to a fiber-producing phase at the appropriate time. However, it remains to be fully elucidated how progression of secondary growth is properly controlled. We focused on phenotypes of hypocotyl vasculatures caused by double mutation in ERECTA (ER) and ER-LIKE1 (ERL1) receptor-kinase genes to elucidate their roles in secondary growth. ER and ERL1 redundantly suppressed excessive radial growth of the hypocotyl vasculature during secondary growth. ER and ERL1 also prevented premature initiation of the fiber differentiation process mediated by the NAC SECONDARY WALL THICKENING PROMOTING FACTORs in the hypocotyl xylem. Upon floral transition, the hypocotyl xylem gained a competency to respond to GA in a BREVIPEDICELLUS-dependent manner, which was a prerequisite for fiber differentiation. However, even after the floral transition, ER and ERL1 prevented precocious initiation of the GA-mediated fiber formation. Collectively, our findings reveal that ER and ERL1 redundantly prevent premature progression of sequential events in secondary growth.

  20. Variation in the checkpoint kinase 2 gene is associated with type 2 diabetes in multiple populations

    PubMed Central

    Franceschini, Nora; Avery, Christy L.; Baird, Lisa; Graff, Mariaelisa; Leppert, Mark; Chung, Jay H.; Zhang, Jinghui; Hanis, Craig; Boerwinkle, Eric; Volcik, Kelly A.; Grove, Megan L.; Mosley, Thomas H.; Gu, Charles; Heiss, Gerardo; Pankow, James S.; Couper, David J.; Ballantyne, Christie M.; Linda Kao, W. H.; Weder, Alan B.; Cooper, Richard S.; Ehret, Georg B.; O'Connor, Ashley A.; Chakravarti, Aravinda; Hunt, Steven C.

    2010-01-01

    Identification and characterization of the genetic variants underlying type 2 diabetes susceptibility can provide important understanding of the etiology and pathogenesis of type 2 diabetes. We previously identified strong evidence of linkage for type 2 diabetes on chromosome 22 among 3,383 Hypertension Genetic Epidemiology Network (HyperGEN) participants from 1,124 families. The checkpoint 2 (CHEK2) gene, an important mediator of cellular responses to DNA damage, is located 0.22 Mb from this linkage peak. In this study, we tested the hypothesis that the CHEK2 gene contains one or more polymorphic variants that are associated with type 2 diabetes in HyperGEN individuals. In addition, we replicated our findings in two other Family Blood Pressure Program (FBPP) populations and in the population-based Atherosclerosis Risk in Communities (ARIC) study. We genotyped 1,584 African-American and 1,531 white HyperGEN participants, 1,843 African-American and 1,569 white GENOA participants, 871 African-American and 1,009 white GenNet participants, and 4,266 African-American and 11,478 white ARIC participants for four single nucleotide polymorphisms (SNPs) in CHEK2. Using additive models, we evaluated the association of CHEK2 SNPs with type 2 diabetes in participants within each study population stratified by race, and in a meta-analysis, adjusting for age, age2, sex, sex-by-age interaction, study center, and relatedness. One CHEK2 variant, rs4035540, was associated with an increased risk of type 2 diabetes in HyperGEN participants, two replication samples, and in the meta-analysis. These results may suggest a new pathway in the pathogenesis of type 2 diabetes that involves pancreatic beta-cell damage and apoptosis. PMID:19855918

  1. Investigation and analysis of single nucleotide polymorphisms in Janus kinase/signal transducer and activator of transcription genes with leukemia.

    PubMed

    Zhong, Yuejiao; Wu, Jianzhong; Chen, Baoan; Ma, Rong; Cao, Haixia; Wang, Zhuo; Cheng, Lu; Ding, Jiahua; Feng, Jifeng

    2012-06-01

    Aberrant activation of the Janus kinase/signal transducer and activator of transcription (JAK/STAT) pathway may predispose to leukemia due to deregulation of proliferation, differentiation or apoptosis. This study was conducted to investigate whether any association exists between genetic polymorphisms in the JAK2, STAT3 and STAT5 genes and individual susceptibility to leukemia. A case-control study was carried out using a Chinese sample set with 344 cases of leukemia and 346 controls matched by age and ethnicity. Genomic DNA was assayed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF) on 13 single nucleotide polymorphisms (SNPs). Genotype analyses showed that two SNPs, namely rs17886724 and rs2293157 located in STAT3 and STAT5, respectively, were significantly associated with leukemia (p < 0.05 for all). Interaction analyses of SNPs (rs17886724|rs2293157; rs11079041| rs2293157) showed that there were inferior associations in chronic lymphocytic leukemia (CLL) and acute myeloid leukemia (AML) compared to the control group (0.1 > p > 0.05). Linkage disequilibrium existed between rs11079041 and rs2293157 in both leukemia and control groups (r(2) = 0.7). The haplotypes displayed significant association between rs11079041 and rs2293157 in both leukemia and control groups (p < 0.05). The accuracy rate of the support vector machine (SVM) classification model in making a prediction of leukemia was 97%. The results indicated that STAT3 and STAT5 gene SNPs may be prognostic of leukemia.

  2. Conditional Mutagenesis of a Novel Choline Kinase Demonstrates Plasticity of Phosphatidylcholine Biogenesis and Gene Expression in Toxoplasma gondii*

    PubMed Central

    Sampels, Vera; Hartmann, Anne; Dietrich, Isabelle; Coppens, Isabelle; Sheiner, Lilach; Striepen, Boris; Herrmann, Andreas; Lucius, Richard; Gupta, Nishith

    2012-01-01

    The obligate intracellular and promiscuous protozoan parasite Toxoplasma gondii needs an extensive membrane biogenesis that must be satisfied irrespective of its host-cell milieu. We show that the synthesis of the major lipid in T. gondii, phosphatidylcholine (PtdCho), is initiated by a novel choline kinase (TgCK). Full-length (∼70-kDa) TgCK displayed a low affinity for choline (Km ∼0.77 mm) and harbors a unique N-terminal hydrophobic peptide that is required for the formation of enzyme oligomers in the parasite cytosol but not for activity. Conditional mutagenesis of the TgCK gene in T. gondii attenuated the protein level by ∼60%, which was abolished in the off state of the mutant (Δtgcki). Unexpectedly, the mutant was not impaired in its growth and exhibited a normal PtdCho biogenesis. The parasite compensated for the loss of full-length TgCK by two potential 53- and 44-kDa isoforms expressed through a cryptic promoter identified within exon 1. TgCK-Exon1 alone was sufficient in driving the expression of GFP in E. coli. The presence of a cryptic promoter correlated with the persistent enzyme activity, PtdCho synthesis, and susceptibility of T. gondii to a choline analog, dimethylethanolamine. Quite notably, the mutant displayed a regular growth in the off state despite a 35% decline in PtdCho content and lipid synthesis, suggesting a compositional flexibility in the membranes of the parasite. The observed plasticity of gene expression and membrane biogenesis can ensure a faithful replication and adaptation of T. gondii in disparate host or nutrient environments. PMID:22451671

  3. Fibrinopeptide A binds Gly-Pro-Arg-Pro.

    PubMed Central

    Root-Bernstein, R S; Westall, F C

    1984-01-01

    The tetrapeptide Gly-Pro-Arg-Pro inhibits fibrinogen aggregation, probably by binding to the same sites used during initiation of fibrin formation. The Gly-Pro-Arg-Pro binding sites have not yet been identified. However, their possible sequence and locations have been predicted on the basis of the amino acid pairing hypothesis. One of these predicted sites is on fibrinopeptide A. We report here that nuclear magnetic resonance studies indicate that Gly-Pro-Arg-Pro binds to fibrinopeptide A with a binding constant, K, of ca. 10(4) per mol. We also report results of 19 related peptide combinations used as controls. PMID:6589598

  4. Molecular characterisation of the STRUBBELIG-RECEPTOR FAMILY of genes encoding putative leucine-rich repeat receptor-like kinases in Arabidopsis thaliana

    PubMed Central

    Eyüboglu, Banu; Pfister, Karen; Haberer, Georg; Chevalier, David; Fuchs, Angelika; Mayer, Klaus FX; Schneitz, Kay

    2007-01-01

    Background Receptor-like kinases are a prominent class of surface receptors that regulate many aspects of the plant life cycle. Despite recent advances the function of most receptor-like kinases remains elusive. Therefore, it is paramount to investigate these receptors. The task is complicated by the fact that receptor-like kinases belong to a large monophyletic family with many sub-clades. In general, functional analysis of gene family members by reverse genetics is often obscured by several issues, such as redundancy, subtle or difficult to detect phenotypes in mutants, or by decision problems regarding suitable biological and biochemical assays. Therefore, in many cases additional strategies have to be employed to allow inference of hypotheses regarding gene function. Results We approached the function of genes encoding the nine-member STRUBBELIG-RECEPTOR FAMILY (SRF) class of putative leucine-rich repeat receptor-like kinases. Sequence comparisons show overall conservation but also divergence in predicted functional domains among SRF proteins. Interestingly, SRF1 undergoes differential splicing. As a result, SRF1 is predicted to exist in a standard receptor configuration and in a membrane-anchored receptor-like version that lacks most of the intracellular domain. Furthermore, SRF1 is characterised by a high degree of polymorphism between the Ler and Col accessions. Two independent T-DNA-based srf4 mutants showed smaller leaves while 35S::SRF4 plants displayed enlarged leaves. This is in addition to the strubbelig phenotype which has been described before. Additional single and several key double mutant combinations did not reveal obvious mutant phenotypes. Ectopic expression of several SRF genes, using the 35S promoter, resulted in male sterility. To gain possible insights into SRF gene function we employed a computational analysis of publicly available microarray data. We performed global expression profiling, coexpression analysis, and an analysis of the

  5. Modulation of the Chromatin Phosphoproteome by the Haspin Protein Kinase*

    PubMed Central

    Maiolica, Alessio; de Medina-Redondo, Maria; Schoof, Erwin M.; Chaikuad, Apirat; Villa, Fabrizio; Gatti, Marco; Jeganathan, Siva; Lou, Hua Jane; Novy, Karel; Hauri, Simon; Toprak, Umut H.; Herzog, Franz; Meraldi, Patrick; Penengo, Lorenza; Turk, Benjamin E.; Knapp, Stefan; Linding, Rune; Aebersold, Ruedi

    2014-01-01

    Recent discoveries have highlighted the importance of Haspin kinase activity for the correct positioning of the kinase Aurora B at the centromere. Haspin phosphorylates Thr3 of the histone H3 (H3), which provides a signal for Aurora B to localize to the centromere of mitotic chromosomes. To date, histone H3 is the only confirmed Haspin substrate. We used a combination of biochemical, pharmacological, and mass spectrometric approaches to study the consequences of Haspin inhibition in mitotic cells. We quantified 3964 phosphorylation sites on chromatin-associated proteins and identified a Haspin protein-protein interaction network. We determined the Haspin consensus motif and the co-crystal structure of the kinase with the histone H3 tail. The structure revealed a unique bent substrate binding mode positioning the histone H3 residues Arg2 and Lys4 adjacent to the Haspin phosphorylated threonine into acidic binding pockets. This unique conformation of the kinase-substrate complex explains the reported modulation of Haspin activity by methylation of Lys4 of the histone H3. In addition, the identification of the structural basis of substrate recognition and the amino acid sequence preferences of Haspin aided the identification of novel candidate Haspin substrates. In particular, we validated the phosphorylation of Ser137 of the histone variant macroH2A as a target of Haspin kinase activity. MacroH2A Ser137 resides in a basic stretch of about 40 amino acids that is required to stabilize extranucleosomal DNA, suggesting that phosphorylation of Ser137 might regulate the interactions of macroH2A and DNA. Overall, our data suggest that Haspin activity affects the phosphorylation state of proteins involved in gene expression regulation and splicing. PMID:24732914

  6. A soluble factor from Trypanosoma cruzi inhibits transforming growth factor-ß-induced MAP kinase activation and gene expression in dermal fibroblasts.

    PubMed

    Mott, G Adam; Costales, Jaime A; Burleigh, Barbara A

    2011-01-01

    The protozoan parasite Trypanosoma cruzi, which causes human Chagas' disease, exerts a variety of effects on host extracellular matrix (ECM) including proteolytic degradation of collagens and dampening of ECM gene expression. Exposure of primary human dermal fibroblasts to live infective T. cruzi trypomastigotes or their shed/secreted products results in a rapid down-regulation of the fibrogenic genes collagenIα1, fibronectin and connective tissue growth factor (CTGF/CCN2). Here we demonstrate the ability of a secreted/released T. cruzi factor to antagonize ctgf/ccn2 expression in dermal fibroblasts in response to TGF-ß, lysophosphatidic acid or serum, where agonist-induced phosphorylation of the mitogen-activated protein (MAP) kinases Erk1/2, p38 and JNK was also inhibited. Global analysis of gene expression in dermal fibroblasts identified a discrete subset of TGF-ß-inducible genes involved in cell proliferation, wound repair, and immune regulation that are inhibited by T. cruzi secreted/released factors, where the genes exhibiting the highest sensitivity to T. cruzi are known to be regulated by MAP kinase-activated transcription factors. Consistent with this observation, the Ets-family transcription factor binding site in the proximal promoter region of the ctgf/ccn2 gene (-91 bp to -84 bp) was shown to be required for T. cruzi-mediated down-regulation of ctgf/ccn2 reporter expression. The cumulative data suggest a model in which T. cruzi-derived molecules secreted/released early in the infective process dampen MAP kinase signaling and the activation of transcription factors that regulate expression of fibroblast genes involved in wound repair and tissue remodelling, including ctgf/ccn2. These findings have broader implications for local modulation of ECM synthesis/remodelling by T. cruzi during the early establishment of infection in the mammalian host and highlight the potential for pathogen-derived molecules to be exploited as tools to modulate the

  7. Draft Genome Sequence of Methylobacterium sp. Strain ARG-1 Isolated from the White-Rot Fungus Armillaria gallica

    PubMed Central

    Collins, Caitlin; Kowalski, Caitlin; Zebrowski, Jessica; Tulchinskaya, Yevgeniya; Tai, Albert K.; James-Pederson, Magdalena

    2016-01-01

    Methylobacterium sp. strain ARG-1 was isolated from a cell culture of hyphal tips of the white-rot fungus Armillaria gallica. We describe here the sequencing, assembly, and annotation of its genome, confirming the presence of genes involved in methylotrophy. This is the first genome announcement of a strain of Methylobacterium associated with A. gallica. PMID:27257212

  8. Structures of Arg- and Gln-type bacterial cysteine dioxygenase homologs: Arg- and Gln-type Bacterial CDO Homologs

    DOE PAGES

    Driggers, Camden M.; Hartman, Steven J.; Karplus, P. Andrew

    2015-01-01

    In some bacteria, cysteine is converted to cysteine sulfinic acid by cysteine dioxygenases (CDO) that are only ~15–30% identical in sequence to mammalian CDOs. Among bacterial proteins having this range of sequence similarity to mammalian CDO are some that conserve an active site Arg residue (“Arg-type” enzymes) and some having a Gln substituted for this Arg (“Gln-type” enzymes). Here, we describe a structure from each of these enzyme types by analyzing structures originally solved by structural genomics groups but not published: a Bacillus subtilis “Arg-type” enzyme that has cysteine dioxygenase activity (BsCDO), and a Ralstonia eutropha “Gln-type” CDO homolog ofmore » uncharacterized activity (ReCDOhom). The BsCDO active site is well conserved with mammalian CDO, and a cysteine complex captured in the active site confirms that the cysteine binding mode is also similar. The ReCDOhom structure reveals a new active site Arg residue that is hydrogen bonding to an iron-bound diatomic molecule we have interpreted as dioxygen. Notably, the Arg position is not compatible with the mode of Cys binding seen in both rat CDO and BsCDO. As sequence alignments show that this newly discovered active site Arg is well conserved among “Gln-type” CDO enzymes, we conclude that the “Gln-type” CDO homologs are not authentic CDOs but will have substrate specificity more similar to 3-mercaptopropionate dioxygenases.« less

  9. Structures of Arg- and Gln-type bacterial cysteine dioxygenase homologs: Arg- and Gln-type Bacterial CDO Homologs

    SciTech Connect

    Driggers, Camden M.; Hartman, Steven J.; Karplus, P. Andrew

    2015-01-01

    In some bacteria, cysteine is converted to cysteine sulfinic acid by cysteine dioxygenases (CDO) that are only ~15–30% identical in sequence to mammalian CDOs. Among bacterial proteins having this range of sequence similarity to mammalian CDO are some that conserve an active site Arg residue (“Arg-type” enzymes) and some having a Gln substituted for this Arg (“Gln-type” enzymes). Here, we describe a structure from each of these enzyme types by analyzing structures originally solved by structural genomics groups but not published: a Bacillus subtilis “Arg-type” enzyme that has cysteine dioxygenase activity (BsCDO), and a Ralstonia eutropha “Gln-type” CDO homolog of uncharacterized activity (ReCDOhom). The BsCDO active site is well conserved with mammalian CDO, and a cysteine complex captured in the active site confirms that the cysteine binding mode is also similar. The ReCDOhom structure reveals a new active site Arg residue that is hydrogen bonding to an iron-bound diatomic molecule we have interpreted as dioxygen. Notably, the Arg position is not compatible with the mode of Cys binding seen in both rat CDO and BsCDO. As sequence alignments show that this newly discovered active site Arg is well conserved among “Gln-type” CDO enzymes, we conclude that the “Gln-type” CDO homologs are not authentic CDOs but will have substrate specificity more similar to 3-mercaptopropionate dioxygenases.

  10. Structural analysis of APOB variants, p.(Arg3527Gln), p.(Arg1164Thr) and p.(Gln4494del), causing Familial Hypercholesterolaemia provides novel insights into variant pathogenicity.

    PubMed

    Fernández-Higuero, J A; Etxebarria, A; Benito-Vicente, A; Alves, A C; Arrondo, J L R; Ostolaza, H; Bourbon, M; Martin, C

    2015-12-08

    Familial hypercholesterolaemia (FH) is an inherited autosomal dominant disorder resulting from defects in the low-density lipoprotein receptor (LDLR), in the apolipoprotein B (APOB) or in the proprotein convertase subtilisin/kexin type 9 (PCSK9) genes. In the majority of the cases FH is caused by mutations occurring within LDLR, while only few mutations in APOB and PCSK9 have been proved to cause disease. p.(Arg3527Gln) was the first mutation in APOB being identified and characterized. Recently two novel pathogenic APOB variants have been described: p.(Arg1164Thr) and p.(Gln4494del) showing impaired LDLR binding capacity, and diminished LDL uptake. The objective of this work was to analyse the structure of p.(Arg1164Thr) and p.(Gln4494del) variants to gain insight into their pathogenicity. Secondary structure of the human ApoB100 has been investigated by infrared spectroscopy (IR) and LDL particle size both by dynamic light scattering (DLS) and electron microscopy. The results show differences in secondary structure and/or in particle size of p.(Arg1164Thr) and p.(Gln4494del) variants compared with wild type. We conclude that these changes underlie the defective binding and uptake of p.(Arg1164Thr) and p.(Gln4494del) variants. Our study reveals that structural studies on pathogenic variants of APOB may provide very useful information to understand their role in FH disease.

  11. Structural analysis of APOB variants, p.(Arg3527Gln), p.(Arg1164Thr) and p.(Gln4494del), causing Familial Hypercholesterolaemia provides novel insights into variant pathogenicity

    PubMed Central

    Fernández-Higuero, J. A.; Etxebarria, A.; Benito-Vicente, A.; Alves, A. C.; Arrondo, J. L. R.; Ostolaza, H.; Bourbon, M.; Martin, C.

    2015-01-01

    Familial hypercholesterolaemia (FH) is an inherited autosomal dominant disorder resulting from defects in the low-density lipoprotein receptor (LDLR), in the apolipoprotein B (APOB) or in the proprotein convertase subtilisin/kexin type 9 (PCSK9) genes. In the majority of the cases FH is caused by mutations occurring within LDLR, while only few mutations in APOB and PCSK9 have been proved to cause disease. p.(Arg3527Gln) was the first mutation in APOB being identified and characterized. Recently two novel pathogenic APOB variants have been described: p.(Arg1164Thr) and p.(Gln4494del) showing impaired LDLR binding capacity, and diminished LDL uptake. The objective of this work was to analyse the structure of p.(Arg1164Thr) and p.(Gln4494del) variants to gain insight into their pathogenicity. Secondary structure of the human ApoB100 has been investigated by infrared spectroscopy (IR) and LDL particle size both by dynamic light scattering (DLS) and electron microscopy. The results show differences in secondary structure and/or in particle size of p.(Arg1164Thr) and p.(Gln4494del) variants compared with wild type. We conclude that these changes underlie the defective binding and uptake of p.(Arg1164Thr) and p.(Gln4494del) variants. Our study reveals that structural studies on pathogenic variants of APOB may provide very useful information to understand their role in FH disease. PMID:26643808

  12. Antitumor activities of the targeted multi-tyrosine kinase inhibitor lenvatinib (E7080) against RET gene fusion-driven tumor models.

    PubMed

    Okamoto, Kiyoshi; Kodama, Kotaro; Takase, Kazuma; Sugi, Naoko Hata; Yamamoto, Yuji; Iwata, Masao; Tsuruoka, Akihiko

    2013-10-28

    RET gene fusions are recurrent oncogenes identified in thyroid and lung carcinomas. Lenvatinib is a multi-tyrosine kinase inhibitor currently under evaluation in several clinical trials. Here we evaluated lenvatinib in RET gene fusion-driven preclinical models. In cellular assays, lenvatinib inhibited auto-phosphorylation of KIF5B-RET, CCDC6-RET, and NcoA4-RET. Lenvatinib suppressed the growth of CCDC6-RET human thyroid and lung cancer cell lines, and as well, suppressed anchorage-independent growth and tumorigenicity of RET gene fusion-transformed NIH3T3 cells. These results demonstrate that lenvatinib can exert antitumor activity against RET gene fusion-driven tumor models by inhibiting oncogenic RET gene fusion signaling.

  13. Genome-Wide Analyses Identify Recurrent Amplifications of Receptor Tyrosine Kinases and Cell-Cycle Regulatory Genes in Diffuse Intrinsic Pontine Glioma

    PubMed Central

    Paugh, Barbara S.; Broniscer, Alberto; Qu, Chunxu; Miller, Claudia P.; Zhang, Junyuan; Tatevossian, Ruth G.; Olson, James M.; Geyer, J. Russell; Chi, Susan N.; da Silva, Nasjla Saba; Onar-Thomas, Arzu; Baker, Justin N.; Gajjar, Amar; Ellison, David W.; Baker, Suzanne J.

    2011-01-01

    Purpose Long-term survival for children with diffuse intrinsic pontine glioma (DIPG) is less than 10%, and new therapeutic targets are urgently required. We evaluated a large cohort of DIPGs to identify recurrent genomic abnormalities and gene expression signatures underlying DIPG. Patients and Methods Single-nucleotide polymorphism arrays were used to compare the frequencies of genomic copy number abnormalities in 43 DIPGs and eight low-grade brainstem gliomas with data from adult and pediatric (non-DIPG) glioblastomas, and expression profiles were evaluated using gene expression arrays for 27 DIPGs, six low-grade brainstem gliomas, and 66 nonbrainstem low-grade gliomas. Results Frequencies of specific large-scale and focal imbalances varied significantly between DIPGs and nonbrainstem pediatric glioblastomas. Focal amplifications of genes within the receptor tyrosine kinase–Ras–phosphoinositide 3-kinase signaling pathway were found in 47% of DIPGs, the most common of which involved PDGFRA and MET. Thirty percent of DIPGs contained focal amplifications of cell-cycle regulatory genes controlling retinoblastoma protein (RB) phosphorylation, and 21% had concurrent amplification of genes from both pathways. Some tumors showed heterogeneity in amplification patterns. DIPGs showed distinct gene expression signatures related to developmental processes compared with nonbrainstem pediatric high-grade gliomas, whereas expression signatures of low-grade brainstem and nonbrainstem gliomas were similar. Conclusion DIPGs comprise a molecularly related but distinct subgroup of pediatric gliomas. Genomic studies suggest that targeted inhibition of receptor tyrosine kinases and RB regulatory proteins may be useful therapies for DIPG. PMID:21931021

  14. Protein kinase Cmu downregulation of tumor-necrosis-factor-induced apoptosis correlates with enhanced expression of nuclear-factor-kappaB-dependent protective genes.

    PubMed

    Johannes, F J; Horn, J; Link, G; Haas, E; Siemienski, K; Wajant, H; Pfizenmaier, K

    1998-10-01

    Protein kinase Cmu (PKCmu) represents a new subtype of the PKC family characterized by the presence of a pleckstrin homology (PH) domain and an amino-terminal hydrophobic region. In order to analyse the potential role of PKCmu in signal-transduction pathways, stable PKCmu transfectants were established with human and murine cell lines. All transfectants showed a reduced sensitivity to tumor-necrosis-factor (TNF)-induced apoptosis, which correlated with the amount of transgene expressed and with an enhanced basal transcription rate of NF-kappaB-driven genes including the inhibitor of apoptosis protein 2 (cIAP2) and TNF-receptor-associated protein 1 (TRAF1). Sensitivity to apoptosis induced by the lipid mediator ceramide was unchanged in PKCmu transfectants. In support of a PKCmu action on NF-kappaB, we show enhancement and downregulation of TNF-induced expression of a NF-kappaB-dependent reporter gene by transient overexpression of wild-type and kinase-negative mutants of PKCmu, respectively. Interestingly, no significant changes were found in an electrophoretic mobility shift assay, indicative of PKCmu action downstream of IkappaB degradation, probably by modulation of the transactivation capacity of NF-kappaB. The dominant negative action of the kinase-negative mutant further suggest a regulatory role of PKCmu for NF-kappaB-dependent gene expression.

  15. Pb2+ induces gastrin gene expression by extracellular signal-regulated kinases 1/2 and transcription factor activator protein 1 in human gastric carcinoma cells.

    PubMed

    Chan, Chien-Pin; Tsai, Yao-Ting; Chen, Yao-Li; Hsu, Yu-Wen; Tseng, Joseph T; Chuang, Hung-Yi; Shiurba, Robert; Lee, Mei-Hsien; Wang, Jaw-Yuan; Chang, Wei-Chiao

    2015-02-01

    Divalent lead ions (Pb(2+) ) are toxic environmental pollutants known to cause serious health problems in humans and animals. Absorption of Pb(2+) from air, water, and food takes place in the respiratory and digestive tracts. The ways in which absorbed Pb(2+) affects cell physiology are just beginning to be understood at the molecular level. Here, we used reverse transcription PCR and Western blotting to analyze cultures of human gastric carcinoma cells exposed to 10 μM lead nitrate. We found that Pb(2+) induces gastrin hormone gene transcription and translation in a time-dependent manner. Promoter deletion analysis revealed that activator protein 1 (AP1) was necessary for gastrin gene transcription in cells exposed to Pb(2+) . MitogIen-activated protein kinase (MAPK)/ERK kinase inhibitor PD98059 suppressed the Pb(2+) -induced increase in messenger RNA. Epidermal growth factor receptor (EGFR) inhibitors AG1478 and PD153035 reduced both transcription and phosphorylation by extracellular signal-regulated kinase (ERK1/2). Cells exposed to Pb(2+) also increased production of c-Jun protein, a component of AP1, and over-expression of c-Jun enhanced activation of the gastrin promoter. In sum, the findings suggest the EGFR-ERK1/2-AP1 pathway mediates the effects of Pb(2+) on gastrin gene activity in cell culture.

  16. Conserved serine/threonine kinase encoded by CBK1 regulates expression of several hypha-associated transcripts and genes encoding cell wall proteins in Candida albicans.

    PubMed

    McNemar, Mark D; Fonzi, William A

    2002-04-01

    The opportunistic fungal pathogen, Candida albicans, is reported to have several potential virulence factors. A potentially significant factor is the ability to undergo morphological transition from yeast to hypha. This alteration of form is accompanied by many changes within the cell, including alterations in gene expression and cell wall composition. We have isolated a gene that encodes a highly conserved serine/threonine kinase that appears to be involved in the regulation of proteins associated with the cell wall. We have assigned the designation CBK1 (cell wall biosynthesis kinase 1) to this gene. Mutants lacking CBK1 form large aggregates of round cells under all growth conditions and lack the ability to undergo morphological differentiation. Additionally, these mutants show an altered pattern of expression of several transcripts encoding proteins associated with the cell wall. The results suggest that the kinase encoded by CBK1 plays a general role in the maintenance and alteration of the cell wall of C. albicans in all morphologies.

  17. FGFR4 Arg388 allele correlates with tumour thickness and FGFR4 protein expression with survival of melanoma patients.

    PubMed

    Streit, S; Mestel, D S; Schmidt, M; Ullrich, A; Berking, C

    2006-06-19

    A single nucleotide polymorphism in the gene for FGFR4 (-Arg388) has been associated with progression in various types of human cancer. Although fibroblast growth factors (FGFs) belong to the most important growth factors in melanoma, expression of FGF receptor subtype 4 has not been investigated yet. In this study, the protein expression of this receptor was analysed in 137 melanoma tissues of different progression stages by immunohistochemistry. FGFR4 protein was expressed in 45% of the specimens and correlated with pTNM tumour stages (UICC, P = 0.023 and AJCC, P = 0.046), presence of microulceration (P = 0.009), tumour vascularity (P = 0.001), metastases (P = 0.025), number of primary tumours (P = 0.022), overall survival (P = 0.047) and disease-free survival (P = 0.024). Furthermore, FGFR4 Arg388 polymorphism was analysed in 185 melanoma patients by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP). The Arg388 allele was detected in 45% of the melanoma patients and was significantly associated with tumour thickness (by Clark's level of invasion (P = 0.004) and by Breslow in mm (P = 0.02)) and the tumour subtype nodular melanoma (P = 0.002). However, there was no correlation of the FGFR4 Arg388 allele with overall and disease-free survival. In conclusion, the Arg388 genotype and the protein expression of FGFR4 may be potential markers for progression of melanoma.

  18. Co-Expression of Wild-Type P2X7R with Gln460Arg Variant Alters Receptor Function

    PubMed Central

    Aprile-Garcia, Fernando; Metzger, Michael W.; Paez-Pereda, Marcelo; Stadler, Herbert; Acuña, Matías; Liberman, Ana C.; Senin, Sergio A.; Gerez, Juan; Hoijman, Esteban; Refojo, Damian; Mitkovski, Mišo; Panhuysen, Markus; Stühmer, Walter; Holsboer, Florian; Deussing, Jan M.; Arzt, Eduardo

    2016-01-01

    The P2X7 receptor is a member of the P2X family of ligand-gated ion channels. A single-nucleotide polymorphism leading to a glutamine (Gln) by arginine (Arg) substitution at codon 460 of the purinergic P2X7 receptor (P2X7R) has been associated with mood disorders. No change in function (loss or gain) has been described for this SNP so far. Here we show that although the P2X7R-Gln460Arg variant per se is not compromised in its function, co-expression of wild-type P2X7R with P2X7R-Gln460Arg impairs receptor function with respect to calcium influx, channel currents and intracellular signaling in vitro. Moreover, co-immunoprecipitation and FRET studies show that the P2X7R-Gln460Arg variant physically interacts with P2X7R-WT. Specific silencing of either the normal or polymorphic variant rescues the heterozygous loss of function phenotype and restores normal function. The described loss of function due to co-expression, unique for mutations in the P2RX7 gene so far, explains the mechanism by which the P2X7R-Gln460Arg variant affects the normal function of the channel and may represent a mechanism of action for other mutations. PMID:26986975

  19. The ubiquitous mitochondrial creatine kinase gene maps to a conserved region on human chromosome 15q15 and mouse chromosome 2 bands F1-F3

    SciTech Connect

    Steeghs, K.; Wieringa, B.; Merkx, G.

    1994-11-01

    Members of the creatine kinase isoenzyme family (CKs; EC 2.7.3.2) are found in mitochondria and specialized subregions of the cytoplasm and catalyze the reversible exchange of high-energy phosphoryl between ATP and phosphocreatine. At least four functionally active genes, which encode the distinct CK subunits CKB, CKM, CKMT1 (ubiquitous), and CKMT2 (sarcomeric), and a variable number of CKB pseudogenes have been identified. Here, we report the use of a CKMT1 containing phage to map the CKMT1 gene by in situ hybridization on both human and mouse chromosomes.

  20. Monocyte chemoattractant protein-1 gene delivery enhances antitumor effects of herpes simplex virus thymidine kinase/ganciclovir system in a model of colon cancer.

    PubMed

    Kagaya, T; Nakamoto, Y; Sakai, Y; Tsuchiyama, T; Yagita, H; Mukaida, N; Kaneko, S

    2006-04-01

    Suicide gene therapy using the herpes simplex virus thymidine kinase/ganciclovir (HSV-tk/GCV) system is a well-characterized tool for cancer gene therapy; however, it does not yet exhibit sufficient efficacy to cure patients of malignancies. We have reported that adenovirally delivered monocyte chemoattractant protein (MCP)-1 augmented the antitumor effects of the HSV-tk/GCV system in an athymic nude mouse model. The current study, which uses an immunocompetent mouse model of colon cancer, was designed to evaluate the antitumor effects of MCP-1 gene delivery in conjunction with this suicide gene therapy system. Subcutaneous tumor foci were directly transduced with both recombinant adenoviruses (rAds) expressing an HSV-tk gene and either of the MCP-1, CD80 and LacZ genes, followed by GCV administration. The growth of tumors was markedly suppressed by codelivery of HSV-tk and MCP-1 genes, which was exclusively associated with the recruitment of monocytes/macrophages, T helper 1 (Th1) cytokine gene expression and cytotoxic activity of the splenocytes. Furthermore, the antitumor effects were more efficient than that obtained by the combination of HSV-tk and CD80 genes. These results suggest an immunomodulatory effect of MCP-1 in the context of suicide gene therapy of colon cancer via orchestration of innate and acquired immune responses.

  1. Modeling of tRNA-assisted mechanism of Arg activation based on a structure of Arg-tRNA synthetase, tRNA, and an ATP analog (ANP).

    PubMed

    Konno, Michiko; Sumida, Tomomi; Uchikawa, Emiko; Mori, Yukie; Yanagisawa, Tatsuo; Sekine, Shun-ichi; Yokoyama, Shigeyuki; Yokoyama, Shigeuki

    2009-09-01

    The ATP-pyrophosphate exchange reaction catalyzed by Arg-tRNA, Gln-tRNA and Glu-tRNA synthetases requires the assistance of the cognate tRNA. tRNA also assists Arg-tRNA synthetase in catalyzing the pyrophosphorolysis of synthetic Arg-AMP at low pH. The mechanism by which the 3'-end A76, and in particular its hydroxyl group, of the cognate tRNA is involved with the exchange reaction catalyzed by those enzymes has yet to be established. We determined a crystal structure of a complex of Arg-tRNA synthetase from Pyrococcus horikoshii, tRNA(Arg)(CCU) and an ATP analog with Rfactor = 0.213 (Rfree = 0.253) at 2.0 A resolution. On the basis of newly obtained structural information about the position of ATP bound on the enzyme, we constructed a structural model for a mechanism in which the formation of a hydrogen bond between the 2'-OH group of A76 of tRNA and the carboxyl group of Arg induces both formation of Arg-AMP (Arg + ATP --> Arg-AMP + pyrophosphate) and pyrophosphorolysis of Arg-AMP (Arg-AMP + pyrophosphate --> Arg + ATP) at low pH. Furthermore, we obtained a structural model of the molecular mechanism for the Arg-tRNA synthetase-catalyzed deacylation of Arg-tRNA (Arg-tRNA + AMP --> Arg-AMP + tRNA at high pH), in which the deacylation of aminoacyl-tRNA bound on Arg-tRNA synthetase and Glu-tRNA synthetase is catalyzed by a quite similar mechanism, whereby the proton-donating group (-NH-C+(NH2)2 or -COOH) of Arg and Glu assists the aminoacyl transfer from the 2'-OH group of tRNA to the phosphate group of AMP at high pH.

  2. Duplication and Divergence of Leucine-Rich Repeat Receptor-Like Protein Kinase (LRR-RLK) Genes in Basal Angiosperm Amborella trichopoda

    PubMed Central

    Liu, Ping-Li; Xie, Lu-Lu; Li, Peng-Wei; Mao, Jian-Feng; Liu, Hui; Gao, Shu-Min; Shi, Peng-Hao; Gong, Jun-Qing

    2016-01-01

    Leucine-rich repeat receptor-like protein kinases (LRR-RLKs) are the largest group of receptor-like kinases, which are one of the largest protein superfamilies in plants, and play crucial roles in development and stress responses. Although the evolution of LRR-RLK families has been investigated in some eudicot and monocot plants, no comprehensive evolutionary studies have been performed for these genes in basal angiosperms like Amborella trichopoda. In this study, we identified 94 LRR-RLK genes in the genome of A. trichopoda. The number of LRR-RLK genes in the genome of A. trichopoda is only 17–50% of that of several eudicot and monocot species. Tandem duplication and whole-genome duplication have made limited contributions to the expansion of LRR-RLK genes in A. trichopoda. According to the phylogenetic analysis, all A. trichopoda LRR-RLK genes can be organized into 18 subfamilies, which roughly correspond to the LRR-RLK subfamilies defined in Arabidopsis thaliana. Most LRR-RLK subfamilies are characterized by highly conserved protein structures, motif compositions, and gene structures. The unique gene structure, protein structures, and protein motif compositions of each subfamily provide evidence for functional divergence among LRR-RLK subfamilies. Moreover, the expression data of LRR-RLK genes provided further evidence for the functional diversification of them. In addition, selection analyses showed that most LRR-RLK protein sites are subject to purifying selection. Our results contribute to a better understanding of the evolution of LRR-RLK gene family in angiosperm and provide a framework for further functional investigation on A. trichopoda LRR-RLKs. PMID:28066499

  3. Duplication and Divergence of Leucine-Rich Repeat Receptor-Like Protein Kinase (LRR-RLK) Genes in Basal Angiosperm Amborella trichopoda.

    PubMed

    Liu, Ping-Li; Xie, Lu-Lu; Li, Peng-Wei; Mao, Jian-Feng; Liu, Hui; Gao, Shu-Min; Shi, Peng-Hao; Gong, Jun-Qing

    2016-01-01

    Leucine-rich repeat receptor-like protein kinases (LRR-RLKs) are the largest group of receptor-like kinases, which are one of the largest protein superfamilies in plants, and play crucial roles in development and stress responses. Although the evolution of LRR-RLK families has been investigated in some eudicot and monocot plants, no comprehensive evolutionary studies have been performed for these genes in basal angiosperms like Amborella trichopoda. In this study, we identified 94 LRR-RLK genes in the genome of A. trichopoda. The number of LRR-RLK genes in the genome of A. trichopoda is only 17-50% of that of several eudicot and monocot species. Tandem duplication and whole-genome duplication have made limited contributions to the expansion of LRR-RLK genes in A. trichopoda. According to the phylogenetic analysis, all A. trichopoda LRR-RLK genes can be organized into 18 subfamilies, which roughly correspond to the LRR-RLK subfamilies defined in Arabidopsis thaliana. Most LRR-RLK subfamilies are characterized by highly conserved protein structures, motif compositions, and gene structures. The unique gene structure, protein structures, and protein motif compositions of each subfamily provide evidence for functional divergence among LRR-RLK subfamilies. Moreover, the expression data of LRR-RLK genes provided further evidence for the functional diversification of them. In addition, selection analyses showed that most LRR-RLK protein sites are subject to purifying selection. Our results contribute to a better understanding of the evolution of LRR-RLK gene family in angiosperm and provide a framework for further functional investigation on A. trichopoda LRR-RLKs.

  4. MGMT-independent temozolomide resistance in pediatric glioblastoma cells associated with a PI3-kinase-mediated HOX/stem cell gene signature.

    PubMed

    Gaspar, Nathalie; Marshall, Lynley; Perryman, Lara; Bax, Dorine A; Little, Suzanne E; Viana-Pereira, Marta; Sharp, Swee Y; Vassal, Gilles; Pearson, Andrew D J; Reis, Rui M; Hargrave, Darren; Workman, Paul; Jones, Chris

    2010-11-15

    Sensitivity to temozolomide is restricted to a subset of glioblastoma patients, with the major determinant of resistance being a lack of promoter methylation of the gene encoding the repair protein DNA methyltransferase MGMT, although other mechanisms are thought to be active. There are, however, limited preclinical data in model systems derived from pediatric glioma patients. We screened a series of cell lines for temozolomide efficacy in vitro, and investigated the differential mechanisms of resistance involved. In the majority of cell lines, a lack of MGMT promoter methylation and subsequent protein overexpression were linked to temozolomide resistance. An exception was the pediatric glioblastoma line KNS42. Expression profiling data revealed a coordinated upregulation of HOX gene expression in resistant lines, especially KNS42, which was reversed by phosphoinositide 3-kinase pathway inhibition. High levels of HOXA9/HOXA10 gene expression were associated with a shorter survival in pediatric high-grade glioma patient samples. Combination treatment in vitro of pathway inhibition and temozolomide resulted in a highly synergistic interaction in KNS42 cells. The resistance gene signature further included contiguous genes within the 12q13-q14 amplicon, including the Akt enhancer PIKE, significantly overexpressed in the KNS42 line. These cells were also highly enriched for CD133 and other stem cell markers. We have thus shown an in vitro link between phosphoinositide 3-kinase-mediated HOXA9/HOXA10 expression, and a drug-resistant, progenitor cell phenotype in MGMT-independent pediatric glioblastoma.

  5. Effects of doxepin on brain-derived neurotrophic factor, tumor necrosis factor alpha, mitogen-activated protein kinase 14, and AKT1 genes expression in rat hippocampus

    PubMed Central

    Eidelkhani, Nastaran; Radahmadi, Maryam; Kazemi, Mohammad; Rafiee, Laleh; Alaei, Hojjatallah; Reisi, Parham

    2015-01-01

    Background: It has been suggested that doxepin in addition to enhancement of noradrenaline and serotonin levels may have neuroprotective effects. Therefore, this study investigated the effect of doxepin on gene expression of brain-derived neurotrophic factor (BDNF), tumor necrosis factor alpha (TNF-α), mitogen-activated protein kinase 14 (MAPK14), and serine-threonine protein kinase AKT1 in rat hippocampus. Materials and Methods: Male rats were divided randomly into three groups: Control, doxepin 1 mg/kg, and doxepin 5 mg/kg. Rats received an i.p injection of doxepin for 21 days. Then the hippocampi were dissected for the measurement of the expression of BDNF, TNF-α, MAPK14, and AKT1 genes. Results: Our results showed no significant effects of doxepin on gene expression of BDNF, TNF-α, MAPK14, and AKT1 genes in the hippocampus. Conclusions: These results did not show significant effects of doxepin on the genes that affect the neuronal survival in intact animals. However, more studies need to be done, especially in models associated with neuronal damage. PMID:26601091

  6. LcMKK, a MAPK kinase from Lycium chinense, confers cadmium tolerance in transgenic tobacco by transcriptional upregulation of ethylene responsive transcription factor gene.

    PubMed

    Guan, Chunfeng; Ji, Jing; Li, Xiaozhou; Jin, Chao; Wang, Gang

    2016-12-01

    Cadmium (Cd) is a highly toxic element to plants. Ethylene is an important phytohormone in the regulation of plant growth, development and stress response. Mitogen-activated protein kinase (MAPK) activation has been observed in plants exposed to Cd stress and was suggested to be involved in ethylene biosynthesis. We hypothesized that there may be a link between MAPK cascades and ethylene signalling in Cd-stressed plants. To test this hypothesis, the expression of LcMKK, LchERF and LcGSH1 genes, endogenous ethylene accumulation, GSH content and Cd concentration in Lycium chinense with or without Cd stress treatment were studied. Our results showed that LcMKK gene expression can be induced by the treatment of Cd in L. chinense. The transgenic tobacco expressing 35S::LcMKK showed greater tolerance to Cd stress and enhanced expression of NtERF and NtGSH1 genes, indicating that LcMKK is associated with the enhanced expression level of ERF and GSH synthesis-related genes in tobacco. We also found that endogenous ethylene and GSH content can be induced by Cd stress in L. chinense, and inhibited by cotreatment with PD98059, an inhibitor of MAPK kinase. Evidences presented here suggest that under Cd stress, GSH accumulation occurred at least partially by enhanced LcMKK gene expression and the ethylene signal transduction pathways might be involved in this accumulation.

  7. A dominant truncation allele identifies a gene, STE20, that encodes a putative protein kinase necessary for mating in Saccharomyces cerevisiae.

    PubMed Central

    Ramer, S W; Davis, R W

    1993-01-01

    This work reports the identification, characterization, and nucleotide sequence of STE20, a newly discovered gene involved in the Saccharomyces cerevisiae mating response pathway, to date one of the best understood signal transduction pathways. STE20 encodes a putative serine/threonine-specific protein kinase with a predicted molecular mass of 102 kDa. Its expression pattern is similar to that of several other protein kinases in the mating response pathway. Deletion of the kinase domain of STE20 causes sterility in both haploid mating types. This sterility can be partially suppressed by high-level production of STE12 but is not suppressible by high levels of STE4 or a dominant STE11 truncation allele. A truncation allele of STE20 was isolated that can activate the mating response pathway in the absence of exogenous mating pheromone. This allele causes dominant growth arrest that cannot be suppressed by deletions of STE4, STE5, STE7, STE11, or STE12. The allele is able to suppress the mating defect of a strain in which the STE20 kinase domain has been deleted, but not the mating defects of strains carrying mutations in STE4, STE5, STE7, STE11, or STE12. Images PMID:8421676

  8. Role of FAST Kinase Domains 3 (FASTKD3) in Post-transcriptional Regulation of Mitochondrial Gene Expression.

    PubMed

    Boehm, Erik; Zornoza, María; Jourdain, Alexis A; Delmiro Magdalena, Aitor; García-Consuegra, Inés; Torres Merino, Rebeca; Orduña, Antonio; Martín, Miguel A; Martinou, Jean-Claude; De la Fuente, Miguel A; Simarro, María

    2016-12-09

    The Fas-activated serine/threonine kinase (FASTK) family of proteins has recently emerged as a central regulator of mitochondrial gene expression through the function of an unusual RNA-binding domain named RAP (for RNA-binding domain abundant in Apicomplexans), shared by all six members of the family. Here we describe the role of one of the less characterized members, FASTKD3, in mitochondrial RNA metabolism. First, we show that, in contrast to FASTK, FASTKD2, and FASTKD5, FASTKD3 does not localize in mitochondrial RNA granules, which are sites of processing and maturation of mtRNAs and ribosome biogenesis. Second, we generated FASTKD3 homozygous knock-out cell lines by homologous recombination and observed that the absence of FASTKD3 resulted in increased steady-state levels and half-lives of a subset of mature mitochondrial mRNAs: ND2, ND3, CYTB, COX2, and ATP8/6. No aberrant processing of RNA precursors was observed. Rescue experiments demonstrated that RAP domain is required for FASTKD3 function in mRNA stability. Besides, we describe that FASTKD3 is required for efficient COX1 mRNA translation without altering mRNA levels, which results in a decrease in the steady-state levels of COX1 protein. This finding is associated with reduced mitochondrial complex IV assembly and activity. Our observations suggest that the function of this family of proteins goes beyond RNA processing and ribosome assembly and includes RNA stability and translation regulation within mitochondria.

  9. Liposomal insulin promoter-thymidine kinase gene therapy followed by ganciclovir effectively ablates human pancreatic cancer in mice.

    PubMed

    Wu, James X; Liu, Shi-He; Nemunaitis, John J; Brunicardi, F Charles

    2015-04-10

    PDX1 is overexpressed in pancreatic cancer, and activates the insulin promoter (IP). Adenoviral IP-thymidine kinase and ganciclovir (TK/GCV) suppresses human pancreatic ductal carcinoma (PDAC) in mice, but repeated doses carry significant toxicity. We hypothesized that multiple cycles of liposomal IP-TK/GCV ablate human PDAC in SCID mice with minimal toxicity compared to adenoviral IP-TK/GCV. SCID mice with intraperitoneal human pancreatic cancer PANC-1 tumor implants were given a single cycle of 35 µg iv L-IP-TK, or four cycles of 1, 10, 20, 30, or 35 µg iv L-IP-TK (n = 20 per group), followed by intraperitoneal GCV. Insulin and glucose levels were monitored in mice treated with four cycles of 35 µg iv L-IP-TK. We found that four cycles of 10-35 µg L-IP-TK/GCV ablated more PANC-1 tumor volume compared to a single cycle with 35 µg. Mice that received four cycles of 10 µg L-IP-TK demonstrated the longest survival (P < 0.05), with a median survival of 126 days. In comparison, mice that received a single cycle of 35 µg L-IP-TK/GCV or GCV alone survived a median of 92 days and 68.7 days, respectively. There were no significant changes in glucose or insulin levels following treatment. In conclusion, multiple cycles of liposomal IP-TK/GCV ablate human PDAC in SCID mice with minimal toxicity, suggesting non-viral vectors are superior to adenoviral vectors for IP-gene therapy.

  10. Identification of the glycerol kinase gene and its role in diapause embryo restart and early embryo development of Artemia sinica.

    PubMed

    Cheng, Cheng; Yao, Feng; Chu, Bing; Li, Xuejie; Liu, Yan; Wu, Yang; Mei, Yanli; Wang, Peisheng; Hou, Lin; Zou, Xiangyang

    2014-03-01

    Glycerol kinase (GK) catalyzes the rate-limiting step in glycerol utilization by transferring a phosphate from ATP to glycerol, yielding glycerol 3-phosphate, which is an important intermediate for both energy metabolism and glycerolipid production. Artemia sinica has an unusual diapause process under stress conditions of high salinity, low temperature and lack of food. In the process, diapause embryos of A. sinica (brine shrimp) accumulate high concentrations of glycerol as a cryoprotectant to prevent low temperature damage to embryos. Upon embryo restart, glycerol is converted into glucose and other carbohydrates. Therefore, GK plays an important role in the diapause embryo restart process. However, the role of GK in diapause termination of embryo development in A. sinica remains unknown. In the present study, a 2096 bp full-length cDNA of gk from A. sinica (As-gk) was obtained, encoding putative 551 amino acids, 60.6 kDa protein. As a crucial enzyme in glycerol uptake and metabolism, GK has been conserved structurally and functionally during evolution. The expression pattern of As-gk was investigated by quantitative real-time PCR and Western blotting. Expression locations of As-gk were analyzed using in situ hybridization. As-gk was widely distributed in the early embryo and several main parts of Artemia after differentiation. The expression of As-GK was also induced by stresses such as cold exposure and high salinity. This initial research into the expression pattern and stress response of GK in Artemia provides a sound basis for further understanding of the function and regulation of genes in early embryonic development in A. sinica and the stress response.

  11. A large germline deletion in the Chek2 kinase gene is associated with an increased risk of prostate cancer

    PubMed Central

    Cybulski, C; Wokołorczyk, D; Huzarski, T; Byrski, T; Gronwald, J; Górski, B; Dębniak, T; Masojć, B; Jakubowska, A; Gliniewicz, B; Sikorski, A; Stawicka, M; Godlewski, D; Kwias, Z; Antczak, A; Krajka, K; Lauer, W; Sosnowski, M; Sikorska‐Radek, P; Bar, K; Klijer, R; Zdrojowy, R; Małkiewicz, B; Borkowski, A; Borkowski, T; Szwiec, M; Narod, S A; Lubiński, J

    2006-01-01

    Background Germline mutations in the Chek2 kinase gene (CHEK2) have been associated with a range of cancer types. Recently, a large deletion of exons 9 and 10 of CHEK2 was identified in several unrelated patients with breast cancer of Czech or Slovak origin. The geographical and ethnic extent of this founder allele has not yet been determined. Participants and methods We assayed for the presence of this deletion, and of three other CHEK2 founder mutations, in 1864 patients with prostate cancer and 5496 controls from Poland. Results The deletion was detected in 24 of 5496 (0.4%) controls from the general population, and is the most common CHEK2 truncating founder allele in Polish patients. The deletion was identified in 15 of 1864 (0.8%) men with unselected prostate cancer (OR 1.9; 95% CI 0.97 to 3.5; p = 0.09) and in 4 of 249 men with familial prostate cancer (OR 3.7; 95% CI 1.3 to 10.8; p = 0.03). These ORs were similar to those associated with the other truncating mutations (IVS2+1G→A, 1100delC). Conclusion A large deletion of exons 9 and 10 of CHEK2 confers an increased risk of prostate cancer in Polish men. The del5395 founder deletion might be present in other Slavic populations, including Ukraine, Belarus, Russia, Baltic and Balkan countries. It will be of interest to see to what extent this deletion is responsible for the burden of prostate cancer in other populations. PMID:17085682

  12. Selective killing of lung cancer cells using carcinoembryonic antigen promoter and double suicide genes, thymidine kinase and cytosine deaminase (pCEA-TK/CD).

    PubMed

    Qiu, Yuan; Peng, Gui-Lin; Liu, Qi-Cai; Li, Fu-Li; Zou, Xu-Sen; He, Jian-Xing

    2012-03-01

    The application of gene therapy in cancer treatment is limited by non-specific targeting. In the present study, we constructed a recombinant plasmid, containing a carcinoembryonic antigen (CEA) promoter and double suicide genes thymidine kinase (TK) and cytosine deaminase (CD), henceforth referred to as pCEA-TK/CD. Our results showed that the CEA promoter can specifically drive target gene expression in CEA-positive lung cancer cells. In the presence of prodrugs 5-flucytosine and ganciclovir, pCEA-TK/CD transfection decreased inhibitory concentration 50 and increased apoptosis and cyclomorphosis. Our result suggests that gene therapy using pCEA-TK/CD may be a promising new approach for treating lung cancer.

  13. Mapping of the NEP receptor tyrosine kinase gene to human chromosome 6p21.3 and mouse chromosome 17C

    SciTech Connect

    Edelhoff, S.; Disteche, C.M.; Sweetser, D.A.

    1995-01-01

    The mouse receptor tyrosine kinase (RTK) NEP, also called Ptk-3, is widely expressed, with high levels in proliferating neuroepithelia of mouse embryos. The recently described human discoidin domain receptor (DDR) has a predicted amino acid sequence 93% identical to that of murine NEP and may be its human homologue. We have mapped the gene encoding NEP in human and mouse by fluorescence in situ hybridization using a mouse cDNA probe. The NEP/Nep gene maps to human chromosome 6p21.3 and mouse chromosome 17C, respectively. This places the NEP/Nep gene at, or near, the major histocompatibility (MHC) locus-HLA in human and H2 in mouse, respectively. Based on its pattern of expression during development, NEP and Nep represent candidate genes for several MHC-linked developmental abnormalities in human and mouse. 19 refs., 1 fig.

  14. Organization and nucleotide sequences of the Spiroplasma citri genes for ribosomal protein S2, elongation factor Ts, spiralin, phosphofructokinase, pyruvate kinase, and an unidentified protein.

    PubMed Central

    Chevalier, C; Saillard, C; Bové, J M

    1990-01-01

    The gene for spiralin, the major membrane protein of the helical mollicute Spiroplasma citri, was cloned in Escherichia coli as a 5-kilobase-pair (kbp) DNA fragment. The complete nucleotide sequence of the 5.0-kbp spiroplasmal DNA fragment was determined (GenBank accession no. M31161). The spiralin gene was identified by the size and amino acid composition of its translational product. Besides the spiralin gene, the spiroplasmal DNA fragment was found to contain five additional open reading frames (ORFs). The translational products of four of these ORFs were identified by their amino acid sequence homologies with known proteins: ribosomal protein S2, elongation factor Ts, phosphofructokinase, and pyruvate kinase, respectively encoded by the genes rpsB, tsf, pfk, and pyk. The product of the fifth ORF remains to be identified and was named protein X (X gene). The order of the above genes was tsf--X--spiralin gene--pfk--pyk. These genes were transcribed in one direction, while the gene for ribosomal protein S2 (rpsB) was transcribed in the opposite direction. Images PMID:2139649

  15. Phosphate Concentration and the Putative Sensor Kinase Protein CckA Modulate Cell Lysis and Release of the Rhodobacter capsulatus Gene Transfer Agent

    PubMed Central

    Westbye, A. B.; Leung, M. M.; Florizone, S. M.; Taylor, T. A.; Johnson, J. A.; Fogg, P. C.

    2013-01-01

    The gene transfer agent of Rhodobacter capsulatus (RcGTA) is a bacteriophage-like genetic element with the sole known function of horizontal gene transfer. Homologues of RcGTA genes are present in many members of the alphaproteobacteria and may serve an important role in microbial evolution. Transcription of RcGTA genes is induced as cultures enter the stationary phase; however, little is known about cis-active sequences. In this work, we identify the promoter of the first gene in the RcGTA structural gene cluster. Additionally, gene transduction frequency depends on the growth medium, and the reason for this is not known. We report that millimolar concentrations of phosphate posttranslationally inhibit the lysis-dependent release of RcGTA from cells in both a complex medium and a defined medium. Furthermore, we found that cell lysis requires the genes rcc00555 and rcc00556, which were expressed and studied in Escherichia coli to determine their predicted functions as an endolysin and holin, respectively. Production of RcGTA is regulated by host systems, including a putative histidine kinase, CckA, and we found that CckA is required for maximal expression of rcc00555 and for maturation of RcGTA to yield gene transduction-functional particles. PMID:23995641

  16. Involvement of adenosine monophosphate-activated protein kinase in the influence of timed high-fat evening diet on the hepatic clock and lipogenic gene expression in mice.

    PubMed

    Huang, Yan; Zhu, Zengyan; Xie, Meilin; Xue, Jie

    2015-09-01

    A high-fat diet may result in changes in hepatic clock gene expression, but potential mechanisms are not yet elucidated. Adenosine monophosphate-activated protein kinase (AMPK) is a serine/threonine protein kinase that is recognized as a key regulator of energy metabolism and certain clock genes. Therefore, we hypothesized that AMPK may be involved in the alteration of hepatic clock gene expression under a high-fat environment. This study aimed to examine the effects of timed high-fat evening diet on the activity of hepatic AMPK, clock genes, and lipogenic genes. Mice with hyperlipidemic fatty livers were induced by orally administering high-fat milk via gavage every evening (19:00-20:00) for 6 weeks. Results showed that timed high-fat diet in the evening not only decreased the hepatic AMPK protein expression and activity but also disturbed its circadian rhythm. Accordingly, the hepatic clock genes, including clock, brain-muscle-Arnt-like 1, cryptochrome 2, and period 2, exhibited prominent changes in their expression rhythms and/or amplitudes. The diurnal rhythms of the messenger RNA expression of peroxisome proliferator-activated receptorα, acetyl-CoA carboxylase 1α, and carnitine palmitoyltransferase 1 were also disrupted; the amplitude of peroxisome proliferator-activated receptorγcoactivator 1α was significantly decreased at 3 time points, and fatty liver was observed. These findings demonstrate that timed high-fat diet at night can change hepatic AMPK protein levels, activity, and circadian rhythm, which may subsequently alter the circadian expression of several hepatic clock genes and finally result in the disorder of hepatic lipogenic gene expression and the formation of fatty liver.

  17. Different structural alterations upregulate in vitro tyrosine kinase activity and transforming potency of the erbB-2 gene.

    PubMed Central

    Segatto, O; King, C R; Pierce, J H; Di Fiore, P P; Aaronson, S A

    1988-01-01

    Compared with normal erbB-2 gp185, mutant erbB-2 proteins generated by mutations either in the transmembrane domain or by NH2-terminal deletion are able to transform NIH 3T3 cells at a 10- to 100-fold greater efficiency. Mutant proteins of both classes show increased tyrosine kinase activity, suggesting that an abnormal level of receptor-associated tyrosine kinase activity is a major determinant of erbB-2 oncogenic potential. Images PMID:2907606

  18. Leucine-rich Repeat Kinase 2 (LRRK2) Pharmacological Inhibition Abates α-Synuclein Gene-induced Neurodegeneration.

    PubMed

    Daher, João P L; Abdelmotilib, Hisham A; Hu, Xianzhen; Volpicelli-Daley, Laura A; Moehle, Mark S; Fraser, Kyle B; Needle, Elie; Chen, Yi; Steyn, Stefanus J; Galatsis, Paul; Hirst, Warren D; West, Andrew B

    2015-08-07

    Therapeutic approaches to slow or block the progression of Parkinson disease (PD) do not exist. Genetic and biochemical studies implicate α-synuclein and leucine-rich repeat kinase 2 (LRRK2) in late-onset PD. LRRK2 kinase activity has been linked to neurodegenerative pathways. However, the therapeutic potential of LRRK2 kinase inhibitors is not clear because significant toxicities have been associated with one class of LRRK2 kinase inhibitors. Furthermore, LRRK2 kinase inhibitors have not been tested previously for efficacy in models of α-synuclein-induced neurodegeneration. To better understand the therapeutic potential of LRRK2 kinase inhibition in PD, we evaluated the tolerability and efficacy of a LRRK2 kinase inhibitor, PF-06447475, in preventing α-synuclein-induced neurodegeneration in rats. Both wild-type rats as well as transgenic G2019S-LRRK2 rats were injected intracranially with adeno-associated viral vectors expressing human α-synuclein in the substantia nigra. Rats were treated with PF-06447475 or a control compound for 4 weeks post-viral transduction. We found that rats expressing G2019S-LRRK2 have exacerbated dopaminergic neurodegeneration and inflammation in response to the overexpression of α-synuclein. Both neurodegeneration and neuroinflammation associated with G2019S-LRRK2 expression were mitigated by LRRK2 kinase inhibition. Furthermore, PF-06447475 provided neuroprotection in wild-type rats. We could not detect adverse pathological indications in the lung, kidney, or liver of rats treated with PF-06447475. These results demonstrate that pharmacological inhibition of LRRK2 is well tolerated for a 4-week period of time in rats and can counteract dopaminergic neurodegeneration caused by acute α-synuclein overexpression.

  19. The Arabidopsis TOR Kinase Specifically Regulates the Expression of Nuclear Genes Coding for Plastidic Ribosomal Proteins and the Phosphorylation of the Cytosolic Ribosomal Protein S6

    PubMed Central

    Dobrenel, Thomas; Mancera-Martínez, Eder; Forzani, Céline; Azzopardi, Marianne; Davanture, Marlène; Moreau, Manon; Schepetilnikov, Mikhail; Chicher, Johana; Langella, Olivier; Zivy, Michel; Robaglia, Christophe; Ryabova, Lyubov A.; Hanson, Johannes; Meyer, Christian

    2016-01-01

    Protein translation is an energy consuming process that has to be fine-tuned at both the cell and organism levels to match the availability of resources. The target of rapamycin kinase (TOR) is a key regulator of a large range of biological processes in response to environmental cues. In this study, we have investigated the effects of TOR inactivation on the expression and regulation of Arabidopsis ribosomal proteins at different levels of analysis, namely from transcriptomic to phosphoproteomic. TOR inactivation resulted in a coordinated down-regulation of the transcription and translation of nuclear-encoded mRNAs coding for plastidic ribosomal proteins, which could explain the chlorotic phenotype of the TOR silenced plants. We have identified in the 5′ untranslated regions (UTRs) of this set of genes a conserved sequence related to the 5′ terminal oligopyrimidine motif, which is known to confer translational regulation by the TOR kinase in other eukaryotes. Furthermore, the phosphoproteomic analysis of the ribosomal fraction following TOR inactivation revealed a lower phosphorylation of the conserved Ser240 residue in the C-terminal region of the 40S ribosomal protein S6 (RPS6). These results were confirmed by Western blot analysis using an antibody that specifically recognizes phosphorylated Ser240 in RPS6. Finally, this antibody was used to follow TOR activity in plants. Our results thus uncover a multi-level regulation of plant ribosomal genes and proteins by the TOR kinase. PMID:27877176

  20. The Arabidopsis TOR Kinase Specifically Regulates the Expression of Nuclear Genes Coding for Plastidic Ribosomal Proteins and the Phosphorylation of the Cytosolic Ribosomal Protein S6.

    PubMed

    Dobrenel, Thomas; Mancera-Martínez, Eder; Forzani, Céline; Azzopardi, Marianne; Davanture, Marlène; Moreau, Manon; Schepetilnikov, Mikhail; Chicher, Johana; Langella, Olivier; Zivy, Michel; Robaglia, Christophe; Ryabova, Lyubov A; Hanson, Johannes; Meyer, Christian

    2016-01-01

    Protein translation is an energy consuming process that has to be fine-tuned at both the cell and organism levels to match the availability of resources. The target of rapamycin kinase (TOR) is a key regulator of a large range of biological processes in response to environmental cues. In this study, we have investigated the effects of TOR inactivation on the expression and regulation of Arabidopsis ribosomal proteins at different levels of analysis, namely from transcriptomic to phosphoproteomic. TOR inactivation resulted in a coordinated down-regulation of the transcription and translation of nuclear-encoded mRNAs coding for plastidic ribosomal proteins, which could explain the chlorotic phenotype of the TOR silenced plants. We have identified in the 5' untranslated regions (UTRs) of this set of genes a conserved sequence related to the 5' terminal oligopyrimidine motif, which is known to confer translational regulation by the TOR kinase in other eukaryotes. Furthermore, the phosphoproteomic analysis of the ribosomal fraction following TOR inactivation revealed a lower phosphorylation of the conserved Ser240 residue in the C-terminal region of the 40S ribosomal protein S6 (RPS6). These results were confirmed by Western blot analysis using an antibody that specifically recognizes phosphorylated Ser240 in RPS6. Finally, this antibody was used to follow TOR activity in plants. Our results thus uncover a multi-level regulation of plant ribosomal genes and proteins by the TOR kinase.

  1. Epidermal growth factor-dependent association of phosphatidylinositol 3-kinase with the erbB3 gene product.

    PubMed

    Kim, H H; Sierke, S L; Koland, J G

    1994-10-07

    The ErbB3 protein is a member of the ErbB subfamily of receptor protein tyrosine kinases. In the present study, the mechanism by which the ErbB3 protein is phosphorylated and the signal-transducing functions of this phosphorylated protein were investigated. When phosphorylated by the epidermal growth factor receptor in vitro, the ErbB3 protein strongly associated with the regulatory p85 subunit and the catalytic activity of phosphatidylinositol (PI) 3-kinase. The association of PI 3-kinase with ErbB3 in human breast cancer cells was found to be correlated with the constitutive phosphorylation of ErbB3 on tyrosine residues. In MDA-MB-468 breast cancer cells in which the ErbB3 protein is not constitutively phosphorylated, stimulation with epidermal growth factor led to the phosphorylation of ErbB3 on tyrosine residues and the formation of a functional signal transduction complex involving the ErbB3 protein and PI 3-kinase. These results suggest that the ErbB3 protein can be phosphorylated on tyrosine residues by a cross-phosphorylation mechanism and that the phosphorylated ErbB3 protein can couple other growth factor receptor protein tyrosine kinases to the PI 3-kinase pathway in a manner similar to the insulin receptor substrate 1 protein.

  2. Dominant and recessive mutations in the Raf-like kinase HT1 gene completely disrupt stomatal responses to CO2 in Arabidopsis

    PubMed Central

    Hashimoto-Sugimoto, Mimi; Negi, Juntaro; Monda, Keina; Higaki, Takumi; Isogai, Yasuhiro; Nakano, Toshiaki; Hasezawa, Seiichiro; Iba, Koh

    2016-01-01

    HT1 (HIGH LEAF TEMPERATURE 1) is the first component associated with changes in stomatal aperture in response to CO2 to be isolated by forward genetic screening. The HT1 gene encodes a protein kinase expressed mainly in guard cells. The loss-of-function ht1-1 and ht1-2 mutants in Arabidopsis thaliana have CO2-hypersensitive stomatal closure with concomitant reductions in their kinase activities in vitro. In addition to these mutants, in this study we isolate or obtaine five new ht1 alleles (ht1-3, ht1-4, ht1-5, ht1-6, and ht1-7). Among the mutants, only ht1-3 has a dominant mutant phenotype and has widely opened stomata due to CO2 insensitivity. The ht1-3 mutant has a missense mutation affecting a non-conserved residue (R102K), whereas the other six recessive mutants have mutations in highly conserved residues in the catalytic domains required for kinase activity. We found that the dominant mutation does not affect the expression of HT1 or the ability to phosphorylate casein, a universal kinase substrate, but it does affect autophosphorylation activity in vitro. A 3D structural model of HT1 also shows that the R102 residue protrudes from the surface of the kinase, implying a role for the formation of oligomers and/or interaction with its targets. We demonstrate that both the loss-of-function and gain-of-function ht1 mutants have completely disrupted CO2 responses, although they have normal responses to ABA. Furthermore, light-induced stomatal opening is smaller in ht1-3 and much smaller in ht1-2. Taken together, these results indicate that HT1 is a critical regulator for CO2 signaling and is partially involved in the light-induced stomatal opening pathway. PMID:27034327

  3. Identification of the In Vivo Casein Kinase II Phosphorylation Site within the Homeodomain of the Cardiac Tisue-Specifying Homeobox Gene Product Csx/Nkx2.5

    PubMed Central

    Kasahara, Hideko; Izumo, Seigo

    1999-01-01

    Csx/Nkx2.5, a member of the homeodomain-containing transcription factors, serves critical developmental functions in heart formation in vertebrates and nonvertebrates. In this study the putative nuclear localization signal (NLS) of Csx/Nkx2.5 was identified by site-directed mutagenesis to the amino terminus of the homeodomain, which is conserved in almost all homeodomain proteins. When the putative NLS of Csx/Nkx2.5 was mutated a significant amount of the cytoplasmically localized Csx/Nkx2.5 was unphosphorylated, in contrast to the nuclearly localized Csx/Nkx2.5, which is serine- and threonine-phosphorylated, suggesting that Csx/Nkx2.5 phosphorylation is regulated, at least in part, by intracellular localization. Tryptic phosphopeptide mapping indicated that Csx/Nkx2.5 has at least five phosphorylation sites. Using in-gel kinase assays, we detected a Csx/Nkx2.5 kinase whose molecular mass is approximately 40 kDa in both cytoplasmic and nuclear extracts. Mutational analysis and in vitro kinase assays suggested that this 40-kDa Csx/Nkx2.5 kinase is a catalytic subunit of casein kinase II (CKII) that phosphorylates the serine residue between the first and second helix of the homeodomain. This CKII site is phosphorylated in vivo. CKII-dependent phosphorylation of the homeodomain increased Csx/Nkx2.5 DNA binding. Serine-to-alanine mutation at the CKII phosphorylation site reduced transcriptional activity when the carboxyl-terminal repressor domain was deleted. Although the precise biological function of Csx/Nkx2.5 phosphorylation by CKII remains to be determined, it may play an important role, as this CKII phosphorylation site within the homeodomain is fully conserved in all known members of the NK2 family of the homeobox genes. PMID:9858576

  4. ZmCPK1, a calcium-independent kinase member of the Zea mays CDPK gene family, functions as a negative regulator in cold stress signalling.

    PubMed

    Weckwerth, Philipp; Ehlert, Britta; Romeis, Tina

    2015-03-01

    Calcium-dependent protein kinases (CDPKs) have been shown to play important roles in plant environmental stress signal transduction. We report on the identification of ZmCPK1 as a member of the maize (Zea mays) CDPK gene family involved in the regulation of the maize cold stress response. Based upon in silico analysis of the Z. mays cv. B73 genome, we identified that the maize CDPK gene family consists of 39 members. Two CDPK members were selected whose gene expression was either increased (Zmcpk1) or decreased (Zmcpk25) in response to cold exposure. Biochemical analysis demonstrated that ZmCPK1 displays calcium-independent protein kinase activity. The C-terminal calcium-binding domain of ZmCPK1 was sufficient to mediate calcium independency of a previously calcium-dependent enzyme in chimeric ZmCPK25-CPK1 proteins. Furthermore, co-transfection of maize mesophyll protoplasts with active full-length ZmCPK1 suppressed the expression of a cold-induced marker gene, Zmerf3 (ZmCOI6.21). In accordance, heterologous overexpression of ZmCPK1 in Arabidopsis thaliana yielded plants with altered acclimation-induced frost tolerance. Our results identify ZmCPK1 as a negative regulator of cold stress signalling in maize.

  5. Sulfotransferase 1A1 Arg(213)His polymorphism and prostate cancer risk.

    PubMed

    Arslan, Serdal; Silig, Yavuz; Pinarbasi, Hatice

    2011-11-01

    Sulfotransferase 1A1 (SULT1A1) is a member of the sulfotransferase family that plays an important role in the biotransformation of numerous carcinogenic and mutagenic compounds through sulfation. A transition, G to A at position 638, in the SULT1A1 gene, results in the Arg(213)His change. This single nucleotide polymorphism reduces the activity and thermostability of the SULT1A1 enzyme. In the present study, the relationship between the SULT1A1 Arg(213)His polymorphism and prostate cancer was investigated using PCR-RFLP. No significant difference in genotype and allele distribution was noted between the prostate cancer and control populations (P=0.072; P=0.099, respectively). The risk of prostate cancer in individuals carrying the SULT1A1(*)2 allele (His(213) allele) was determined by combining the SULT1A1(*)1/SULT1A1(*)2 (Arg/His(213)) and SULT1A1(*)2/SULT1A1(*)2 (His/His(213)) genotypes. No association was observed between SULT1A1 Arg(213)His polymorphism and prostate cancer incidence (P=0.24; OR, 1.36; 95% CI, 0.84-2.25). However, the His(213) allele was found to increase the risk of prostate cancer by 1.36-fold. In smoker and non-smoker populations, no significant relationship was determined between the prostate cancer and control population (P=0.45; P=0.34, respectively).

  6. Non-small cell lung cancer as a target disease for herpes simplex type 1 thymidine kinase-ganciclovir gene therapy.

    PubMed

    Määttä, Ann-Marie; Tenhunen, Anni; Pasanen, Tiina; Meriläinen, Outi; Pellinen, Riikka; Mäkinen, Kimmo; Alhava, Esko; Wahlfors, Jarmo

    2004-04-01

    Lung cancer is a group of diseases that are difficult to cure and new treatment modalities, like gene therapy are actively tested to find alternatives for currently used strategies. Herpes simplex virus thymidine kinase/ganciclovir (HSV-TK/GCV) method is one of the most frequently utilized forms of gene therapy and it has been tested on lung cancer, but no systematic study with comparison of different lung cancer types has been published. In this study, we examined in vitro and in vivo how good targets non-small cell lung cancer (NSCLC) cell lines representing adenocarcinoma, squamous cell lung cancer and large cell lung cancer are for adenovirus-mediated HSV-TK/GCV gene therapy. By using an adenovirus vector carrying a fusion gene of HSV-TK and green fluorescent protein (GFP), we found that: a) adenoviruses were efficient gene transfer vehicles for all types of NSCLCs; b) all adenocarcinoma and large cell lung cancer cells were good targets for HSV-TK/GCV therapy, whereas one of the squamous cell carcinoma cell lines was not responsive to the treatment; c) bystander effect played a major role in the success of this gene therapy form; d) subcutaneous tumors representing all three NSCLC types were efficiently treated with adenovirus-mediated HSV-TK/GCV gene therapy. In summary, this form of gene therapy appeared to be efficient treatment for human NSCLC and these results warrant further studies with primary lung cancer cells and orthotopic lung tumor models.

  7. Molecular cloning and characterization of two novel genes from hexaploid wheat that encode double PR-1 domains coupled with a receptor-like protein kinase.

    PubMed

    Lu, Shunwen; Faris, Justin D; Edwards, Michael C

    2017-04-01

    Hexaploid wheat (Triticum aestivum L.) contains at least 23 TaPr-1 genes encoding the group 1 pathogenesis-related (PR-1) proteins as identified in our previous work. Here, we report the cloning and characterization of TaPr-1-rk1 and TaPr-1-rk2, two novel genes closely related to the wheat PR-1 family. The two TaPr-1-rk genes are located on homoeologous chromosomes 3D and 3A, respectively, and each contains a large open reading frame (7385 or 6060 bp) that is interrupted by seven introns and subjected to alternative splicing (AS) with five or six isoforms of mRNA transcripts. The deduced full-length TaPR-1-RK1 and TaPR-1-RK2 proteins (95% identity) contain two repeat PR-1 domains, the second of which is fused via a transmembrane helix to a serine/threonine kinase catalytic (STKc) domain characteristic of receptor-like protein kinases. Phylogenetic analysis indicated that the two PR-1 domains of the TaPR-1-RK proteins form sister clades with their homologues identified in other monocot plants and are well separated from stand-alone PR-1 proteins, whereas the STKc domains may have originated from cysteine-rich receptor-like kinases (CRKs). Reverse-transcriptase-PCR analysis revealed that the TaPr-1-rk genes are predominantly expressed in wheat leaves and their expression levels are elevated in response to pathogen attack, such as infection by barley stripe mosaic virus (BSMV), and also to stress conditions, most obviously, to soil salinity. This is the first report of PR-1-CRK hybrid proteins in wheat. The data may shed new insights into the function/evolutionary origin of the PR-1 family and the STKc-mediated defense/stress response pathways in plants.

  8. Transcriptional responses to loss or gain of function of the leucine-rich repeat kinase 2 (LRRK2) gene uncover biological processes modulated by LRRK2 activity

    PubMed Central

    Nikonova, Elena V.; Xiong, Yulan; Tanis, Keith Q.; Dawson, Valina L.; Vogel, Robert L.; Finney, Eva M.; Stone, David J.; Reynolds, Ian J.; Kern, Jonathan T.; Dawson, Ted M.

    2012-01-01

    Mutations in the leucine-rich repeat kinase 2 gene (LRRK2) are the most common genetic cause of Parkinson's disease (PD) and cause both autosomal dominant familial and sporadic PD. Currently, the physiological and pathogenic activities of LRRK2 are poorly understood. To decipher the biological functions of LRRK2, including the genes and pathways modulated by LRRK2 kinase activity in vivo, we assayed genome-wide mRNA expression in the brain and peripheral tissues from LRRK2 knockout (KO) and kinase hyperactive G2019S (G2019S) transgenic mice. Subtle but significant differences in mRNA expression were observed relative to wild-type (WT) controls in the cortex, striatum and kidney of KO animals, but only in the striatum in the G2019S model. In contrast, robust, consistent and highly significant differences were identified by the direct comparison of KO and G2019S profiles in the cortex, striatum, kidney and muscle, indicating opposite effects on mRNA expression by the two models relative to WT. Ribosomal and glycolytic biological functions were consistently and significantly up-regulated in LRRK2 G2019S compared with LRRK2 KO tissues. Genes involved in membrane-bound organelles, oxidative phosphorylation, mRNA processing and the endoplasmic reticulum were down-regulated in LRRK2 G2019S mice compared with KO. We confirmed the expression patterns of 35 LRRK2-regulated genes using quantitative reverse transcription polymerase chain reaction. These findings provide the first description of the transcriptional responses to genetically modified LRRK2 activity and provide preclinical target engagement and/or pharmacodynamic biomarker strategies for LRRK2 and may inform future therapeutic strategies for LRRK2-associated PD. PMID:21972245

  9. β Adrenergic Receptor Kinase C-Terminal Peptide Gene-Therapy Improves β2-Adrenergic Receptor-Dependent Neoangiogenesis after Hindlimb Ischemia.

    PubMed

    Cannavo, Alessandro; Liccardo, Daniela; Lymperopoulos, Anastasios; Gambino, Giuseppina; D'Amico, Maria Loreta; Rengo, Franco; Koch, Walter J; Leosco, Dario; Ferrara, Nicola; Rengo, Giuseppe

    2016-02-01

    After hindlimb ischemia (HI), increased catecholamine levels within the ischemic muscle can cause dysregulation of β2-adrenergic receptor (β2AR) signaling, leading to reduced revascularization. Indeed, in vivo β2AR overexpression via gene therapy enhances angiogenesis in a rat model of HI. G protein-coupled receptor kinase 2 (GRK2) is a key regulator of βAR signaling, and β adrenergic receptor kinase C-terminal peptide (βARKct), a peptide inhibitor of GRK2, has been shown to prevent βAR down-regulation and to protect cardiac myocytes and stem cells from ischemic injury through restoration of β2AR protective signaling (i.e., protein kinase B/endothelial nitric oxide synthase). Herein, we tested the potential therapeutic effects of adenoviral-mediated βARKct gene transfer in an experimental model of HI and its effects on βAR signaling and on endothelial cell (EC) function in vitro. Accordingly, in this study, we surgically induced HI in rats by femoral artery resection (FAR). Fifteen days of ischemia resulted in significant βAR down-regulation that was paralleled by an approximately 2-fold increase in GRK2 levels in the ischemic muscle. Importantly, in vivo gene transfer of the βARKct in the hindlimb of rats at the time of FAR resulted in a marked improvement of hindlimb perfusion, with increased capillary and βAR density in the ischemic muscle, compared with control groups. The effect of βARKct expression was also assessed in vitro in cultured ECs. Interestingly, ECs expressing the βARKct fenoterol, a β2AR-agonist, induced enhanced β2AR proangiogenic signaling and increased EC function. Our results suggest that βARKct gene therapy and subsequent GRK2 inhibition promotes angiogenesis in a model of HI by preventing ischemia-induced β2AR down-regulation.

  10. Effects of doxepin on gene expressions of Bcl-2 family, TNF-α, MAP kinase 14, and Akt1 in the hippocampus of rats exposed to stress.

    PubMed

    Reisi, Parham; Eidelkhani, Nastaran; Rafiee, Laleh; Kazemi, Mohammad; Radahmadi, Maryam; Alaei, Hojjatallah

    2017-02-01

    Stress is one of the effective factors in the development of depressive disorders that performs some parts of its effects by affecting hippocampus. Since doxepin has been shown to have neuroprotective effects, in this study, we focused on the effects of doxepin on the expression of involved genes in neuronal survival and plasticity in the rat hippocampus following chronic stress. Male Wistar rats were divided into four groups, the control, the stress, the stress-doxepin 1 mg/kg and the stress-doxepin 5 mg/kg, respectively. To induce stress, the rats were placed within adjustable restraint chambers for 6 h/day, for 21 days. Before daily induction of the stress, rats received an i.p. injection of doxepin. At the end of experiments, expression of Bax, Bad, Bcl-2, tumor necrosis factor alpha (TNF-α), mitogen-activated protein kinase 14 (MAPK14) and serine-threonine protein kinase AKT1 genes were detected by reverse transcription polymerase chain reaction (RT-PCR) in the hippocampus. Results showed significant enhancements in expression of Bax, Bad and Bcl-2 genes in the stressed rats, whereas expression of TNF-α, MAPK14, and AKT1 genes didn't show significant differences. Doxepin could decrease the expression of Bax and Bad genes in the stress group, but had no significant effects on the expression of other genes. The present findings indicated that doxepin can probably change the pattern of gene expression in the hippocampus to maintain neurons against destructive effects of stress.

  11. Effects of doxepin on gene expressions of Bcl-2 family, TNF-α, MAP kinase 14, and Akt1 in the hippocampus of rats exposed to stress

    PubMed Central

    Reisi, Parham; Eidelkhani, Nastaran; Rafiee, Laleh; Kazemi, Mohammad; Radahmadi, Maryam; Alaei, Hojjatallah

    2017-01-01

    Stress is one of the effective factors in the development of depressive disorders that performs some parts of its effects by affecting hippocampus. Since doxepin has been shown to have neuroprotective effects, in this study, we focused on the effects of doxepin on the expression of involved genes in neuronal survival and plasticity in the rat hippocampus following chronic stress. Male Wistar rats were divided into four groups, the control, the stress, the stress-doxepin 1 mg/kg and the stress-doxepin 5 mg/kg, respectively. To induce stress, the rats were placed within adjustable restraint chambers for 6 h/day, for 21 days. Before daily induction of the stress, rats received an i.p. injection of doxepin. At the end of experiments, expression of Bax, Bad, Bcl-2, tumor necrosis factor alpha (TNF-α), mitogen-activated protein kinase 14 (MAPK14) and serine-threonine protein kinase AKT1 genes were detected by reverse transcription polymerase chain reaction (RT-PCR) in the hippocampus. Results showed significant enhancements in expression of Bax, Bad and Bcl-2 genes in the stressed rats, whereas expression of TNF-α, MAPK14, and AKT1 genes didn’t show significant differences. Doxepin could decrease the expression of Bax and Bad genes in the stress group, but had no significant effects on the expression of other genes. The present findings indicated that doxepin can probably change the pattern of gene expression in the hippocampus to maintain neurons against destructive effects of stress. PMID:28255309

  12. Synergism between paraoxonase Arg 192 and the angiotensin converting enzyme D allele is associated with severity of coronary artery disease.

    PubMed

    Vaisi-Raygani, Asad; Rahimi, Zohreh; Tavilani, Haidar; Vaisi-Raygani, Hadiss; Kiani, A; Aminian, M; Shakiba, E; Shakiba, Y; Pourmotabbed, Tayebeh

    2012-03-01

    We have previously shown that angiotensin-converting enzyme (ACE) gene D allele is an independent risk factor for early onset coronary artery disease (CAD). Little is known about the concomitant presence of the ACE gene D allele and paraoxonase (PON1) codon 192 arginine (Arg) on the severity of CAD. Regarding the high rate of CAD among Iranians the aim of present study was to examine the hypothesis of synergistic effects between ACE-D and PON1-Arg alleles on predisposition and the severity of CAD in our population. The PON1 192 and ACE insertion/deletion (I/D) genotypes were detected by PCR-RFLP and PCR, respectively in 414 individuals undergoing their first coronary angiography. Patients were placed into one of two groups: CAD and control without CAD or diabetes. We mentioned the synergistic effects of both genes and not ACE gene alone is a risk factor for CAD. We found that PON1 Arg 192 and ACE D allele act synergistically to increase the risk of CAD (OR 1.3, P = 0.044). Our results showed a significant correlation between the possession of both PON1 192 Arg and the ACE D allele and the extent of CAD in CAD patients and CAD subjects without diabetes, represented by the increased frequency of three-vessel disease with OR 2.7, P = 0.046; χ(2) = 4, P = 0.046 and OR 2.4, P = 0.051; χ(2) = 3.8, P = 0.051, respectively. We found that PON1 Arg 192 and ACE D alleles act synergistically to increase the risk of CAD in CAD patients and CAD subjects without diabetes from west of Iran, who have high frequency of three-vessel disease. Our data suggest that PON1 192 Arg and the ACE D allele in combination with each other can be important independent risk factor for severity of CAD in patients carrying both PON1 192 Arg and the ACE D allele in a west population of Iran.

  13. P53 codon 72 Arg/Pro polymorphism and lung cancer risk in Asians: an updated meta-analysis.

    PubMed

    Wang, Siyang; Lan, Xingang; Tan, Sheng; Wang, Siwen; Li, Yu

    2013-10-01

    The polymorphism of p53 codon 72, a transversion of G to C (Arg to Pro), has been demonstrated to be associated with the risk for lung cancer. However, individual studies conducted in Asians have provided conflicting and inconclusive findings. Thus, we performed a meta-analysis by pooling all currently available case-control studies to estimate the effect of p53 codon 72 Arg/Pro polymorphism on the development of lung cancer. The pooled odds ratios (ORs) with the corresponding 95 % confidence intervals (95 %CIs) were calculated to assess this effect. A total of 14 individual studies involving 7,929 cases and 5,924 controls were included into this meta-analysis according to the inclusion criteria. The overall OR for the dominant genetic model indicated that the p53 codon 72 Arg/Pro variant was positively correlated with lung cancer risk (ORArg/Pro + Pro/Pro vs. Arg/Arg = 1.14, 95 %CI 1.07-1.23, P OR < 0.001). Similar results were found in the stratified analysis of population-based studies. The histological types of lung cancer and smoking status seemed to exert no effect on the lung cancer risk. Sensitivity analysis confirmed the stability of the above findings. The updated meta-analysis suggests that the p53 codon 72 Arg/Pro polymorphism is a risk factor for lung cancer in the Asian population. However, the potential role of gene-environment interaction in lung cancer susceptibility needs further investigation in future studies with high quality.

  14. Effect of ZIP2 Gln/Arg/Leu (rs2234632) polymorphism on zinc homeostasis and inflammatory response following zinc supplementation.

    PubMed

    Giacconi, Robertina; Costarelli, Laura; Malavolta, Marco; Cardelli, Maurizio; Galeazzi, Roberta; Piacenza, Francesco; Gasparini, Nazzarena; Basso, Andrea; Mariani, Erminia; Fulop, Tamas; Rink, Lothar; Dedoussis, George; Herbein, Georges; Jajte, Jolanta; Provinciali, Mauro; Busco, Franco; Mocchegiani, Eugenio

    2015-01-01

    Zinc dyshomeostasis may lead to an augmented production of proinflammatory cytokines promoting chronic inflammation and increasing the susceptibility to age-related diseases. Several studies suggest that the zinc transporter protein ZIP2 may play a relevant role in the immune system especially during zinc deficiency, while a polymorphism on the coding region of ZIP2 gene (Gln/Arg/Leu) has been associated with severe carotid artery disease. The aim of this study is to investigate the role of ZIP2 SNP on zinc and inflammatory status in 1090 elderly healthy free-living subjects enrolled in the ZincAge project and to assess the effect of zinc supplementation on zinc status, inflammatory mediators, and zinc transporter expression depending on ZIP2 genotype. ZIP2 Leu- (Arg43Arg) carriers showed enhanced IL-6, TNF-α, and RANTES plasma levels associated with decreased free cytosolic zinc in PBMCs and an upregulation of zinc transporters ZIP2, ZIP8, and Znt1. Moreover, Leu- subjects displayed significant decrement of inflammatory mediators such as MCP-1, TNF-α, and RANTES following zinc supplementation. In summary, this investigation provides new evidence on the effect of ZIP2 Gln/Arg/Leu polymorphism on proinflammatory mediators and zinc homeostasis in elderly population with a more pronounced anti-inflammatory effect of zinc supplementation in subjects carrying ZIP2 Leu- (Arg43Arg) genotype. These novel findings could be useful in identifying elderly subjects who may benefit of zinc intervention to decrease the inflammatory status and to prevent or delay the development of age-related diseases.

  15. GATA2 germline mutations impair GATA2 transcription, causing haploinsufficiency: functional analysis of the p.Arg396Gln mutation.

    PubMed

    Cortés-Lavaud, Xabier; Landecho, Manuel F; Maicas, Miren; Urquiza, Leire; Merino, Juana; Moreno-Miralles, Isabel; Odero, María D

    2015-03-01

    Germline GATA2 mutations have been identified as the cause of familial syndromes with immunodeficiency and predisposition to myeloid malignancies. GATA2 mutations appear to cause loss of function of the mutated allele leading to haploinsufficiency; however, this postulate has not been experimentally validated as the basis of these syndromes. We hypothesized that mutations that are translated into abnormal proteins could affect the transcription of GATA2, triggering GATA2 deficiency. Chromatin immunoprecipitation and luciferase assays showed that the human GATA2 protein activates its own transcription through a specific region located at -2.4 kb, whereas the p.Thr354Met, p.Thr355del, and p.Arg396Gln germline mutations impair GATA2 promoter activation. Accordingly, GATA2 expression was decreased to ∼58% in a patient with p.Arg396Gln, compared with controls. p.Arg396Gln is the second most common mutation in these syndromes, and no previous functional analyses have been performed. We therefore analyzed p.Arg396Gln. Our data show that p.Arg396Gln is a loss-of-function mutation affecting DNA-binding ability and, as a consequence, it fails to maintain the immature characteristics of hematopoietic stem and progenitor cells, which could result in defects in this cell compartment. In conclusion, we show that human GATA2 binds to its own promoter, activating its transcription, and that the aforementioned mutations impair the transcription of GATA2. Our results indicate that they can affect other GATA2 target genes, which could partially explain the variability of symptoms in these diseases. Moreover, we show that p.Arg396Gln is a loss-of-function mutation, which is unable to retain the progenitor phenotype in cells where it is expressed.

  16. Expression analysis of sugarcane shaggy-like kinase (SuSK) gene identified through cDNA subtractive hybridization in sugarcane (Saccharum officinarum L.).

    PubMed

    Patade, Vikas Yadav; Rai, Archana Neeraj; Suprasanna, Penna

    2011-07-01

    Identification of genes whose expression enables plants to adapt to any kind of stresses is integral to developing stress tolerance in crop plants. In this study, PCR-based cDNA suppression subtractive hybridization technique was used to construct sugarcane salt (NaCl) stress specific forward and reverse subtracted cDNA library. For this, mRNAs were pooled from the shoot and root tissues stressed with NaCl (200 mM) for various time intervals (0.5 to 18 h). Sequencing the clones from the forward subtracted cDNA library, we identified shaggy-like protein kinase (hereafter referred as sugarcane shaggy-like protein kinase, SuSK; NCBI GenBank EST database Acc: FG804674). The sequence analysis of the SuSK revealed homology to Arabidopsis thaliana shaggy-related protein kinase delta (E value, 1e(-108)), dzeta and iota. Alignment of the catalytic domain sequence of GSK-3/shaggy-like kinase with partial sequence of SuSK performed using ClustalW tool indicated kinase active-site signature sequence. Spatial and temporal transcript expression profiling of the SuSK gene based on Real-Time PCR revealed significant induction of transcript expression in response to short-term salt (NaCl 200 mM) or polyethylene glycol-8,000 (PEG; 20% w/v) induced osmotic stress in leaves and shoots of sugarcane plants. The transcript expression increased progressively under salt stress and reached to 1.5-fold of the control up to 8 h treatment. In response to PEG stress, the transcript expression increased by 1.5-fold over the control in 2-h treatment in leaf, whereas in shoots, the expression remained unchanged in response to the various treatments. Differences in growth parameters, relative water content, and membrane damage rate were statistically insignificant in the short-term salt or PEG-stressed plants as compared to the control, non-stressed plants. Expression analysis revealed the differential and temporal regulation of this gene under salt and PEG stress and that its early induction may

  17. Transcription of the protein kinase C-δ gene is activated by JNK through c-Jun and ATF2 in response to the antican