Science.gov

Sample records for gene arg kinase

  1. Global Regulation of Differential Gene Expression by c-Abl/Arg Oncogenic Kinases.

    PubMed

    Dong, Qincai; Li, Chenggong; Qu, Xiuhua; Cao, Cheng; Liu, Xuan

    2017-05-30

    BACKGROUND Studies have found that c-Abl oncogenic kinases may regulate gene transcription by RNA polymerase II phosphorylation or by direct regulation of specific transcription factors or coactivators. However, the global regulation of differential gene expression by c-Abl/Arg is largely unknown. In this study, differentially expressed genes (DEGs) regulated by c-Abl/Arg were identified, and related cellular functions and associated pathways were investigated. MATERIAL AND METHODS RNA obtained from wild-type and c-Abl/Arg gene-silenced MCF-7 cells was analyzed by RNA-Seq. DEGs were identified using edgeR software and partially validated by qRT-PCR. Gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses were used to explore the potential functions of these DEGs. RESULTS A total of 1,034 DEGs were significantly regulated by c-Abl/Arg (399 were up-regulated and 635 were down-regulated after c-Abl/Arg double knockdown). GO and KEGG analyses showed that the DEGs were primarily involved in cellular metabolic processes, neurodegenerative disease, the metabolic process and signaling pathway of cAMP, angiogenesis, and cell proliferation. CONCLUSIONS Our data collectively support the hypothesis that c-Abl/Arg regulate differential gene expression, providing new insights into the biological functions of c-Abl and Arg.

  2. 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.

  3. 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.

  4. 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.

  5. 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.

  6. 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.

  7. 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. © 2012 Elsevier Inc. All rights reserved.

  8. FGFR-4 Arg388 enhances prostate cancer progression via extracellular signal-related kinase and serum response factor signaling

    PubMed Central

    Yu, Wendong; Feng, Shu; Dakhova, Olga; Creighton, Chad J.; Cai, Yi; Wang, Jianghua; Li, Rile; Frolov, Anna; Ayala, Gustavo; Ittmann, Michael

    2011-01-01

    Purpose Increased expression of FGFR-4 and its ligands have been linked to lethal prostate cancer (PCa). Furthermore, a germline polymorphism in the FGFR-4 gene, resulting in arginine at codon 388 (Arg388) instead of glycine (Gly388), is associated with aggressive disease. The FGFR-4 Arg388 variant results in increased receptor stability, sustained receptor activation and increased motility and invasion compared to Gly388. However, the impact of sustained signaling on cellular signal transduction pathways is unknown. Experimental Design Expression microarray analysis of immortalized prostatic epithelial cells lines expressing FGFR-4 Arg388 or Gly388 was used to establish a gene signature associated with FGFR-4 Arg388 expression. Transient transfection of reporters and inhibitors were used to establish the pathways activated by FGFR-4 Arg388 expression. The impact of pathway knockdown in vitro and in an orthotopic model was assessed using inhibitors and/or shRNA. Results Expression of the FGFR-4 Arg388 protein leads to increased activity of the extracellular signal-related kinase (ERK) pathway, increased activity of serum response factor (SRF) and AP1 and transcription of multiple genes which are correlated with aggressive clinical behavior in PCa. Increased expression of SRF is associated with biochemical recurrence in men undergoing radical prostatectomy. Consistent with these observations, knockdown of FGFR-4 Arg388 in PCa cells decreases proliferation and invasion in vitro and primary tumor growth and metastasis in vivo. Conclusions These studies define a signal transduction pathway downstream of FGFR-4 Arg388 that acts via ERK and SRF to promote prostate cancer progression. PMID:21622724

  9. 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.

  10. Occurrence and temporal variation of antibiotic resistance genes (ARGs) in shrimp aquaculture: ARGs dissemination from farming source to reared organisms.

    PubMed

    Su, Haochang; Liu, Shan; Hu, Xiaojuan; Xu, Xiangrong; Xu, Wujie; Xu, Yu; Li, Zhuojia; Wen, Guoliang; Liu, Yousheng; Cao, Yucheng

    2017-12-31

    Considerable attention has been paid to the occurrence and abundance of antibiotic resistance genes (ARGs) in aquatic environments. However, the temporal variation and dissemination of ARGs in aquaculture environments and reared organisms need further study. This study investigated the abundance and diversity of ARGs and bacterial community in water source, shrimp pond water, sediment, and shrimps during the rearing period in Pearl River Delta region, South China. The results showed that sul1, qnrD, cmlA, and floR were the predominant ARGs in the aquaculture samples. A trend of decreasing abundance of ARGs was observed for pond water samples during the rearing period, whereas an increasing trend was observed in the sediment and shrimp samples. The total concentration of ARGs in water source was significantly higher than that in shrimp pond water (p<0.05). A significant negative correlation was found between the total concentrations of ARGs in pond waters and sediments (p<0.01). The total abundances of ARGs in intestinal tract of adult shrimps were 4.48-19.0 times higher than those in juvenile shrimps. Similar to water source and pond water, cmlA and sul1 were the predominant ARGs in shrimp intestinal tract. The bacterial community in the shrimp intestinal tract changed greatly from juvenile to adult. The results of the present study indicated that the abundances of ARGs in aquaculture varied temporally during the rearing period. Water source was an important medium disseminating ARGs to the aquaculture environments and reared organisms. Sul1 could be used as a potential indicator for ARGs in both water and sediment in aquaculture in the estuary of the Pearl River Delta, South China. This study represents a case study for the temporal variation of abundance and dissemination of ARGs in aquaculture and is a reference for potential risks to food safety and human health. Copyright © 2017 Elsevier B.V. All rights reserved.

  11. 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…

  12. 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…

  13. 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.

  14. FGFR-4 Arg³⁸⁸ enhances prostate cancer progression via extracellular signal-related kinase and serum response factor signaling.

    PubMed

    Yu, Wendong; Feng, Shu; Dakhova, Olga; Creighton, Chad J; Cai, Yi; Wang, Jianghua; Li, Rile; Frolov, Anna; Ayala, Gustavo; Ittmann, Michael

    2011-07-01

    Increased expression of FGFR-4 and its ligands have been linked to lethal prostate cancer (PCa). Furthermore, a germ line polymorphism in the FGFR-4 gene, resulting in arginine at codon 388 (Arg³⁸⁸) instead of glycine (Gly³⁸⁸), is associated with aggressive disease. The FGFR-4 Arg³⁸⁸ variant results in increased receptor stability, sustained receptor activation, and increased motility and invasion compared with Gly³⁸⁸. However, the impact of sustained signaling on cellular signal transduction pathways is unknown. Expression microarray analysis of immortalized prostatic epithelial cells lines expressing FGFR-4 Arg³⁸⁸ or Gly³⁸⁸ was used to establish a gene signature associated with FGFR-4 Arg³⁸⁸ expression. Transient transfection of reporters and inhibitors was used to establish the pathways activated by FGFR-4 Arg³⁸⁸ expression. The impact of pathway knockdown in vitro and in an orthotopic model was assessed using inhibitors and/or short hairpin RNA (shRNA). Expression of the FGFR-4 Arg³⁸⁸ protein leads to increased activity of the extracellular signal-related kinase (ERK) pathway, increased activity of serum response factor (SRF) and AP1, and transcription of multiple genes that are correlated with aggressive clinical behavior in PCa. Increased expression of SRF is associated with biochemical recurrence in men undergoing radical prostatectomy. Consistent with these observations, knockdown of FGFR-4 Arg³⁸⁸ in PCa cells decreases proliferation and invasion in vitro and primary tumor growth and metastasis in vivo. These studies define a signal transduction pathway downstream of FGFR-4 Arg³⁸⁸ that acts via ERK and SRF to promote PCa progression.

  15. 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.

  16. 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.

  17. ARGs-OAP: online analysis pipeline for antibiotic resistance genes detection from metagenomic data using an integrated structured ARG-database.

    PubMed

    Yang, Ying; Jiang, Xiaotao; Chai, Benli; Ma, Liping; Li, Bing; Zhang, Anni; Cole, James R; Tiedje, James M; Zhang, Tong

    2016-08-01

    Environmental dissemination of antibiotic resistance genes (ARGs) has become an increasing concern for public health. Metagenomics approaches can effectively detect broad profiles of ARGs in environmental samples; however, the detection and subsequent classification of ARG-like sequences are time consuming and have been severe obstacles in employing metagenomic methods. We sought to accelerate quantification of ARGs in metagenomic data from environmental samples. A Structured ARG reference database (SARG) was constructed by integrating ARDB and CARD, the two most commonly used databases. SARG was curated to remove redundant sequences and optimized to facilitate query sequence identification by similarity. A database with a hierarchical structure (type-subtype-reference sequence) was then constructed to facilitate classification (assigning ARG-like sequence to type, subtype and reference sequence) of sequences identified through similarity search. Utilizing SARG and a previously proposed hybrid functional gene annotation pipeline, we developed an online pipeline called ARGs-OAP for fast annotation and classification of ARG-like sequences from metagenomic data. We also evaluated and proposed a set of criteria important for efficiently conducting metagenomic analysis of ARGs using ARGs-OAP. Perl script for ARGs-OAP can be downloaded from https://github.com/biofuture/Ublastx_stageone ARGs-OAP can be accessed through http://smile.hku.hk/SARGs zhangt@hku.hk or tiedjej@msu.edu Supplementary data are available at Bioinformatics online. © The Author 2016. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

  18. 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.

  19. Characterization of Aspartate Kinase from Corynebacterium pekinense and the Critical Site of Arg169

    PubMed Central

    Min, Weihong; Li, Huiying; Li, Hongmei; Liu, Chunlei; Liu, Jingsheng

    2015-01-01

    Aspartate kinase (AK) is the key enzyme in the biosynthesis of aspartate-derived amino acids. Recombinant AK was efficiently purified and systematically characterized through analysis under optimal conditions combined with steady-state kinetics study. Homogeneous AK was predicted as a decamer with a molecular weight of ~48 kDa and a half-life of 4.5 h. The enzymatic activity was enhanced by ethanol and Ni2+. Moreover, steady-state kinetic study confirmed that AK is an allosteric enzyme, and its activity was inhibited by allosteric inhibitors, such as Lys, Met, and Thr. Theoretical results indicated the binding mode of AK and showed that Arg169 is an important residue in substrate binding, catalytic domain, and inhibitor binding. The values of the kinetic parameter Vmax of R169 mutants, namely, R169Y, R169P, R169D, and R169H AK, with l-aspartate as the substrate, were 4.71-, 2.25-, 2.57-, and 2.13-fold higher, respectively, than that of the wild-type AK. Furthermore, experimental and theoretical data showed that Arg169 formed a hydrogen bond with Glu92, which functions as the entrance gate. This study provides a basis to develop new enzymes and elucidate the corresponding amino acid production. PMID:26633359

  20. Characterization of Aspartate Kinase from Corynebacterium pekinense and the Critical Site of Arg169.

    PubMed

    Min, Weihong; Li, Huiying; Li, Hongmei; Liu, Chunlei; Liu, Jingsheng

    2015-11-27

    Aspartate kinase (AK) is the key enzyme in the biosynthesis of aspartate-derived amino acids. Recombinant AK was efficiently purified and systematically characterized through analysis under optimal conditions combined with steady-state kinetics study. Homogeneous AK was predicted as a decamer with a molecular weight of ~48 kDa and a half-life of 4.5 h. The enzymatic activity was enhanced by ethanol and Ni(2+). Moreover, steady-state kinetic study confirmed that AK is an allosteric enzyme, and its activity was inhibited by allosteric inhibitors, such as Lys, Met, and Thr. Theoretical results indicated the binding mode of AK and showed that Arg169 is an important residue in substrate binding, catalytic domain, and inhibitor binding. The values of the kinetic parameter Vmax of R169 mutants, namely, R169Y, R169P, R169D, and R169H AK, with l-aspartate as the substrate, were 4.71-, 2.25-, 2.57-, and 2.13-fold higher, respectively, than that of the wild-type AK. Furthermore, experimental and theoretical data showed that Arg169 formed a hydrogen bond with Glu92, which functions as the entrance gate. This study provides a basis to develop new enzymes and elucidate the corresponding amino acid production.

  1. Association of Arg194Trp, Arg280His and Arg399Gln Polymorphisms in X-ray Repair Cross-Complementing Group 1 Gene and Risk of Differentiated Thyroid Carcinoma in Iran

    PubMed Central

    Fard-Esfahani, Pezhman; Fard-Esfahani, Armaghan; Fayaz, Shima; Ghanbarzadeh, Bahareh; Saidi, Parinaz; Mohabati, Reyhaneh; Bidoki, Seyed Kazem; Majdi, Mina

    2011-01-01

    Background: X-ray repair cross-complementing group 1 (XRCC1) gene is a DNA repair gene and its non-synonymous single nucleotide polymorphisms (SNP) may influence DNA repair capacity which has been considered as a modifying risk factor for cancer development. Methods: A case-control study was conducted to investigate impact of three frequently studied polymorphisms (Arg194Trp, Arg280His and Arg399Gln) on developing differentiated thyroid carcinoma (DTC). Results: Increased risks for DTC were shown in homozygous (odds ratio [OR]: 3.66, 95% confidence interval [CI]: 0.38-35.60) and in dominant trait (OR: 1.22, 95% CI: 1.64-2.32) of Arg194Trp genotype. Also, for Arg280His genotype, an increased risk for DTC was shown in dominant trait (OR: 1.42, 95% confidence interval [CI]: 0.76-2.68), while a mildly reduction of risk for DTC (OR: 0.77, 95% [CI]: 0.50-1.17) was estimated in dominant Gln genotype of Arg399Gln. Considering combinatory effects of Arg194Trp and Arg280His genotypes on DTC, the calculated OR and 95% CI for being heterozygous for one of Arg194Trp or Arg280His genotypes were 1.57 and 0.90-2.74, respectively. Conclusion: Genotyping of codons 194, 280 and 399 in XRCC1 gene may use in risk assessment of DTC. PMID:21987112

  2. 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.

  3. 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

  4. 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.

  5. [Distribution, dissemination and removal of antibiotic resistant genes (ARGs) in the aquatic environment].

    PubMed

    Wen, Han-qing; Shi, Jun; Xun, Hao; Deng, Hui-ping

    2015-02-01

    In recent years, the intensive use of antibiotics induces the development of antibiotic resistant genes (ARGs), which is an increasingly critical problem affecting human health, and the potential toxic effects of the ARGs have drawn great attention all over the world. This review gave an overview of the occurrence, potential sources, fate and ecological risks of ARGs in the environment. What's more, the removal of ARGs by different treatment processes such as sludge digestion, constructed wetland, disinfection and advanced treatments were assessed, and the improving directions of different treatment processes were also pointed out. Additionally, the highlights in need for further research were proposed based on the current pollution status.

  6. The Abl and Arg Kinases Mediate Distinct Modes of Phagocytosis and Are Required for Maximal Leishmania Infection

    PubMed Central

    McMahon-Pratt, Diane; Koleske, Anthony J.

    2012-01-01

    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 (Mϕs). 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 Mϕs, while that of C3bi-coated beads is unaffected. Conversely, uptake of C3bi-coated beads is decreased in Abl-deficient Mϕs, but that of IgG-coated beads is unaffected. Consistent with these results, Abl-deficient Mϕs are inefficient at C3bi-opsonized promastigote uptake, and Arg-deficient Mϕs 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. PMID:22665498

  7. 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.

  8. 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.

  9. 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

  10. IP3-4 kinase Arg1 regulates cell wall homeostasis and surface architecture to promote clearance of Cryptococcus neoformans infection in a mouse model.

    PubMed

    Li, Cecilia; Lev, Sophie; Desmarini, Desmarini; Kaufman-Francis, Keren; Saiardi, Adolfo; Silva, Ana P G; Mackay, Joel P; Thompson, Philip E; Sorrell, Tania C; Djordjevic, Julianne T

    2017-10-04

    We previously identified a series of inositol polyphosphate kinases (IPKs), Arg1, Ipk1, Kcs1 and Asp1, in the opportunistic fungal pathogen Cryptococcus neoformans. Using gene deletion analysis, we characterized Arg1, Ipk1 and Kcs1 and showed that they act sequentially to convert IP3 to PP-IP5 (IP7), a key metabolite promoting stress tolerance, metabolic adaptation and fungal dissemination to the brain. We have now directly characterized the enzymatic activity of Arg1, demonstrating that it is a dual specificity (IP3/IP4) kinase producing IP5. We showed previously that IP5 is further phosphorylated by Ipk1 to produce IP6, which is a substrate for the synthesis of PP-IP5 by Kcs1. Phenotypic comparison of the arg1Δ and kcs1Δ deletion mutants (both PP-IP5-deficient) reveals that arg1Δ has the most deleterious phenotype: while PP-IP5 is essential for metabolic and stress adaptation in both mutant strains, PP-IP5 is dispensable for virulence-associated functions such as capsule production, cell wall organization, and normal N-linked mannosylation of the virulence factor, phospholipase B1, as these phenotypes were defective only in arg1Δ. The more deleterious arg1Δ phenotype correlated with a higher rate of arg1Δ phagocytosis by human peripheral blood monocytes and rapid arg1Δ clearance from lung in a mouse model. This observation is in contrast to kcs1Δ, which we previously reported establishes a chronic, confined lung infection. In summary, we show that Arg1 is the most crucial IPK for cryptococcal virulence, conveying PP-IP5-dependent and novel PP-IP5-independent functions.

  11. 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.

  12. Effect of the ARG1 gene on arsenic resistance of 293T cells.

    PubMed

    Mu, X L; Li, L; Li, H

    2013-12-19

    To study the relationship between arsenic resistance of 293T cells and overexpression of ARG1, the ARG1 gene in a recombinant plasmid was transfected into 293T cells via liposomes, and then ARG1 overexpression was examined by real-time PCR and immunocytochemistry. The survival rate, arsenic accumulation and arsenic efflux, GSH level, and GST activity of 293T cells overexpressing ARG1 were assayed by MTT, atomic absorption spectrophotometry, and DTNB, and expression of MRP-2 was detected by Western blot analysis. Compared to that in the control cells, the survival rate of ARG1 gene-overexpressing cells was much higher following exposure to lower sodium arsenite (≤ 8 µM). When cells were exposed to lower sodium arsenite for 24 h, the arsenite content of ARG1 gene-overexpressing cells decreased and arsenic efflux increased. After 48 h, the GSH level, GST activity, and expression of MRP2 increased in a concentration-dependent manner. We conclude that the ARG1 gene increases arsenic resistance of 293T cells.

  13. 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

  14. Arg-16 and Arg-21 in the N-terminal region of the triple-gene-block protein 1 of Bamboo mosaic virus are essential for virus movement.

    PubMed

    Lin, Ming-Kuem; Chang, Ban-Yang; Liao, Jia-Teh; Lin, Na-Sheng; Hsu, Yau-Heiu

    2004-01-01

    The protein encoded by the first gene of the triple gene block (TGBp1) of potexviruses is required for movement of the viruses. It has been reported that single Arg-->Ala substitutions at position 11, 16 or 21 of TGBp1 of Bamboo mosaic virus (BaMV) eliminate its RNA-binding activity, while substitutions at position 16 or 21 only affect its NTPase activity (Liou et al., Virology 277, 336-344, 2000). However, it remains unclear whether these Arg-->Ala substitutions also affect the movement of BaMV in plants. To address this question, six mutants of BaMV, each containing either a single- or a double-alanine substitution at Arg-11, Arg-16 and Arg-21 of TGBp1, were constructed and used to infect Chenopodium quinoa and Nicotiana benthamiana. We found that all of the BaMV mutants were able to replicate in protoplasts of N. benthamiana. However, only the mutant with an Arg-11-->Ala substitution in TGBp1 remained capable of movement from cell to cell in plants. Mutants with Arg-16, Arg-21 or both Arg-16 and Arg-21 of TGBp1 replaced with alanine were defective in virus movement. This defect was suppressed when a wild-type TGBp1 allele was co-introduced into the cells using a novel satellite replicon. The ability to trans-complement the movement defect by the wild-type TGBp1 strongly suggests that the Arg-->Ala substitution at position 16 or 21 of TGBp1, which diminishes the RNA-binding and NTPase activities of TGBp1, also eliminates the capability of BaMV to move from cell to cell in host plants.

  15. 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

  16. 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.

  17. 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.

  18. Expression of the Rice Arginase Gene OsARG in Cotton Influences the Morphology and Nitrogen Transition of Seedlings

    PubMed Central

    Zhang, Rui; Liang, Chengzhen; Wan, Jianmin; Wang, Yanling; Zhai, Honghong; Guo, Sandui

    2015-01-01

    Arginase is the only enzyme capable of producing urea in plants. This enzyme also contributes to many important biological functions during plant growth and development, such as seed development, root development and plant nitrogen using. The unique rice arginase gene OsARG is known to affect nitrogen use efficiency and is also associated with higher yields in rice. In this study, we transformed OsARG into upland cotton R18 by Agrobacterium-mediated genetic transformation and analyzed the function of OsARG in transgenic cotton. Two independent OsARG expression transgenic cotton lines, ARG-26 and ARG-38, were obtained via transformation. Southern blot analysis indicated that two copies and one copy of the OsARG gene were integrated into the ARG-26 and ARG-38 genomes, respectively. Enzyme activity and RNA transcription analysis revealed that the OsARG gene is highly expressed in cotton. The nitric oxide content and the morphology of ARG-26 and ARG-38 seedlings were both affected by expression of the OsARG gene. Field experiments indicated that the polyamine and nitrogen content increased by more than two-fold in the T3 generation plants of the transgenic cotton lines ARG-26-2, ARG-26-7, ARG-38-8, and ARG-38-11, as compared with the control plants. After harvesting cotton fibers grown in field conditions, we analyzed the quality of fiber and found that the fiber length was increased in the transgenic lines. The average cotton fiber length for all of the transgenic cotton lines was two millimeters longer than the fibers of the control plants; the average cotton fiber lengths were 31.94 mm, 32.00 mm, 32.68 mm and 32.84 mm in the ARG-26ARG-26-2, ARG-26-7, ARG-38-8 and ARG-38-11 lines, respectively, but the average fiber length of the control plants was 29.36mm. Our results indicate that the OsARG gene could potentially be used to improve cotton fiber length traits. PMID:26528551

  19. [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.

  20. 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

  1. Analysis of expression of the argC and argD genes in the cyanobacterium Anabaena sp. strain PCC 7120.

    PubMed Central

    Floriano, B; Herrero, A; Flores, E

    1994-01-01

    A cloned DNA fragment from Anabaena sp. strain PCC 7120 that complements an arginine auxotrophic mutant from the same organism was found to include an open reading frame encoding a 427-residue polypeptide that is homologous to N-acetylornithine aminotransferase from Bacillus subtilis, Escherichia coli, and Saccharomyces cerevisiae. The gene encoding N-acetylornithine aminotransferase in bacteria has been named argD. The expression of Anabaena sp. strain PCC 7120 argD, as well as of argC, was analyzed at the mRNA level. Both genes were transcribed as monocistronic mRNAs, and their expression was not affected by exogenously added arginine. Primer extension analysis identified transcription start points for both genes which were preceded by sequences similar to that of the E. coli RNA polymerase sigma 70 consensus promoter. A second transcription start point for the argD gene that is not preceded by a sigma 70 consensus promoter was detected in dinitrogen-grown cultures. Images PMID:7929012

  2. 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.

  3. 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.

  4. Cloning, characterization, and nucleotide sequence analysis of the argH gene from Campylobacter jejuni TGH9011 encoding argininosuccinate lyase.

    PubMed Central

    Hani, E K; Chan, V L

    1994-01-01

    The complete structural gene for argininosuccinate lyase (argH) from Campylobacter jejuni TGH9011 has been cloned into Escherichia coli by complementation of an E. coli argH auxotrophic mutant. The gene has been subcloned for sequencing on a 4.1-kb DNA segment and localized by the complementing activity of deletion mutants. The complete DNA sequence of the C. jejuni argH gene was determined. The transcription start point for argH mRNA was determined by primer extension analysis and found to be within the coding sequence of the upstream gene, identified as the phosphoenolpyruvate carboxykinase gene (ppc). The argininosuccinate lyase and the phosphoenolpyruvate carboxykinase reading frames overlap by one base, the second example of this phenomenon in C. jejuni chromosomal genes. The enzyme has a deduced subunit molecular weight of 51,831. Recombinant plasmids containing the argH gene generate a 56-kDa protein and a 43-kDa protein in E. coli maxicells. An alternate translation initiation producing a polypeptide with a deduced molecular mass of 42 kDa may account for the smaller protein observed in sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The C. jejuni argH gene shows nucleotide homology to both yeast and human argininosuccinate lyase genes, and conserved amino acid domains are evident between the corresponding proteins. Images PMID:8144452

  5. The Gly(972)Arg Variant of Human IRS1 Gene Is Associated With Variation in Glomerular Filtration Rate Likely Through Impaired Insulin Receptor Signaling

    PubMed Central

    Thameem, Farook; Puppala, Sobha; Schneider, Jennifer; Bhandari, Basant; Arya, Rector; Arar, Nedal H.; Vasylyeva, Tetyana L.; Farook, Vidya S.; Fowler, Sharon; Almasy, Laura; Blangero, John; Duggirala, Ravindranath; Abboud, Hanna E.

    2012-01-01

    The objective of this study is to identify and characterize the genetic variants related to the glomerular filtration rate (GFR) linkage on 2q37. Of the positional candidate genes, we selected IRS1 and resequenced its 2-kb promoter region and exons for sequence variants in 32 subjects. A total of 11 single nucleotide polymorphisms (SNPs) were identified. To comprehensively cover the 59-kb-long intron-1, eight additional tagging SNPs were selected from the HapMap. All the 19 SNPs were genotyped by TaqMan Assay in the entire data set (N = 670; 39 families). Association analyses between the SNPs and GFR and type 2 diabetes–related traits were performed using the measured genotype approach. Of the SNPs examined for association, only the Gly(972)Arg variant of IRS1 exhibited a significant association with GFR (P = 0.0006) and serum triglycerides levels (P = 0.003), after accounting for trait-specific covariate effects. Carriers of Arg972 had significantly decreased GFR values. Gly(972)Arg contributed to 26% of the linkage signal on 2q. Expression of IRS1 mutant Arg972 in human mesangial cells significantly reduced the insulin-stimulated phosphorylation of IRS1 and Akt kinase. Taken together, the data provide the first evidence that genetic variation in IRS1 may influence variation in GFR probably through impaired insulin receptor signaling. PMID:22617042

  6. 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.

  7. 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

  8. A Strong Promoter Provided with the Gene Encoding Arginyl-tRNA Synthetase(argS) from Escherichia coli.

    PubMed

    Liu, Mo-Fang; Li, Tong; Yin, Zhao-Bao; Xu, Min-Gang; Wang, En-Duo; Wang, Yin-Lai

    2000-01-01

    Previous studies showed that the gene argS encoding the arginyl-tRNA synthetase(ArgRS) from Escherichia coli(E.coli), was overexpressed 1 000 folds in the E.coli transformant TG1/pUC-argS, while the gene leuS, encoding the leucyl-tRNA synthetase(LeuRS) from E.coli, was only overproduced 35-fold in the same case. To investigate why the expression of these two aminoacyl-tRNA synthetase genes is so different, a fused gene (termed parg-leuS) was constructed by replacement of the 5' flanking region of leuS to 5' flanking region of argS. In the E.coli transformant TG1/pUC-parg-leuS, the activity of LeuRS was only improved 8.5-fold, which was much lower than that of the transformant harboring the recombinant plasmid pUC18-leuS or pKK-leuS. However, by RNA dot hybridization the amount of mRNA produced in the transcription of parg-leuS was about 5 times than that of the wild type leuS, and was similar to that of pKK-leuS, suggesting that the promoter of argS is very strong. Analysis of the secondary structure around the initiation codon among three mRNAs showed that the secondary structure of the mRNA from parg-leuS was the strongest of the three mRNAs. From the results, it could be deduced that expression of the fused gene parg-leuS might be controlled at the translational level and the strong secondary structure of this mRNA may hinder translation initiation and result in a low translation efficiency.

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

    PubMed Central

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

    2010-01-01

    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. PMID:20737438

  10. 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

  11. 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.

  12. 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.

  13. 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.

  14. Fate of potential indicator antimicrobial resistance genes (ARGs) and bacterial community diversity in simulated manure-soil microcosms.

    PubMed

    Wang, Mianzhi; Liu, Peng; Xiong, Wenguang; Zhou, Qin; Wangxiao, Junyi; Zeng, Zhenling; Sun, Yongxue

    2017-09-24

    The aim of this study was to investigate the fate of nine potential indicator antimicrobial resistance genes (ARGs) (sul1, sul2, tetB, tetM, ermB, ermF, fexA, cfr, intI1) and the diversity of bacterial communities in response to poultry manure applications to arable soil over a 90 day period. Quantitative real time PCR and Illumina high-throughput sequencing of 16S rDNA gene were used to quantify and trace ARG fate. The levels of all genes dramatically decreased over time and intI1, sul1, sul2 and tetM always had the greatest abundance and lowest dissipation rates. This indicated that more effort should be focused on the ARG elimination from manure rather than waiting for subsequent attenuation in the environment. Our sequencing results documented dramatic changes in the microbial community structure and diversity during these experiments. In poultry manure groups, Bacteroidetes and Actinobacteria were the two dominant phyla while Acidobacteria dominated the control groups. Moreover, the relative abundance of genera Corynebacterium, Pseudomonas, Ochrobactrum, Actinomadura and Bacillus, which contained potential opportunistic pathogens, changed over time suggesting that poultry manure not only strongly influenced bacterial community composition, but also selected specific bacterial communities. This study provides a glimpse of ARG fates and bacterial community diversity in soil after the application of poultry manure. Copyright © 2017 Elsevier Inc. All rights reserved.

  15. [Regional features of obesity-associated gene polymorphism (rs9939609 FTO gene and gene Trp64Arg ADRB3) in Russian population].

    PubMed

    Baturin, A K; Sorokina, E Iu; Pogozheva, A V; Peskova, E V; Makurina, O N; Tutel'ian, V A

    2014-01-01

    Recent studies have shown a significant association with obesity polymorphisms: rs9939609 gene due to fat mass and obesity FTO in European and some Asian and African American populations Trp64Arg ADRB3 gene in several European populations. Association of variants rs9939609 and Trp64Arg obesity was studied in 1244 the inhabitants of Moscow and Sverdlovsk regions. Genotyping was performed using allele-specific amplification, detection results in real time using TaqMan-probes complementary DNA polymorphic sites. The frequency of the mutant allele of the FTO gene in the population of Moscow and Sverdlovsk region was 45.1%, with the TT genotype was detected in 30.2% of cases, AT--49.5%, AA--20.3%. Women had the presence of the mutant allele more likely than men (48.4 vs. 42.5%). People with obesity were more genotypes AA (26.3%) and AT (52.8%) compared to the surveyed with a BMI of less than 30 kg/m2 (respectively 18.1 and 50.7%). A significantly higher incidence of risk allele A was found in individuals with obesity (52.6 and 43.4%). The presence of the mutant allele of the gene ADRB3 among the population of Moscow and Sverdlovsk regions was noted in 7.4% of cases. While 15.5% of patients had a heterozygous genotype Trp64Arg ADRB3, that is consistent with international research. The frequency of the risk allele and genotype Arg64 Trp64Arg in women (9.3 and 18.5%) was significantly higher than men (6.2 and 12.2%). The presence of the mutant allele and genotype Trp64Arg ADRB3 (respectively, 9.1 and 18.1%) were significantly more marked in the examined obese compared with those with a body mass index less than 30 kg/m2 (7.4 and 14.9%), but these differences were not statistically significant. The results of these studies suggest that genetic variants of the FTO gene rs9939609 genotype and Trp64Arg ADRB3 contribute to the development of obesity among residents of Moscow and Sverdlovsk Region of Russia. The risk of obesity increases in the case of combined polymorphisms in

  16. 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.

  17. 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

  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. Searching for Arg201 mutations in the GNAS1 gene in Italian patients with McCune-Albright syndrome.

    PubMed

    de Sanctis, Luisa; Romagnolo, Damiano; Greggio, Nella; Genitori, Lorenzo; Lala, Roberto; de Sanctis, Carlo

    2002-01-01

    McCune-Albright syndrome (MAS) is a rare disease caused by somatic postzygotic mutations at Arg201 in the GNAS1 gene that encodes for the Gsalpha protein. Arg201 mutations are gain-of-function mutations in affected tissues (including bone, skin, endocrine glands and other tissues) that result in the activation of cAMP. We used a polymerase chain reaction(PCR)-based technique for the selective enrichment and analysis of the Arg201 mutant allele in 27 different tissues from 24 Italian patients with one or more signs of MAS. Arg201 mutations were identified in 13 different tissues (48.1%) from 11 patients (45.8%). Mutation detection rates differed across the various types of tissue samples, and the mutation was not always found in every tissue sample from the same patient. To overcome problems in the analysis of mutations in somatic mosaicism, as occurs in MAS, a highly sensitive molecular technique should be applied, the most appropriate tissue source selected, and various affected tissues from the same patient analyzed.

  20. 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).

  1. Relation of Trp64Arg polymorphism of beta 3 adrenoreceptor gene with metabolic syndrome and insulin resistance in obese women.

    PubMed

    De Luis Román, Daniel Antonio; Primo, David; Izaola, Olatz; Aller, Rocío

    2017-03-30

    Trp64Arg variant in beta 3 adrenoreceptor has been reported to be associated with increased body weight and insulin resistance. These risk factors are the ones that make up the so-called metabolic syndrome. The aim of our study was to investigate the relationship between metabolic syndrome and Trp64Arg polymorphism in the beta3 adrenoreceptor gene in obese women. A population of 531 obese women was analyzed in cross-sectional survey. A bioimpedance, blood pressure, a serial assessment of nutritional intake with 3 days written food records and biochemical analysis were performed. Genotype of beta 3 adrenoreceptor gene polymorphism (Trp64Arg) was studied. Prevalence of metabolic syndrome (MS) with ATP III definition was 47.1% (250 patients) and 52.9% patients without MS (n = 281 patients). Prevalence of beta 3 genotypes was similar in patients with metabolic syndrome (87.6% wild genotype and 12.4% mutant genotype) and without metabolic syndrome (87.9% wild genotype and 12.1% mutant genotype). Insulin and HOMA levels were higher in patients with mutant genotype than wild type, in patients with and without metabolic syndrome. In mutant group of beta3 adrenoreceptor gene patients have higher insulin and HOMA levels than wild type group, without relation with metabolic syndrome.

  2. 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

  3. 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.

  4. 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

  5. Trp64Arg polymorphism of the ADRB3 gene associated with maximal fat oxidation and LDL-C levels in non-obese adolescents.

    PubMed

    Jesus, Íncare Correa de; Alle, Lupe Furtado; Munhoz, Eva Cantalejo; Silva, Larissa Rosa da; Lopes, Wendell Arthur; Tureck, Luciane Viater; Purim, Katia Sheylla Malta; Titski, Ana Claudia Kapp; Leite, Neiva

    2017-09-20

    To analyze the association between the Trp64Arg polymorphism of the ADRB3 gene, maximal fat oxidation rates (FATMAX) and the lipid profile levels in non-obese adolescents. 72 schoolchildren, of both genders, aged between 11 and 17 years, participated in the study. The anthropometric and body composition variables, in addition to total cholesterol (TC), high density lipoprotein (HDL-c), low density lipoprotein (LDL-c), triglycerides (TG), insulin (INS), and basal glycemia (GLIC), were evaluated. The sample was divided into two groups according to the presence or absence of the polymorphism: non-carriers of the Arg64 allele, i.e., homozygous (Trp64Trp: n=54), and carriers of the Arg64 allele (Trp64Arg+Arg64Arg: n=18), in which the frequency of the Arg64 allele was 15.2%. The maximal oxygen uptake and peak of oxygen uptake during exercise were obtained through the symptom-limited, submaximal treadmill test. Maximal fat oxidation (FATMAX) was determined according to the ventilatory ratio proposed in Lusk's table. Adolescents carrying the less frequent allele (Trp64Arg and Arg64Arg) had higher LDL-c levels (p=0.031) and lower FATMAX rates (p=0.038) when compared with non-carriers (Trp64Trp). Although the physiological processes related to lipolysis and lipid metabolism are complex, the presence of the Arg 64 allele was associated with lower rates of FATMAX during aerobic exercise, as well as with higher levels of LDL-c in adolescents. Copyright © 2017 Sociedade Brasileira de Pediatria. Published by Elsevier Editora Ltda. All rights reserved.

  6. 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

  7. Identification of ArgP and Lrp as Transcriptional Regulators of lysP, the Gene Encoding the Specific Lysine Permease of Escherichia coli▿†

    PubMed Central

    Ruiz, Jimena; Haneburger, Ina; Jung, Kirsten

    2011-01-01

    Expression of lysP, which encodes the lysine-specific transporter LysP in Escherichia coli, is regulated by the concentration of exogenous available lysine. In this study, the LysR-type transcriptional regulator ArgP was identified as the activator of lysP expression. At lysine concentrations higher than 25 μM, lysP expression was shut off and phenocopied an argP deletion mutant. Purified ArgP-His6 bound to the lysP promoter/control region at a sequence containing a conserved T-N11-A motif. Its affinity increased in the presence of lysine but not in the presence of the other known coeffector, arginine. In vivo data suggest that lysine-loaded ArgP and arginine-loaded ArgP compete at the lysP promoter. We propose that lysine-loaded ArgP prevents lysP transcription at the promoter clearance step, as described for the lysine-dependent regulation of argO (R. S. Laishram and J. Gowrishankar, Genes Dev. 21:1258-1272, 2007). The global regulator Lrp also bound to the lysP promoter/control region. An lrp mutant exhibited reduced lysP expression in the absence of external lysine. These results indicate that ArgP is a major regulator of lysP expression but that Lrp modulates lysP transcription under lysine-limiting conditions. PMID:21441513

  8. 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.

  9. 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

  10. 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.

  11. XRCC-1 Gene Polymorphism (Arg399Gln) and Susceptibility to Development of Lung Cancer in Cohort of North Indian Population: A Pilot Study

    PubMed Central

    Mehndiratta, Mohit; Mohapatra, Debabratta; Grover, Rajesh K; Puri, Dinesh

    2014-01-01

    Background: Smoking has been considered to be the major cause of lung cancer. However, only a fraction of cigarette smokers develop this disease. This suggests the importance of genetic constitution in predicting the individual’s susceptibility towards lung cancer. This genetic susceptibility may result from inherited polymorphisms in genes controlling carcinogen metabolism and repair of damaged deoxyribonucleic acid (DNA). These repair systems are fundamental to the maintenance of genomic integrity. X-ray repair cross complimenting group I (XRCC1), a major DNA repair gene in the base excision repair (BER) pathway. It is involved in repair by interacting with components of DNA at the site of damage. Inconsistent results have been reported regarding the associations between the Arg399Gln polymorphism of XRCC1. This study demonstrates the importance of recognition of this relationship of lung carcinoma and genetic constitution of the person which will help guide clinicians on the optimal screening of this disease. Aim: To assess the role of XRCC1 gene polymorphism (Arg399Gln) directly on the variation in susceptibility to development of lung cancer in North Indian subjects. Materials and Methods: One hundred males with diagnosed cases of lung cancer were recruited from Delhi State Cancer Institute (DSCI). Hundred healthy volunteers were taken as controls. DNA isolation was done and Polymerase chain reaction–Restriction Fragment Length Polymorphism (PCR–RFLP) procedure undertaken to amplify the region containing Arg/Gln substitution at codon 399 (in exon 10). Results: XRCC1 gene polymorphism is associated with increased risk of lung cancer when the Arg/Arg genotype was used as the reference group. The Arg/Gln and Gln/Gln was associated with statistically increased risk for cancer. Conclusion: Arg399Gln polymorphism in XRCC1 gene polymorphism is associated with lung cancer in North Indian subjects and screening for this polymorphism will help in targeting

  12. 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.

  13. Development and dissection of diagnostic SNP markers for the downy mildew resistance genes PlArg and Pl8 and maker-assisted gene pyramiding in sunflower (Helianthus annuus L.)

    USDA-ARS?s Scientific Manuscript database

    Downy mildew, which is caused by fungus Plasmopara halstedii (Farl.) Berlese & de Toni, is one of the most important diseases that affect sunflower production globally. Two downy mildew resistance genes, PlArg and Pl8, were discovered in the late 1980s. Over two decades, PlArg is still effective aga...

  14. 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

  15. 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.

  16. 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.

  17. 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.

  18. Gene encoding γ-carbonic anhydrase is cotranscribed with argC and induced in response to stationary phase and high CO2 in Azospirillum brasilense Sp7

    PubMed Central

    2010-01-01

    Background Carbonic anhydrase (CA) is a ubiquitous enzyme catalyzing the reversible hydration of CO2 to bicarbonate, a reaction underlying diverse biochemical and physiological processes. Gamma class carbonic anhydrases (γ-CAs) are widespread in prokaryotes but their physiological roles remain elusive. At present, only γ-CA of Methanosarcina thermophila (Cam) has been shown to have CA activity. Genome analysis of a rhizobacterium Azospirillum brasilense, revealed occurrence of ORFs encoding one β-CA and two γ-CAs. Results One of the putative γ-CA encoding genes of A. brasilense was cloned and overexpressed in E. coli. Electrometric assays for CA activity of the whole cell extracts overexpressing recombinant GCA1 did not show CO2 hydration activity. Reverse transcription-PCR analysis indicated that gca1 in A. brasilense is co-transcribed with its upstream gene annotated as argC, which encodes a putative N-acetyl-γ-glutamate-phosphate reductase. 5'-RACE also demonstrated that there was no transcription start site between argC and gca1, and the transcription start site located upstream of argC transcribed both the genes (argC-gca1). Using transcriptional fusions of argC-gca1 upstream region with promoterless lacZ, we further demonstrated that gca1 upstream region did not have any promoter and its transcription occurred from a promoter located in the argC upstream region. The transcription of argC-gca1 operon was upregulated in stationary phase and at elevated CO2 atmosphere. Conclusions This study shows lack of CO2 hydration activity in a recombinant protein expressed from a gene predicted to encode a γ-carbonic anhydrase in A. brasilense although it cross reacts with anti-Cam antibody raised against a well characterized γ-CA. The organization and regulation of this gene along with the putative argC gene suggests its involvement in arginine biosynthetic pathway instead of the predicted CO2 hydration. PMID:20598158

  19. 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.

  20. 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

  1. A novel mutation Thr162Arg of the melanocortin 4 receptor gene in a Spanish children and adolescent population.

    PubMed

    Ochoa, M C; Azcona, C; Biebermann, H; Brumm, H; Razquin, C; Wermter, A-K; Martínez, J A; Hebebrand, J; Hinney, A; Moreno-Aliaga, M J; Marti, A; Patiño, A; Chueca, M; Oyarzabal, M; Pelach, R

    2007-05-01

    The melanocortin 4 receptor gene (MC4R) is involved in body weight regulation. While many studies associated MC4R mutations with childhood obesity, information on MC4R mutations in Spanish children and adolescents is lacking. Our objective was to screen a population of children and adolescents from the north of Spain (Navarra) for MC4R mutations and to study the phenotypes of carriers and their families. In addition, functional assays were performed for a novel MC4R mutation. The study was composed of 451 Spanish children and adolescents (49% boys), aged 5-18 year. According to the International Obesity Task Force (IOTF) criteria, the groups included 160 obese, 132 overweight and 159 normal-weight control subjects. One novel (Thr162Arg) and three known nonsynonymous mutations in the MC4R gene (Ser30Phe, Thr150Ile, Ala244Glu) were detected heterozygously. The MC4R mutations were found in three male (one obese and two overweight) and two female subjects (one obese and one overweight). The novel mutation did not appear to lead to an impaired receptor function. An unequivocal relationship of MC4R mutations with obesity in pedigrees together with an impaired function of the encoded receptor could not be established for any of the mutations. The presence of heterozygous MC4R mutations in obese and overweight subjects indicates that these mutations may be a susceptibility factor for obesity development, but lifestyle factors, such as exercise or sedentary activities, may modify their effect.

  2. 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.

  3. 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.

  4. 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.

  5. 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.

  6. 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.

  7. [A frame shift mutation, Arg346fs mutation, is identified in cardiac myosin-binding protein C gene in a Chinese family with hypertrophic cardiomyopathy].

    PubMed

    Xie, Wen-li; Liu, Wen-ling; Hu, Da-yi; Cui, Wei; Zhu, Tian-gang; Li, Cui-lan; Sun, Yi-hong; Li, Lei; Bian, Hong

    2005-04-13

    To explore the disease-causing gene mutation in Chinese with hypertrophic cardiomyopathy (HCM). The peripheral venous blood samples were collected from 5 HCM families without consanguinity, including 5 probands, 2 males and 3 females, 28 sporadic HCM patients, 18 males and 10 females, and 80 healthy controls. The exons in the functional regions of cardiac myosin-binding protein C (MYBPC3) were amplified with PCR and the amplified products were sequenced. A frame shift mutation-Arg346fs mutation in exon 13, the first mutation identified in Chinese-was discovered in one family with HCM. However, the members of the same HCM family with the Arg346fs mutation showed differences in phenotype and prognosis. Cardiac myosin-binding protein C (MYBPC3) may be one of the main disease-causing genes. The heterogeneity of phenotype suggests that multiple factors may be involved in the pathogenesis.

  8. [Prevalence of alleles of polymorphic variants Leu33Pro and Leu66Arg gene ITGB3 among inhabitants of Siberia].

    PubMed

    Goncharova, I A; Babushkina, N P; Minaĭcheva, L I; Markova, V V; Kulish, E V; Salakhov, R R; Makeeva, O A; Puzyrev, V P

    2013-08-01

    The frequency of the polymorphic variant T196C (Leu33Pro, rs5918) of ITGB3 gene was studied in several groups of inhabitants of Siberia, including pregnant women with reproductive disorders (n = 186), patients with acute coronary syndrome (n = 330), and population control (n = 858). The frequency of the rare PLA2 allele among residents of Tomsk and Kemerovo was 14.7 and 15.0% respectively. There were no differences in the allele and genotype frequencies of polymorphic variant between patients with acute coronary syndrome and the control group (p = 0.925, p = 0.622). The highest frequency of abnormal PLA2 allele (22.1%) and the PLA2/PLA2 genotype (8.8%) was observed among women, who had miscarried, which was significantly different from the frequency of this allele and genotype in the control group (14.7%, p = 0.017; 2.1%, p = 0.0009). Sequencing showed that all samples with the nonspecific band had the polymorphic rs5918 variant and rs36080296 mutations (T216G, Leu66Arg). The frequency of the rs36080296 mutation among the residents of Siberia was 0.51%. Among the women with reproductive disorders, the frequency of rs36080296 was 2.7%, while in the group who suffered from miscarriages, it was 4.4%; this was different from the frequency in the control group (0.08%, p = 0.2 x 10(-6)). The accumulation of mutations was also observed among men with acute coronary syndrome (0.6%), but the differences from the control group (0%) had no statistical significance.

  9. 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.

  10. [Polymorphic markers Ala455Val of the THBD gene and Arg353Gln of the F7 gene and association with unfavorable outcomes of coronary atherosclerosis in patients with a history of acute ischemic heart disease].

    PubMed

    Pushkov, A A; Blagodatskikh, K A; Nikitin, A G; Agapkina, Iu V; Brovkin, A N; Chudakova, D A; Evdokimova, M A; Aseĭcheva, O Iu; Osmolovskaia, V S; Minushkina, L O; Baklanova, T N; Talyzin, P A; Donetskaia, O P; Tereshchenko, S N; Dzhaiani, N A; Akatova, E A; Glezer, M G; Galiavich, A S; Zakirova, V B; Koziolova, N A; Iagoda, A V; Boeva, O I; Horolets, E V; Shlyk, S V; Volkova, E G; Margarian, M P; Guz', I O; Konstantinov, V O; Sidorenko, B A; Zateĭshchikov, D A; Nosikov, V V

    2011-10-01

    The polymorphic markers Ala455Val of the THBD gene and Arg353Gln of the F7 gene were tested for association with the frequency of unfavorable outcomes in patients with a history of acute ischemic heart disease. The study involved 1145 patients hospitalized in cardiology clinics of Moscow, St. Petersburg, Kazan, Chelyabinsk, Perm, Stavropol, and Rostov-on-Don because of acute ischemic heart disease. The patients were followed up for up to 62.5 months. None of the markers displayed a significant association with the time to an endpoint. The patients were then grouped by sex. In females, the frequency of unfavorable outcomes (fatal or nonfatal myocardial infarction and fatal or nonfatal stroke) was higher in carriers of allele Val of the Ala344Val polymorphic marker of the THBD gene and carriers of genotype Arg/Arg of the Arg353Gln polymorphic marker of the F7 gene, but the difference was not statistically significant. Such an increase in frequency was not observed in males. To study the combined effect of the polymorphic markers of the THBD and F7 genes, the course of ischemic heart disease was compared for two female subgroups. One included carriers of allele Val of the Ala344Val polymorphic marker of the THBD gene and genotype Arg/Arg of the Arg353Gln polymorphic marker of the F7 gene; the other subgroup included carriers ofgenotype Ala/Ala of the Ala455Val polymorphic marker of the THBD gene and allele Gln of the Arg353Gln polymorphic marker of the F7 gene. The frequency of unfavorable outcomes in the first subgroup was higher than in the second one. The time to an endpoin was 40.5 months (95% confidence interval (CI) 33.5-47.6) in the first subgroup and 51.6 months (95% CI 45.0-58.1) in the second subgroup (chi2 = 4.15, P = 0.042). The results made it possible to assume that the F7 and THBD genes play an important role in genetic predisposition to unfavorable outcomes in patients with a history of acute ischemic heart disease.

  11. nArgBP2 regulates excitatory synapse formation by controlling dendritic spine morphology.

    PubMed

    Lee, Sang-Eun; Kim, Yoonju; Han, Jeong-Kyu; Park, Hoyong; Lee, Unghwi; Na, Myeongsu; Jeong, Soomin; Chung, ChiHye; Cestra, Gianluca; Chang, Sunghoe

    2016-06-14

    Neural Abelson-related gene-binding protein 2 (nArgBP2) was originally identified as a protein that directly interacts with synapse-associated protein 90/postsynaptic density protein 95-associated protein 3 (SAPAP3), a postsynaptic scaffolding protein critical for the assembly of glutamatergic synapses. Although genetic deletion of nArgBP2 in mice leads to manic/bipolar-like behaviors resembling many aspects of symptoms in patients with bipolar disorder, the actual function of nArgBP2 at the synapse is completely unknown. Here, we found that the knockdown (KD) of nArgBP2 by specific small hairpin RNAs (shRNAs) resulted in a dramatic change in dendritic spine morphology. Reintroducing shRNA-resistant nArgBP2 reversed these defects. In particular, nArgBP2 KD impaired spine-synapse formation such that excitatory synapses terminated mostly at dendritic shafts instead of spine heads in spiny neurons, although inhibitory synapse formation was not affected. nArgBP2 KD further caused a marked increase of actin cytoskeleton dynamics in spines, which was associated with increased Wiskott-Aldrich syndrome protein-family verprolin homologous protein 1 (WAVE1)/p21-activated kinase (PAK) phosphorylation and reduced activity of cofilin. These effects of nArgBP2 KD in spines were rescued by inhibiting PAK or activating cofilin combined with sequestration of WAVE. Together, our results suggest that nArgBP2 functions to regulate spine morphogenesis and subsequent spine-synapse formation at glutamatergic synapses. They also raise the possibility that the aberrant regulation of synaptic actin filaments caused by reduced nArgBP2 expression may contribute to the manifestation of the synaptic dysfunction observed in manic/bipolar disorder.

  12. 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.

  13. 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.

  14. [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.

  15. 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

  16. 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

  17. 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. © The Author 2016. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

  18. 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.

  19. No association of the neuropeptide Y (Leu7Pro) and ghrelin gene (Arg51Gln, Leu72Met, Gln90Leu) single nucleotide polymorphisms with eating disorders.

    PubMed

    Kindler, Jochen; Bailer, Ursula; de Zwaan, Martina; Fuchs, Karoline; Leisch, Friedrich; Grün, Bettina; Strnad, Alexandra; Stojanovic, Mirjana; Windisch, Julia; Lennkh-Wolfsberg, Claudia; El-Giamal, Nadja; Sieghart, Werner; Kasper, Siegfried; Aschauer, Harald

    2011-06-01

    Genetic factors likely contribute to the biological vulnerability of eating disorders. Case-control association study on one neuropeptide Y gene (Leu7Pro) polymorphism and three ghrelin gene (Arg51Gln, Leu72Met and Gln90Leu) polymorphisms. 114 eating disorder patients (46 with anorexia nervosa, 30 with bulimia nervosa, 38 with binge eating disorder) and 164 healthy controls were genotyped. No differences were detected between patients and controls for any of the four polymorphisms in allele frequency and genotype distribution (P > 0.05). Allele frequencies and genotypes had no significant influence on body mass index (P > 0.05) in eating disorder patients. Positive findings of former case-control studies of associations between ghrelin gene polymorphisms and eating disorders could not be replicated. Neuropeptide Y gene polymorphisms have not been investigated in eating disorders before.

  20. 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.

  1. Deep sequencing of the LRRK2 gene in 14,002 individuals reveals evidence of purifying selection and independent origin of the p.Arg1628Pro mutation in Europe

    PubMed Central

    Rubio, Justin P.; Topp, Simon; Warren, Liling; St Jean, Pamela L.; Wegmann, Daniel; Kessner, Darren; Novembre, John; Shen, Judong; Fraser, Dana; Aponte, Jennifer; Nangle, Keith; Cardon, Lon R.; Ehm, Margaret G.; Chissoe, Stephanie L.; Whittaker, John C.; Nelson, Matthew R.; Mooser, Vincent E.

    2012-01-01

    Genetic variation in LRRK2 predisposes to Parkinson disease (PD), which underpins its development as a therapeutic target. Here, we aimed to identify novel genotype-phenotype associations that might support developing LRRK2 therapies for other conditions. We sequenced the 51 exons of LRRK2 in cases comprising 12 common diseases (n = 9,582), and in 4,420 population controls. We identified 739 single nucleotide variants (SNVs), 62% of which were observed in only one person, including 316 novel exonic variants. We found evidence of purifying selection for the LRRK2 gene and a trend suggesting that this is more pronounced in the central (ROC-COR-kinase) core protein domains of LRRK2 than the flanking domains. Population genetic analyses revealed that LRRK2 is not especially polymorphic or differentiated in comparison to 201 other drug target genes. Amongst Europeans, we identified 17 carriers (0.13%) of pathogenic LRRK2 mutations that were not significantly enriched within any disease or in those reporting a family history of PD. Analysis of pathogenic mutations within Europe reveals that the p.Arg1628Pro (c4883G>C) mutation arose independently in Europe and Asia. Taken together, these findings demonstrate how targeted deep sequencing can help to reveal fundamental characteristics of clinically important loci. PMID:22415848

  2. 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.

  3. Association of Gly972Arg polymorphism of IRS1 gene with type 2 diabetes mellitus in lean participants of a national health survey in Mexico: a candidate gene study.

    PubMed

    Burguete-Garcia, Ana I; Cruz-Lopez, Miguel; Madrid-Marina, Vicente; Lopez-Ridaura, Ruy; Hernández-Avila, Mauricio; Cortina, Bernardo; Gómez, Rosa E; Velasco-Mondragón, Eduardo

    2010-01-01

    Type 2 diabetes mellitus (T2D) is a main public health problem in the Mexican population. It is characterized by insulin resistance in peripheral tissues and a relative deficiency in the pancreatic beta-cell functions. Diverse single nucleotide polymorphisms (SNPs) of the IRS1 gene have been associated with insulin resistance and T2D risk. The aim of this study was to identify the association between known IRS1 polymorphisms (Pro512Ala, Asn1137Asp, Gly972Arg, and Arg158Pro) in a sample of diabetic patients compared with healthy controls selected from Mexico's 2000 National Health Survey, both with normal body mass index (BMI). We identified 444 diabetes cases that were age matched with the same number of controls. Genotypic and allelic frequencies were evaluated, and conditional logistic regression was used to evaluate the association between the SNPs and diabetes risk. Of the 4 SNPs studied, only Gly972Arg showed significant differences between cases and controls, with allele frequency of 2.6% in controls as compared with 7.9% in cases. Subjects with at least 1 copy of the Gly972Arg polymorphism of the IRS1 gene showed a greater risk for diabetes, with a crude odds ratio of 3.26 (95% confidence interval, 2.00-5.33); after adjusting for BMI, age, family history of T2D, and sex, the odds ratio was 2.91 (95% confidence interval, 1.73-4.90). Our results suggest the participation of Gly972Arg polymorphism of IRS1 in the genetic susceptibility to TD2 in Mexican population. The restriction of including only participants with normal BMI might increase the power to detect genetic determinants of T2D.

  4. 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

  5. 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

  6. 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

  7. Ophthalmic findings in a family with early-onset isolated ectopia lentis and the p.Arg62Cys mutation of the fibrillin-1 gene (FBN1).

    PubMed

    Zhao, Jun-Hong; Jin, Tian-Bo; Liu, Qing-Bo; Chen, Chao; Hu, Hai-Tao

    2013-01-01

    The purpose of this paper is to describe ophthalmic findings in a family with isolated ectopia lentis (EL) caused by a specific FBN1 mutation. Detailed family histories and clinical data were recorded for six isolated EL patients of 11 family members. The ophthalmological and systematic examinations were performed on patients and unaffected members of the investigated family. The detailed ocular examinations included visual acuity, anterior chamber depth, pupil size, lens location, optometry, central corneal thickness, keratometry, slitlamp examination, fundus examination, axial length, ocular B-ultrasound, gonioscope checking, ultrasound biomicroscopy (UBM) and intraocular pressure (IOP; Goldmann applanation tonometer). Systematic examinations included the measurement of echocardiogram, height, arm span, skull, face, jaw, tooth, breast bone, spinal column, and skin. Genomic DNA was extracted using the phenol-chloroform extraction method for all subjects, and sequencing was carried out on an ABI Prism 3730 Genetic Analyzer. A heterozygous mutation, c.184C>T (p.Arg62Cys) in exon 2 of FBN1 was identified in all affected members but was not found in any unaffected member of the family. Our study presented detailed clinical manifestations, including some novel ophthalmic findings, such as pupillary abnormality, different types of glaucoma, and progressive hyperopia. Ophthalmic findings and the p.Arg62Cys mutation of FBN1 gene were reported in a family with early-onset isolated ectopia lentis.

  8. 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

  9. 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

  10. 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.

  11. Genome-wide identification of rubber tree (Hevea brasiliensis Muell. Arg.) aquaporin genes and their response to ethephon stimulation in the laticifer, a rubber-producing tissue.

    PubMed

    Zou, Zhi; Gong, Jun; An, Feng; Xie, Guishui; Wang, Jikun; Mo, Yeyong; Yang, Lifu

    2015-11-25

    Natural rubber, an important industrial raw material, is specifically synthesized in laticifers located inside the rubber tree (Hevea brasiliensis Muell. Arg.) trunk. Due to the absence of plasmodesmata, the laticifer water balance is mediated by aquaporins (AQPs). However, to date, the characterization of H. brasiliensis AQPs (HbAQPs) is still in its infancy. In this study, 51 full-length AQP genes were identified from the rubber tree genome. The phylogenetic analysis assigned these AQPs to five subfamilies, including 15 plasma membrane intrinsic proteins (PIPs), 17 tonoplast intrinsic proteins (TIPs), 9 NOD26-like intrinsic proteins (NIPs), 4 small basic intrinsic proteins (SIPs) and 6 X intrinsic proteins (XIPs). Functional prediction based on the analysis of the aromatic/arginine (ar/R) selectivity filter, Froger's positions and specificity-determining positions (SDPs) showed a remarkable difference in substrate specificity among subfamilies. Homology analysis supported the expression of 44 HbAQP genes in at least one of the examined tissues. Furthermore, deep sequencing of the laticifer transcriptome in the form of latex revealed a key role of several PIP subfamily members in the laticifer water balance, and qRT-PCR analysis showed diverse expression patterns of laticifer-expressed HbAQP genes upon ethephon treatment, a widely-used practice for the stimulation of latex yield. This study provides an important genetic resource of HbAQP genes, which will be useful to improve the water use efficiency and latex yield of Hevea.

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

    USDA-ARS?s Scientific Manuscript database

    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. Copyright © 2016 Elsevier Ltd. All rights reserved.

  14. 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.

  15. 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

  16. Early socio-emotional experience induces expression of the immediate-early gene Arc/arg3.1 (activity-regulated cytoskeleton-associated protein/activity-regulated gene) in learning-relevant brain regions of the newborn chick.

    PubMed

    Bock, J; Thode, C; Hannemann, O; Braun, K; Darlison, M G

    2005-01-01

    We have cloned a full-length complementary DNA from the chicken (Gallus gallus domesticus), which encodes a polypeptide that exhibits approximately 75% identity to the product of the mammalian gene Arc (activity-regulated cytoskeleton-associated protein), also known as arg3.1 (activity-regulated gene). Since this gene is an immediate-early gene that has been suggested to play a role in synaptic plasticity and learning and memory processes, its expression has been analyzed in a juvenile form of learning, namely, filial imprinting. Our results demonstrate that Arc/arg3.1 mRNA is detectable in the newborn chick brain, and that at this early age the level of this transcript can be altered by brief sensory/emotional experience. After postnatal exposure to a novel 30-min auditory imprinting stimulus, Arc/arg3.1 mRNA was found to be significantly increased in two higher associative areas, the mesopallium intermediomediale (P = 0.002) and the nidopallium dorso-caudale (P = 0.031), compared with naïve controls. The transcript level was also significantly elevated after imprinting in Area L pallii (P=0.045), which is analogous to the mammalian auditory cortex. In addition, increases were seen in the medio-rostral nidopallium/mesopallium (P = 0.054), which is presumed to be the analog of the mammalian prefrontal cortex, and the hyperpallium intercalatum (P = 0.054), but these did not quite reach significance. We discuss these data in the light of those obtained in an earlier study, in the same paradigm, for the avian immediate-early gene, zenk (an acronym for zif-268, egr-1, ngfi-a and krox-24, which are different names for the orthologous mammalian gene). We conclude that, although both the Arc/arg3.1 and zenk genes are induced by auditory imprinting, they are significantly up-regulated in different learning-relevant brain regions. It is, therefore, evident that they must be activated by different mechanisms.

  17. [A new hereditary cause of portal vein thrombosis: the abnormal resistance to activated protein C by the Arg 506-->Gln mutation of the gene of factor V].

    PubMed

    Levoir, D; Aubertin, J M; Alhenc-Gelas, M; Bloch, F; Becheur, H; Petite, J P

    1995-01-01

    We report 2 cases of portal vein thrombosis associated with a single point mutation in the factor V gene that replaces arginine in residue 506 with glutamine. This mutation induces abnormal resistance to anticoagulant activity of activated protein C and increases the risk of deep vein thrombosis. Both patients had a personal and familial history of deep vein thrombosis. Intraabdominal neoplasia or infection, myeloproliferative disorder, antiphospholipid syndrome, paroxysmal nocturnal hemoglobinuria and coagulation inhibitor deficiency (antithrombin, proteins C and S) were excluded by exhaustive investigation. However, an abnormal resistance to activated protein C was found, and DNA analysis showed the factor V Arg506 to Gln mutation in both cases. Anticoagulant treatment was begun. A study of family history made in one case, showed the same genetic disease in one of the relatives. Resistance to activated protein C with factor V gene mutation should be investigated in patients with portal vein thrombosis. A study of family history, and anticoagulant treatment are justified for symptomatic patients.

  18. The Serum Response Factor and a Putative Novel Transcription Factor Regulate Expression of the Immediate-Early Gene Arc/Arg3.1 in Cultured Cortical Neurons

    PubMed Central

    Pintchovski, Sean A.; Peebles, Carol L.; Kim, Hong Joo; Verdin, Eric; Finkbeiner, Steven

    2010-01-01

    The immediate-early effector gene Arc/Arg3.1 is robustly upregulated by synaptic activity associated with learning and memory. Here we show in primary cortical neuron culture that diverse stimuli induce Arc expression through new transcription. Searching for regulatory regions important for Arc transcription, we found nine DNaseI-sensitive nucleosome-depleted sites at this genomic locus. A reporter gene encompassing these sites responded to synaptic activity in an NMDA receptor–dependent manner, consistent with endogenous Arc mRNA. Responsiveness mapped to two enhancer regions ∼6.5 kb and ∼1.4 kb upstream of Arc. We dissected these regions further and found that the proximal enhancer contains a functional and conserved “Zeste-like” response element that binds a putative novel nuclear protein in neurons. Therefore, activity regulates Arc transcription partly by a novel signaling pathway. We also found that the distal enhancer has a functional and highly conserved serum response element. This element binds serum response factor, which is recruited by synaptic activity to regulate Arc. Thus, Arc is the first target of serum response factor that functions at synapses to mediate plasticity. PMID:19193899

  19. 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.

  20. In vitro transcription of the Escherichia coli K-12 argA, argE, and argCBH operons.

    PubMed Central

    Sens, D; Natter, W; James, E

    1977-01-01

    Deoxyribonucleic acid isolated from argA and argECBH transducing phages was utilized to study the in vitro synthesis of argA, argE, and argCBH messenger ribonucleic acid. The specific regulation of these operons by the arginine holorepressor was demonstrated, providing evidence that the majority, if not all, of the control of these operons is exercised at the transcriptional level. Data are presented which indicate that the arginine holorepressor functions by binding to the operator region and concomitantly prevents the binding of ribonucleic acid polymerase to the corresponding promoter region. PMID:400784

  1. 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.

  2. Safe and Efficient Gene Therapy for Pyruvate Kinase Deficiency

    PubMed Central

    Garcia-Gomez, Maria; Calabria, Andrea; Garcia-Bravo, Maria; Benedicenti, Fabrizio; Kosinski, Penelope; López-Manzaneda, Sergio; Hill, Collin; del Mar Mañu-Pereira, María; Martín, Miguel A; Orman, Israel; Vives-Corrons, Joan-LLuis; Kung, Charles; Schambach, Axel; Jin, Shengfang; Bueren, Juan A; Montini, Eugenio; Navarro, Susana; Segovia, Jose C

    2016-01-01

    Pyruvate kinase deficiency (PKD) is a monogenic metabolic disease caused by mutations in the PKLR gene that leads to hemolytic anemia of variable symptomatology and that can be fatal during the neonatal period. PKD recessive inheritance trait and its curative treatment by allogeneic bone marrow transplantation provide an ideal scenario for developing gene therapy approaches. Here, we provide a preclinical gene therapy for PKD based on a lentiviral vector harboring the hPGK eukaryotic promoter that drives the expression of the PKLR cDNA. This therapeutic vector was used to transduce mouse PKD hematopoietic stem cells (HSCs) that were subsequently transplanted into myeloablated PKD mice. Ectopic RPK expression normalized the erythroid compartment correcting the hematological phenotype and reverting organ pathology. Metabolomic studies demonstrated functional correction of the glycolytic pathway in RBCs derived from genetically corrected PKD HSCs, with no metabolic disturbances in leukocytes. The analysis of the lentiviral insertion sites in the genome of transplanted hematopoietic cells demonstrated no evidence of genotoxicity in any of the transplanted animals. Overall, our results underscore the therapeutic potential of the hPGK-coRPK lentiviral vector and provide high expectations toward the gene therapy of PKD and other erythroid metabolic genetic disorders. PMID:27138040

  3. Safe and Efficient Gene Therapy for Pyruvate Kinase Deficiency.

    PubMed

    Garcia-Gomez, Maria; Calabria, Andrea; Garcia-Bravo, Maria; Benedicenti, Fabrizio; Kosinski, Penelope; López-Manzaneda, Sergio; Hill, Collin; Del Mar Mañu-Pereira, María; Martín, Miguel A; Orman, Israel; Vives-Corrons, Joan-LLuis; Kung, Charles; Schambach, Axel; Jin, Shengfang; Bueren, Juan A; Montini, Eugenio; Navarro, Susana; Segovia, Jose C

    2016-08-01

    Pyruvate kinase deficiency (PKD) is a monogenic metabolic disease caused by mutations in the PKLR gene that leads to hemolytic anemia of variable symptomatology and that can be fatal during the neonatal period. PKD recessive inheritance trait and its curative treatment by allogeneic bone marrow transplantation provide an ideal scenario for developing gene therapy approaches. Here, we provide a preclinical gene therapy for PKD based on a lentiviral vector harboring the hPGK eukaryotic promoter that drives the expression of the PKLR cDNA. This therapeutic vector was used to transduce mouse PKD hematopoietic stem cells (HSCs) that were subsequently transplanted into myeloablated PKD mice. Ectopic RPK expression normalized the erythroid compartment correcting the hematological phenotype and reverting organ pathology. Metabolomic studies demonstrated functional correction of the glycolytic pathway in RBCs derived from genetically corrected PKD HSCs, with no metabolic disturbances in leukocytes. The analysis of the lentiviral insertion sites in the genome of transplanted hematopoietic cells demonstrated no evidence of genotoxicity in any of the transplanted animals. Overall, our results underscore the therapeutic potential of the hPGK-coRPK lentiviral vector and provide high expectations toward the gene therapy of PKD and other erythroid metabolic genetic disorders.

  4. 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.

  5. [Therapeutic effect of focal adhesion kinase gene silence on leukemia].

    PubMed

    Xu, Lü-Hong; Fang, Jian-Pei; Weng, Wen-Jun; Xu, Hong-Gui; Zhang, Ya-Ting

    2011-06-01

    This study was aimed to investigate the effects of focal adhesion kinase (FAK) gene silence on leukemia cell growth, leukemogenesis and efficacy of chemotherapy drug. Vector containing lentiviral-FAK-shRNA was constructed and transfected into BCR/ABL-BaF3 leukemic cells, the cell growth and apoptosis were detected in vitro. The effect of FAK shRNA on leukemogenesis was studied in a murine model with leukemia. The apoptosis of leukemia cells and survival of leukemic mice treated by FAK shRNA combined with drug STI571 were monitored. The results showed that FAK gene expression was knocked down by lentiviral-FAK-shRNA. FAK gene silencing inhibited leukemia cell growth in vitro. The apoptosis test results showed that the percentages of Annexin V(+) cells in vector control group and FAK shRNA group were (3.46 ± 0.56)% and (7.3 ± 0.79)%, respectively, and the difference was statistically significant (p < 0.05). The mice in vector control group died at day 21 to 27, while the mice in FAK shRNA group died between day 52 and 60, and the difference was statistically significant (p < 0.05). Moreover, FAK gene silence combined with drug STI571 could enhance the apoptosis of leukemia cells and prolong survival time of leukemic mice. It is concluded that FAK gene silence inhibits leukemogenesis and promotes efficacy of chemotherapy drug on leukemia cells, indicating FAK gene silence may be considered as a new therapeutic strategy for leukemia.

  6. 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

  7. 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.

  8. Novel Compound Heterozygous Mutations in the Pantothenate Kinase 2 Gene in a Korean Patient with Atypical Pantothenate Kinase Associated Neurodegeneration

    PubMed Central

    Kim, Sung-Hyouk; Sung, Young-Hee; Park, Kee-Hyung; Lee, Yeung-Bae; Park, Hyeon-Mi; Shin, Dong Jin; Kim, Gu-Hwan

    2009-01-01

    Pantothenate kinase-associated neurodegeneration (PKAN) is an autosomal recessive disorder that is characterized by mutations in the pantothenate kinase 2 gene (PANK2) and typical magnetic resonance imaging findings. We report a case of atypical PKAN presenting with generalized dystonia. Our patient had compound heterozygous mutations in the PANK2 gene, including mutation in exon 3 (p.D268G) and exon 4 (p.R330P). To our knowledge, this patient is the first to have the p.R330P mutation and the second to have the p.D268G mutation. PMID:24868354

  9. Novel compound heterozygous mutations in the pantothenate kinase 2 gene in a korean patient with atypical pantothenate kinase associated neurodegeneration.

    PubMed

    Kim, Sung-Hyouk; Sung, Young-Hee; Park, Kee-Hyung; Lee, Yeung-Bae; Park, Hyeon-Mi; Shin, Dong Jin; Kim, Gu-Hwan

    2009-05-01

    Pantothenate kinase-associated neurodegeneration (PKAN) is an autosomal recessive disorder that is characterized by mutations in the pantothenate kinase 2 gene (PANK2) and typical magnetic resonance imaging findings. We report a case of atypical PKAN presenting with generalized dystonia. Our patient had compound heterozygous mutations in the PANK2 gene, including mutation in exon 3 (p.D268G) and exon 4 (p.R330P). To our knowledge, this patient is the first to have the p.R330P mutation and the second to have the p.D268G mutation.

  10. 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.

  11. 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.

  12. 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

  13. 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.

  14. 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.

  15. 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.

  16. 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

  17. 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

  18. 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.

  19. 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

  20. 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.

  1. 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.

  2. Genome-wide identification, phylogeny and expressional profiles of mitogen activated protein kinase kinase kinase (MAPKKK) gene family in bread wheat (Triticum aestivum L.).

    PubMed

    Wang, Meng; Yue, Hong; Feng, Kewei; Deng, Pingchuan; Song, Weining; Nie, Xiaojun

    2016-08-22

    Mitogen-activated protein kinase kinase kinases (MAPKKKs) are the important components of MAPK cascades, which play the crucial role in plant growth and development as well as in response to diverse stresses. Although this family has been systematically studied in many plant species, little is known about MAPKKK genes in wheat (Triticum aestivum L.), especially those involved in the regulatory network of stress processes. In this study, we identified 155 wheat MAPKKK genes through a genome-wide search method based on the latest available wheat genome information, of which 29 belonged to MEKK, 11 to ZIK and 115 to Raf subfamily, respectively. Then, chromosome localization, gene structure and conserved protein motifs and phylogenetic relationship as well as regulatory network of these TaMAPKKKs were systematically investigated and results supported the prediction. Furthermore, a total of 11 homologous groups between A, B and D sub-genome and 24 duplication pairs among them were detected, which contributed to the expansion of wheat MAPKKK gene family. Finally, the expression profiles of these MAPKKKs during development and under different abiotic stresses were investigated using the RNA-seq data. Additionally, 10 tissue-specific and 4 salt-responsive TaMAPKKK genes were selected to validate their expression level through qRT-PCR analysis. This study for the first time reported the genome organization, evolutionary features and expression profiles of the wheat MAPKKK gene family, which laid the foundation for further functional analysis of wheat MAPKKK genes, and contributed to better understanding the roles and regulatory mechanism of MAPKKKs in wheat.

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

    SciTech Connect

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

    1996-08-01

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

  4. 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

  5. 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

  6. 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

  7. 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.

  8. Possible peptide chain termination mutants in thymide kinase gene of a mammalian virus, herpes simplex virus.

    PubMed

    Summers, W P; Wagner, M; Summers, W C

    1975-10-01

    Mutations in the viral gene coding for the thymidine kinase (ATP:thymidine 5'-phosphotransferase, EC 2.7.1.75) induced by herpes simplex virus have been obtained by selection of virus resistant to bromodeoxyuridine when grown in thymidine-kinase-deficient LMTK- mouse cells. Proteins labeled after infection of Vero (monkey) cells with herpes simplex virus were analyzed by gel electrophoresis and one protein of about 40,000 daltons was consistently altered in a number of thymidine-kinase-deficient mutants. Many viral mutants lacked this peptide and one class of these mutants induced the synthesis of new shorter peptides. Revertant virus could be selected which simultaneously regained the ability to induce thymidine kinase activity, regained the intact thymidine kinase peptide, and lost the ability to synthesize the shorter peptide fragment. These mutants comprise a class of animal virus mutants which have the properties expected of peptide chain termination mutants.

  9. Possible peptide chain termination mutants in thymide kinase gene of a mammalian virus, herpes simplex virus.

    PubMed Central

    Summers, W P; Wagner, M; Summers, W C

    1975-01-01

    Mutations in the viral gene coding for the thymidine kinase (ATP:thymidine 5'-phosphotransferase, EC 2.7.1.75) induced by herpes simplex virus have been obtained by selection of virus resistant to bromodeoxyuridine when grown in thymidine-kinase-deficient LMTK- mouse cells. Proteins labeled after infection of Vero (monkey) cells with herpes simplex virus were analyzed by gel electrophoresis and one protein of about 40,000 daltons was consistently altered in a number of thymidine-kinase-deficient mutants. Many viral mutants lacked this peptide and one class of these mutants induced the synthesis of new shorter peptides. Revertant virus could be selected which simultaneously regained the ability to induce thymidine kinase activity, regained the intact thymidine kinase peptide, and lost the ability to synthesize the shorter peptide fragment. These mutants comprise a class of animal virus mutants which have the properties expected of peptide chain termination mutants. Images PMID:172894

  10. 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

  11. Conservation of MAP kinase activity and MSP genes in parthenogenetic nematodes

    PubMed Central

    2010-01-01

    Background MAP (mitogen-activated protein) kinase activation is a prerequisite for oocyte maturation, ovulation and fertilisation in many animals. In the hermaphroditic nematode Caenorhabditis elegans, an MSP (major sperm protein) dependent pathway is utilised for MAP kinase activation and successive oocyte maturation with extracellular MSP released from sperm acting as activator. How oocyte-to-embryo transition is triggered in parthenogenetic nematode species that lack sperm, is not known. Results We investigated two key elements of oocyte-to-embryo transition, MSP expression and MAP kinase signaling, in two parthenogenetic nematodes and their close hermaphroditic relatives. While activated MAP kinase is present in all analysed nematodes irrespective of the reproductive mode, MSP expression differs. In contrast to hermaphroditic or bisexual species, we do not find MSP expression at the protein level in parthenogenetic nematodes. However, genomic sequence analysis indicates that functional MSP genes are present in several parthenogenetic species. Conclusions We present three alternative interpretations to explain our findings. (1) MSP has lost its function as a trigger of MAP kinase activation and is not expressed in parthenogenetic nematodes. Activation of the MAP kinase pathway is achieved by another, unknown mechanism. Functional MSP genes are required for occasionally emerging males found in some parthenogenetic species. (2) Because of long-term disadvantages, parthenogenesis is of recent origin. MSP genes remained intact during this short intervall although they are useless. As in the first scenario, an unknown mechanism is responsible for MAP kinase activation. (3) The molecular machinery regulating oocyte-to-embryo transition in parthenogenetic nematodes is conserved with respect to C. elegans, thus requiring intact MSP genes. However, MSP expression has been shifted to non-sperm cells and is reduced below the detection limits, but is still sufficient to

  12. 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.

  13. 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.

  14. Mutational analysis of the kinase domain of MYLK2 gene in common human cancers.

    PubMed

    Soung, Young Hwa; Lee, Jong Woo; Kim, Su Young; Nam, Suk Woo; Park, Won Sang; Lee, Jung Young; Yoo, Nam Jin; Lee, Sug Hyung

    2006-01-01

    Genetic alterations of the genes encoding protein kinases have been implicated in the development of human cancers. Myosin light chain kinase 2, skeletal muscle (MYLK2) encodes a calcium/calmodulin-dependent serine/threonine kinase. In a recent study, MYLK2 gene was somatically mutated in colorectal carcinomas. The aim of this study was to explore the possibility that other common human carcinomas besides colorectal carcinomas harbored MYLK2 mutations in the kinase domain. We analyzed exons 6 and 7 eccoding the kinase domain of MYLK2 for somatic mutations in 60 gastric, 104 colorectal, 79 non-small cell lung, and 54 breast cancers using a polymerase chain reaction (PCR)-based single-strand conformation polymorphism (SSCP). We found one MYLK2 mutation in lung adenocarcinomas, but not in other cancers. The MYLK2 mutation detected was a missense mutation that would substitute an amino acid (E374D) However, there was no somatic mutation of the MYLK2 gene. These data suggest that the kinase domain of MYLK2 is rarely mutated in common human carcinomas and that it does not play a dominant role in cancer pathogenesis.

  15. The Saccharomyces cerevisiae LSB6 gene encodes phosphatidylinositol 4-kinase activity.

    PubMed

    Han, Gil-Soo; Audhya, Anjon; Markley, Daniel J; Emr, Scott D; Carman, George M

    2002-12-06

    The LSB6 gene product was identified from the Saccharomyces Genome Data Base (locus YJL100W) as a putative member of a novel type II phosphatidylinositol (PI) 4-kinase family. Cell extracts lacking the LSB6 gene had a reduced level of PI 4-kinase activity. In addition, multicopy plasmids containing the LSB6 gene directed the overexpression of PI 4-kinase activity in cell extracts of wild-type cells, in an lsb6Delta mutant, in a pik1(ts) stt4(ts) double mutant, and in an pik1(ts) stt4(ts) lsb6Delta triple mutant. The heterologous expression of the S. cerevisiae LSB6 gene in Escherichia coli resulted in the expression of a protein that possessed PI 4-kinase activity. Although the lsb6Delta mutant did not exhibit a growth phenotype and failed to exhibit a defect in phosphoinositide synthesis in vivo, the overexpression of the LSB6 gene could partially suppress the lethal phenotype of an stt4Delta mutant defective in the type III STT4-encoded PI 4-kinase indicating that Lsb6p functions as a PI 4-kinase in vivo. Lsb6p was localized to the membrane fraction of the cell, and when overexpressed, GFP-tagged Lsb6p was observed on both the plasma membrane and the vacuole membrane. The enzymological properties (pH optimum, dependence on magnesium or manganese as a cofactor, the dependence of activity on Triton X-100, the dependence on the PI surface concentration, and temperature sensitivity) of the LSB6-encoded enzyme were very similar to the membrane-associated 55-kDa PI 4-kinase previously purified from S. cerevisiae.

  16. 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.

  17. 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.

  18. 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.

  19. 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.

  20. [Effects of overexpression of NADH kinase gene on ethanol fermentation by Saccharomyces cerevisiae].

    PubMed

    Wang, Han; Zhang, Liang; Shi, Guiyang

    2014-09-01

    Glycerol is the main byproduct in ethanol production by Saccharomyces cerevisiae. In order to improve ethanol yield and the substrate conversion, a cassette about 4.5 kb for gene homologous recombination, gpd2Δ::PGK1(PT)-POS5-HyBR, was constructed and transformed into the haploid strain S. cerevisiae S1 (MATa) to replace the GPD2 gene by POS5 gene. The NADH kinase gene POS5 was successfully over expressed in the recombinant strain S. cerevisiae S3. Comparing with the parent strain, the recombinant strain S. cerevisiae S3 exhibited an 8% increase in ethanol production and a 33.64% decrease in glycerol production in the conical flask fermentation with an initiatory glucose concentration of 150 g/L. Overexpression of NADH kinase gene seems effective in reducing glycerol production and increasing ethanol yield.

  1. 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.

  2. 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

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

    PubMed

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

    1989-06-01

    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.

  4. 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

  5. Identification of Novel Gene Targets and Functions of p21-Activated Kinase 1 during DNA Damage by Gene Expression Profiling

    PubMed Central

    Motwani, Mona; Li, Da-Qiang; Horvath, Anelia; Kumar, Rakesh

    2013-01-01

    P21-activated kinase 1 (PAK1), a serine/threonine protein kinase, modulates many cellular processes by phosphorylating its downstream substrates. In addition to its role in the cytoplasm, PAK1 also affects gene transcription due to its nuclear localization and association with chromatin. It is now recognized that PAK1 kinase activity and its nuclear translocation are rapidly stimulated by ionizing radiation (IR), and that PAK1 activation is a component of the DNA damage response. Owing to the role of PAK1 in the cell survival, its association with the chromatin, and now, stimulation by ionizing radiation, we hypothesize that PAK1 may be contributing to modulation of genes with roles in cellular processes that might be important in the DNA damage response. The purpose of this study was to identify new PAK1 targets in response to ionizing radiation with putative role in the DNA damage response. We examined the effect of IR on the gene expression patterns in the murine embryonic fibroblasts with or without Pak1 using microarray technology. Differentially expressed transcripts were identified using Gene Spring GX 10.0.2. Pathway, network, functional analyses and gene family classification were carried out using Kyoto Encyclopedia of Genes and Genomes (KEGG), Ingenuity Pathway, Gene Ontology and PANTHER respectively. Selective targets of PAK1 were validated by RT-qPCR. For the first time, we provide a genome-wide analysis of PAK1 and identify its targets with potential roles in the DNA damage response. Gene Ontology analysis identified genes in the IR-stimulated cells that were involved in cell cycle arrest and cell death. Pathway analysis revealed p53 pathway being most influenced by IR responsive, PAK1 targets. Gene family of transcription factors was over represented and gene networks involved in DNA replication, repair and cellular signaling were identified. In brief, this study identifies novel PAK1 dependent IR responsive genes which reveal new aspects of PAK1

  6. Disruption of the mouse inositol 1,3,4,5,6-pentakisphosphate 2-kinase gene, associated lethality, and tissue distribution of 2-kinase expression.

    PubMed

    Verbsky, John; Lavine, Kory; Majerus, Philip W

    2005-06-14

    Many functions have been suggested for inositol 1,2,3,4,5,6-hexakisphosphate (InsP6), including mRNA export, nonhomologous end-joining, endocytosis, and ion channel regulation. However, it remains to be demonstrated that InsP6 is necessary for in vivo survival. We previously isolated a cDNA encoding the mammalian inositol 1,3,4,5,6-pentakisphosphate (InsP5) 2-kinase (2-kinase), the enzyme that converts InsP5 to InsP6. We used the sequence to search the BayGenomics databases and identify an ES cell line (XA232) that has a gene trap construct embedded in the 2-kinase gene. We obtained a mouse from this line, produced heterozygotes, and confirmed that the heterozygotes contain the trapping construct and have diminished 2-kinase activity. Breeding the XA232 heterozygotes produced no homozygous offspring; thus, loss of 2-kinase is lethal in mice. Dissections of embryonic day-8.5 uteri yielded no homozygous embryos; thus, the mice die before day 8.5 postcoitum. The gene trap construct contains a beta-galactosidase/neomycin reporter gene, allowing us to stain heterozygotes for beta-galactosidase to determine tissue-specific expression of 2-kinase protein. 2-kinase is expressed in the hippocampus, the cortex, the Purkinje layer of the cerebellum in the brain, in cardiomyocytes, and in the testes of adult mice. At day 9.5 postcoitum, 2-kinase was expressed in the notochord, the ventricular layer of the neural tube, and the myotome of the somites. Intense staining was also seen in the yolk sac, suggesting that InsP6 is necessary for yolk sac development or function. Furthermore, failure of yolk sac development or function is consistent with the early lethality of 2-kinase embryos.

  7. Identification and characterization of a protein kinase gene in the Lymantria dispar nultinucleocapsid nuclear polyhedrosis virus

    Treesearch

    David S. Bischoff; James M. Slavicek

    1994-01-01

    The Lymantria dispar multinucleocapsid nuclear polyhedrosis virus (LdMNPV) gene encoding vPK has been cloned and sequenced. LdMNPV vPK shows a 24% amino acid identity to the catalytic domains of the eucaryotic protein kinases nPKC from rabbits, HSPKCE from humans, APLPKCB from Aplysia californica, and dPKC98F from ...

  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. Analysis of kinase gene expression patterns across 5681 human tissue samples reveals functional genomic taxonomy of the kinome.

    PubMed

    Kilpinen, Sami; Ojala, Kalle; Kallioniemi, Olli

    2010-12-03

    Kinases play key roles in cell signaling and represent major targets for drug development, but the regulation of their activation and their associations with health and disease have not been systematically analyzed. Here, we carried out a bioinformatic analysis of the expression levels of 459 human kinase genes in 5681 samples consisting of 44 healthy and 55 malignant human tissues. Defining the tissues where the kinase genes were transcriptionally active led to a functional genomic taxonomy of the kinome and a classification of human tissues and disease types based on the similarity of their kinome gene expression. The co-expression network around each of the kinase genes was defined in order to determine the functional context, i.e. the biological processes that were active in the cells and tissues where the kinase gene was expressed. Strong associations for individual kinases were found for mitosis (69 genes, including AURKA and BUB1), cell cycle control (73 genes, including PLK1 and AURKB), DNA repair (49 genes, including CHEK1 and ATR), immune response (72 genes, including MATK), neuronal (131 genes, including PRKCE) and muscular (72 genes, including MYLK2) functions. We then analyzed which kinase genes gain or lose transcriptional activity in the development of prostate and lung cancers and elucidated the functional associations of individual cancer associated kinase genes. In summary, we report here a systematic classification of kinases based on the bioinformatic analysis of their expression in human tissues and diseases, as well as grouping of tissues and tumor types according to the similarity of their kinome transcription.

  10. Analysis of Kinase Gene Expression Patterns across 5681 Human Tissue Samples Reveals Functional Genomic Taxonomy of the Kinome

    PubMed Central

    Kilpinen, Sami; Ojala, Kalle; Kallioniemi, Olli

    2010-01-01

    Kinases play key roles in cell signaling and represent major targets for drug development, but the regulation of their activation and their associations with health and disease have not been systematically analyzed. Here, we carried out a bioinformatic analysis of the expression levels of 459 human kinase genes in 5681 samples consisting of 44 healthy and 55 malignant human tissues. Defining the tissues where the kinase genes were transcriptionally active led to a functional genomic taxonomy of the kinome and a classification of human tissues and disease types based on the similarity of their kinome gene expression. The co-expression network around each of the kinase genes was defined in order to determine the functional context, i.e. the biological processes that were active in the cells and tissues where the kinase gene was expressed. Strong associations for individual kinases were found for mitosis (69 genes, including AURKA and BUB1), cell cycle control (73 genes, including PLK1 and AURKB), DNA repair (49 genes, including CHEK1 and ATR), immune response (72 genes, including MATK), neuronal (131 genes, including PRKCE) and muscular (72 genes, including MYLK2) functions. We then analyzed which kinase genes gain or lose transcriptional activity in the development of prostate and lung cancers and elucidated the functional associations of individual cancer associated kinase genes. In summary, we report here a systematic classification of kinases based on the bioinformatic analysis of their expression in human tissues and diseases, as well as grouping of tissues and tumor types according to the similarity of their kinome transcription. PMID:21151926

  11. 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

  12. 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

  13. 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

  14. 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.

  15. Identification of genes from pattern formation, tyrosine kinase, and potassium channel families by DNA amplification

    SciTech Connect

    Kamb, A.; Weir, M.; Rudy, B.; Varmus, H.; Kenyon, C. )

    1989-06-01

    The study of gene family members has been aided by the isolation of related genes on the basis of DNA homology. The authors have adapted the polymerase chain reaction to screen animal genomes very rapidly and reliably for likely gene family members. Using conserved amino acid sequences to design degenerate oligonucleotide primers, they have shown that the genome of the nematode Caenorhabditis elegans contains sequences homologous to many Drosophila genes involved in pattern formation, including the segment polarity gene wingless (vertebrate int-1), and homeobox sequences characteristic of the Antennapedia, engrailed, and paired families. In addition, they have used this method to show that C. elegans contains at least five different sequences homologous to genes in the tyrosine kinase family. Lastly, they have isolated six potassium channel sequences from humans, a result that validates the utility of the method with large genomes and suggests that human potassium channel gene diversity may be extensive.

  16. 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.

  17. 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

  18. 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.

  19. 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

  20. Cloning and characterization of two genes encoding dihydroxyacetone kinase from Schizosaccharomyces pombe IFO 0354.

    PubMed

    Kimura, T; Takahashi, M; Yoshihara, K; Furuichi, T; Suzuki, K; Imai, K; Karita, S; Sakka, K; Ohmiya, K

    1998-11-08

    We report the cloning and characterization of two genes encoding dihydroxyacetone kinase (EC 2.7.1.29), SpDAK1 and SpDAK2, from Schizosaccharomyces pombe IFO 0354. The open reading frames of both genes encode 591 amino acids and have Mrs of 62158 and 62170, respectively. Both predicted amino acid sequences exhibited a high identity to each other (99.8%) and relatively high identities (30% to 76%) to other putative dihydroxyacetone kinase gene products. A Western blot analysis showed that these enzymes are induced by glycerol and repressed by glucose. A genomic Southern blot analysis indicated the presence of SpDAK1 and the absence of SpDAK2 in a standard laboratory strain, S. pombe 972h-.

  1. 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.

  2. 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

  3. High throughput screening of tyrosine kinase inhibitor resistant genes in CML

    PubMed Central

    Ma, Leyuan; Roderick, Justine; Kelliher, Michelle A.; Green, Michael R.

    2017-01-01

    Summary Genome-wide RNA interference (RNAi) screening in mammalian cells has proven to be a powerful tool for identifying new genes and molecular pathways relevant to many cellular processes and diseases. For example, screening for genes that, when inactivated, lead to resistance to cancer therapeutic drugs can reveal new mechanisms for how resistance develops, and identify potential targetable strategies to overcome drug resistance. Here, we describe a detailed procedure for performing a high throughput RNAi screen using a genome-wide human short hairpin RNA (shRNA) library for identifying tyrosine kinase inhibitor (TKI)-resistance genes in a human CML cell line model. PMID:27581147

  4. 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.

  5. Anaplasma phagocytophilum infection modulates expression of megakaryocyte cell cycle genes through phosphatidylinositol-3-kinase signaling.

    PubMed

    Khanal, Supreet; Sultana, Hameeda; Catravas, John D; Carlyon, Jason A; Neelakanta, Girish

    2017-01-01

    Anaplasma phagocytophilum, the agent of human granulocytic anaplasmosis infects neutrophils and other cells from hematopoietic origin. Using human megakaryocytic cell line, MEG-01, we show that expression of cell cycle genes in these cells are altered upon A. phagocytophilum infection. Expression of several cell cycle genes in MEG-01 cells was significantly up regulated at early and then down regulated at later stages of A. phagocytophilum infection. Lactate dehydrogenase (LDH) assays revealed reduced cellular cytotoxicity in MEG-01 cells upon A. phagocytophilum infection. The levels of both PI3KCA (p110 alpha, catalytic subunit) and PI3KR1 (p85, regulatory subunit) of Class I PI3 kinases and phosphorylated protein kinase B (Akt/PKB) and inhibitory kappa B (IκB) were elevated at both early and late stages of A. phagocytophilum infection. Inhibition of PI3 kinases with LY294002 treatment resulted in significant reduction in the expression of tested cell cycle genes, A. phagocytophilum burden and phosphorylated Akt levels in these MEG-01 cells. Collectively, these results suggest a role for PI3K-Akt-NF-κB signaling pathway in the modulation of megakaryocyte cell cycle genes upon A. phagocytophilum infection.

  6. 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.

  7. K-sam gene encodes secreted as well as transmembrane receptor tyrosine kinase.

    PubMed Central

    Katoh, M; Hattori, Y; Sasaki, H; Tanaka, M; Sugano, K; Yazaki, Y; Sugimura, T; Terada, M

    1992-01-01

    K-sam was first identified as a gene amplified in the stomach cancer cell line KATO-III. The size of the major transcript of the K-sam gene was 3.5 kilobases in KATO-III cells, and we have previously shown that K-sam encodes a receptor tyrosine kinase that belongs to the heparin-binding growth factor receptor, or fibroblast growth factor receptor, gene family. The K-sam gene expresses multiple sizes of mRNAs in brain tissue, the immature teratoma cell line NCC-IT, and KATO-III. RNA blot analyses with a variety of K-sam probes indicate that there are at least four classes of K-sam mRNAs. Three types of K-sam cDNAs in addition to the previously reported type of K-sam cDNA were isolated, and their nucleotide sequences encode a full-length transmembrane receptor, a secreted receptor with a tyrosine kinase domain, and a secreted receptor without a tyrosine kinase domain. Images PMID:1313574

  8. Transcriptional analysis of three major putative phosphatidylinositol kinase genes in a parasitic protozoan, Giardia lamblia.

    PubMed

    Hernandez, Yunuen; Zamora, Gus; Ray, Suparna; Chapoy, Jaime; Chavez, Edna; Valvarde, Robert; Williams, Ebonye; Aley, Stephen B; Das, Siddhartha

    2007-01-01

    The current investigation evaluates the expression of phosphatidylinositol kinase (PIK) genes in the parasitic protozoan, Giardia lamblia. The G. lamblia Genome Database revealed the presence of two putative phosphatidylinositol-3-kinase (gPI3K) and one phosphatidylinositol-4-kinase (gPI4K) genes resembling the catalytic subunit of eukaryotic PIKs. Primers, designed to amplify mRNA of these three genes, were used to measure transcription by quantitative reverse-transcriptase polymerase chain reactions. Results suggest that all three PIK genes are expressed in non-encysting and encysting trophozoites. The relative levels of the mRNA were highest in parasites cultured in pre-encysting medium that contained no bile. Two inhibitors of PI3K, LY 294002 and wortmannin were found to inhibit the growth of the trophozoite in culture. However, wortmannin was more effective than LY294002. Altogether, the present study indicates that Giardia is capable of expressing PIKs that are necessary for the growth and differentiation of this pathogen.

  9. Physcomitrella patens Has Kinase-LRR R Gene Homologs and Interacting Proteins

    PubMed Central

    Tanigaki, Yusuke; Ito, Kenji; Obuchi, Yoshiyuki; Kosaka, Akiko; Yamato, Katsuyuki T.; Okanami, Masahiro; Lehtonen, Mikko T.; Valkonen, Jari P. T.; Akita, Motomu

    2014-01-01

    Plant disease resistance gene (R gene)-like sequences were screened from the Physcomitrella patens genome. We found 603 kinase-like, 475 Nucleotide Binding Site (NBS)-like and 8594 Leucine Rich Repeat (LRR)-like sequences by homology searching using the respective domains of PpC24 (Accession No. BAD38895), which is a candidate kinase-NBS-LRR (kinase-NL) type R-like gene, as a reference. The positions of these domains in the genome were compared and 17 kinase-NLs were predicted. We also found four TIR-NBS-LRR (TIR-NL) sequences with homology to Arabidopsis TIR-NL (NM_001125847), but three out of the four TIR-NLs had tetratricopeptide repeats or a zinc finger domain in their predicted C-terminus. We also searched for kinase-LRR (KLR) type sequences by homology with rice OsXa21 and Arabidopsis thaliana FLS2. As a result, 16 KLRs with similarity to OsXa21 were found. In phylogenetic analysis of these 16 KLRs, PpKLR36, PpKLR39, PpKLR40, and PpKLR43 formed a cluster with OsXa21. These four PpKLRs had deduced transmembrane domain sequences and expression of all four was confirmed. We also found 14 homologs of rice OsXB3, which is known to interact with OsXa21 and is involved in signal transduction. Protein–protein interaction was observed between the four PpKLRs and at least two of the XB3 homologs in Y2H analysis. PMID:24748046

  10. 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.

  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. 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.

  13. 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

  14. 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

  15. 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

  16. Bile acid signal-induced phosphorylation of small heterodimer partner by protein kinase Cζ is critical for epigenomic regulation of liver metabolic genes.

    PubMed

    Seok, Sunmi; Kanamaluru, Deepthi; Xiao, Zhen; Ryerson, Daniel; Choi, Sung-E; Suino-Powell, Kelly; Xu, H Eric; Veenstra, Timothy D; Kemper, Jongsook Kim

    2013-08-09

    Bile acids (BAs) are recently recognized key signaling molecules that control integrative metabolism and energy expenditure. BAs activate multiple signaling pathways, including those of nuclear receptors, primarily farnesoid X receptor (FXR), membrane BA receptors, and FXR-induced FGF19 to regulate the fed-state metabolism. Small heterodimer partner (SHP) has been implicated as a key mediator of these BA signaling pathways by recruitment of chromatin modifying proteins, but the key question of how SHP transduces BA signaling into repressive histone modifications at liver metabolic genes remains unknown. Here we show that protein kinase Cζ (PKCζ) is activated by BA or FGF19 and phosphorylates SHP at Thr-55 and that Thr-55 phosphorylation is critical for the epigenomic coordinator functions of SHP. PKCζ is coimmunopreciptitated with SHP and both are recruited to SHP target genes after bile acid or FGF19 treatment. Activated phosphorylated PKCζ and phosphorylated SHP are predominantly located in the nucleus after FGF19 treatment. Phosphorylation at Thr-55 is required for subsequent methylation at Arg-57, a naturally occurring mutation site in metabolic syndrome patients. Thr-55 phosphorylation increases interaction of SHP with chromatin modifiers and their occupancy at selective BA-responsive genes. This molecular cascade leads to repressive modifications of histones at metabolic target genes, and consequently, decreased BA pools and hepatic triglyceride levels. Remarkably, mutation of Thr-55 attenuates these SHP-mediated epigenomic and metabolic effects. This study identifies PKCζ as a novel key upstream regulator of BA-regulated SHP function, revealing the role of Thr-55 phosphorylation in epigenomic regulation of liver metabolism.

  17. 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

  18. Isolation of an Aspergillus terreus mutant impaired in arginine biosynthesis and its complementation with the argB gene from Aspergillus nidulans.

    PubMed

    Ventura, L; Ramón, D; Pérez-González, J A

    1992-12-01

    Using filtration enrichment techniques, an Aspergillus terreus arginine auxotrophic strain which contains a mutation that abolishes ornithine transcarbamylase (OTCase) activity has been isolated. This mutant has been genetically transformed with the cloned Aspergillus nidulans OTCase gene. Prototrophic transformants arose at a frequency of about 50 transformants per microgram of plasmid DNA. Southern blot analysis of DNA from the transformants showed that the transforming DNA was ectopically integrated at different locations in the A. terreus genome, often in multiple tandem copies. The transformants were phenotypically stable for several mitotic divisions and retained their capacity to produce extracellular enzymes.

  19. 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.

  20. 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

  1. 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

  2. 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

  3. 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.

  4. 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

  5. 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.

  6. 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

  7. Rapid induction by wounding and bacterial infection of an S gene family receptor-like kinase gene in Brassica oleracea.

    PubMed

    Pastuglia, M; Roby, D; Dumas, C; Cock, J M

    1997-01-01

    A receptor-like kinase, SRK, has been implicated in the autoincompatible response that leads to the rejection of self-pollen in Brassica plants. SRK is encoded by one member of a multigene family, which includes several receptor-like kinase genes with patterns of expression very different from that of SRK but of unknown function. Here, we report the characterization of a novel member of the Brassica S gene family, SFR2. RNA gel blot analysis demonstrated that SFR2 mRNA accumulated rapidly in response both to wounding and to infiltration with either of two bacteria: Xanthomonas campestris, a pathogen, and Escherichia coli, a saprophyte. SFR2 mRNA also accumulated rapidly after treatment with salicylic acid, a molecule that has been implicated in plant defense response signaling pathways. A SFR2 promoter and reporter gene fusion was introduced into tobacco and was shown to be induced by bacteria of another genus, Ralstonia (Pseudomonas) solanacearum. The accumulation of SFR2 mRNA in response to wounding and pathogen invasion is typical of a gene involved in the defense responses of the plant. The rapidity of SFR2 mRNA accumulation is consistent with SFR2 playing a role in the signal transduction pathway that leads to induction of plant defense proteins, such as pathogenesis-related proteins or enzymes of phenylpropanoid metabolism.

  8. Rapid induction by wounding and bacterial infection of an S gene family receptor-like kinase gene in Brassica oleracea.

    PubMed Central

    Pastuglia, M; Roby, D; Dumas, C; Cock, J M

    1997-01-01

    A receptor-like kinase, SRK, has been implicated in the autoincompatible response that leads to the rejection of self-pollen in Brassica plants. SRK is encoded by one member of a multigene family, which includes several receptor-like kinase genes with patterns of expression very different from that of SRK but of unknown function. Here, we report the characterization of a novel member of the Brassica S gene family, SFR2. RNA gel blot analysis demonstrated that SFR2 mRNA accumulated rapidly in response both to wounding and to infiltration with either of two bacteria: Xanthomonas campestris, a pathogen, and Escherichia coli, a saprophyte. SFR2 mRNA also accumulated rapidly after treatment with salicylic acid, a molecule that has been implicated in plant defense response signaling pathways. A SFR2 promoter and reporter gene fusion was introduced into tobacco and was shown to be induced by bacteria of another genus, Ralstonia (Pseudomonas) solanacearum. The accumulation of SFR2 mRNA in response to wounding and pathogen invasion is typical of a gene involved in the defense responses of the plant. The rapidity of SFR2 mRNA accumulation is consistent with SFR2 playing a role in the signal transduction pathway that leads to induction of plant defense proteins, such as pathogenesis-related proteins or enzymes of phenylpropanoid metabolism. PMID:9014364

  9. Insulin-induced reactivation of an inactive herpes simplex thymidine kinase gene.

    PubMed Central

    Clough, D W; Morse, B S; Kucherlapati, R S; Davidson, R L

    1984-01-01

    A line of mouse cells transformed with ultraviolet-irradiated herpes simplex virus type 1 and containing a methylated and inactive viral thymidine kinase (TK) gene was treated with insulin in an attempt to induce expression of the inactive gene. Insulin was found to be capable of inducing the inactive TK gene in these cells. The induction of the TK+ phenotype was dose dependent (from 1-100 micrograms of insulin per ml), and the TK activity induced was shown to be of viral origin. Analysis of the methylation pattern of the viral TK gene by using the methylation-sensitive restriction endonucleases Sma I, Hpa II, and Hha I revealed that the active viral TK gene in the parental transformed cells was hypomethylated, whereas the inactive TK gene in the uninduced TK- cells was methylated. The active TK gene in three insulin-induced TK+ lines also was methylated, but the methylation patterns in the insulin-induced lines all were different from the uninduced TK- line. These data suggest that extensive hypomethylation of the inactive TK gene is not required for insulin induction. Four other transformed lines containing an inactive viral TK gene were tested for insulin inducibility, but insulin was unable to induce expression of the TK gene in any of the other lines. Thus, insulin inducibility does not seem to be a function of the viral TK gene itself. These results suggest that insulin inducibility of the viral TK gene may be a reflection of the region of the host genome into which the TK gene was integrated. Images PMID:6322172

  10. 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

  11. 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.

  12. 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

  13. 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.

  14. 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.

  15. 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.

  16. 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.

  17. 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.

  18. 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

  19. 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

  20. 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.

  1. 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.

  2. Gene structure of the two-domain taurocyamine kinase from Paragonimus westermani: evidence for a distinct lineage of trematode phosphagen kinases.

    PubMed

    Jarilla, Blanca R; Tokuhiro, Shinji; Nagataki, Mitsuru; Uda, Kouji; Suzuki, Tomohiko; Acosta, Luz P; Agatsuma, Takeshi

    2013-07-11

    Taurocyamine kinase (TK) is an enzyme that catalyzes the reversible transfer of a phosphate between ATP and taurocyamine. Annelid TKs were suggested to have evolved from a CK ancestor. However, TKs from the lung fluke Paragonimus westermani comprised another lineage. Construction of phylogenetic tree and comparison of exon/intron organization showed that P. westermani TK and other trematode TKs evolved from a molluscan arginine kinase (AK) gene. Exon shuffling probably caused the changes in amino acid sequence thereby changing the affinity from AK to TK. The present study provides new insights on the evolution of phosphagen kinases found in trematodes. Copyright © 2013 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

  3. A kinase-START gene confers temperature-dependent resistance to wheat stripe rust.

    PubMed

    Fu, Daolin; Uauy, Cristobal; Distelfeld, Assaf; Blechl, Ann; Epstein, Lynn; Chen, Xianming; Sela, Hanan; Fahima, Tzion; Dubcovsky, Jorge

    2009-03-06

    Stripe rust is a devastating fungal disease that afflicts wheat in many regions of the world. New races of Puccinia striiformis, the pathogen responsible for this disease, have overcome most of the known race-specific resistance genes. We report the map-based cloning of the gene Yr36 (WKS1), which confers resistance to a broad spectrum of stripe rust races at relatively high temperatures (25 degrees to 35 degrees C). This gene includes a kinase and a putative START lipid-binding domain. Five independent mutations and transgenic complementation confirmed that both domains are necessary to confer resistance. Yr36 is present in wild wheat but is absent in modern pasta and bread wheat varieties, and therefore it can now be used to improve resistance to stripe rust in a broad set of varieties.

  4. 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.

  5. 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.

  6. 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

  7. 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.

  8. Identification by targeted differential display of an immediate early gene encoding a putative serine/threonine kinase.

    PubMed

    Donohue, P J; Alberts, G F; Guo, Y; Winkles, J A

    1995-04-28

    Fibroblast growth factor (FGF)-1 mitogenic signal transduction is mediated in part by gene products that are specifically expressed in response to cell surface receptor binding and activation. We have used a targeted differential display method to identify FGF-1-inducible genes in murine NIH 3T3 fibroblasts. Here we report that one of these genes is predicted to encode a novel serine/threonine-specific protein kinase. This putative kinase has been named Fnk, for FGF-inducible kinase. The deduced Fnk amino acid sequence has 49, 36, 33, 32, and 22% overall identity to mouse serum-inducible kinase (Snk), mouse polo-like kinase (Plk), Drosophila polo, Saccharomyces Cdc5, and mouse Snk/Plk-akin kinase (Sak), respectively. These proteins are all members of the polo subfamily of structurally related serine/threonine kinases. The Plk, polo, Cdc5, and Sak kinases are required for cell division. FGF-1 induction of Fnk mRNA expression is first detected at 30 min after mitogen addition, reflects transcriptional activation, and does not require de novo protein synthesis. FGF-2, platelet-derived growth factor-BB, calf serum, or phorbol myristate acetate treatment of quiescent cells also induces fnk gene expression. Fnk mRNA is expressed in vivo in a tissue-specific manner, with relatively high levels detected in newborn and adult mouse skin. These results indicate that Fnk may be a transiently expressed protein kinase involved in the early signaling events required for growth factor-stimulated cell cycle progression.

  9. Pyruvate Kinase and Fcγ Receptor Gene Copy Numbers Associated With Malaria Phenotypes.

    PubMed

    Faik, Imad; van Tong, Hoang; Lell, Bertrand; Meyer, Christian G; Kremsner, Peter G; Velavan, Thirumalaisamy P

    2017-07-15

    Genetic factors are associated with susceptibility to many infectious diseases and may be determinants of clinical progression. Gene copy number variation (CNV) has been shown to be associated with phenotypes of numerous diseases, including malaria. We quantified gene copy numbers of the pyruvate kinase, liver, and red blood cell (PKLR) gene as well as of the Fcγ receptor 2A and Fcγ receptor 2C (FCGR2A, FCGR2C) and Fcγ receptor 3 (FCGR3) genes using real-time quantitative polymerase chain reaction (RT-qPCR) assays in Gabonese children with severe (n = 184) or and mild (n = 189) malaria and in healthy Gabonese and white individuals (n = 76 each). The means of PKLR, FCGR2A, FCGR2C, and FCGR3 copy numbers were significantly higher among children with severe malaria compared to those with mild malaria (P < .002), indicating that a surplus of copies of those genes is significantly associated with malaria severity. Copy numbers of the FCGR2A and FCGR2C genes were significantly lower (P = .005) in Gabonese individuals compared with white individuals. In conclusion, CNV of the PKLR, FCGR2A, FCGR2C, and FCGR3 genes is associated with malaria severity, and our results provide evidence for a role of CNV in host responses to malaria. © The Author 2017. Published by Oxford University Press for the Infectious Diseases Society of America. All rights reserved. For permissions, e-mail: journals.permissions@oup.com.

  10. MAP Kinase Phosphatase 3 (MKP3) Preserves Norepinephrine Transporter Activity by Modulating ERK1/2 Kinase-Mediated Gene Expression

    PubMed Central

    Mortensen, Ole V.; Larsen, Mads B.; Amara, Susan G.

    2017-01-01

    The norepinephrine transporter (NET) mediates the clearance of norepinephrine (NE) from the extracellular space and is a target of therapeutic antidepressants and psychostimulants. Previously we identified a MAP kinase phosphatase 3 (MKP3), as an important modulator of protein kinase C (PKC) mediated internalization of the related dopamine transporter (DAT). Here we show that MKP3 decreases PKC-mediated down regulation of NET expressed in PC12 cells. We demonstrate that this process involves a PKC-stimulated decrease of NET surface expression that is dependent on dynamin. Surprisingly, MAP kinase inhibitors have no effect on the PKC-mediated regulation of NET activity, suggesting that, like PKC-mediated regulation of the DAT, the acute activation of MAP kinases is not likely to be involved. To elucidate potential mechanisms we used a substrate trap-based assay to identify extracellular-signal-regulated kinase (ERK)1/2 as the predominant substrate of MKP3. Furthermore we also established that brief chemical stabilization of a modified destabilized MKP3 does not alter PKC-mediated down regulation of NET. Finally, the expression of a dominant negative version of H-Ras, an upstream activator of ERK1/2, abolishes phorbol 12-myristate 13-acetate (PMA)-mediated down regulation of NET in a manner similar to MKP3. Taken together we propose that chronic MKP3 expression regulates surface NET through the sustained inhibition of ERK1/2 MAP kinase signaling that alters gene expression in PC12 cells. This is supported by gene expression data from naïve and MKP3-expressing PC12 cells that reveal robust decreases in gene expression of several genes in the MKP3-tranfected cells. Interestingly, caveolin-1, a protein with a critical role in membrane protein trafficking is down regulated by MKP3 expression. We further show that selective silencing of the caveolin-1 gene in naïve PC12 cells attenuates PKC-mediated downregulation of NET activity, consistent with a potential role for

  11. 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

  12. 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.

  13. 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.

  14. Characterization of an S-locus receptor protein kinase-like gene from peach.

    PubMed

    Bassett, Carole L; Nickerson, Michael L; Farrell, Robert E; Artlip, Timothy S; El Ghaouth, Ahmed; Wilson, Charles L; Wisniewski, Michael E

    2005-04-01

    A receptor-like protein kinase gene (Ppsrkl1) was isolated from a peach (Prunus persica (L.) Batsch.) bark cDNA library prepared with RNAs isolated from bark collected in December (cold acclimated). Sequence analysis indicated that this gene is related to the S-locus family of receptor protein kinases (SRKs) and that it shares greatest homology with ZMPK1 from maize and At4g32300 from Arabidopsis, both of which are intron-less genes. In bark tissues, Ppsrkl1 is induced by water deficit treatment, repressed by short-day photoperiods and showed no response to cold treatment. The Ppsrkl1 mRNA also increased in roots in response to water deficit. In fruit, Ppsrkl1 shows no response up to 6 h after wounding, but at 12 and 24 h after wounding, Ppsrkl1 mRNA shows an abrupt decline. This decline was prevented by the addition of salicylic acid to the wound site. The Ppsrkl1 mRNA rapidly decreased in fruit after 10-min exposure to UV-C radiation, followed by a return to normal levels within 1.5 h. Taken together, these experiments indicate that Ppsrkl1 is negatively regulated by light and positively influenced by salicylic acid treatment in fruit and water stress in bark and roots.

  15. 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

  16. Disruption of the acetate kinase (ack) gene of Clostridium acetobutylicum results in delayed acetate production.

    PubMed

    Kuit, Wouter; Minton, Nigel P; López-Contreras, Ana M; Eggink, Gerrit

    2012-05-01

    In microorganisms, the enzyme acetate kinase (AK) catalyses the formation of ATP from ADP by de-phosphorylation of acetyl phosphate into acetic acid. A mutant strain of Clostridium acetobutylicum lacking acetate kinase activity is expected to have reduced acetate and acetone production compared to the wild type. In this work, a C. acetobutylicum mutant strain with a selectively disrupted ack gene, encoding AK, was constructed and genetically and physiologically characterized. The ack (-) strain showed a reduction in acetate kinase activity of more than 97% compared to the wild type. The fermentation profiles of the ack (-) and wild-type strain were compared using two different fermentation media, CGM and CM1. The latter contains acetate and has a higher iron and magnesium content than CGM. In general, fermentations by the mutant strain showed a clear shift in the timing of peak acetate production relative to butyrate and had increased acid uptake after the onset of solvent formation. Specifically, in acetate containing CM1 medium, acetate production was reduced by more than 80% compared to the wild type under the same conditions, but both strains produced similar final amounts of solvents. Fermentations in CGM showed similar peak acetate and butyrate levels, but increased acetoin (60%), ethanol (63%) and butanol (16%) production and reduced lactate (-50%) formation by the mutant compared to the wild type. These findings are in agreement with the proposed regulatory function of butyryl phosphate as opposed to acetyl phosphate in the metabolic switch of solventogenic clostridia.

  17. Stimulation of sky receptor tyrosine kinase by the product of growth arrest-specific gene 6.

    PubMed

    Ohashi, K; Nagata, K; Toshima, J; Nakano, T; Arita, H; Tsuda, H; Suzuki, K; Mizuno, K

    1995-09-29

    Sky (also called Rse, Brt, and Tyro3) is a member of a subfamily of related receptor tyrosine kinases, including Axl/Ufo/Ark and c-Eyk/Mer. We obtained evidence that Gas6 (the product of growth arrest-specific gene 6) is a ligand of the Sky receptor tyrosine kinase. Gas6, but not protein S (an anticoagulant protein structurally similar to Gas6), specifically bound to the soluble form of Sky (Sky-Fc), composed of the extracellular domain of Sky fused to the Fc domain of human immunoglobulin G1. The native and recombinant Gas6, but not protein S, stimulated tyrosine phosphorylation of Sky ectopically expressed in Chinese hamster ovary cells. Stimulation of Sky in response to Gas6 was inhibited by Sky-Fc. The half-maximal concentration of Gas6 that stimulated Sky was about 1 nM. Thus, Gas6 as a ligand for Sky specifically binds to and stimulates Sky receptor tyrosine kinase.

  18. 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.

  19. 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. © 2015 Japanese Dermatological Association.

  20. Gene transfer of arginine kinase to skeletal muscle using adeno-associated virus.

    PubMed

    Forbes, S C; Bish, L T; Ye, F; Spinazzola, J; Baligand, C; Plant, D; Vandenborne, K; Barton, E R; Sweeney, H L; Walter, G A

    2014-04-01

    In this study, we tested the feasibility of non-invasively measuring phosphoarginine (PArg) after gene delivery of arginine kinase (AK) using an adeno-associated virus (AAV) to murine hindlimbs. This was achieved by evaluating the time course, regional distribution and metabolic flux of PArg using (31)phosphorus magnetic resonance spectroscopy ((31)P-MRS). AK gene was injected into the gastrocnemius of the left hindlimb of C57Bl10 mice (age 5 weeks, male) using self-complementary AAV, type 2/8 with desmin promoter. Non-localized (31)P-MRS data were acquired over 9 months after injection using 11.1-T and 17.6-T Bruker Avance spectrometers. In addition, (31)P two-dimensional chemical shift imaging and saturation transfer experiments were performed to examine the spatial distribution and metabolic flux of PArg, respectively. PArg was evident in each injected mouse hindlimb after gene delivery, increased until 28 weeks, and remained elevated for at least 9 months (P<0.05). Furthermore, PArg was primarily localized to the injected posterior hindimb region and the metabolite was in exchange with ATP. Overall, the results show the viability of AAV gene transfer of AK gene to skeletal muscle, and provide support of PArg as a reporter that can be used to non-invasively monitor the transduction of genes for therapeutic interventions.

  1. 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.

  2. 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

  3. Assignment of the human diacylglycerol kinase 4 (DAGK4) gene to chromosome 4p16.3

    SciTech Connect

    Endele, S.; Zabel, B.; Winterpacht, A.

    1996-04-01

    This report describes the localization of the human gene for diacylglycerol kinase 4 (DAGK4) to human chromosome 4p16.3 using an exon amplification scheme. It also discusses the possible implications of the chromosomal location of this gene in certain hereditary malignancies. 9 refs., 1 fig.

  4. 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.

  5. Evolutionary history of mitogen-activated protein kinase (MAPK) genes in Lotus, Medicago, and Phaseolus.

    PubMed

    Neupane, Achal; Nepal, Madhav P; Benson, Benjamin V; Macarthur, Kenton J; Piya, Sarbottam

    2013-11-01

    Mitogen-Activated Protein Kinase (MAPK) genes encode proteins that mediate various signaling pathways associated with biotic and abiotic stress responses in eukaryotes. The MAPK genes form a 3-tier signal transduction cascade between cellular stimuli and physiological responses. Recent identification of soybean MAPKs and availability of genome sequences from other legume species allowed us to identify their MAPK genes. The main objectives of this study were to identify MAPKs in 3 legume species, Lotus japonicus, Medicago truncatula, and Phaseolus vulgaris, and to assess their phylogenetic relationships. We used approaches in comparative genomics for MAPK gene identification and named the newly identified genes following Arabidopsis MAPK nomenclature model. We identified 19, 18, and 15 MAPKs and 7, 4, and 9 MAPKKs in the genome of Lotus japonicus, Medicago truncatula, and Phaseolus vulgaris, respectively. Within clade placement of MAPKs and MAPKKs in the 3 legume species were consistent with those in soybean and Arabidopsis. Among 5 clades of MAPKs, 4 founder clades were consistent to MAPKs of other plant species and orthologs of MAPK genes in the fifth clade-"Clade E" were consistent with those in soybean. Our results also indicated that some gene duplication events might have occurred prior to eudicot-monocot divergence. Highly diversified MAPKs in soybean relative to those in 3 other legume species are attributable to the polyploidization events in soybean. The identification of the MAPK genes in the legume species is important for the legume crop improvement; and evolutionary relationships and functional divergence of these gene members provide insights into plant genome evolution.

  6. Systematic analysis of human kinase genes: a large number of genes and alternative splicing events result in functional and structural diversity

    PubMed Central

    Milanesi, Luciano; Petrillo, Mauro; Sepe, Leandra; Boccia, Angelo; D'Agostino, Nunzio; Passamano, Myriam; Di Nardo, Salvatore; Tasco, Gianluca; Casadio, Rita; Paolella, Giovanni

    2005-01-01

    Background Protein kinases are a well defined family of proteins, characterized by the presence of a common kinase catalytic domain and playing a significant role in many important cellular processes, such as proliferation, maintenance of cell shape, apoptosys. In many members of the family, additional non-kinase domains contribute further specialization, resulting in subcellular localization, protein binding and regulation of activity, among others. About 500 genes encode members of the kinase family in the human genome, and although many of them represent well known genes, a larger number of genes code for proteins of more recent identification, or for unknown proteins identified as kinase only after computational studies. Results A systematic in silico study performed on the human genome, led to the identification of 5 genes, on chromosome 1, 11, 13, 15 and 16 respectively, and 1 pseudogene on chromosome X; some of these genes are reported as kinases from NCBI but are absent in other databases, such as KinBase. Comparative analysis of 483 gene regions and subsequent computational analysis, aimed at identifying unannotated exons, indicates that a large number of kinase may code for alternately spliced forms or be incorrectly annotated. An InterProScan automated analysis was perfomed to study domain distribution and combination in the various families. At the same time, other structural features were also added to the annotation process, including the putative presence of transmembrane alpha helices, and the cystein propensity to participate into a disulfide bridge. Conclusion The predicted human kinome was extended by identifiying both additional genes and potential splice variants, resulting in a varied panorama where functionality may be searched at the gene and protein level. Structural analysis of kinase proteins domains as defined in multiple sources together with transmembrane alpha helices and signal peptide prediction provides hints to function assignment

  7. DNA-dependent kinase (p350) as a candidate gene for the murine SCID defect.

    PubMed

    Kirchgessner, C U; Patil, C K; Evans, J W; Cuomo, C A; Fried, L M; Carter, T; Oettinger, M A; Brown, J M

    1995-02-24

    Severe combined immunodeficient (SCID) mice are deficient in a recombination process utilized in both DNA double-strand break repair and in V(D)J recombination. The phenotype of these mice involves both cellular hypersensitivity to ionizing radiation and a lack of B and T cell immunity. The catalytic subunit of DNA-dependent protein kinase, p350, was identified as a strong candidate for the murine gene SCID. Both p350 and a gene complementing the SCID defect colocalize to human chromosome 8q11. Chromosomal fragments expressing p350 complement the SCID phenotype, and p350 protein levels are greatly reduced in cells derived from SCID mice compared to cells from wild-type mice.

  8. 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.

  9. 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

  10. 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

  11. 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.

  12. High fat diet induced obesity alters ovarian phosphatidylinositol-3 kinase signaling gene expression.

    PubMed

    Nteeba, J; Ross, J W; Perfield, J W; Keating, A F

    2013-12-01

    Insulin regulates ovarian phosphatidylinositol-3-kinase (PI3 K) signaling, important for primordial follicle viability and growth activation. This study investigated diet-induced obesity impacts on: (1) insulin receptor (Insr) and insulin receptor substrate 1 (Irs1); (2) PI3K components (Kit ligand (Kitlg), kit (c-Kit), protein kinase B alpha (Akt1) and forkhead transcription factor subfamily 3 (Foxo3a)); (3) xenobiotic biotransformation (microsomal epoxide hydrolase (Ephx1), Cytochrome P450 isoform 2E1 (Cyp2e1), Glutathione S-transferase (Gst) isoforms mu (Gstm) and pi (Gstp)) and (4) microRNA's 184, 205, 103 and 21 gene expression. INSR, GSTM and GSTP protein levels were also measured. Obese mouse ovaries had decreased Irs1, Foxo3a, Cyp2e1, MiR-103, and MiR-21 but increased Kitlg, Akt1, and miR-184 levels relative to lean littermates. These results support that diet-induced obesity potentially impairs ovarian function through aberrant gene expression. Copyright © 2013 Elsevier Inc. All rights reserved.

  13. High fat diet induced obesity alters ovarian phosphatidylinositol-3 kinase signaling gene expression

    PubMed Central

    Nteeba, J.; Ross, J.W.; Perfield, J.W.; Keating, A.F.

    2013-01-01

    Insulin regulates ovarian phosphatidylinositol-3-kinase (PI3K) signaling, important for primordial follicle viability and growth activation. This study investigated diet-induced obesity impacts on: 1) insulin receptor (Insr) and insulin receptor substrate 1 (Irs1); 2) PI3K components (Kit ligand (Kitlg), kit (c-Kit), protein kinase B alpha (Akt1) and forkhead transcription factor subfamily 3 (Foxo3a)); 3) xenobiotic biotransformation (microsomal epoxide hydrolase (Ephx1), Cytochrome P450 isoform 2E1 (Cyp2e1), Glutathione S-transferase (Gst) isoforms mu (Gstm) and pi (Gstp)) and 4) microRNA’s 184, 205, 103 and 21 gene expression. INSR, GSTM and GSTP protein levels were also measured. Obese mouse ovaries had decreased Irs1, Foxo3a, Cyp2e1, MiR-103, and MiR-21 but increased Kitlg, Akt1, and miR-184 levels relative to lean littermates. These results support that diet-induced obesity potentially impairs ovarian function through aberrant gene expression. PMID:23954404

  14. Genomic structure and chromosomal localization of the human deoxycytidine kinase gene

    SciTech Connect

    Song, J.J.; Walker, S.; Gribbin, T. ); Chen, E. Univ. of North Carolina, Chapel Hill ); Johnson, E.E.; Spychala, J.; Mitchell, B.S. )

    1993-01-15

    Deoxycytidine kinase (NTP:deoxycytidine 5[prime]-phosphotransferase, EC 2.7.1.74) is an enzyme that catalyzes phosphorylation of deoxyribonucleosides and a number of nucleoside analogs that are important in antiviral and cancer chemotherapy. Deficiency of this enzyme activity is associated with resistance to these agents, whereas increased enzyme activity is associated with increased activation of such compounds to cytotoxic nucleoside triphosphate derivatives. To characterize the regulation of expression of this gene, we have isolated genomic clones encompassing its entire coding and 5[prime] flanking regions and delinated all the exon/intron boundaries. The gene extends over more than 34 kilobases on chromosome 4 and the coding region is composed of 7 exons ranging in size from 90 to 1544 base pairs (bp). The 5[prime] flanking region is highly G+C-rich and contains four regions that are potential Sp1 binding sites. A 697-bp fragment encompassing 386 bp of 5[prime] upstream region, the 250-bp first exon, and 61 bp of the first intron was demonstrated to promote chloramphenicol acetyltransferase activity in a T-lymphoblast cell line and to have >6-fold greater activity in a Jurkat T-lymphoblast than in a Raji B-lymphoblast cell line. Our data suggest that these 5[prime] sequences may contain elements that are important for the tissue-specific differences in deoxycytidine kinase expression. 32 refs., 4 figs., 2 tabs.

  15. 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.

  16. 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.

  17. Mutation of CERKL, a Novel Human Ceramide Kinase Gene, Causes Autosomal Recessive Retinitis Pigmentosa (RP26)

    PubMed Central

    Tuson, Miquel; Marfany, Gemma; Gonzàlez-Duarte, Roser

    2004-01-01

    Retinitis pigmentosa (RP), the main cause of adult blindness, is a genetically heterogeneous disorder characterized by progressive loss of photoreceptors through apoptosis. Up to now, 39 genes and loci have been implicated in nonsyndromic RP, yet the genetic bases of >50% of the cases, particularly of the recessive forms, remain unknown. Previous linkage analysis in a Spanish consanguineous family allowed us to define a novel autosomal recessive RP (arRP) locus, RP26, within an 11-cM interval (17.4 Mb) on 2q31.2-q32.3. In the present study, we further refine the RP26 locus down to 2.5 Mb, by microsatellite and single-nucleotide polymorphism (SNP) homozygosity mapping. After unsuccessful mutational analysis of the nine genes initially reported in this region, a detailed gene search based on expressed-sequence-tag data was undertaken. We finally identified a novel gene encoding a ceramide kinase (CERKL), which encompassed 13 exons. All of the patients from the RP26 family bear a homozygous mutation in exon 5, which generates a premature termination codon. The same mutation was also characterized in another, unrelated, Spanish pedigree with arRP. Human CERKL is expressed in the retina, among other adult and fetal tissues. A more detailed analysis by in situ hybridization on adult murine retina sections shows expression of Cerkl in the ganglion cell layer. Ceramide kinases convert the sphingolipid metabolite ceramide into ceramide-1-phosphate, both key mediators of cellular apoptosis and survival. Ceramide metabolism plays an essential role in the viability of neuronal cells, the membranes of which are particularly rich in sphingolipids. Therefore, CERKL deficiency could shift the relative levels of the signaling sphingolipid metabolites and increase sensitivity of photoreceptor and other retinal cells to apoptotic stimuli. This is the first genetic report suggesting a direct link between retinal neurodegeneration in RP and sphingolipid-mediated apoptosis. PMID

  18. 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.

  19. 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.

  20. 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.

  1. Organization and evolution of multifunctional Ca(2+)/CaM-dependent protein kinase genes.

    PubMed

    Tombes, Robert M; Faison, M Omar; Turbeville, J M

    2003-12-11

    The "multi-functional" Ca(2+) and calmodulin-dependent protein kinase, type II (CaMK-II) is an evolutionarily conserved protein. It has been found as a single gene in the horseshoe crab, marine sponge, sea urchin, nematode, and fruit fly, whereas most vertebrates possess four genes (alpha, beta, gamma, and delta). Species from fruit flies to humans encode alternative splice variants which are differentially targeted to phosphorylate diverse downstream targets of Ca(2+) signaling. By comparing known CaMK-II protein and nucleotide sequences, we have now provided evidence for the evolutionary relatedness of CaMK-IIs. Parsimony analyses unambiguously indicate that the four vertebrate CaMK-II genes arose via repeated duplications. Nucleotide phylogenies show consistent but moderate support for the placement of the vertebrate delta CaMK-II as the earliest diverging vertebrate gene. delta CaMK-II is the only gene with both central and C-terminal variable domains and has three to four times more intronic sequence than the other three genes. beta and gamma CaMK-II genes show strong sequence similarity and have comparable exon and intron organization and utilization. alpha CaMK-II is absent from amphibians (Xenopus laevis) and has the most restricted tissue specificity in mammals, whereas beta, gamma, and delta CaMK-IIs are expressed in most tissues. All 38 known mammalian CaMK-II splice variants were compiled with their tissue specificity and exon usage. Some of these variants use alternative 5' and 3' donors within a single exon as well as alternative promoters. These findings serve as an important benchmark for future phylogenetic, developmental, or biochemical studies on this important, conserved, and highly regulated gene family.

  2. 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.

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

    PubMed Central

    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.

    2016-01-01

    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. PMID:26796753

  4. 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.

  5. 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

  6. 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.

  7. 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.

  8. Structural and functional studies of the intracellular tyrosine kinase MATK gene and its translated product.

    PubMed

    Avraham, S; Jiang, S; Ota, S; Fu, Y; Deng, B; Dowler, L L; White, R A; Avraham, H

    1995-01-27

    We recently cloned the cDNA which encodes a novel megakaryocyte-associated tyrosine kinase termed MATK. In this study, we have cloned and characterized the human MATK gene as well as the murine homolog of human MATK cDNA and performed functional studies of its translated product. Comparison of the deduced amino acid sequences of human and murine MATK cDNAs revealed 85% homology, indicating that MATK is highly conserved in mouse and human. The human gene consists of 13 exons interrupted by 12 introns. The genetic units which encode the SH3 and SH2 domains are located on separate exons. The putative ATP binding site (GXGXXG) is localized on exon 7, and the entire catalytic domain is subdivided into seven exons (7-13). Somatic cell hybrid analysis indicated that human MATK gene is located on chromosome 19 while the murine Matk gene is located on chromosome 10. The immediate 5'-flanking region was highly rich in GC sequences, and potential cis-acting elements were identified including several SP1, GATA-1, APRE, and APRE1. Antisense oligonucleotides directed against MATK mRNA sequences significantly inhibited megakaryocyte progenitor proliferation. Functional studies indicated that MATK can phosphorylate the carboxyl-terminal conserved tyrosine of the Src protein. These results support the notion that MATK acts as a regulator of p60c-src in megakaryocytic cells and participates in the pathways regulating growth of cells of this lineage.

  9. 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.

  10. 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

  11. 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.

  12. 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

  13. An ant-plant mutualism through the lens of cGMP-dependent kinase genes.

    PubMed

    Malé, Pierre-Jean G; Turner, Kyle M; Doha, Manjima; Anreiter, Ina; Allen, Aaron M; Sokolowski, Marla B; Frederickson, Megan E

    2017-09-13

    In plant-animal mutualisms, how an animal forages often determines how much benefit its plant partner receives. In many animals, foraging behaviour changes in response to foraging gene expression or activation of the cGMP-dependent protein kinase (PKG) that foraging encodes. Here, we show that this highly conserved molecular mechanism affects the outcome of a plant-animal mutualism. We studied the two PKG genes of Allomerus octoarticulatus, an Amazonian ant that defends the ant-plant Cordia nodosa against herbivores. Some ant colonies are better 'bodyguards' than others. Working in the field in Peru, we found that colonies fed with a PKG activator recruited more workers to attack herbivores than control colonies. This resulted in less herbivore damage. PKG gene expression in ant workers correlated with whether an ant colony discovered an herbivore and how much damage herbivores inflicted on leaves in a complex way; natural variation in expression levels of the two genes had significant interaction effects on ant behaviour and herbivory. Our results suggest a molecular basis for ant protection of plants in this mutualism. © 2017 The Author(s).

  14. 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

  15. Sphingosine kinase 1 gene transfer reduces postoperative peritoneal adhesion in an experimental model.

    PubMed

    Guo, Q; Li, Q-F; Liu, H-J; Li, R; Wu, C-T; Wang, L-S

    2008-02-01

    Recovery of the surgically damaged mesothelial cell layer is a major process in reducing postoperative peritoneal adhesions. Sphingosine kinase (SPK) 1 is a signalling molecule involved in the regulation of proliferation and migration of various cell types. This study determined the effect of SPK-1 gene transfer on the recovery of damaged mesothelial cells and on peritoneal adhesion formation after surgery. Rat mesothelial cells were isolated and characterized by their expression of cytokeratin and vimentin. Their migration was determined by scratch wound motility assay. Cellular SPK-1 activity was measured by [gamma-32P]adenosine 5'-triphosphate incorporation. Wistar rats underwent laparotomy with subsequent caecum or uterine horn abrasion. Rats were randomized to either SPK-1 gene (Ad-SPK-1) transfer or control groups. The animals were killed 14 days after operation and peritoneal adhesions were graded. Adenovirus-mediated SPK-1 gene transfer increased the cellular SPK-1 activity of mesothelial cells, leading to enhanced migration. Median adhesion scores were significantly lower in the Ad-SPK-1 group than in controls in both rat caecum (0.98 versus 2.60; P < 0.001) and rat uterine horn (0.28 versus 1.83; P < 0.001) models. Adenovirus-mediated SPK-1 gene transfer promotes recovery of the surgically damaged mesothelial cell layer and prevents postoperative peritoneal adhesion formation. 2007 British Journal of Surgery Society Ltd. Published by John Wiley & Sons, Ltd.

  16. 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.

  17. 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.

  18. 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.

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

    PubMed

    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

    2015-05-15

    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.

  20. 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

  1. 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.

  2. 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

  3. 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

  4. 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.

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

    PubMed

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

    2015-06-01

    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

  6. 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

  7. 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.

  8. Transcriptional profiling of GBM invasion genes identifies effective inhibitors of the LIM kinase-Cofilin pathway

    PubMed Central

    Golbourn, Brian; Bertrand, Kelsey C.; Luck, Amanda; Sabha, Nesrin; Smith, Christian A.; Byron, Sara; Zadeh, Gelareh; Croul, Sidney; Berens, Michael; Rutka, James T.

    2014-01-01

    Malignant gliomas are highly proliferative and invasive neoplasms where total surgical resection is often impossible and effective local radiation therapy difficult. Consequently, there is a need to develop a greater understanding of the molecular events driving invasion and to identify novel treatment targets. Using microarray analysis comparing normal brain samples and mesenchymal glioblastoma multiforme (GBM), we identified over 140 significant genes involved in cell migration and invasion. The cofilin (CFL) pathway, which disassembles actin filaments, was highly up-regulated compared to normal brain. Up-regulation of LIM domain kinase 1 and 2 (LIMK1/2), that phosphorylates and inactivates cofilin, was confirmed in an additional independent data set comparing normal brain to GBM. We identified and utilized two small molecule inhibitors BMS-5 and Cucurbitacin I directed against the cofilin regulating kinases, LIMK1 and LIMK2, to target this pathway. Significant decreases in cell viability were observed in glioma cells treated with BMS-5 and Cucurbitacin I, while no cytotoxic effects were seen in normal astrocytes that lack LIMK. BMS-5 and Cucurbitacin I promoted increased adhesion in GBM cells, and decreased migration and invasion. Collectively, these data suggest that use of LIMK inhibitors may provide a novel way to target the invasive machinery in GBM. PMID:25237832

  9. [Gene expression and activity regulation of two calmodulin binding protein kinases in tobacco seedling].

    PubMed

    Hua, Wei; Li, Rong-Jun; Liang, Shu-Ping; Lu, Ying-Tang

    2005-06-01

    Two different calmodulin-binding protein kinase cDNAs (NtCBK1/2) have been isolated from tobacco. To understand the CBK protein activity regulation, we compared the activity regulation of NtCBK1 and NtCBK2 by pH, Mg(2+) concentration and Na(+) concentration. We found the autophosphorylation of NtCBK1/2 reached the maximum in pH 7.5 and 8 respectively; Mg(2+) and Na(+) shown different effects on the activity of NtCBKs, high and low Mg(2+) concentrations both inhibited the activity of NtCBKs, but Na+ had little effect on the kinase activity. In addition, to obtain further insight about the physiological roles of individual NtCBKs, we detected the expression profiles of CBKs. The results revealed different patterns of expression of NtCBK1 and NtCBK2. Both are largely expressed in leaf and flower; but in stem and root, NtCBK1 gene had stronger expression than NtCBK2. NtCBK2 expression was induced by GA treatment, while NtCBK1 expression remained unchanged under GA treatment. Expression of both NtCBK1 and NtCBK2 increased in response to salt stress, the former to a greater extent, and both expressions did not change under high/low temperature, drought, NAA and ABA treatments.

  10. 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

  11. HDA18 Affects Cell Fate in Arabidopsis Root Epidermis via Histone Acetylation at Four Kinase Genes[W

    PubMed Central

    Liu, Cui; Li, Lin-Chen; Chen, Wen-Qian; Chen, Xian; Xu, Zhi-Hong; Bai, Shu-Nong

    2013-01-01

    The differentiation of hair (H) and non-hair (N) cells in the Arabidopsis thaliana root epidermis is dependent on positional relationships with underlying cortical cells. We previously found that histone acetylation relays positional information and that a mutant altered in the histone deacetylase gene family member HISTONE DEACETYLASE 18 (HDA18) exhibits altered H and N epidermal cell patterning. Here, we report that HDA18 has in vitro histone deacetylase activity and that both mutation and overexpression of HDA18 led to cells at the N position having H fate. The HDA18 protein physically interacted with histones related to a specific group of kinase genes, which are demonstrated in this study to be components of a positional information relay system. Both down- and upregulation of HDA18 increased transcription of the targeted kinase genes. Interestingly, the acetylation levels of histone 3 lysine 9 (H3K9), histone 3 lysine 14 (H3K14) and histone 3 lysine 18 (H3K18) at the kinase genes were differentially affected by down- or upregulation of HDA18, which explains why the transcription levels of the four HDA18-target kinase genes increased in all lines with altered HDA18 expression. Our results reveal the surprisingly complex mechanism by which HDA18 affects cellular patterning in Arabidopsis root epidermis. PMID:23362208

  12. 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

  13. 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.

  14. 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.

  15. The Medicago truncatula Lysine Motif-Receptor-Like Kinase Gene Family Includes NFP and New Nodule-Expressed Genes1[W

    PubMed Central

    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

    2006-01-01

    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 lysine 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. PMID:16844829

  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. Genetic analysis of the Caenorhabditis elegans MAP kinase gene mpk-1.

    PubMed Central

    Lackner, M R; Kim, S K

    1998-01-01

    The Caenorhabditis elegans mpk-1 gene encodes a MAP kinase protein that plays an important role in Ras-mediated induction of vulval cell fates. We show that mutations that eliminate mpk-1 activity result in a highly penetrant, vulvaless phenotype. A double mutant containing a gain-of-function mpk-1 mutation and a gain-of-function mek mutation (MEK phosphorylates and activates MPK-1) exhibits a multivulva phenotype. These results suggest that mpk-1 may transduce most or all of the anchor cell signal. Epistasis analysis suggests that mpk-1 acts downstream of mek-2 (encodes a MEK homolog) and upstream of lin-1 (encodes an Ets transcription factor) in the anchor cell signaling pathway. Finally, mpk-1 may act together with let-60 ras in multiple developmental processes, as mpk-1 mutants exhibit nearly the same range of developmental phenotypes as let-60 ras mutants. PMID:9725833

  19. Genetic analysis of the Caenorhabditis elegans MAP kinase gene mpk-1.

    PubMed

    Lackner, M R; Kim, S K

    1998-09-01

    The Caenorhabditis elegans mpk-1 gene encodes a MAP kinase protein that plays an important role in Ras-mediated induction of vulval cell fates. We show that mutations that eliminate mpk-1 activity result in a highly penetrant, vulvaless phenotype. A double mutant containing a gain-of-function mpk-1 mutation and a gain-of-function mek mutation (MEK phosphorylates and activates MPK-1) exhibits a multivulva phenotype. These results suggest that mpk-1 may transduce most or all of the anchor cell signal. Epistasis analysis suggests that mpk-1 acts downstream of mek-2 (encodes a MEK homolog) and upstream of lin-1 (encodes an Ets transcription factor) in the anchor cell signaling pathway. Finally, mpk-1 may act together with let-60 ras in multiple developmental processes, as mpk-1 mutants exhibit nearly the same range of developmental phenotypes as let-60 ras mutants.

  20. 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.

  1. 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.

  2. The Role of Focal Adhesion Kinase in Keratinocyte Fibrogenic Gene Expression

    PubMed Central

    Januszyk, Michael; Kwon, Sun Hyung; Wong, Victor W.; Padmanabhan, Jagannath; Maan, Zeshaan N.; Whittam, Alexander J.; Major, Melanie R.; Gurtner, Geoffrey C.

    2017-01-01

    Abnormal skin scarring causes functional impairment, psychological stress, and high socioeconomic cost. Evidence shows that altered mechanotransduction pathways have been linked to both inflammation and fibrosis, and that focal adhesion kinase (FAK) is a key mediator of these processes. We investigated the importance of keratinocyte FAK at the single cell level in key fibrogenic pathways critical for scar formation. Keratinocytes were isolated from wildtype and keratinocyte-specific FAK-deleted mice, cultured, and sorted into single cells. Keratinocytes were evaluated using a microfluidic-based platform for high-resolution transcriptional analysis. Partitive clustering, gene enrichment analysis, and network modeling were applied to characterize the significance of FAK on regulating keratinocyte subpopulations and fibrogenic pathways important for scar formation. Considerable transcriptional heterogeneity was observed within the keratinocyte populations. FAK-deleted keratinocytes demonstrated increased expression of genes integral to mechanotransduction and extracellular matrix production, including Igtbl, Mmpla, and Col4a1. Transcriptional activities upon FAK deletion were not identical across all single keratinocytes, resulting in higher frequency of a minor subpopulation characterized by a matrix-remodeling profile compared to wildtype keratinocyte population. The importance of keratinocyte FAK signaling gene expression was revealed. A minor subpopulation of keratinocytes characterized by a matrix-modulating profile may be a keratinocyte subset important for mechanotransduction and scar formation. PMID:28880199

  3. 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

  4. 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

  5. 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

  6. 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

  7. 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.

  8. 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

  9. 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.

  10. 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.

  11. 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.

  12. 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.

  13. Variant Alleles in XRCC1 Arg194Trp and Arg399Gln Polymorphisms Increase Risk of Gastrointestinal Cancer in Sabah, North Borneo.

    PubMed

    Halim, Noor Hanis Abu; Chong, Eric Tzyy Jiann; Goh, Lucky Poh Wah; Chuah, Jitt Aun; See, Edwin Un Hean; Chua, Kek Heng; Lee, Ping-Chin

    2016-01-01

    The XRCC1 protein facilitates various DNA repair pathways; single-nucleotide polymorphisms (SNPs) in this gene are associated with a risk of gastrointestinal cancer (GIC) with inconsistent results, but no data have been previously reported for the Sabah, North Borneo, population. We accordingly investigated the XRCC1 Arg194Trp and Arg399Gln SNPs in terms of GIC risk in Sabah. We performed genotyping for both SNPs for 250 GIC patients and 572 healthy volunteers using a polymerase chain reaction- restriction fragment length polymorphism approach. We validated heterozygosity and homozygosity for both SNPs using direct sequencing. The presence of a variant 194Trp allele in the Arg194Trp SNP was significantly associated with a higher risk of GIC, especially with gastric and colorectal cancers. We additionally found that the variant 399Gln allele in Arg399Gln SNP was associated with a greater risk of developing gastric cancer. Our combined analysis revealed that inheritance of variant alleles in both SNPs increased the GIC risk in Sabah population. Based on our etiological analysis, we found that subjects ≥50 years and males who carrying the variant 194Trp allele, and Bajau subjects carrying the 399Gln allele had a significantly increased risk of GIC. Our findings suggest that inheritance of variant alleles in XRCC1 Arg194Trp and Arg399Gln SNPs may act as biomarkers for the early detection of GIC, especially for gastric and colorectal cancers in the Sabah population.

  14. 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.

  15. 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. Copyright © 2013 Elsevier B.V. All rights reserved.

  16. 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

  17. 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.

  18. 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

  19. 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...

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

    USDA-ARS?s Scientific Manuscript database

    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 ...

  1. 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...

  2. Rubber Tree (Hevea brasiliensis Muell. Arg).

    PubMed

    Venkatachalam, Perumal; Jayashree, Radha; Rekha, Karumamkandathil; Sushmakumari, Sreedharannair; Sobha, Sankaren; Kumari Jayasree, Parukkuttyamma; Kala, Radha Gopikkuttanunithan; Thulaseedharan, Arjunan

    2006-01-01

    Rubber tree (Hevea brasiliensis Muell. Arg.) is an important industrial crop for natural rubber production. At present, more than 9.5 million hectares in about 40 countries are devoted to rubber tree cultivation with a production about 6.5 million tons of dry rubber each year. The world supply of natural rubber is barely keeping up with a global demand for 12 million tons of natural rubber in 2020. Tapping panel dryness (TPD) is a complex physiological syndrome widely found in rubber tree plantations, which causes severe yield and crop losses in natural rubber producing countries. Currently, there is no effective prevention or treatment for this serious malady. As it is a perennial tree crop, the integration of specific desired traits through conventional breeding is both time-consuming and labour-intensive. Genetic transformation with conventional breeding is certainly a more promising tool for incorporation of agronomically important genes that could improve existing Hevea genotype. This chapter provides an Agrobacterium-mediated transformation protocol for rubber tree using immature anther-derived calli as initial explants. We have applied this protocol to generate genetically engineered plants from a high yielding Indian clone RRII 105 of Hevea brasiliensis (Hb). Calli were co-cultured with Agrobacterium tumefaciens harboring a plasmid vector containing the Hb superoxide dismutase (SOD) gene and the reporter gene used was beta-glucuronidase (GUS) gene (uidA). The selectable marker gene used was neomycin phosphotransferase (nptII) and kanamycin was used as selection agent. We found that a suitable transformation protocol for Hevea consists of a 3-d co-cultivation with Agrobacterium in the presence of 20 mM acetosyringone, 15 mM betaine HCl, and 11.55 mM proline followed by selection on medium containing 300 mg/L kanamycin. Transformed calli surviving on medium containing 300 mg/L kanamycin showed a strong GUS-positive reaction. Upon subsequent subculture into

  3. 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.

  4. Interleukin-1 Receptor–Associated Kinase 3 Gene Associates with Susceptibility to Acute Lung Injury

    PubMed Central

    Pino-Yanes, María; Ma, Shwu-Fan; Sun, Xiaoguang; Tejera, Paula; Corrales, Almudena; Blanco, Jesús; Pérez-Méndez, Lina; Espinosa, Elena; Muriel, Arturo; Blanch, Lluis; Garcia, Joe G. N.; Villar, Jesús

    2011-01-01

    Sepsis is the most common cause of acute lung injury (ALI), leading to organ dysfunction and death in critically ill patients. Previous studies associated variants of interleukin-1 receptor–associated kinase genes (IRAKs) with differential immune responses to pathogens and with outcomes during sepsis, and revealed that increased expression levels of the IRAK3 gene were correlated with poor outcomes during sepsis. Here we explored whether common variants of the IRAK3 gene were associated with susceptibility to, and outcomes of, severe sepsis. After our discovery of polymorphism, we genotyped a subset of seven single-nucleotide polymorphisms (SNPs) in 336 population-based control subjects and 214 patients with severe sepsis, collected as part of a prospective study of adults from a Spanish network of intensive care units. Whereas IRAK3 SNPs were not associated with susceptibility to severe sepsis, rs10506481 showed a significant association with the development of ALI among patients with sepsis (P = 0.007). The association remained significant after adjusting for multiple comparisons, population stratification, and clinical variables (odds ratio, 2.50; 95% confidence interval, 1.15–5.47; P = 0.021). By imputation, we revealed three additional SNPs independently associated with ALI (P < 0.01). One of these (rs1732887) predicted the disruption of a putative human–mouse conserved transcription factor binding site, and demonstrated functional effects in vitro (P = 0.017). Despite the need for replication in independent studies, our data suggest that common SNPs in the IRAK3 gene may be determinants of sepsis-induced ALI. PMID:21297081

  5. Prospects for herpes-simplex-virus thymidine-kinase and cytokine gene transduction as immunomodulatory gene therapy for prostate cancer.

    PubMed

    Hassan, W; Sanford, M A; Woo, S L; Chen, S H; Hall, S J

    2000-04-01

    In completed and ongoing clinical trials, adenovirus-mediated (Ad.) expression of herpes-simplex-virus thymidine-kinase (HSV-tk) gene transduction followed by ganciclovir (GCV) therapy has produced limited toxicity and evidence of antitumor activity following injection of the prostate. Furthermore, this system has been shown to direct systemic antitumor activity in several experimental cancer models, including that of prostate cancer, which may serve as the basis for in-situ immunomodulatory gene therapy. In a mouse model of prostate cancer, natural killer (NK) cells have been identified as the mediator of antimetastatic activity following Ad.HSV-tk + GCV, resulting in the combination of Ad.HSV-tk and adenovirus-mediated expression of interleukin 12 (Ad.IL-12) to exploit this cytokine's ability to enhance NK proliferation and cytotoxicity. Combination therapy demonstrated superior local and systemic growth suppression over that obtained with either therapy alone. Importantly, when the metastatic tumor burden was increased to an extent that negated the growth-suppressive activity directed by Ad.HSV-tk + GCV or Ad.IL-12 alone, combination therapy continued to demonstrate significant growth suppression. Examination of tumor-infiltrating lymphocytes documented enhanced NK lytic activity following combination therapy. Therefore, it appears that the combination of Ad.HSV-tk and Ad.IL-12 should be validated in a clinical trial for the treatment of prostate cancer.

  6. The Maize Low-Phytic Acid Mutant lpa2 Is Caused by Mutation in an Inositol Phosphate Kinase Gene

    PubMed Central

    Shi, Jinrui; Wang, Hongyu; Wu, Yunsheng; Hazebroek, Jan; Meeley, Robert B.; Ertl, David S.

    2003-01-01

    Reduced phytic acid content in seeds is a desired goal for genetic improvement in several crops. Low-phytic acid mutants have been used in genetic breeding, but it is not known what genes are responsible for the low-phytic acid phenotype. Using a reverse genetics approach, we found that the maize (Zea mays) low-phytic acid lpa2 mutant is caused by mutation in an inositol phosphate kinase gene. The maize inositol phosphate kinase (ZmIpk) gene was identified through sequence comparison with human and Arabidopsis Ins(1,3,4)P3 5/6-kinase genes. The purified recombinant ZmIpk protein has kinase activity on several inositol polyphosphates, including Ins(1,3,4)P3, Ins(3,5,6)P3, Ins(3,4,5,6)P4, and Ins(1,2,5,6)P4. The ZmIpk mRNA is expressed in the embryo, the organ where phytic acid accumulates in maize seeds. The ZmIpk Mutator insertion mutants were identified from a Mutator F2 family. In the ZmIpk Mu insertion mutants, seed phytic acid content is reduced approximately 30%, and inorganic phosphate is increased about 3-fold. The mutants also accumulate myo-inositol and inositol phosphates as in the lpa2 mutant. Allelic tests showed that the ZmIpk Mu insertion mutants are allelic to the lpa2. Southern-blot analysis, cloning, and sequencing of the ZmIpk gene from lpa2 revealed that the lpa2-1 allele is caused by the genomic sequence rearrangement in the ZmIpk locus and the lpa2-2 allele has a nucleotide mutation that generated a stop codon in the N-terminal region of the ZmIpk open reading frame. These results provide evidence that ZmIpk is one of the kinases responsible for phytic acid biosynthesis in developing maize seeds. PMID:12586875

  7. Glycerol Hypersensitivity in a Drosophila Model for Glycerol Kinase Deficiency Is Affected by Mutations in Eye Pigmentation Genes

    PubMed Central

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

    2012-01-01

    Glycerol kinase plays a critical role in metabolism by converting glycerol to glycerol 3-phosphate in an ATP dependent reaction. In humans, glycerol kinase deficiency results in a wide range of phenotypic variability; patients can have severe metabolic and CNS abnormalities, while others possess hyperglycerolemia and glyceroluria with no other apparent phenotype. In an effort to help understand the pathogenic mechanisms underlying the phenotypic variation, we have created a Drosophila model for glycerol kinase deficiency by RNAi targeting of dGyk (CG18374) and dGK (CG7995). As expected, RNAi flies have reduced glycerol kinase RNA expression, reduced phosphorylation activity and elevated glycerol levels. Further investigation revealed these flies to be hypersensitive to fly food supplemented with glycerol. Due to the hygroscopic nature of glycerol, we predict glycerol hypersensitivity is a result of greater susceptibility to desiccation, suggesting glycerol kinase to play an important role in desiccation resistance in insects. To evaluate a role for genetic modifier loci in determining severity of the glycerol hypersensitivity observed in knockdown flies, we performed a preliminary screen of lethal transposon insertion mutant flies using a glycerol hypersensitive survivorship assay. We demonstrate that this type of screen can identify both enhancer and suppressor genetic loci of glycerol hypersensitivity. Furthermore, we found that the glycerol hypersensitivity phenotype can be enhanced or suppressed by null mutations in eye pigmentation genes. Taken together, our data suggest proteins encoded by eye pigmentation genes play an important role in desiccation resistance and that eye pigmentation genes are strong modifiers of the glycerol hypersensitive phenotype identified in our Drosophila model for glycerol kinase deficiency. PMID:22427807

  8. 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

  9. 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.

  10. 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

  11. 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.

  12. Measuring herpes simplex virus thymidine kinase reporter gene expression in vitro.

    PubMed

    Yaghoubi, Shahriar S; Gambhir, Sanjiv S

    2006-01-01

    The herpes simplex 1 virus thymidine kinase (HSV1-tk) positron emission tomography (PET) reporter gene (PRG) or its mutant HSV1-sr39tk are used to investigate intracellular molecular events in cultured cells and for imaging intracellular molecular events and cell trafficking in living subjects. Two in vitro methods are available to assay gene expression of HSV1-tk or HSV1-sr39tk in cells or tissues. One method determines the level of HSV1-TK or HSV1-sr39TK enzyme activity in cell or tissue lysates by measuring the amount of the radiolabeled substrates that have been phosphorylated by these enzymes in a fixed amount of cell lysate protein after a fixed incubation time. The other method, called the 'cell-uptake assay', takes into account the natural uptake and efflux characteristics of the radiolabeled substrate by specific cells, in addition to the level of HSV1-TK or HSV1-sr39TK activity. Both of these assays can be used to validate molecular models in cultured cells, prior to studying them in living research subjects. Each of these assays can be completed in one day.

  13. 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.

  14. 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.

  15. 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.

  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. The polymorphism XRCC1 Arg194Trp and 8-hydroxydeoxyguanosine increased susceptibility to arsenic-related renal cell carcinoma.

    PubMed

    Hsueh, Yu-Mei; Lin, Ying-Chin; Chen, Wei-Jen; Huang, Chao-Yuan; Shiue, Horng-Sheng; Pu, Yeong-Shiau; Chen, Chi-Hung; Su, Chien-Tien

    2017-10-01

    This study was designed to explore the relationship between X-ray repair cross-complementing group 1 (XRCC1) gene polymorphisms and renal cell carcinoma (RCC) and to investigate whether individuals with an XRCC1 risk genotype, a high level of 8-OHdG or a high urinary total arsenic concentration have a modified odds ratio (OR) of RCC. We recruited 180 RCC patients and 360 age- and sex-matched controls from a hospital-based pool. Image-guided biopsy or surgical resection of renal tumors was performed on RCC patients for pathological verification. Genomic DNA was used to examine the genotype of XRCC1(Arg399Gln), XRCC1(Arg194Trp), XRCC3(Thr241Met) and XPD(Lys751Gln) by PCR-RFLP. Liquid chromatography with tandem mass spectrometry was used to determine urinary 8-OHdG levels. A HPLC-HG-AAS was used to determine the concentrations of urinary arsenic species. Participants with the genotype XRCC1(Arg194Trp) Arg/Trp+Trp/Trp had a significantly higher OR of RCC than those with the Arg/Arg genotype; the OR and 95% confidence interval was 0.66 (0.45-0.97) after multivariate adjustment. The OR of RCC for the combined effect of high urinary 8-OHdG levels and high urinary total arsenic concentration in individuals with a XRCC1(Arg194Trp) Arg/Trp+Trp/Trp genotype was higher than in patients with an Arg/Arg genotype, which was evident in a dose response manner. In conclusion, this is the first study to show that the XRCC1 Arg194 allele is a predicting factor for RCC. The more risk factors (high urinary 8-OHdG levels, high urinary total arsenic concentrations, and XRCC1 Arg194 allele) that were present, the higher the OR of RCC. Copyright © 2017 Elsevier Inc. All rights reserved.

  18. 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.

  19. 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

  20. 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.

  1. 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.

  2. 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

  3. 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

  4. 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.

  5. Siderophore production of a periplasmic transport binding protein kinase gene defective mutant of Magnetospirillum magneticum AMB-1.

    PubMed

    Calugay, Ronie J; Okamura, Yoshiko; Wahyudi, Aris Tri; Takeyama, Haruko; Matsunaga, Tadashi

    2004-10-22

    A non-magnetic mutant, NMA61, of the magnetic bacterium Magnetospirillum magneticum AMB-1 was generated by transposon mutagenesis to identify genes involved in magnetosome synthesis. The genomic region of NMA61 interrupted by a Mini-Tn5 transposon was analyzed. The transposon was inserted in an open reading frame (ORF) coding for a periplasmic transport binding protein kinase gene homologue. Three adjacent ORFs and a promoter were identified upstream, indicating that the sequences comprised an operon. Phenotype characterizations showed that the growth inhibition imposed by the exogenous non-assimilable iron chelator nitrilotriacetate was relieved in wild type but not in NMA61, by the addition of the isolated wild type siderophore. Higher concentration of siderophores accumulated in the culture medium of NMA61 than in wild type. These data suggest that the interrupted periplasmic transport binding protein kinase gene homologue is required for siderophore transport into M. magneticum AMB-1.

  6. 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.

  7. Pancreatic β-Cell Dysfunction in Diet-Induced Obese Mice: Roles of AMP-Kinase, Protein Kinase Cε, Mitochondrial and Cholesterol Metabolism, and Alterations in Gene Expression.

    PubMed

    Pepin, Émilie; Al-Mass, Anfal; Attané, Camille; Zhang, Kezhuo; Lamontagne, Julien; Lussier, Roxane; Madiraju, S R Murthy; Joly, Erik; Ruderman, Neil B; Sladek, Robert; Prentki, Marc; Peyot, Marie-Line

    2016-01-01

    Diet induced obese (DIO) mice can be stratified according to their weight gain in response to high fat diet as low responders (LDR) and high responders (HDR). This allows the study of β-cell failure and the transitions to prediabetes (LDR) and early diabetes (HDR). C57BL/6N mice were fed for 8 weeks with a normal chow diet (ND) or a high fat diet and stratified as LDR and HDR. Freshly isolated islets from ND, LDR and HDR mice were studied ex-vivo for mitochondrial metabolism, AMPK activity and signalling, the expression and activity of key enzymes of energy metabolism, cholesterol synthesis, and mRNA profiling. Severely compromised glucose-induced insulin secretion in HDR islets, as compared to ND and LDR islets, was associated with suppressed AMP-kinase activity. HDR islets also showed reduced acetyl-CoA carboxylase activity and enhanced activity of 3-hydroxy-3-methylglutaryl-CoA reductase, which led respectively to elevated fatty acid oxidation and increased cholesterol biosynthesis. HDR islets also displayed mitochondrial membrane hyperpolarization and reduced ATP turnover in the presence of elevated glucose. Expression of protein kinase Cε, which reduces both lipolysis and production of signals for insulin secretion, was elevated in DIO islets. Genes whose expression increased or decreased by more than 1.2-fold were minor between LDR and ND islets (17 differentially expressed), but were prominent between HDR and ND islets (1508 differentially expressed). In HDR islets, particularly affected genes were related to cell cycle and proliferation, AMPK signaling, mitochondrial metabolism and cholesterol metabolism. In conclusion, chronically reduced AMPK activity, mitochondrial dysfunction, elevated cholesterol biosynthesis in islets, and substantial alterations in gene expression accompany β-cell failure in HDR islets. The β-cell compensation process in the prediabetic state (LDR) is largely independent of transcriptional adaptive changes, whereas the transition

  8. Pancreatic β-Cell Dysfunction in Diet-Induced Obese Mice: Roles of AMP-Kinase, Protein Kinase Cε, Mitochondrial and Cholesterol Metabolism, and Alterations in Gene Expression

    PubMed Central

    Pepin, Émilie; Al-Mass, Anfal; Attané, Camille; Zhang, Kezhuo; Lamontagne, Julien; Lussier, Roxane; Madiraju, S. R. Murthy; Joly, Erik; Ruderman, Neil B.; Sladek, Robert; Prentki, Marc; Peyot, Marie-Line

    2016-01-01

    Diet induced obese (DIO) mice can be stratified according to their weight gain in response to high fat diet as low responders (LDR) and high responders (HDR). This allows the study of β-cell failure and the transitions to prediabetes (LDR) and early diabetes (HDR). C57BL/6N mice were fed for 8 weeks with a normal chow diet (ND) or a high fat diet and stratified as LDR and HDR. Freshly isolated islets from ND, LDR and HDR mice were studied ex-vivo for mitochondrial metabolism, AMPK activity and signalling, the expression and activity of key enzymes of energy metabolism, cholesterol synthesis, and mRNA profiling. Severely compromised glucose-induced insulin secretion in HDR islets, as compared to ND and LDR islets, was associated with suppressed AMP-kinase activity. HDR islets also showed reduced acetyl-CoA carboxylase activity and enhanced activity of 3-hydroxy-3-methylglutaryl-CoA reductase, which led respectively to elevated fatty acid oxidation and increased cholesterol biosynthesis. HDR islets also displayed mitochondrial membrane hyperpolarization and reduced ATP turnover in the presence of elevated glucose. Expression of protein kinase Cε, which reduces both lipolysis and production of signals for insulin secretion, was elevated in DIO islets. Genes whose expression increased or decreased by more than 1.2-fold were minor between LDR and ND islets (17 differentially expressed), but were prominent between HDR and ND islets (1508 differentially expressed). In HDR islets, particularly affected genes were related to cell cycle and proliferation, AMPK signaling, mitochondrial metabolism and cholesterol metabolism. In conclusion, chronically reduced AMPK activity, mitochondrial dysfunction, elevated cholesterol biosynthesis in islets, and substantial alterations in gene expression accompany β-cell failure in HDR islets. The β-cell compensation process in the prediabetic state (LDR) is largely independent of transcriptional adaptive changes, whereas the transition

  9. Anaplastic lymphoma kinase (ALK) gene alteration in signet ring cell carcinoma of the gastrointestinal tract.

    PubMed

    Alese, Olatunji B; El-Rayes, Bassel F; Sica, Gabriel; Zhang, Guojing; Alexis, Dianne; La Rosa, Francisco G; Varella-Garcia, Marileila; Chen, Zhengjia; Rossi, Michael R; Adsay, Nazim V; Khuri, Fadlo R; Owonikoko, Taofeek K

    2015-03-01

    ALK-EML4 translocation is an established driver aberration in non-small cell lung cancer (NSCLC), with reported predilection for cases with signet ring histology. We assessed the presence of anaplastic lymphoma kinase (ALK) gene rearrangements in signet ring cancers arising in the stomach and colon. Histologically confirmed cases of signet ring adenocarcinoma of the stomach or the colon were identified. The presence of the classic ALK and EML4 fusion gene was initially determined by fluorescence in-situ hybridization (FISH) technique. Immunohistochemistry (IHC) was performed using two previously validated antibodies, ALK1 clone (1:100; DAKO) and 5A4 (Novocastra, Leica Biosystems) along with positive controls of ALK-translocated lung cancer. We employed 42 cases of signet ring carcinoma diagnosed between 2001 and 2011; 25 gastric and 17 colon cancer. Median age 63.3 years; male/female 17/25; race, black 47.5%, white 47.5%, others, 5%; stage I, 21.4%; stage II, 31%; stage III, 26.2%; stage IV, 21.4%. One of 42 cases (2.3%) was positive for ALK translocation by FISH using the standard criteria of at least 15% positive cells for the break-apart signal (50-70 cells enumerated per case). Using a less restrictive cut-off of 10% positive cells, 7 cases (16%) were considered possibly positive. None of the 'possibly positive' cases was found to harbor ALK translocation by another molecular testing approach (IHC). IHC with two previously validated monoclonal antibodies showed 0 of 42 (0%) cases positive. ALK gene rearrangement is very rare in gastrointestinal cancers and enrichment strategy focusing on signet ring cell histology did not significantly improve the detection rate.

  10. 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

  11. Nuclear Gene 33/Mig6 regulates the DNA damage response through an ATM serine/threonine kinase-dependent mechanism.

    PubMed

    Li, Cen; Park, Soyoung; Zhang, Xiaowen; Eisenberg, Leonard M; Zhao, Hong; Darzynkiewicz, Zbigniew; Xu, Dazhong

    2017-10-06

    Gene 33 (Mig6, ERRFI1) is an adaptor protein with multiple cellular functions. We recently linked Gene 33 to the DNA damage response (DDR) induced by hexavalent chromium (Cr(VI)), but the molecular mechanism remains unknown. Here we show that ectopic expression of Gene 33 triggers DDR in an ATM serine/threonine kinase (ATM)-dependent fashion and through pathways dependent or not dependent on ABL proto-oncogene 1 non-receptor tyrosine kinase (c-Abl). We observed the clear presence of Gene 33 in the nucleus and chromatin fractions of the cell. We also found that the nuclear localization of Gene 33 is regulated by its 14-3-3-binding domain and that the chromatin localization of Gene 33 is partially dependent on its ErbB-binding domain. Our data further indicated that Gene 33 may regulate the targeting of c-Abl to chromatin. Moreover, we observed a clear association of Gene 33 with histone H2AX and that ectopic expression of Gene 33 promotes the interaction between ATM and histone H2AX without triggering DNA damage. In summary, our results reveal nuclear functions of Gene 33 that regulate DDR. The nuclear localization of Gene 33 also provides a spatial explanation of the previously reported regulation of apoptosis by Gene 33 via the c-Abl/p73 pathway. On the basis of these findings and our previous studies, we propose that Gene 33 is a proximal regulator of DDR that promotes DNA repair. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

  12. 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.

  13. 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.

  14. Direct and indirect effects of RNA interference against pyridoxal kinase and pyridoxine 5'-phosphate oxidase genes in Bombyx mori.

    PubMed

    Huang, ShuoHao; Yao, LiLi; Zhang, JianYun; Huang, LongQuan

    2016-08-01

    Vitamin B6 comprises six interconvertible pyridine compounds (vitamers), among which pyridoxal 5'-phosphate is a coenzyme involved in a high diversity of biochemical reactions. Humans and animals obtain B6 vitamers from diet, and synthesize pyridoxal 5'-phosphate by pyridoxal kinase and pyridoxine 5'-phosphate oxidase. Currently, little is known on how pyridoxal 5'-phosphate biosynthesis is regulated, and pyridoxal 5'-phosphate is supplied to meet their requirement in terms of cofactor. Bombyx mori is a large silk-secreting insect, in which protein metabolism is most active, and the vitamin B6 demand is high. In this study, we successfully down-regulated the gene expression of pyridoxal kinase and pyridoxine 5'-phosphate oxidase by body cavity injection of synthesized double-stranded small interfering RNA to 5th instar larvae of Bombyx mori, and analyzed the gene transcription levels of pyridoxal 5'-phosphate dependent enzymes, phosphoserine aminotransferase and glutamic-oxaloacetic transaminase. Results show that the gene expression of pyridoxal kinase and pyridoxine 5'-phosphate oxidase has a greater impact on the gene transcription of enzymes using pyridoxal 5'-phosphate as a cofactor in Bombyx mori. Our study suggests that pyridoxal 5'-phosphate biosynthesis and dynamic balance may be regulated by genetic networks. Copyright © 2016 Elsevier B.V. All rights reserved.

  15. 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.

  16. 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

  17. 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.

  18. 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.

  19. 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.

  20. 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.

  1. 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

  2. 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

  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. Accelerated Evolution of PAK3- and PIM1-like Kinase Gene Families in the Zebra Finch, Taeniopygia guttata

    PubMed Central

    Kong, Lesheng; Lovell, Peter V.; Heger, Andreas; Mello, Claudio V.; Ponting, Chris P.

    2010-01-01

    Genes encoding protein kinases tend to evolve slowly over evolutionary time, and only rarely do they appear as recent duplications in sequenced vertebrate genomes. Consequently, it was a surprise to find two families of kinase genes that have greatly and recently expanded in the zebra finch (Taeniopygia guttata) lineage. In contrast to other amniotic genomes (including chicken) that harbor only single copies of p21-activated serine/threonine kinase 3 (PAK3) and proviral integration site 1 (PIM1) genes, the zebra finch genome appeared at first to additionally contain 67 PAK3-like (PAK3L) and 51 PIM1-like (PIM1L) protein kinase genes. An exhaustive analysis of these gene models, however, revealed most to be incomplete, owing to the absence of terminal exons. After reprediction, 31 PAK3L genes and 10 PIM1L genes remain, and all but three are predicted, from the retention of functional sites and open reading frames, to be enzymatically active. PAK3L, but not PIM1L, gene sequences show evidence of recurrent episodes of positive selection, concentrated within structures spatially adjacent to N- and C-terminal protein regions that have been discarded from zebra finch PAK3L genes. At least seven zebra finch PAK3L genes were observed to be expressed in testis, whereas two sequences were found transcribed in the brain, one broadly including the song nuclei and the other in the ventricular zone and in cells resembling Bergmann's glia in the cerebellar Purkinje cell layer. Two PIM1L sequences were also observed to be expressed with broad distributions in the zebra finch brain, one in both the ventricular zone and the cerebellum and apparently associated with glial cells and the other showing neuronal cell expression and marked enrichment in midbrain/thalamic nuclei. These expression patterns do not correlate with zebra finch-specific features such as vocal learning. Nevertheless, our results show how ancient and conserved intracellular signaling molecules can be co

  5. 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.

  6. 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

  7. 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.

  8. Cdk12 Is A Gene-Selective RNA Polymerase II Kinase That Regulates a Subset of the Transcriptome, Including Nrf2 Target Genes

    PubMed Central

    Li, Xuan; Chatterjee, Nirmalya; Spirohn, Kerstin; Boutros, Michael; Bohmann, Dirk

    2016-01-01

    The Nrf2 transcription factor is well conserved throughout metazoan evolution and serves as a central regulator of adaptive cellular responses to oxidative stress. We carried out an RNAi screen in Drosophila S2 cells to better understand the regulatory mechanisms governing Nrf2 target gene expression. This paper describes the identification and characterization of the RNA polymerase II (Pol II) kinase Cdk12 as a factor that is required for Nrf2 target gene expression in cell culture and in vivo. Cdk12 is, however, not essential for bulk mRNA transcription and cells lacking CDK12 function are viable and able to proliferate. Consistent with previous findings on the DNA damage and heat shock responses, it emerges that Cdk12 may be specifically required for stress activated gene expression. Transcriptome analysis revealed that antioxidant gene expression is compromised in flies with reduced Cdk12 function, which makes them oxidative stress sensitive. In addition to supporting Reactive Oxygen Species (ROS) induced gene activation, Cdk12 suppresses genes that support metabolic functions in stressed conditions. We suggest that Cdk12 acts as a gene-selective Pol II kinase that engages a global shift in gene expression to switch cells from a metabolically active state to “stress-defence mode” when challenged by external stress. PMID:26911346

  9. Cdk12 Is A Gene-Selective RNA Polymerase II Kinase That Regulates a Subset of the Transcriptome, Including Nrf2 Target Genes.

    PubMed

    Li, Xuan; Chatterjee, Nirmalya; Spirohn, Kerstin; Boutros, Michael; Bohmann, Dirk

    2016-02-25

    The Nrf2 transcription factor is well conserved throughout metazoan evolution and serves as a central regulator of adaptive cellular responses to oxidative stress. We carried out an RNAi screen in Drosophila S2 cells to better understand the regulatory mechanisms governing Nrf2 target gene expression. This paper describes the identification and characterization of the RNA polymerase II (Pol II) kinase Cdk12 as a factor that is required for Nrf2 target gene expression in cell culture and in vivo. Cdk12 is, however, not essential for bulk mRNA transcription and cells lacking CDK12 function are viable and able to proliferate. Consistent with previous findings on the DNA damage and heat shock responses, it emerges that Cdk12 may be specifically required for stress activated gene expression. Transcriptome analysis revealed that antioxidant gene expression is compromised in flies with reduced Cdk12 function, which makes them oxidative stress sensitive. In addition to supporting Reactive Oxygen Species (ROS) induced gene activation, Cdk12 suppresses genes that support metabolic functions in stressed conditions. We suggest that Cdk12 acts as a gene-selective Pol II kinase that engages a global shift in gene expression to switch cells from a metabolically active state to "stress-defence mode" when challenged by external stress.

  10. 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.

  11. 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.

  12. 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.

  13. [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.

  14. 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

  15. 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).

  16. 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.

  17. Comparison of the expression patterns of two small gene families of S gene family receptor kinase genes during the defence response in Brassica oleracea and Arabidopsis thaliana.

    PubMed

    Pastuglia, Martine; Swarup, Ranjan; Rocher, Anne; Saindrenan, Patrick; Roby, Dominique; Dumas, Christian; Cock, J Mark

    2002-01-09

    SFR2, a member of the S gene family of receptor kinases, has been shown to be rapidly induced by wounding and bacterial infection suggesting that this gene may play a role in the defence response in Brassica. In this study we have compared the response of SFR2 to that of two other members of the SFR gene family in Brassica (SFR1 and SFR3) and to the closely-related ARK genes of Arabidopsis. Different patterns of mRNA accumulation were observed for different members of these families. SFR1 transcripts only accumulated in response to bacterial infection and their abundance was not significantly affected by wounding. Neither treatment induced accumulation of SFR3 transcripts. ARK1 and ARK3 resembled SFR2 in that their mRNAs accumulated in response to both wounding and bacterial infection. Both SFR1 and SFR2 mRNAs accumulated in response to exogenously applied salicylic acid (SA) and SA was shown to be required for induction of expression from the SFR2 promoter in Arabidopsis. However, the timing of the increase in endogenous SA levels following bacterial infiltration in Brassica indicates that the accumulation of SFR mRNA in the first few hours after infiltration does not occur in response to an increase in SA levels. We discuss the possibility that induction of SFR gene expression by SA may contribute to potentialization of the defence response. Taken together with previous studies that indicate a possible role during development, the data presented here suggest that the SFR and ARK gene families may have overlapping roles in both defence and during development.

  18. 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

  19. Transcriptional response of a novel HpCDPK1 kinase gene from Hippeastrum x hybr. to wounding and fungal infection.

    PubMed

    Pawełek, Agnieszka; Duszyn, Maria; Świeżawska, Brygida; Szmidt-Jaworska, Adriana; Jaworski, Krzysztof

    2017-09-01

    Calcium dependent protein kinases (CDPK) are well established plant sensor and effectors for calcium ions and participate in regulation of multiple abiotic and biotic stress responses in plant cells. Here we present the identification and characterization of a new CDPK kinase gene from bulbous plant Hippeastrum x hybr. and examine the role of this kinase in stress responses leading to phytoalexin (PA) production in plant tissues. In the previous research, it was shown that Hippeastrum bulbs mechanically wounded or infected with Peyronellaea curtisii (=Phoma narcissi) are inducted to an antifungal red substance synthesis. In this research, we demonstrated Ca(2+) dependence of the phytoalexin production by wounded bulbs. Furthermore, the isolated HpCDPK1 cDNA for ORF was found to be 1596bp long and encoded 531 amino acid protein with CDPK kinase activity, as was shown by recombinant GST-HpCDPK1 enzyme production and analysis. HpCDPK1 transcript was present in all vegetative and chosen generative organs of Hippeastrum plant. The dynamics of the observed HpCDPK1 mRNA changes in bulbs depended on stressor type. The mechanical injury caused one wave of transcript increase while more complex transcript changes were observed within 48h after Peyronellaea inoculation. In plant bulbs already accumulating red phytoalexin, increases in HpCDPK1 mRNA level were observed at certain intervals within 48h whereas, in the case of fungal infection, only one big increment in the transcript amount at the 10th minute after inoculation was detected. The observed transcriptional response of HpCDPK1 gene to wounding and pathogen infection stress suggests a positive correlation with phytoalexin synthesis and maintenance in bulb tissues and puts more light on CDPK kinase role in the plant stress response regulation. This also bears some potential for understanding the mechanism of a phytoalexin formation. Copyright © 2017 Elsevier GmbH. All rights reserved.

  20. 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

  1. 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

  2. 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.

  3. Origin and diversification of leucine-rich repeat receptor-like protein kinase (LRR-RLK) genes in plants.

    PubMed

    Liu, Ping-Li; Du, Liang; Huang, Yuan; Gao, Shu-Min; Yu, Meng

    2017-02-07

    Leucine-rich repeat receptor-like protein kinases (LRR-RLKs) are the largest group of receptor-like kinases in plants and play crucial roles in development and stress responses. The evolutionary relationships among LRR-RLK genes have been investigated in flowering plants; however, no comprehensive studies have been performed for these genes in more ancestral groups. The subfamily classification of LRR-RLK genes in plants, the evolutionary history and driving force for the evolution of each LRR-RLK subfamily remain to be understood. We identified 119 LRR-RLK genes in the Physcomitrella patens moss genome, 67 LRR-RLK genes in the Selaginella moellendorffii lycophyte genome, and no LRR-RLK genes in five green algae genomes. Furthermore, these LRR-RLK sequences, along with previously reported LRR-RLK sequences from Arabidopsis thaliana and Oryza sativa, were subjected to evolutionary analyses. Phylogenetic analyses revealed that plant LRR-RLKs belong to 19 subfamilies, eighteen of which were established in early land plants, and one of which evolved in flowering plants. More importantly, we found that the basic structures of LRR-RLK genes for most subfamilies are established in early land plants and conserved within subfamilies and across different plant lineages, but divergent among subfamilies. In addition, most members of the same subfamily had common protein motif compositions, whereas members of different subfamilies showed variations in protein motif compositions. The unique gene structure and protein motif compositions of each subfamily differentiate the subfamily classifications and, more importantly, provide evidence for functional divergence among LRR-RLK subfamilies. Maximum likelihood analyses showed that some sites within four subfamilies were under positive selection. Much of the diversity of plant LRR-RLK genes was established in early land plants. Positive selection contributed to the evolution of a few LRR-RLK subfamilies.

  4. A receptor kinase gene of the LysM type is involved in legume perception of rhizobial signals.

    PubMed

    Madsen, Esben Bjørn; Madsen, Lene Heegaard; Radutoiu, Simona; Olbryt, Magdalena; Rakwalska, Magdalena; Szczyglowski, Krzysztof; Sato, Shusei; Kaneko, Takakazu; Tabata, Satoshi; Sandal, Niels; Stougaard, Jens

    2003-10-09

    Plants belonging to the legume family develop nitrogen-fixing root nodules in symbiosis with bacteria commonly known as rhizobia. The legume host encodes all of the functions necessary to build the specialized symbiotic organ, the nodule, but the process is elicited by the bacteria. Molecular communication initiates the interaction, and signals, usually flavones, secreted by the legume root induce the bacteria to produce a lipochitin-oligosaccharide signal molecule (Nod-factor), which in turn triggers the plant organogenic process. An important determinant of bacterial host specificity is the structure of the Nod-factor, suggesting that a plant receptor is involved in signal perception and signal transduction initiating the plant developmental response. Here we describe the cloning of a putative Nod-factor receptor kinase gene (NFR5) from Lotus japonicus. NFR5 is essential for Nod-factor perception and encodes an unusual transmembrane serine/threonine receptor-like kinase required for the earliest detectable plant responses to bacteria and Nod-factor. The extracellular domain of the putative receptor has three modules with similarity to LysM domains known from peptidoglycan-binding proteins and chitinases. Together with an atypical kinase domain structure this characterizes an unusual receptor-like kinase.

  5. 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.

  6. 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.

  7. 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

  8. 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.

  9. GSA-1/ARG1 protects root gravitropism in Arabidopsis under ammonium stress.

    PubMed

    Zou, Na; Li, Baohai; Chen, Hao; Su, Yanhua; Kronzucker, Herbert J; Xiong, Liming; Baluška, František; Shi, Weiming

    2013-10-01

    Gravitropism plays a critical role in plant growth and development, plant stability and acclimation to changes in water and nutrient availability. Ammonium (NH4(+)) is well known to have profound effects on root growth, but its impacts on gravitropism are poorly understood. To determine which genes are essential for the maintenance of root gravitropism under NH4(+) stress, we isolated and identified an NH4 (+)-sensitive mutant, gsa-1 (gravitropism sensitive to ammonium-1), in Arabidopsis thaliana, using an agar plate root reorientation assay. We found that, under NH4(+) stress, gsa-1 displayed increased loss of root gravitropism. Gene cloning and sequencing revealed that gsa-1 contains a G to C transversion mutation at the highly conserved 5'-GT splice position of intron 10 of ARG1 (ALTERED RESPONSE TO GRAVITY1), known to participate in the transduction of the root gravity signal. Genetic complement tests established the locus of GSA-1/ARG1 and its role in resistance to NH4 (+) inhibition on root gravitropism. GSA-1/ARG1 is required for normal AUX1 expression and basipetal auxin transport in root apices. In addition, PIN-FORMED2 (PIN2) is proposed as a target in the reduction of root gravitropism under NH4(+) stress, a response which can be antagonized by the GSA-1/ARG1-dependent pathway. These results suggest that GSA-1/ARG1 protects root gravitropism in Arabidopsis thaliana under ammonium stress. © 2013 The Authors. New Phytologist © 2013 New Phytologist Trust.

  10. 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

  11. 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.

  12. 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.

  13. 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

  14. Targeting Abl kinases to regulate vascular leak during sepsis and acute respiratory distress syndrome.

    PubMed

    Rizzo, Alicia N; Aman, Jurjan; van Nieuw Amerongen, Geerten P; Dudek, Steven M

    2015-05-01

    The vascular endothelium separates circulating fluid and inflammatory cells from the surrounding tissues. Vascular leak occurs in response to wide-spread inflammatory processes, such as sepsis and acute respiratory distress syndrome, because of the formation of gaps between endothelial cells. Although these disorders are leading causes of mortality in the intensive care unit, no medical therapies exist to restore endothelial cell barrier function. Recent evidence highlights a key role for the Abl family of nonreceptor tyrosine kinases in regulating vascular barrier integrity. These kinases have well-described roles in cancer progression and neuronal morphogenesis, but their functions in the vasculature have remained enigmatic until recently. The Abl family kinases, c-Abl (Abl1) and Abl related gene (Arg, Abl2), phosphorylate several cytoskeletal effectors that mediate vascular permeability, including nonmuscle myosin light chain kinase, cortactin, vinculin, and β-catenin. They also regulate cell-cell and cell-matrix junction dynamics, and the formation of actin-based cellular protrusions in multiple cell types. In addition, both c-Abl and Arg are activated by hyperoxia and contribute to oxidant-induced endothelial cell injury. These numerous roles of Abl kinases in endothelial cells and the current clinical usage of imatinib and other Abl kinase inhibitors have spurred recent interest in repurposing these drugs for the treatment of vascular barrier dysfunction. This review will describe the structure and function of Abl kinases with an emphasis on their roles in mediating vascular barrier integrity. We will also provide a critical evaluation of the potential for exploiting Abl kinase inhibition as a novel therapy for inflammatory vascular leak syndromes. © 2015 American Heart Association, Inc.

  15. Characterization of a murine gene encoding a developmentally regulated cytoplasmic dual-specificity mitogen-activated protein kinase phosphatase.

    PubMed Central

    Dickinson, Robin J; Williams, David J; Slack, David N; Williamson, Jill; Seternes, Ole-Morten; Keyse, Stephen M

    2002-01-01

    Mitogen-activated protein kinases (MAPKs) play a vital role in cellular growth control, but far less is known about these signalling pathways in the context of embryonic development. Duration and magnitude of MAPK activation are crucial factors in cell fate decisions, and reflect a balance between the activities of upstream activators and specific MAPK phosphatases (MKPs). Here, we report the isolation and characterization of the murine Pyst3 gene, which encodes a cytosolic dual-specificity MKP. This enzyme selectively interacts with, and is catalytically activated by, the 'classical' extracellular signal-regulated kinases (ERKs) 1 and 2 and, to a lesser extent, the stress-activated MAPK p38alpha. These properties define the ability of this enzyme to dephosphorylate and inactivate ERK1/2 and p38alpha, but not JNK (c-Jun N-terminal kinase) in vivo. When expressed in mammalian cells, the Pyst3 protein is predominantly cytoplasmic. Furthermore, leptomycin B causes a complete redistribution of the protein to the nucleus, implicating a CRM (chromosomal region maintenance)1/exportin 1-dependent nuclear export signal in determining the subcellular localization of this enzyme. Finally, whole-mount in situ hybridization studies in mouse embryos reveal that the Pyst3 gene is expressed specifically in the placenta, developing liver and in migratory muscle cells. Our results suggest that this enzyme may have a critical role in regulating the activity of MAPK signalling during early development and organogenesis. PMID:11988087

  16. 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

  17. Isolation and characterization of a novel wheat cysteine-rich receptor-like kinase gene induced by Rhizoctonia cerealis

    NASA Astrophysics Data System (ADS)

    Yang, Kun; Rong, Wei; Qi, Lin; Li, Jiarui; Wei, Xuening; Zhang, Zengyan

    2013-10-01

    Cysteine-rich receptor kinases (CRKs) belong to the receptor-like kinase family. Little is known about CRK genes in wheat. We isolated a wheat CRK gene TaCRK1 from Rhizoctonia cerealis-resistant wheat CI12633 based on a differentially expressed sequence identified by RNA-Sequencing (RNA-Seq) analysis. TaCRK1 was more highly expressed in CI12633 than in susceptible Wenmai 6. Transcription of TaCRK1 in wheat was induced in CI12633 after R. cerealis infection and exogenous abscisic acid (ABA) treatment. The deduced TaCRK1 protein contained a signal peptide, two DUF26 domains, a transmembrane domain, and a serine/threonine protein kinase domain. Transient expression of a green fluorescence protein fused with TaCRK1 in wheat and onion indicated that TaCRK1 may localize to plasma membranes. Characterization of TaCRK1 silencing induced by virus-mediated method in CI12633 showed that the downregulation of TaCRK1 transcript did not obviously impair resistance to R. cerealis. This study paves the way to further CRK research in wheat.

  18. Association of p21 Ser31Arg and p53 Arg72Pro polymorphisms with lung cancer risk in CAPUA study

    PubMed Central

    Souto-García, Ana; Fernández-Somoano, Ana; Pascual, Teresa; Álvarez-Avellón, Sara M; Tardón, Adonina

    2012-01-01

    Background The aim of this study was to investigate how Ser31Arg polymorphisms in p21 may modify lung cancer susceptibility. Because p21 is the major downstream mediator of p53, we analyzed the combined effect of two polymorphisms, p21 Ser31Arg and TP53 Arg72Pro, to elucidate whether polymorphic variants determine the risk of lung cancer. Methods This was designed as a hospital-based case-control study, and included 675 cases and 675 control subjects matched by ethnicity, gender, and age. Genotypes were determined by polymerase chain reaction restriction fragment length polymorphism, and multivariate unconditional logistic regression was performed to analyze the results. Results Subjects who carried the p21 Ser31Arg allele had a higher risk of lung cancer (adjusted odds ratio [OR] 1.38; 95% confidence interval [CI] 0.99–2.03). This risk was increased in men aged younger than 55 years (adjusted OR 2.35; 95% CI 1.00–5.51). Smokers had an increased risk of lung cancer (adjusted OR 2.23; 95% CI 1.24–4.02). Men younger than 55 years carrying risk alleles for both genes (p21 Ser31Arg and TP53 Arg72Pro) had an increased risk (adjusted OR 5.78; 95% CI 1.38–24.19), as did smokers with both risk alleles (adjusted OR 4.52; 95% CI 1.52–13.50). Conclusion The presence of both variant alleles increased the risk of developing lung cancer in men, particularly in smokers younger than 55 years. PMID:28210126

  19. 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

  20. 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.

  1. 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.

  2. Differential regulation of polo-like kinase 1, 2, 3, and 4 gene expression in mammalian cells and tissues.

    PubMed

    Winkles, Jeffrey A; Alberts, Gregory F

    2005-01-10

    The four mammalian polo-like kinase (Plk) family members are critical regulators of cell cycle progression, mitosis, cytokinesis, and the DNA damage response. Research conducted to date has primarily investigated the expression patterns, structural features, substrates, and subcellular distribution of these important serine-threonine kinases. Here, we review the published data describing the regulation of Plk1, 2, 3, or 4 gene expression either during mammalian cell cycle progression or in tissue samples. These studies have demonstrated that the Plk family genes are differentially expressed following growth factor stimulation of quiescent fibroblasts. Furthermore, although Plk1 and Plk2 mRNA and protein levels are coordinately regulated during cell cycle progression, this is not the case for Plk3. In addition, the Plk1, 2 and 4 proteins have relatively short intracellular half-lives, but Plk3 is very stable. The Plk family genes are also differentially regulated in stressed cells; for example, when DNA-damaging agents are added to cycling cells, Plk1 expression decreases, but Plk2 and Plk3 expression increases. Finally, Plk1, 2, 3, and 4 are expressed to varying degrees in different human tissue types and it has been reported that Plk1 expression is increased and Plk3 expression is decreased in tumor specimens. These results indicate that the differential regulation of Plk family member gene expression is one cellular strategy for controlling Plk activity in mammalian cells.

  3. The codon 399 Arg/Gln XRCC1 polymorphism is associated with lung cancer in Indians.

    PubMed

    Natukula, Kirmani; Jamil, Kaiser; Pingali, Usha Rani; Attili, Venkata Satya Suresh; Madireddy, Umamaheshwar Rao Naidu

    2013-01-01

    The XRCC1 (X-ray repair cross complimenting group-I) gene in BER (base excision repair) pathway is essential for DNA repair process. Polymorphisms in this gene are associated with variations in the repair efficiency which might predispose individuals to development of various cancers. Two variants of XRCC1gene (at codon 399), Gln/Gln and Arg/Gln, have been shown to be related to lowered DNA repair capacity and increased genomic instability in multiple studies. Hence our investigation focused on genotyping these variants to correlate with other multiple risk factors in lung cancer (NSCLC) patients since we hypothesized that these variants of the XRCC1 gene might influence disease susceptibility. We examined the frequency of the polymorphism in one hundred cases and an almost equal number of controls after recording their demographics with a structured questionnaire. Genomic DNA from blood samples was extracted for PCR studies, followed by RFLP to determine the variants. The significance of the data was statistically analyzed. The three genotypes in cases and controls were Arg/Arg (40% and 54.45%); Gln/Gln (19% and 9.90%), and Arg/Gln (41.0% and 35.64%) respectively. Among these 3 genotypes, we found Gln/Gln and Arg/Gln to show association with lung cancer. Correlating these genotypes with several parameters, we also found that these two variants were associated with risk in males (p<0.05) and with smoking habits (p<0.05). In females Arg/Gln genotype showed association with stage of the disease (p=0.04). This is the first report in South Indian scenario where Arg399Gln genotypes were found to be associated with stage of the disease in females. It is concluded that XRCC1 genotypes Gln/Gln and Arg/Gln may influence cancer susceptibility in patients with smoking habits and these functional SNPs in XRCC1 gene may act as attractive candidate biomarkers in lung cancer for diagnosis and prognosis.

  4. Novel Compound Heterozygous Mutations in the PANK2 Gene in a Chinese Patient With Atypical Pantothenate Kinase-Associated Neurodegeneration

    PubMed Central

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

    2007-01-01

    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. PMID:15747360

  5. 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. Copyright 2005 Movement Disorder Society.

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

    PubMed

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

    2016-08-01

    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. 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. 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. 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.

  7. 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

  8. 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.

  9. Sucrose regulated expression of a Ca2+-dependent protein kinase (TaCDPK1) gene in excised leaves of wheat.

    PubMed

    Martínez-Noël, Giselle; Nagaraj, Vinay J; Caló, Gonzalo; Wiemken, Andres; Pontis, Horacio G

    2007-01-01

    Sucrose (Suc) can influence the expression of a large number of genes and thereby regulates many metabolic and developmental processes. However, the Suc sensing and the components of the ensuing signaling transduction pathway leading to the regulation of gene expression are not fully understood. We have shown that protein kinases and phosphatases are involved in the Suc induced expression of fructosyltransferase (FT) genes and fructan accumulation by an hexokinase independent pathway in wheat (Triticum aestivum). In the present study, using an RT-PCR based strategy, we have cloned a calcium-dependent protein kinase (TaCDPK1) cDNA that is upregulated during Suc treatment of excised wheat leaves. The deduced amino-acid sequence of CDPK1 has high sequence similarity (>70%) to known CDPKs from both monocots and dicots. Based on sequence homology, TaCDPK1 sequence shows a variable domain preceding a catalytic domain, an autoinhibitory function domain, and a C-terminal calmodulin-domain containing 4 EF-hand calcium-binding motifs, along with a N-myristoylation motif in the N-terminal variable domain. The recombinant Escherichia coli expressed TaCDPK1 was able to phosphorylate histone III-S in a calcium dependent manner in in vitro assays. The TaCDPK1 gene expression, as determined by quantitative RT-PCR, is induced by Suc and this effect is repressed by the inhibitors of the putative components of the Suc signal transduction pathway (calcium, Ser/Thr protein kinases and protein phosphatases). We propose that TaCDPK1 is involved in the Suc induced signaling pathway in wheat leaves.

  10. 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

  11. LY294002 inhibits glucocorticoid-induced COX-2 gene expression in cardiomyocytes through a phosphatidylinositol 3 kinase-independent mechanism

    SciTech Connect

    Sun Haipeng; Xu Beibei; Sheveleva, Elena; Chen, Qin M.

    2008-10-01

    Glucocorticoids induce COX-2 expression in rat cardiomyocytes. While investigating whether phosphatidylinositol 3 kinase (PI3K) plays a role in corticosterone (CT)-induced COX-2, we found that LY294002 (LY29) but not wortmannin (WM) attenuates CT from inducing COX-2 gene expression. Expression of a dominant-negative mutant of p85 subunit of PI3K failed to inhibit CT from inducing COX-2 expression. CT did not activate PI3K/AKT signaling pathway whereas LY29 and WM decreased the activity of PI3K. LY303511 (LY30), a structural analogue and a negative control for PI3K inhibitory activity of LY29, also suppressed COX-2 induction. These data suggest PI3K-independent mechanisms in regulating CT-induced COX-2 expression. LY29 and LY30 do not inhibit glucocorticoid receptor transactivity. Both compounds have been reported to inhibit Casein Kinase 2 activity and modulate potassium and calcium levels independent of PI3K, while LY29 has been reported to inhibit mammalian Target of Rapamycin (mTOR), and DNA-dependent Protein Kinase (DNA-PK). Inhibitor of Casein Kinase 2 (CK2), mTOR or DNA-PK failed to prevent CT from inducing COX-2 expression. Tetraethylammonium (TEA), a potassium channel blocker, and nimodipine, a calcium channel blocker, both attenuated CT from inducing COX-2 gene expression. CT was found to increase intracellular Ca{sup 2+} concentration, which can be inhibited by LY29, TEA or nimodipine. These data suggest a possible role of calcium instead of PI3K in CT-induced COX-2 expression in cardiomyocytes.

  12. 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.

  13. 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.

  14. 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.

  15. Ornithine Transcarbamylase ArgK Plays a Dual role for the Self-defense of Phaseolotoxin Producing Pseudomonas syringae pv. phaseolicola.

    PubMed

    Chen, Li; Li, Pin; Deng, Zixin; Zhao, Changming

    2015-08-10

    Pseudomonas syringae is a phytopathogenic bacterium widely spread on terrestrial plants. Sulfodiaminophosphinyl tripeptide Phaseolotoxins (PHTs), produced by P. syringae pv. phaseolicola and P. syringae pv. actinidiae, represent a kind of antimetabolic phytotoxins. PHTs inhibit host cell Ornithine transcarbamylase (OTCase) activity and induce Arginine auxotrophic phenotype. The biosynthesis of PHT is temperature dependent, being optically produced at around 18 °C, while blocked above 28 °C. PHT resistant OTCase ArgK acts as a functional replacement of housekeeping OTCase ArgF, which is the acting target of PHT, to confer PHT producers with self-resistance. It was postulated that argK might be regulated directly by a PHT biosynthetic precursor and indirectly by temperature with an unknown manner. Neither transcriptional regulator nor thermal regulation related protein encoding gene was detected from PHT biosynthetic gene cluster. The tripeptide, Cit-Ala-hArg, was identified to be a by-product of PHT biosynthetic pathway in this report. Formation of Cit-Ala-hArg was catalyzed by ArgK with tripeptide Orn-Ala-hArg and carbamyl phosphate as substrates. It showed that ArgK not only provided alternative Arginine source as reported previously, but also controlled the production of PHTs by converting PHT biosynthetic precursors to nontoxic Cit-Ala-hArg reservoir for producers' self-defense.

  16. 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.

  17. Effects of lifestyle intervention on weight and metabolic parameters in patients with impaired glucose tolerance related to beta-3 adrenergic receptor gene polymorphism Trp64Arg(C/T): Results from the Japan Diabetes Prevention Program.

    PubMed

    Sakane, Naoki; Sato, Juichi; Tsushita, Kazuyo; Tsujii, Satoru; Kotani, Kazuhiko; Tominaga, Makoto; Kawazu, Shoji; Sato, Yuzo; Usui, Takeshi; Kamae, Isao; Yoshida, Toshihide; Kiyohara, Yutaka; Sato, Shigeaki; Tsuzaki, Kokoro; Takahashi, Kaoru; Kuzuya, Hideshi

    2016-05-01

    The beta-3 adrenergic receptor (ADRB3), primarily expressed in adipose tissue, is involved in the regulation of energy metabolism. The present study hypothesized that ADRB3 (Trp64Arg, rs4994) polymorphisms modulate the effects of lifestyle intervention on weight and metabolic parameters in patients with impaired glucose tolerance. Data were analyzed from 112 patients with impaired glucose tolerance in the Japan Diabetes Prevention Program, a lifestyle intervention trial, randomized to either an intensive lifestyle intervention group or usual care group. Changes in weight and metabolic parameters were measured after the 6-month intervention. The ADRB3 polymorphisms were determined using the polymerase chain reaction restriction fragment length polymorphism method. Non-carriers showed a greater weight reduction compared with the carriers in both the lifestyle intervention group and usual care group, and a greater increase of high-density lipoprotein cholesterol levels than the carriers only in the lifestyle intervention group. ADRB3 polymorphisms could influence the effects of lifestyle interventions on weight and lipid parameters in impaired glucose tolerance patients.

  18. 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.

  19. In Vivo Identification of Solar Radiation-Responsive Gene Network: Role of the p38 Stress-Dependent Kinase

    PubMed Central

    Mouchet, Nicolas; Adamski, Henri; Bouvet, Régis; Corre, Sébastien; Courbebaisse, Yann; Watier, Eric; Mosser, Jean; Chesné, Christophe; Galibert, Marie-Dominique

    2010-01-01

    Solar radiation is one of the most common threats to the skin, with exposure eliciting a specific protective cellular response. To decrypt the underlying mechanism, we used whole genome microarrays (Agilent 44K) to study epidermis gene expression in vivo in skin exposed to simulated solar radiation (SSR). We procured epidermis samples from healthy Caucasian patients, with phototypes II or III, and used two different SSR doses (2 and 4 J/cm2), the lower of which corresponded to the minimal erythemal dose. Analyses were carried out five hours after irradiation to identify early gene expression events in the photoprotective response. About 1.5% of genes from the human genome showed significant changes in gene expression. The annotations of these affected genes were assessed. They indicated a strengthening of the inflammation process and up-regulation of the JAK-STAT pathway and other pathways. Parallel to the p53 pathway, the p38 stress-responsive pathway was affected, supporting and mediating p53 function. We used an ex vivo assay with a specific inhibitor of p38 (SB203580) to investigate genes the expression of which was associated with active p38 kinase. We identified new direct p38 target genes and further characterized the role of p38. Our findings provide further insight into the physiological response to UV, including cell-cell interactions and cross-talk effects. PMID:20505830

  20. 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.

  1. 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.

  2. 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.

  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. 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

  5. Association Pattern of Interleukin-1 Receptor-Associated Kinase-4 Gene Polymorphisms with Allergic Rhinitis in a Han Chinese Population

    PubMed Central

    Zhang, Yuan; Lin, Xiaoping; Desrosiers, Martin; Zhang, Wei; Meng, Na; Zhao, Liping

    2011-01-01

    Objective Interleukin-1 receptor-associated kinase-4 (IRAK-4) encodes a kinase that is essential for NF-kB activation in Toll-like receptor and T-cell receptor signaling pathways, indicating a possible crosstalk between innate and acquired immunities. We attempted to determine whether the polymorphisms in the Interleukin-1 receptor-associated kinase-4 (IRAK-4) gene are associated with allergic rhinitis (AR) in the Han Chinese population. Methods A population of 379 patients with AR and 333 healthy controls was studied. Blood was drawn for DNA extraction and total serum immunoglobulin E (IgE). A total of 11 single nucleotide polymorphisms (SNPs) in IRAK-4 were selected and individually genotyped. Results Significant allelic differences between cases and controls were obtained for the SNP of rs3794262 in the IRAK-4 gene. In the stratified analysis for gender, two SNPs (rs4251431 and rs6582484) in males appeared as significant associations. Subgroup analysis for the presence of different allergen sensitivities displayed associations only in the house dust mite-allergic cohorts (rs3794262, rs4251481). None of the selected SNPs in IRAK-4 was associated with total IgE level. The haplotype analyisis indicated GCCTGCGA was significantly associated with AR. The SNP-SNP interaction information analysis indicated that the selected sets of polymorphisms had no synergistic effect. Conclusions Our findings did not support the potential contribution of the IRAK-4 gene to serum IgE levels. However, the results demonstrated a gender- and allergen-dependant association pattern between polymorphisms in IRAK-4 and AR in Chinese population. PMID:21738793

  6. 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.

  7. [Isolation, prokaryotic expression and activity analysis of thymidylate kinase (tmk) gene from Phytoplasma of wheat blue dwarf].

    PubMed

    Li, Bei; Ji, Lingling; Wu, Yunfeng; Hao, Xing'an

    2008-06-01

    Wheat blue dwarf (WBD) is an important disease in winter wheat district, which causes serious losses in wheat production. Thymidylate kinase (TMK) catalyses the phosphorylation of dTMP to dTDP in the de novo and salvage pathways of dTTP synthesis in both prokaryotes and eukaryotes. In order effectively control this phytoplasma, we isolated the thymidylate kinase gene of WBD phytoplasma, and analyzed the catalytic activity of TMK protein. tmk gene was amplified from the phytoplasma of WBD, the amplicons were digested with EcoR I and Hind III and then inserted into expression vector pET-30a(+). The polyHis-tagged TMK was expressed in E. coli BL21 (DE3) and fusion protein was obtained and purified by Ni-NTA column. The TMK activities were measured by the method of en-zyme-coupled assay involving Mg2+, dTMP and ATP. Two genes, tmk-1 and tmk-2 were obtained, with the molecular weight of 630 bp and 624 bp. Both of them encoded an amino acid sequence with three conserved functional motifs which related with binding NTP/NMP. The fusion protein, TMK-2 had a higher catalytic activity (112.41 U/mg) than TMK-1 (16.4 U/mg), and its optimum catalytic conditions were 32 degrees C, pH7.3, 1.5 mmol/L Mg2+ and 1 mmol/L ATP. TMK-1 and TMK-2 had conserved functional motifs in their primary sequence, and suggested that they may function as TMK enzymes. But, the TMK-1-polyHis fusion protein had very low catalytic activity, the possible reason was that two highly conserved regions were absent in TMK-1, and it might function as another type of kinase in WBD phytoplasma. This experiment lay a foundation for further study of the TMK function in infection and reproduction of WBD phytoplasma.

  8. Targetable kinase gene fusions in high risk B-ALL: a study from the Children's Oncology Group.

    PubMed

    Reshmi, Shalini C; Harvey, Richard C; Roberts, Kathryn G; Stonerock, Eileen; Smith, Amy; Jenkins, Heather; Chen, I-Ming; Valentine, Marc; Liu, Yu; Li, Yongjin; Shao, Ying; Easton, John; Payne-Turner, Debbie; Gu, Zhaohui; Tran, Thai Hoa; Nguyen, Jonathan V; Devidas, Meenakshi; Dai, Yunfeng; Heerema, Nyla A; Carroll, Andrew J; Raetz, Elizabeth A; Borowitz, Michael J; Wood, Brent L; Angiolillo, Anne L; Burke, Michael J; Salzer, Wanda L; Zweidler-McKay, Patrick A; Rabin, Karen R; Carroll, William L; Zhang, Jinghui; Loh, Mignon L; Mullighan, Charles G; Willman, Cheryl L; Gastier-Foster, Julie M; Hunger, Stephen P

    2017-04-13

    Philadelphia chromosome-like acute lymphoblastic leukemia (Ph-like ALL) is a high-risk subtype characterized by genomic alterations that activate cytokine receptor and kinase signaling. We examined the frequency and spectrum of targetable genetic lesions in a retrospective cohort of 1389 consecutively diagnosed childhood B-ALL patients with high-risk clinical features and/or elevated minimal residual disease at the end of remission induction therapy. The Ph-like gene expression profile was identified in 341 of 1389 patients, 57 of which were excluded from additional analysis because of the presence of BCR-ABL1 (n=46) or ETV6-RUNX1 (n=11). Among the remaining 284 (20.4%) patients, overexpression and rearrangement of CRLF2 (IGH-CRLF2 or P2RY8-CRLF2) were identified in 124 (43.7%), with concomitant genomic alterations activating the JAK-STAT pathway (JAK1, JAK2, IL7R) identified in 63 patients (50.8% of CRLF2-rearranged cases). Of the remaining patients, using RT-PCR or transcriptome sequencing, we identified targetable ABL-class fusions (ABL1, ABL2, CSF1R and PDGFRB) in 14.1% of Ph-like ALL cases, EPOR rearrangements or JAK2 fusions (8.8%), alterations activating other JAK-STAT signaling genes (IL7R, SH2B3, JAK1; 6.3%) and other kinases (FLT3, NTRK3, LYN; 4.6%), and mutations involving the Ras pathway (KRAS, NRAS, NF1, PTPN11; 6%). We identified eight new rearrangement partners for four kinase genes previously reported to be rearranged in Ph-like ALL. The current findings provide support for the precision-medicine testing and treatment approach for Ph-like ALL that has been implemented in Children's Oncology Group ALL trials.

  9. Effect of L-arginine supplementation on the hepatic phosphatidylinositol 3-kinase signaling pathway and gluconeogenic enzymes in early intrauterine growth-restricted rats.

    PubMed

    Luo, Kaiju; Chen, Pingyang; Li, Suping; Li, Wen; He, Mingfeng; Wang, Tao; Chen, Juncao

    2017-09-01

    The present study aimed to investigate the response of the phosphatidylinositol 3-kinase (PI3K) signaling pathway and gluconeogenic enzymes in intrauterine growth-restricted rats to dietary L-arginine (L-Arg) supplementation during the lactation period early in life. Pregnant Sprague-Dawley rats were randomly divided into a control group (CON), an intrauterine growth restriction group (IUGR) and an L-Arg group (LA). The pregnant rats in the CON group were fed a 21% protein diet, and those in the IUGR and LA groups were fed a 10% low protein diet, and all rats were fed a 21% protein diet after delivery. Water was available ad libitum to the pregnant rats during the 21-day lactation period, and the water provided to the LA group included 200 mg/kg/day L-Arg. Blood glucose, serum insulin, homeostasis model of assessment for insulin resistance (HOMA-IR), PI3K and protein kinase B (PKB) protein expression, and phosphoenolpyruvate carboxykinase (PEPCK) and glucose-6-phosphatase (G-6-Pase) mRNA expression in the offspring rats were measured postnatally at 1, 3 and 8 weeks. No significant difference in blood glucose, serum insulin and HOMA-IR were identified at any time point among the three groups. PI3K and PKB expression was lower in the IUGR group offspring compared with that in the CON group offspring, but both were increased by dietary L-Arg supplementation. PEPCK mRNA and G-6-Pase mRNA expression levels in the offspring of the IUGR group were higher compared with those in the CON group but were downregulated following L-Arg supplementation. These results suggest that dietary L-Arg supplementation during the early lactation period promoted catch-up growth and reversed abnormalities in hepatic insulin signaling and gene expression of gluconeogenic enzymes in IUGR offspring rats.

  10. 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.

  11. 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.

  12. 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.