Science.gov

Sample records for gene arg kinase

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

    PubMed Central

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

    2015-01-01

    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. PMID:25849507

  2. Iron status in patients with pyruvate kinase deficiency: neonatal hyperferritinaemia associated with a novel frameshift deletion in the PKLR gene (p.Arg518fs), and low hepcidin to ferritin ratios.

    PubMed

    Mojzikova, Renata; Koralkova, Pavla; Holub, Dusan; Zidova, Zuzana; Pospisilova, Dagmar; Cermak, Jaroslav; Striezencova Laluhova, Zuzana; Indrak, Karel; Sukova, Martina; Partschova, Martina; Kucerova, Jana; Horvathova, Monika; Divoky, Vladimir

    2014-05-01

    Pyruvate kinase (PK) deficiency is an iron-loading anaemia characterized by chronic haemolysis, ineffective erythropoiesis and a requirement for blood transfusion in most cases. We studied 11 patients from 10 unrelated families and found nine different disease-causing PKLR mutations. Two of these mutations - the point mutation c.878A>T (p.Asp293Val) and the frameshift deletion c.1553delG (p.(Arg518Leufs*12)) - have not been previously described in the literature. This frameshift deletion was associated with an unusually severe phenotype involving neonatal hyperferritinaemia that is not typical of PK deficiency. No disease-causing mutations in genes associated with haemochromatosis could be found. Inappropriately low levels of hepcidin with respect to iron loading were detected in all PK-deficient patients with increased ferritin, confirming the predominant effect of accelerated erythropoiesis on hepcidin production. Although the levels of a putative hepcidin suppressor, growth differentiation factor-15, were increased in PK-deficient patients, no negative correlation with hepcidin was found. This result indicates the existence of another as-yet unidentified erythroid regulator of hepcidin synthesis in PK deficiency. PMID:24533562

  3. [Investigation of Antibiotic Resistance Genes (ARGs) in Landfill].

    PubMed

    Li, Lei; Xu, Jing; Zhao, You-cai; Song, Li-yan

    2015-05-01

    Antibiotic resistant genes (ARGs), an emerging contaminant, have been detected worldwide in various environments such as sediments and river. However, little is known about ARGs distribution in landfill. In this study, we investigated five ARGs [sulfonamides resistant genes (sulI and sulII), chloramphenicols resistant gene (cat), β-lactams resistant gene (bla-SHV), and tetracyclines resistant gene (tetW)] in refuse samples collected from jiangeungou landfill (Xi'an, China) by real-time PCR. We then correlated the ARGs and physiochemical properties of refuse to examine the link between them. Results showed that all tested ARGs have been detected in all samples, suggesting that landfill served as ARGs reservoir. The highest copies numbers of sulII, sulI, tetW, bla-SHV, and cat were (3.70 ± 0.06) x 10(8) copies · g(-1) ( dry refuse), (9.33 · 0.06) x 10(6) copies · g(-1) (dry refuse), (2.27 0.08) x 10(5) copies · g(-1) (dry refuse), (3.68 ± 0.09) x 10(4) copies · g(-1) (dry refuse), and (1.39 ± 0.10) x 10(4) copies · g(-1) (dry refuse), respectively. Further, sulI, sulII, and cat positively correlated to moisture and sulI and cat negatively correlated to pH. PMID:26314129

  4. Arg Kinase-binding Protein 2 (ArgBP2) Interaction with α-Actinin and Actin Stress Fibers Inhibits Cell Migration*

    PubMed Central

    Anekal, Praju Vikas; Yong, Jeffery; Manser, Ed

    2015-01-01

    Cell migration requires dynamic remodeling of the actomyosin network. We report here that an adapter protein, ArgBP2, is a component of α-actinin containing stress fibers and inhibits migration. ArgBP2 is undetectable in many commonly studied cancer-derived cell lines. COS-7 and HeLa cells express ArgBP2 (by Western analysis), but expression was detectable only in approximately half the cells by immunofluorescence. Short term clonal analysis demonstrated 0.2–0.3% of cells switch ArgBP2 expression (on or off) per cell division. ArgBP2 can have a fundamental impact on the actomyosin network: ArgBP2 positive COS-7 cells, for example, are clearly distinguishable by their denser actomyosin (stress fiber) network. ArgBP2γ binding to α-actinin appears to underlie its ability to localize to stress fibers and decrease cell migration. We map a small α-actinin binding region in ArgBP2 (residues 192–228) that is essential for these effects. Protein kinase A phosphorylation of ArgBP2γ at neighboring Ser-259 and consequent 14-3-3 binding blocks its interaction with α-actinin. ArgBP2 is known to be down-regulated in some aggressively metastatic cancers. Our work provides a biochemical explanation for the anti-migratory effect of ArgBP2. PMID:25429109

  5. 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…

  6. 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.

  7. Isolation and Analysis of the Arg-13 Gene of Neurospora Crassa

    PubMed Central

    Liu, Q.; Dunlap, J. C.

    1996-01-01

    Mutations in arg-13 result in slow growth in minimal medium and can suppress mutations in carbamyl phosphate synthase-aspartate carbamyl transferase within the pyrimidine pathway; the exact biochemical function of the gene product is unknown. To understand the role of arg-13 in arginine metabolism, cosmids rescuing growth in arg-13 mutants were cloned and mapped to the position of arg-13 on LG IR. Northern analysis showed the arg-13 message to contain ~2100 nt, although a 1.4-kb genomic fragment truncated at the 5' and 3' ends of the gene encodes a shortened transcript that can rescue arg-13 function. Expression of mRNA arising from the mutant arg-13 gene is induced by arginine starvation, although wild type (arg-13(+)) is not derepressed in minimal medium. The sequence of the arg-13 gene shows ARG-13 to be a member of the mitochondrial carrier superfamily with three repeats of a ~100-amino acid domain, six putative membrane spanning regions, and three copies of the mitochondrial carrier consensus pattern. This information plus available and new nutritional data are consistent with the hypothesis that arg-13 encodes a mitochondrial basic amino acid carrier whose existence was predicted based upon previous physiological, nutritional and biochemical data. PMID:8807290

  8. 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. PMID:27298228

  9. Metagenomic Analysis Revealing Antibiotic Resistance Genes (ARGs) and Their Genetic Compartments in the Tibetan Environment.

    PubMed

    Chen, Baowei; Yuan, Ke; Chen, Xin; Yang, Ying; Zhang, Tong; Wang, Yawei; Luan, Tiangang; Zou, Shichun; Li, Xiangdong

    2016-07-01

    Comprehensive profiles of antibiotic resistance genes (ARGs) and mobile genetic elements (MGEs) in a minimally impacted environment are essential to understanding the evolution and dissemination of modern antibiotic resistance. Chemical analyses of the samples collected from Tibet demonstrated that the region under investigation was almost devoid of anthropogenic antibiotics. The soils, animal wastes, and sediments were different from each other in terms of bacterial community structures, and in the typical profiles of ARGs and MGEs. Diverse ARGs that encoded resistance to common antibiotics (e.g., beta-lactams, fluoroquinolones, etc.) were found mainly via an efflux mechanism completely distinct from modern antibiotic resistome. In addition, a very small fraction of ARGs in the Tibetan environment were carried by MGEs, indicating the low potential of these ARGs to be transferred among bacteria. In comparison to the ARG profiles in relatively pristine Tibet, contemporary ARGs and MGEs in human-impacted environments have evolved substantially since the broad use of anthropogenic antibiotics. PMID:27111002

  10. 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

  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. PMID:27358479

  12. 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

  13. Study of the roles of Arg-104 and Arg-225 in the 2-kinase domain of 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase by site-directed mutagenesis.

    PubMed Central

    Rider, M H; Crepin, K M; De Cloedt, M; Bertrand, L; Vertommen, D; Hue, L

    1995-01-01

    The roles of Arg-104 and Arg-225 located in the 2-kinase domain of the bifunctional enzyme 6-phosphofructo-2-kinase (PFK-2)/fructose-2,6-bisphosphatase (FBPase-2) have been studied by site-directed mutagenesis. In recombinant rat liver PFK-2/FBPase-2, mutation of Arg-225 to Ser increased the Km of PFK-2 for fructose-6-phosphate (Fru-6-P) 7-fold at pH 6 and decreased PFK-2 activity at suboptimal substrate concentrations between pH 6 and 9.5. The mutation had no effect on the Vmax of PFK-2 or on the Km of PFK-2 for MgATP. The mutation also increased the Vmax. of FBPase-2 4-fold without changing the Km for Fru-2,6-P2 or IC50 of Fru-6-P. These findings are in agreement with a previous study [Rider and Hue (1992) Eur. J. Biochem. 207, 967-972] on the protection by Fru-6-P of the labelling of Arg-225 by phenylglyoxal, and suggest that Arg-225 participates in Fru-6-P binding. In recombinant rat muscle PFK-2/FBPase-2, mutation of Arg-104 to Ser increased the Km for Fru-6-P 60-fold, increased the IC50 of citrate, increased the Vmax. 1.5-3-fold at pH 8.5 and altered the pH profile of PFK-2 activity. It did not affect the Km of PFK-2 for MgATP. The mutation also decreased the Vmax. of FBPase-2 3-fold, increased the Km for Fru-2,6-P2 70-fold and increased the IC50 of Fru-6-P at least 300-fold. Although the dimeric structure was maintained in the mutant, its PFK-2 activity was more sensitive towards inactivation by guanidinium chloride than the wild-type enzyme activity. The findings indicate that Arg-104 is involved in Fru-6-P binding in the PFK-2 domain and that it might also bind citrate. Structural changes resulting from the mutation might be responsible for the changes in kinetic properties of FBPase-2. PMID:7619077

  14. [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. PMID:27506057

  15. 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

  16. 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. PMID:25172854

  17. Anabolic ornithine carbamoyltransferase of Pseudomonas aeruginosa: nucleotide sequence and transcriptional control of the argF structural gene.

    PubMed Central

    Itoh, Y; Soldati, L; Stalon, V; Falmagne, P; Terawaki, Y; Leisinger, T; Haas, D

    1988-01-01

    In Pseudomonas aeruginosa PAO the anabolic ornithine carbamoyltransferase (OTCase, EC 2.1.3.3) is the product of the argF gene and the only arginine biosynthetic enzyme whose synthesis is repressible by arginine. We have determined the complete nucleotide sequence of the argF gene including its promoter-control region. The deduced amino acid sequence of the anabolic OTCase consists of 305 residues (Mr 33,924), and this was confirmed by the N-terminal amino acid sequence, the total amino acid composition, and the subunit Mr of the purified enzyme. The native anabolic OTCase (Mr 110,000 to 125,000) was found to be a trimer by cross-linking experiments. P. aeruginosa also has a catabolic OTCase (the arcB gene product), which catalyzes the reverse reaction of the anabolic conversion. At the nucleotide sequence level, the P. aeruginosa argF gene had 52.4% identity with the arcB gene. The Escherichia coli argF and argI genes, which code for anabolic OTCase isoenzymes, had 47.3 and 44.9% identity, respectively, with the P. aeruginosa argF sequence. This suggests that these four genes have evolved from a common ancestral gene. The arcB gene appears to be more closely related to the E. coli argF gene than to the P. aeruginosa argF gene. Two transcripts (mRNA-1, mRNA-2) of the P. aeruginosa argF gene were identified by S1 mapping. The transcription initiation site for mRNA-1 was preceded by sequences having partial homology with the E. coli -35 and -10 consensus promoter sequences. No sequence similar to consensus promoters of enteric bacteria was found upstream of the 5' end of mRNA-2. E. coli carrying a P. aeruginosa argF+ recombinant plasmid produced mRNA-1 with low efficiency but no (or very little) mRNA-2. Arginine repressed argF transcription in P. aeruginosa. In the argF promoter region no sequence homologous to the "arg box" (arginine operator module) of E. coli was found. The mechanism of arginine repression in P. aeruginosa thus appears to be different from that in

  18. Association between β1 adrenergic receptor gene Arg389Gly polymorphism and risk of heart failure: a meta-analysis.

    PubMed

    Ma, S T; Zhao, W; Liu, B; Jia, R Y; Zhao, C J; Cui, L Q

    2015-01-01

    Numerous studies have evaluated the association between Arg389Gly polymorphism in the β1 adrenergic receptor gene and heart failure risk. However, the specific association is still controversial. We performed a meta-analysis of all case-control studies that evaluated the association between Arg389Gly polymorphism and heart failure in humans. Studies were identified in the PubMed, Embase, and China National Knowledge Infrastructure databases. Two reviewers independently assessed the studies. Six case-control studies with a total of 1736 participants were included in the meta-analysis, including 882 cases with heart failure and 854 controls, and our results showed no association between the Arg389Gly polymorphism and heart failure [ArgArg vs GlyGly: odds ratio (OR) = 0.84, 95% confidence interval (CI) 0.59-1.20; ArgArg vs ArgGly: OR = 0.95, 95%CI 0.78-1.16; dominant model: OR = 1.08, 95%CI 0.89-1.31; recessive model: OR = 0.96, 95%CI 0.69-1.35]. No publication bias was found in the present study (all P values > 0.05). In conclusion, the β1 adrenergic receptor gene Arg389Gly polymorphism might not be associated with heart failure risk. Further large and well-designed studies are needed to confirm this conclusion. PMID:26125791

  19. Assignment of CPS1, OTC, CRYD2, ARG2 and ASS genes to the chicken RH map.

    PubMed

    Shimogiri, Takeshi; Bosak, Natalia; Morisson, Mireille; Okamoto, Shin; Kawabe, Kotaro; Maeda, Yoshizane; Vignal, Alain; Yasue, Hiroshi

    2004-01-01

    An attempt was made to assign five genes, CPS1, OTC, ASS, CRYD2, and ARG2, to chicken chromosomes (GGA) by radiation-hybrid mapping. OTC was assigned to GGA1; ARG2 to GGA5; CPS1 to GGA7; and CRYD2 to GGA19. ASS was not, however, assigned to a specific chromosomal position. PMID:15339635

  20. Association of leptin receptor gene Gln223Arg polymorphism with susceptibility to colorectal cancer

    PubMed Central

    Arkani, Maral; Safaei, Akram; Pourhoseingholi, Mohamad Amin; Mohebbi, Seyed Reza; Fatemi, Seyed Reza; Vafaei, Mohammad

    2011-01-01

    Aim Leptin is a 16 kDa polypeptide hormone which secreted by adipose tissue and has an important role in energy balance, insulin pathway and inflammation, because of that it may play an important role in colorectal cancer (CRC). Leptin exerts its effect through the leptin receptor (LEPR) a member of the class I cytokine receptor family. Background We have investigated whether glutamine to arginine substitution (Gln223Arg) in exon 6 of the leptin receptor gene, has implications for susceptibility to CRC. Patients and methods Polymerase chain reaction (PCR) and restriction enzyme digestion (RFLP) was performed to evaluate the association between the Gln223Arg polymorphism of the LEPR and CRC risk in a case-control study in 346 subjects involving 173 cases with CRC and 173 controls. Results There was no statistically evidence of significant difference in genotype and allele frequencies between the cases with CRC and controls for the Gln223Arg polymorphism of LEPR, before or after adjusting for confounders (age, BMI, sex, and smoking status). Furthermore, no significant difference was observed between the CRC cases and controls by BMI, sex and smoking status. Conclusion Our findings suggest that the LEPR Gln223Arg polymorphism is not associated with the risk of CRC in Iranian population. PMID:24834182

  1. Characterization of the Streptomyces clavuligerus argC gene encoding N-acetylglutamyl-phosphate reductase: expression in Streptomyces lividans and effect on clavulanic acid production.

    PubMed Central

    Ludovice, M; Martin, J F; Carrachas, P; Liras, P

    1992-01-01

    The argC gene of Streptomyces clavuligerus encoding N-acetylglutamyl-phosphate reductase (AGPR) has been cloned by complementation of argC mutants Streptomyces lividans 1674 and Escherichia coli XC33. The gene is contained in an open reading frame of 1,023 nucleotides which encodes a protein of 340 amino acids with a deduced molecular mass of 35,224 Da. The argC gene is linked to argE, as shown by complementation of argE mutants of E. coli. Expression of argC from cloned DNA fragments carrying the gene leads to high levels of AGPR in wild-type S. lividans and in the argC mutant S. lividans 1674. Formation of AGPR is repressed by addition of arginine to the culture medium. The protein encoded by the argC gene is very similar to the AGPRs of Streptomyces coelicolor, Bacillus subtilis, and E. coli and, to a lesser degree, to the homologous enzymes of Saccharomyces cerevisiae and Anabaena spp. A conserved PGCYPT domain present in all the AGPR sequences suggests that this may be the active center of the protein. Transformation of S. clavuligerus 328, an argC auxotroph deficient in clavulanic acid biosynthesis, with plasmid pULML30, carrying the cloned argC gene, restored both prototrophy and antibiotic production. Images PMID:1339424

  2. Characterization of the Saccharomyces cerevisiae ARG7 gene encoding ornithine acetyltransferase, an enzyme also endowed with acetylglutamate synthase activity.

    PubMed

    Crabeel, M; Abadjieva, A; Hilven, P; Desimpelaere, J; Soetens, O

    1997-12-01

    We have cloned by functional complementation and characterized the yeast ARG7 gene encoding mitochondrial ornithine acetyltransferase, the enzyme catalyzing the fifth step in arginine biosynthesis. While forming ornithine, this enzyme regenerates acetylglutamate, also produced in the first step by the ARG2-encoded acetylglutamate synthase. Interestingly, total deletion of the genomic ARG7 ORF resulted in an arginine-leaky phenotype, indicating that yeast cells possess an alternative route for generating ornithine from acetylornithine. Yeast ornithine acetyltransferase has been purified and characterized previously as a heterodimer of two subunits proposed to derive from a single precursor protein [Liu, Y-S., Van Heeswijck R., Hoj, P. & Hoogenraad, N. (1995) Eur. J. Biochem. 228, 291-296]; those authors further suggested that the internal processing of Arg7p, which is a mitochondrial enzyme, might occur in the matrix, while the leader peptide would be of the non-cleavable-type. The characterization of the gene (a) establishes that Arg7p is indeed encoded by a single gene, (b) demonstrates the existence of a cleaved mitochondrial prepeptide of eight residues, and (c) shows that the predicted internal processing site is unlike the mitochondrial proteolytic peptidase target sequence. Yeast Arg7p shares between 32-43% identity in pairwise comparisons with the ten analogous bacterial ArgJ enzymes characterized. Among these evolutionarily related enzymes, some but not all appear bifunctional, being able to produce acetylglutamate not only from acetylornithine but also from acetyl-CoA, thus catalyzing the same reaction as the apparently unrelated acetylglutamate synthase. We have addressed the question of the bifunctionality of the eucaryotic enzyme, showing that overexpressed ARG7 can complement yeast arg2 and Escherichia coli argA mutations (affecting acetylglutamate synthase). Furthermore, Arg7p-linked acetylglutamate synthase activity was measurable in an assay. The

  3. Arg924X homozygous mutation in insulin receptor gene in a Tunisian patient with Donohue syndrome.

    PubMed

    Azzabi, Ons; Jilani, Houweyda; Rejeb, Imen; Siala, Nadia; Elaribi, Yasmina; Hizem, Syrine; Selmi, Ines; Halioui, Sonia; Lascols, Olivier; Jemaa, Lamia Ben; Maherzi, Ahmed

    2016-06-01

    Donohue syndrome (DS) is a rare and lethal autosomal recessive disease caused by mutations in the insulin receptor (INSR) gene, manifesting marked insulin resistance, severe growth retardation, hypertrichosis, and characteristic dysmorphic features. We describe a new case of Donohue syndrome born at 37 weeks' gestation of unrelated parents and presented with intra-uterine growth retardation, nipple hypertrophy, macropenis, distended abdomen, hirsutism and dysmorphic features. The clinical course showed failure to thrive, and episodes of alternating hypoglycemia and hyperglycemia. Laboratory tests revealed direct hyperbilirubinemia. The diagnosis of Donohue syndrome was established based on the above clinical characteristics and determination of the INSR mutation. He was found to have homozygous nonsense mutation c. 2270 C>T (Arg924X) at exon 14 of the INSR gene. He later developed enterocolitis and died at 3 months old. Prenatal diagnosis was performed for the family via chorionic villous biopsy. We try to explain gastrointestinal dysfunction seen in our patient. PMID:26974131

  4. Expression of the gene for resistance to phaseolotoxin (argK) depends on the activity of genes phtABC in Pseudomonas syringae pv. phaseolicola.

    PubMed

    Aguilera, Selene; De la Torre-Zavala, Susana; Hernández-Flores, José Luis; Murillo, Jesús; Bravo, Jaime; Alvarez-Morales, Ariel

    2012-01-01

    The bacterium Pseudomonas syringae pv. phaseolicola produces phaseolotoxin in a temperature dependent manner, being optimally produced between 18°C and 20°C, while no detectable amounts are present above 28°C. Phaseolotoxin is an effective inhibitor of ornithine carbamoyltransferase (OCTase) activity from plant, mammalian and bacterial sources and causes a phenotypic requirement for arginine. To protect the cell from its own toxin, P. syringae pv. phaseolicola synthesizes a phaseolotoxin-resistant OCTase (ROCT). The ROCT is the product of the argK gene and is synthesized only under conditions leading to phaseolotoxin synthesis. The argK gene is included in a chromosomal fragment named Pht cluster, which contains genes involved in the synthesis of phaseolotoxin. The aim of the present work was to investigate the possible involvement of other genes included in the Pht cluster in the regulation of gene argK. We conducted transcriptional analyses of argK in several mutants unable to produce phaseolotoxin, transcriptional fusions and electrophoretic mobility shift assays, which allowed us to determine that genes phtABC, located within the Pht cluster, participate in the transcriptional repression of gene argK at temperatures not permissive for phaseolotoxin biosynthesis. This repression is mediated by a protein present in both toxigenic and nontoxigenic strains of P. syringae and in E. coli, and requires the coordinated participation of phtA, phtB and phtC products in order to carry out an efficient argK repression. PMID:23056465

  5. Expression of the Gene for Resistance to Phaseolotoxin (argK) Depends on the Activity of Genes phtABC in Pseudomonas syringae pv. phaseolicola

    PubMed Central

    Aguilera, Selene; De la Torre-Zavala, Susana; Hernández-Flores, José Luis; Murillo, Jesús; Bravo, Jaime; Alvarez-Morales, Ariel

    2012-01-01

    The bacterium Pseudomonas syringae pv. phaseolicola produces phaseolotoxin in a temperature dependent manner, being optimally produced between 18°C and 20°C, while no detectable amounts are present above 28°C. Phaseolotoxin is an effective inhibitor of ornithine carbamoyltransferase (OCTase) activity from plant, mammalian and bacterial sources and causes a phenotypic requirement for arginine. To protect the cell from its own toxin, P. syringae pv. phaseolicola synthesizes a phaseolotoxin-resistant OCTase (ROCT). The ROCT is the product of the argK gene and is synthesized only under conditions leading to phaseolotoxin synthesis. The argK gene is included in a chromosomal fragment named Pht cluster, which contains genes involved in the synthesis of phaseolotoxin. The aim of the present work was to investigate the possible involvement of other genes included in the Pht cluster in the regulation of gene argK. We conducted transcriptional analyses of argK in several mutants unable to produce phaseolotoxin, transcriptional fusions and electrophoretic mobility shift assays, which allowed us to determine that genes phtABC, located within the Pht cluster, participate in the transcriptional repression of gene argK at temperatures not permissive for phaseolotoxin biosynthesis. This repression is mediated by a protein present in both toxigenic and nontoxigenic strains of P. syringae and in E. coli, and requires the coordinated participation of phtA, phtB and phtC products in order to carry out an efficient argK repression. PMID:23056465

  6. 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

  7. Arginine biosynthesis and regulation in Lactobacillus plantarum: the carA gene and the argCJBDF cluster are divergently transcribed.

    PubMed Central

    Bringel, F; Frey, L; Boivin, S; Hubert, J C

    1997-01-01

    A cluster of citrulline biosynthetic genes has been cloned and sequenced from a fragment of Lactobacillus plantarum CCM 1904 (ATCC 8014) DNA isolated as complementing a Bacillus subtilis argF mutation. The gene order was carA-argCJBDF, with carA transcribed divergently from the arg cluster. Although other gram-positive bacteria show similar arg clusters, this arrangement for carA is thus far unprecedented. Downstream from the arg cluster, two open reading frames (ORF7 and ORF8) having unknown functions were found. Sequence analysis of the end of a 10.5-kb cloned DNA fragment showed that argF was 3.5 kb from the ldhL gene coding for L-(+)-lactate dehydrogenase. A tree representation of amino acid sequence clustering relationships of 31 ornithine carbamoyltransferases (OTCases) from various organisms revealed two prokaryotic groups: one with ArgF of L. plantarum and one with ArgF of B. subtilis, which are paralogous. This divergence was not observed in vivo because an L. plantarum argF mutant (AM 1215) harboring no OTCase activity was complemented by the argF genes of L. plantarum and B. subtilis. No OTCase activity was detectable when L. plantarum was grown in the presence of saturating amounts of arginine or citrulline. Arginine may repress the citrulline biosynthetic genes in L. plantarum by using 11 identified DNA motifs which resemble the Escherichia coli ARG box consensus and which are in most cases separated by multiples of 11 bp, corresponding to a DNA helical turn. The carA and argCJBDF genes are divergently transcribed. Their putative promoters are 6 bp apart and are partially overlapped by putative ARG boxes, suggesting concerted transcription regulation. PMID:9098069

  8. 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. PMID:16753197

  9. 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. PMID:27038206

  10. Budd-Chiari Syndrome with Multiple Thrombi due to a Familial Arg42Ser Mutation in the Protein C Gene

    PubMed Central

    Mizoguchi, Kazuhiro; Ataka, Keiji; Yamamoto, Hiroshi; Fan, Xinping; Miyata, Toshiyuki

    2013-01-01

    A 34-year-old Japanese woman was admitted to our hospital complaining of developing bilateral pedal edema. Imaging studies led to a diagnosis of Budd-Chiari syndrome combined with internal jugular vein thrombus. We investigated the cause of thrombosis and found that the anticoagulant activity of protein C was decreased. Genetic analysis showed the presence of a c.125C>A (Arg42Ser) substitution in the protein C gene (PROC) of the proband, which generates an Arg42Ser mutation that replaces the scissile bond Arg42-Ala43 normally cleaved by a furin-like processing protease. Her father and younger brother also carried this mutation, although they had no evidence of thrombosis. PMID:24250338

  11. [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

  12. DNMT3A Arg882 mutation drives chronic myelomonocytic leukemia through disturbing gene expression/DNA methylation in hematopoietic cells

    PubMed Central

    Xu, Jie; Wang, Yue-Ying; Dai, Yu-Jun; Zhang, Wu; Zhang, Wei-Na; Xiong, Shu-Min; Gu, Zhao-Hui; Wang, Kan-Kan; Zeng, Rong; Chen, Zhu; Chen, Sai-Juan

    2014-01-01

    The gene encoding DNA methyltransferase 3A (DNMT3A) is mutated in ∼20% of acute myeloid leukemia cases, with Arg882 (R882) as the hotspot. Here, we addressed the transformation ability of the DNMT3A-Arg882His (R882H) mutant by using a retroviral transduction and bone marrow transplantation (BMT) approach and found that the mutant gene can induce aberrant proliferation of hematopoietic stem/progenitor cells. At 12 mo post-BMT, all mice developed chronic myelomonocytic leukemia with thrombocytosis. RNA microarray analysis revealed abnormal expressions of some hematopoiesis-related genes, and the DNA methylation assay identified corresponding changes in methylation patterns in gene body regions. Moreover, DNMT3A-R882H increased the CDK1 protein level and enhanced cell-cycle activity, thereby contributing to leukemogenesis. PMID:24497509

  13. Association of XPD (Lys751Gln) and XRCC1 (Arg280His) gene polymorphisms in myelodysplastic syndrome.

    PubMed

    Joshi, Dolly; Korgaonkar, Seema; Shanmukhaiah, Chandrakala; Vundinti, Babu Rao

    2016-01-01

    Myelodysplastic syndromes (MDSs) are heterogeneous hematopoietic disease characterized by ineffective haematopoiesis that frequently transforms into acute leukaemia. Alterations in many individual biologic pathways have been reported in MDS pathophysiology. Disease progression along the MDS, acute myeloid leukemia (AML) continuum is believed to be a consequence of stepwise accumulation of DNA mutations which infers a defect in DNA repair. The present study investigated the association between DNA repair genes (XRCC1, XRCC3, OGG1, XPD and RAD51) and the risk of developing MDS. The study was carried out in 92 primary MDS patients. The genotyping study was carried out by PCR-RFLP technique. We have studied seven single-nucleotide polymorphisms (SNPs) of five DNA repair genes (XRCC1 (Arg194Trp, Arg280His, Arg399Gln), XRCC3, XPD, RAD51 and OGG1). Significantly, a high frequency of DNA repair gene XRCC1 (Arg280His) (p=0.05) and XPD (Lys751Gln) (p=0.01) polymorphism was observed in MDS patients compared to controls. The distribution of polymorphisms in MDS subgroups showed a significant association of XRCC1 with RAEB I compared to other subgroup. Though a high frequency of XRCC1 gene polymorphism was observed in farmers and tobacco chewers, it was not statistically significant. Our study suggests that XRCC1 (Arg280His) and XPD polymorphisms are associated with risk of MDS and XRCC1 polymorphism strongly associated with advanced MDS subgroup. Hence, these polymorphisms can be used as a prognostic marker in MDS. PMID:26482462

  14. [The Trp64Arg polymorphism of beta3-adrenoreceptor gene study in persons with overweight and obesity].

    PubMed

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

    2012-01-01

    The development of obesity is determined by lifestyle and genetic mechanisms. In particular, the polymorphisms in the adrenergic receptor genes (ADRB) have been extensively studied for association with obesity-related phenotypes. ADRB3 is an obvious candidate gene given its involvement in the regulation of lipolysis and thermogenesis. ADRB3 Trp64Arg polymorphism, a missense mutation in the first transmembrane domain of the R3-adrenergic receptor is associated with visceral obesity and insulin resistance in the Pima Indian, French, and Finnish populations. The recent meta-analysis that combined data of 6582 individuals from Japanese populations showed significant association the Arg64 allele with increased BMI. There are tested the polymorphisms in the beta3-Adrenoreceptor (ADRB3) gene in associated with body mass index (BMI), fat mass and biochemical parameters.We have been examined 91 persons from Moscow region with BMI >25 kg/m2. The Trp64Arg polymorphism of ADRB3 genes were genotyped with the use of an allelic discrimination assay. The TaqMan-based real-time PCR method was applied. There have been estimated of anthropometric and biochemicalparameters. The frequencies of the Trp64Trp and Trp64Arggenotypes of ADRB3 gene were 82% and 12%, respectively, the frequencies of mutant allele was 6%. Trp64Arg genotypes of ADRB3 compared to Trp64Trp genotypes had significantly higher body fat percentage (respectively 48,6 +/- 0,96% and 43,8 +/- 1,72%, p<0,05), serum glucose (6,51 +/- 0,18 mmol/l and 5,67 +/- 0,09 mmol/l, p<0,01) and uric acid concentrations (0,46 +/- 0,02 mmol/l and 0,38 +/- 0,01 mmol/l, p<0,05). The test of the ADRB3 gene polymorphisms can be used for the personalization of diet in persons with obesity. PMID:22774474

  15. The Arg399Gln polymorphism in the XRCC1 gene is associated with increased risk of hematological malignancies.

    PubMed

    Du, Liang; Liu, Yuqi; Xue, Pei; Song, Chenxi; Shen, Jiani; He, Qing; Peng, Yuanling; Tong, Xiang; Tang, Lizhi; Zhang, Yonggang

    2015-06-01

    The associations between the Arg399Gln polymorphism in X-ray repair cross-complementing gene 1 (XRCC1) gene and the risk of hematological malignancies have been extensively investigated. However, the results were inconsistent. The objective of the current study is to investigate the association by meta-analysis. We searched PubMed database, Embase database, CNKI database, Wanfang database, and Weipu database, covering all studies until August 7, 2013. Statistical analysis was performed by using the Revman4.2 software and the Stata10.0 software. A total of 27 case-control studies concerning the Arg399Gln polymorphism were included from 26 articles. The results suggested that the Arg399Gln polymorphism was not associated with an increased/decreased risk of hematological malignancies in total analysis (OR = 1.15, 95 % confidence interval (CI) = 0.97-1.35, P = 0.10 for Arg/Gln + Gln/Gln vs. Arg/Arg). In the subgroup analysis by ethnicity and cancer types, significant association was found in Asians (OR = 1.35, 95 % CI = 1.04-1.75, P = 0.03) but not in Europeans (OR = 1.07, 95 % CI = 0.86-1.33, P = 0.56), and in leukemia (OR = 1.25, 95 % CI = 1.02-1.54, P = 0.03) but not in lymphoma (OR = 0.98, 95 % CI = 0.80-1.20, P = 0.84) or myeloma (OR = 1.13, 95 % CI = 0.23-5.69, P = 0.88). The current meta-analysis indicated that the Arg399Gln polymorphism in the XRCC1 gene might be a risk factor for hematological malignancies in Asians or for leukemia. In future, more large-scale case-control studies are needed to validate these results. PMID:25619474

  16. Conservation of structure in the human gene encoding argininosuccinate synthetase and the argG genes of the archaebacteria Methanosarcina barkeri MS and Methanococcus vannielii

    SciTech Connect

    Morris, C.J.; Reeve, J.N.

    1988-07-01

    The DNA sequences of the argG genes of Methanosarcina barkeri MS and Methanococcus vannielii were determined. The polypeptide products of these methanogen genes have amino acid sequences which are 50% identical to each other and 38% identical to the amino acid sequence encoded by the exons of the human argininosuccinate synthetase gene. Introns in the human chromosomal gene separate regions which encode amino acids conserved in both the archaebacterial and human gene products. An open reading frame immediately upstream of argG in Methanosarcina barkeri MS codes for an amino acid sequence which is 45 and 31% identical to the sequences of the large subunits of carbamyl phosphate synthetase in Escherichia coli and Saccharomyces cerevisiae, respectively. If this gene encodes carbamyl phosphate synthetase in Methanosarcina barkeri, this is the first example, in an archaebacterium, of physical linkage of genes that encode enzymes which catalyze reactions in the same amino acid biosynthetic pathway.

  17. Human protein kinase CK2 genes.

    PubMed

    Wirkner, U; Voss, H; Lichter, P; Pyerin, W

    1994-01-01

    We have analyzed the genomic structure of human protein kinase CK2. Of the presumably four genes, the gene encoding the regulatory subunit beta and a processed (pseudo)gene of the catalytic subunit alpha have been characterized completely. In addition, a 18.9 kb-long central part of the gene encoding the catalytic subunit alpha has been characterized. The subunit beta gene spans 4.2 kb and is composed of seven exons. Its promoter region shows several features of a "housekeeping gene" and shares common features with the promoter of the regulatory subunit of cAMP-dependent protein kinase. Conforming to the genomic structure, the beta gene transcripts form a band around 1.1 kb. The central part of the subunit alpha gene contains eight exons comprising bases 102 to 824 of the translated region. Within the introns, 16 Alu repeats were identified, some of which arranged in tandems. The structure of both human CK2 coding genes, alpha and beta, is highly conserved. Several introns are located at corresponding positions in the respective genes of the nematode Caenorhabditis elegans. The processed alpha (pseudo)gene has a complete open reading frame and is 99% homologous to the coding region of the CK2 alpha cDNA. Although the gene has a promoter-like upstream region, no transcript could be identified so far. The genomic clones were used for localization in the human genome. The beta gene was mapped to locus 6p21, the alpha gene to locus 20p13 and the alpha (pseudo)gene to locus 11p15. There is no evidence for additional alpha or beta loci in the human genome. PMID:7735323

  18. [Cloning, sequence analysis and expression of N-acetylglutamate kinase gene in Corynebacterium crenatum].

    PubMed

    Hao, Ning; Zhao, Zhi; Wang, Yu; Zhang, Ying-zi; Ding, Jiu-yuan

    2006-02-01

    N-Acetylglutamate kinase (EC 2.7.2.8;NAGK) genes from wild-type Corynebacterium crenatum AS 1.542 and a L-arginine-producing mutant C. crenatum 971.1 were cloned and sequenced. Analysis of argB sequences revealed that only one ORF existed, which used ATG as the initiation codon and coded a peptide of 317 amino acids with a calculated molecular weight of 33.6kDa. Only one nucleotide difference was found in the structure gene and the difference did not cause a change of amino acid by comparison of the gene sequences between the wild type C. crenatum AS 1.542 and the mutant 971.1. The ORF sequence of argB from C. crenatum AS 1.542 showed homologies of 99.89%, 76.62%, 37.94% to those from Corynebacterium glutamicum ATCC 13032, Corynebacterium efficient YS-314 and Escherichia coli k12. And the amino acid sequence deduced from ORF displayed homologies of 100%, 78.55%, 25.25% to those from microorganisms above, respectively. An internal promoter was found in the upstream of the argB gene from C. crenatum. The argB gene from C. crenatum AS 1.542 was expressed both in C. crenatum AS 1.542 and 971.1. The NAGK activity of transformed C. crenatum AS 1.542 was greatly increased by the induction of IPTG. The NAGK activity of transformed C. crenatum 971.1 was almost twice as much as that of C. crenatum 971.1 under the same induction. The amplification of the NAGK activity yielded 25% increase of L-arginine production in C. crenatum 971.1. PMID:16579472

  19. Asthma: Gln27Glu and Arg16Gly polymorphisms of the beta2-adrenergic receptor gene as risk factors

    PubMed Central

    2014-01-01

    Background Asthma is caused by both environmental and genetic factors. The ADRB2 gene, which encodes the beta 2-adrenergic receptor, is one of the most extensively studied genes with respect to asthma prevalence and severity. The Arg16Gly (+46A > G) and Gln27Glu (+79C > G) polymorphisms in the ADRB2 gene cause changes in the amino acids flanking the receptor ligand site, altering the response to bronchodilators and the risk of asthma through complex pathways. The ADRB2 polymorphisms affect beta-adrenergic bronchodilator action and are a tool to identify at-risk populations. Objective To determine the frequency of these two polymorphisms in allergic asthma patients and healthy subjects and to correlate these data with the occurrence and severity of asthma. Methods Eighty-eight allergic asthma patients and 141 healthy subjects were included in this study. The ADRB2 polymorphisms were analyzed using the amplification-refractory mutation system – polymerase chain reaction (ARMS-PCR) technique. The statistical analysis was performed with the SPSS 21.0 software using the Fisher’s Exact and χ2 tests. Results The ADRB2 polymorphisms were associated with asthma occurrence. The Arg16Arg, Gln27Gln and Gln27Glu genotypes were risk factors; the odds ratios were 6.782 (CI = 3.07 to 16.03), 2.120 (CI = 1.22 to 3.71) and 8.096 (CI = 3.90 to 17.77), respectively. For the Gly16Gly and Glu27Glu genotypes, the odds ratios were 0.312 (CI = 0.17 to 0.56) and 0.084 (CI = 0.04 to 0.17), respectively. The haplotype analysis showed that there were associations between the following groups: Arg16Arg-Gln27Gln (OR = 5.108, CI = 1.82 to 16.37), Gly16Gly-Glu27Glu (OR = 2.816, CI = 1.25 to 6.54), Arg16Gly-Gln27Glu (OR = 0.048, CI = 0.01 to 0.14) and Gly16Gly-Gln27Glu (OR = 0.1036, CI = 0.02 to 0.39). The polymorphism Gln27Glu was associated with asthma severity, as the Gln27Gln genotype was a risk factor for severe asthma (OR

  20. 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. PMID:16762971

  1. A novel mutation (Arg169Gln) of the cardiac ryanodine receptor gene causing exercise-induced bidirectional ventricular tachycardia.

    PubMed

    Hsueh, Chia-Hsiang; Weng, Yi-Chun; Chen, Chao-Yu; Lin, Tin-Kwang; Lin, Yen-Hung; Lai, Ling-Ping; Lin, Jiunn-Lee

    2006-04-01

    An 18-year-old woman presented with exercise induced sudden collapse. Series of cardiac work up revealed no structural cardiac abnormalities. Bidirectional ventricular tachycardia occurred during a treadmill exercise test. Under the impression of catecholaminergic polymorphic ventricular tachycardia, we screened the cardiac ryanodine receptor gene for mutation. We identified a novel heterozygous mutation at the 169th amino acid (Arg169Gln). This amino acid is highly conserved among many species and this mutation was not present in 50 normal control subjects. This patient was treated with a beta-block with good response. PMID:16517285

  2. Analysis of ADRB2 (Arg16Gly) Gene Variant with Susceptibility, Pharmacogenetic Response and Disease Severity in South Indian Asthmatics.

    PubMed

    Bandaru, Srinivas; Akka, Jyothy; Marri, Vijaya Kumar; Alvala, Mallika; Ponnala, Deepika; Mundluru, Hema Prasad

    2015-12-01

    β2-Adrenergic receptor (β2-AR) plays a crucial role in asthma pathophysiology by regulating, processes of the lung function, and clinical response to bronchodilators. The +46G>A- Gly16Arg polymorphism in the gene encoding β2 adrenergic receptor (ADRB2) has been associated with receptor non-responsiveness after β2-agonist exposure. In the present study, we sought to evaluate the possible association of Gly16Arg polymorphism with asthma susceptibility, pharmacogenetic response to Salbutamol, and varying degrees of disease severity. Three hundred ninety-eight clinically diagnosed patients and 456 healthy controls were enrolled for the study. Patients were classified into severity classes according to Global Initiative for Asthma guidelines. To assess bronchodilator response, spirometry was performed before and 15 min after Salbutamol (200 μg) delivery. Responders to Salbutamol were categorized if percentage reversibility was greater than or equal to 12% in them, while those showing reversibility less than 12% were classified as non-responders. Genotyping was carried out by ARMS-PCR technique. Statistical methods were applied to test for the significance of the results. In the present study, there was lack of significant association of polymorphism with disease susceptibility as well as with bronchodilator response. The polymorphism was not associated with mild and moderate asthma subtypes; however, there was a notable association with severe asthma subtype. In addition, the polymorphism was associated with severe asthma compared to subtypes of mild and moderate asthma combined. In a South Indian population, the ADRB2 Arg/Gly may not form a susceptible variant to develop asthma nor can be a standard predictive marker to bronchodilator response; nevertheless, the patterns in asthma severity can be predicted by analyzing this variant. PMID:26071206

  3. Tyrosine kinase gene rearrangements in epithelial malignancies

    PubMed Central

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

    2014-01-01

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

  4. 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

  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. The ARG1-LIKE2 Gene of Arabidopsis Functions in a Gravity Signal Transduction Pathway That Is Genetically Distinct from the PGM Pathway1

    PubMed Central

    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. PMID:12970478

  7. Association of beta3-adrenergic receptor (ADRB3) Trp64Arg gene polymorphism with obesity and metabolic syndrome in the Balinese: a pilot study

    PubMed Central

    2011-01-01

    Background Prevalence of obesity is increasing all over the world. ADRB3 Trp64Arg gene polymorphism was proposed to be associated with obesity, although inconsistent findings and differences of the Arg64 allele frequency among various ethnics were reported. Westernization was reported to increase the prevalence of obesity in developing world. In this study we determined the prevalence of obesity and metabolic syndrome among urban and rural Balinese, and studied the association of ADRB3 Trp64Arg polymorphism with obesity and MetS. Findings A total of 528 Balinese (urban 282, rural 246) were recruited. Body mass index (BMI) and waist circumference (WC) were determined; high-density lipoprotein cholesterol (HDL-C), triglyceride (TG), systolic and diastolic blood pressure (SBP and DBP), and fasting plasma glucose (FPG) were measured using standard procedures. BMI and WC classifications were based on WHO classifications for Asian. Metabolic syndrome (MetS) was defined as described in the Joint Interim Statement. Chi-square test was employed to test the association between the ADRB3 Trp64Arg genotype and disease traits. Urban have higher BMI (p = 2.8 × 10-13), WC ( p < 2.2 × 10-16), TG (p = 0.0028), DBP (p = 1.8 × 10-5), and lower HDL-C (p = 0.0376) when compared to rural. Abdominal obesity and MetS prevalence were significantly higher in urban as compared to rural (both p < 0.001). The Arg64 allele frequency was similar between urban (0.06) and rural (0.05). The Arg64 rural female carriers have higher BMI and WC as compared to their Trp64 counterparts (p = 0.041 for BMI and p = 0.012 for WC), and consequently higher abdominal obesity prevalence (p = 0.007). Comparison between male and female, as well as urban and rural, showed different prevalence of MetS co-morbidities. Abdominal obesity and hypertriglyceridaemia were consistently appeared in all groups, suggesting to play a role as determinant of MetS in both urban and rural. Conclusions Prevalence of obesity and

  8. Effect of Inactivation of the Arg- and/or Lys-Gingipain Gene on Selected Virulence and Physiological Properties of Porphyromonas gingivalis

    PubMed Central

    Grenier, Daniel; Roy, Sophie; Chandad, Fatiha; Plamondon, Pascale; Yoshioka, Masami; Nakayama, Koji; Mayrand, Denis

    2003-01-01

    Proteolytic enzymes produced by Porphyromonas gingivalis are thought to play critical roles in the pathogenesis of periodontitis. The aim of this study was to investigate the effect of gingipain cysteine proteinase gene inactivation on selected pathological and physiological functions of P. gingivalis. Our results showed that Arg- and Lys-gingipain activities are critical components for the efficient growth of P. gingivalis in human serum. However, when the serum was supplemented with peptides provided as pancreatic casein hydrolysate, the gingipains did not appear to be essential for growth. The effect of gingipain gene inactivation on the susceptibility of P. gingivalis to serum bactericidal activity was investigated using standardized human serum. The wild-type strain, P. gingivalis ATCC 33277, was largely unaffected by the bactericidal activity of human serum complement. On the other hand, mutants lacking Arg-gingipain A, Arg-gingipain B, or Lys-gingipain activity were susceptible to complement. Since gingipains are mostly located on the outer membrane of P. gingivalis, inactivation of the genes for these enzymes may modify cell surface properties. We showed that gingipain-deficient mutants differed in their capacities to assimilate radiolabeled amino acids, cause hemolysis, express adhesins, hemagglutinate, and form biofilms. Lastly, the gingipains, more specifically Arg-gingipains, were responsible for causing major cell damage to human gingival fibroblasts. In conclusion, our study indicated that, in addition to being critical in the pathogenic process, gingipains may play a variety of physiological roles in P. gingivalis, including controlling the expression and/or processing of virulence factors. Mutations in gingipain genes thus give rise to pleiotropic effects. PMID:12874356

  9. Regulation of the Arginine Deiminase System by ArgR2 Interferes with Arginine Metabolism and Fitness of Streptococcus pneumoniae

    PubMed Central

    Schulz, Christian; Gierok, Philipp; Petruschka, Lothar; Lalk, Michael; Mäder, Ulrike

    2014-01-01

    ABSTRACT Streptococcus pneumoniae is auxotrophic for arginine, and molecular analysis of the pneumococcal genome showed that the gene encoding an arginine-ornithine antiporter (ArcD) is organized in a cluster together with the arcABC genes encoding the arginine deiminase system (ADS) of pneumococci. The ADS consists of the arginine deiminase (AD), the catabolic ornithine carbamoyltransferase (cOCT), and the carbamate kinase (CK). Pneumococcal genomes contain three ArgR-type regulators (ArgR1, ArgR2, and AhrC) that are supposed to be involved in the regulation of arginine metabolism. Here, we identified ArgR2 of TIGR4 as the regulator of the ADS and ArcD. ArgR2 binds to promoter sequences of the arc operon, and the deficiency of ArgR2 in TIGR4 abrogates expression of the ADS, including the arginine-ornithine antiporter ArcD. Intranasal infection of mice and real-time bioimaging revealed that deletion of the arcABCDT genes attenuates TIGR4. However, the acute-pneumonia model and coinfection experiments indicated that the arginine-ornithine antiporter ArcD is essential to maintain fitness, while the deficiency of ADS enzymes has a minor impact on pneumococcal fitness under in vivo conditions. Strikingly, argR2 mutant TIGR4 outcompeted the wild type in the respiratory tract, suggesting an increase in fitness and further regulatory functions of ArgR2. In contrast to TIGR4, other pneumococci, such as D39, lacking expression of ArgR2, constitutively express the ADS with a truncated nonfunctional AD. On the basis of these results, we propose that the arginine-ornithine antiporter is essential to maintain pneumococcal fitness and that the genes of the ADS cluster are positively regulated in a strain-specific manner by ArgR2. PMID:25538192

  10. 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. PMID:27038743

  11. GSTP1 Ile105Val and XRCC1 Arg399Gln gene polymorphisms contribute to the clinical outcome of patients with advanced non-small cell lung cancer.

    PubMed

    Bu, L; Zhang, L B; Mao, X; Wang, P

    2016-01-01

    Glutathione S-transferase P1 (GSTP1) and X-ray repair cross-complementing group 1 (XRCC1) genetic variations may in-fluence the efficacy of chemotherapy in various cancers. We investi-gated the possible roles of GSTP1 Ile105Val and XRCC1 Arg194Trp, and Arg399Gln gene polymorphisms in the prognosis of advanced non-small cell lung carcinoma (NSCLC) patients with cisplatin-based chemotherapy. Between January 2010 and December 2012, this study consecutively recruited 141 patients with advanced NSCLC from the First People's Hospital of Yunnan Province. Logistic regression analy-sis showed that individuals carrying the GG genotype were associated with a better response to chemotherapy than those with the wide-type genotype, with an adjusted odds ratio (95% confidence interval, CI) of 4.07 (1.06-25.06). Moreover, we observed that the AA genotype of XRCC1 Arg399Gln was correlated with a greater complete response + partial response to chemotherapy than that with the GG genotype (odds ratio = 2.71, 95%CI = 1.13-10.08). Based on the Cox hazard proportional model, the GG genotype of GSTP1 Ile105Val was found to be associated with a lower risk of death from all causes as compared to that with the AA genotype (hazard ratio = 0.07, 95%CI = 0.01-0.34). In summary, we suggest that GSTP1 Ile105Val and XRCC1 Arg399Gln polymorphisms could influence the response to chemotherapy and sur-vival of advanced NSCLC. PMID:27323109

  12. Identification of a suppressor gene for the arginine-auxotrophic argJ mutation in Corynebacterium glutamicum.

    PubMed

    Hwang, Gui-Hye; Cho, Jae-Yong

    2010-11-01

    We recently proposed a metabolic engineering strategy for L-ornithine production based on the hypothesis that an increased intracellular supply of N-acetylglutamate may further enhance L-ornithine production in a well-defined recombinant strain of Corynebacterium glutamicum. In this work, an argJ-deficient arginine auxotrophic mutant of C. glutamicum is suppressed by a different locus of C. glutamicum ATCC13032. Overexpression of the NCgl1469 open reading frame (ORF), exhibiting N-acetylglutamate synthase (NAGS) activity, was able to complement the C. glutamicum arginine-auxotrophic argJ strain and showed increased NAGS activity from 0.03 to 0.17 units mg(-1) protein. Additionally, overexpression of the NCgl1469 ORF resulted in a 39% increase in excreted L-ornithine. These results indicate that the intracellular supply of N-acetylglutamate is a rate-limiting step during L-ornithine production in C. glutamicum. PMID:20544254

  13. Insertional activation of a promoterless thymidine kinase gene

    SciTech Connect

    Hiller, S.; Hengstler, M.; Kunze, M.; Knippers, R.

    1988-08-01

    A plasmid carrying a promoterless herpes complex virus thymidine kinase gene was transfected via calcium phosphate precipitation into LM (tk/sup -/) mouse fibroblast cells. The transfected gene was efficiently expressed, as the transfected cells grew perfectly well in selective hypoxanthine-aminopterin-thymidine medium, suggesting that the thymidine kinase-coding region became linked to a promoterlike element on integration into the recipient genome. To investigate the structure of the surrogate promoter, the authors first isolated the integrated gene from a genomic library. The nucleotide sequence of the DNA adjacent to the thymidine kinase-coding sequence was then determined. They found, first, that the integration of the transfected DNA apparently occurred by a blunt end ligation mechanism involving no obvious sequence similarities between integrated and recipient DNA and, second, that the 5'-flanking region included a TATA box, to CCAAT boxes, and a GC box element. However, the TATA box motif and the most proximal CCAAT box appeared to be sufficient of full promoter activity, as determined by the transfection efficiencies of appropriate plasmid constructs. Except for these canonical promoter elements, the surrogate promoter had no obvious similarities to known thymidine kinase gene promoters.

  14. Abnormal movement of tropomyosin and response of myosin heads and actin during the ATPase cycle caused by the Arg167His, Arg167Gly and Lys168Glu mutations in TPM1 gene.

    PubMed

    Borovikov, Yurii S; Rysev, Nikita A; Chernev, Aleksey A; Avrova, Stanislava V; Karpicheva, Olga E; Borys, Danuta; Śliwińska, Małgorzata; Moraczewska, Joanna

    2016-09-15

    Amino acid substitutions: Arg167His, Arg167Gly and Lys168Glu, located in a consensus actin-binding site of the striated muscle tropomyosin Tpm1.1 (TM), were used to investigate mechanisms of the thin filament regulation. The azimuthal movement of TM strands on the actin filament and the responses of the myosin heads and actin subunits during the ATPase cycle were studied using fluorescence polarization of muscle fibres. The recombinant wild-type and mutant TMs labelled with 5-IAF, 1,5-IAEDANS-labelled S1and FITC-phalloidin F-actin were incorporated into the ghost muscle fibres to acquire information on the orientation of the probes relative to the fibre axis. The substitutions Arg167Gly and Lys168Glu shifted TM strands into the actin filament centre, whereas Arg167His moved TM towards the periphery of the filament. In the presence of Arg167Gly-TM and Lys168Glu-TM the fraction of actin monomers that were switched on and the number of the myosin heads strongly bound to F-actin were abnormally high even under conditions close to relaxation. In contrast, Arg167His-TM decreased the fraction of switched on actin and reduced the formation of strongly bound myosin heads throughout the ATPase cycle. We concluded that the altered TM-actin contacts destabilized the thin filament and affected the actin-myosin interactions. PMID:27480605

  15. Pyruvate Kinase M2 Regulates Gene Transcription by Acting as A Protein Kinase

    PubMed Central

    Gao, Xueliang; Wang, Haizhen; Jenny, J. Yang; Liu, Xiaowei; Liu, Zhi-Ren

    2012-01-01

    Summary Pyruvate kinase isoform M2 (PKM2) is a glycolysis enzyme catalyzing conversion of phosphoenolpyruvate (PEP) to pyruvate with transferring a phosphate from PEP to ADP. We report here that PKM2 localizes to the cell nucleus. The levels of nuclear PKM2 correlate with cell proliferation. PKM2 activates transcription of MEK5 by phosphorylating stat3 at Y705. In vitro phosphorylation assays show that PKM2 is a protein kinase using PEP as phosphate donor. ADP competes with the protein substrate binding, indicating that the substrate may bind to the ADP site of PKM2. Our experiments suggest that PKM2 dimer is an active protein kinase, while the tetramer is an active pyruvate kinase. Expression a PKM2 mutant that exists as a dimer promotes cell proliferation, indicating that protein kinase activity of PKM2 plays a role in promoting cell proliferation. Our study reveals an important link between metabolism alteration and gene expression during tumor transformation and progression. PMID:22306293

  16. Phosphoinositide 3-kinase and Bruton's tyrosine kinase regulate overlapping sets of genes in B lymphocytes

    PubMed Central

    Fruman, David A.; Ferl, Gregory Z.; An, Sam S.; Donahue, Amber C.; Satterthwaite, Anne B.; Witte, Owen N.

    2002-01-01

    Bruton's tyrosine kinase (Btk) acts downstream of phosphoinositide 3-kinase (PI3K) in a pathway required for B cell receptor (BCR)-dependent proliferation. We used DNA microarrays to determine what fraction of genes this pathway influences and to investigate whether PI3K and Btk mediate distinct gene regulation events. As complete loss-of-function mutations in PI3K and Btk alter B cell subpopulations and may cause compensatory changes in gene expression, we used B cells with partial loss of function in either PI3K or Btk. Only about 5% of the BCR-dependent gene expression changes were significantly affected by reduced PI3K or Btk. The results indicate that PI3K and Btk share target genes, and that PI3K influences additional genes independently of Btk. These data are consistent with PI3K acting through Btk and other effectors to regulate expression of a critical subset of BCR target genes that determine effective entry into the cell cycle. PMID:11756681

  17. Identification and functional analysis of mitogen-activated protein kinase kinase kinase (MAPKKK) genes in canola (Brassica napus L.)

    PubMed Central

    Sun, Yun; Wang, Chen; Yang, Bo; Jiang, Yuan-Qing

    2014-01-01

    Mitogen-activated protein kinase (MAPK) signalling cascades, consisting of three types of reversibly phosphorylated kinases (MAPKKK, MAPKK, and MAPK), are involved in important processes including plant immunity and hormone responses. The MAPKKKs comprise the largest family in the MAPK cascades, yet only a few of these genes have been associated with physiological functions, even in the model plant Arabidopsis thaliana. Canola (Brassica napus L.) is one of the most important oilseed crops in China and worldwide. To explore MAPKKK functions in biotic and abiotic stress responses in canola, 66 MAPKKK genes were identified and 28 of them were cloned. Phylogenetic analysis of these canola MAPKKKs with homologous genes from representative species classified them into three groups (A–C), comprising four MAPKKKs, seven ZIKs, and 17 Raf genes. A further 15 interaction pairs between these MAPKKKs and the downstream BnaMKKs were identified through a yeast two-hybrid assay. The interactions were further validated through bimolecular fluorescence complementation (BiFC) analysis. In addition, by quantitative real-time reverse transcription–PCR, it was further observed that some of these BnaMAPKKK genes were regulated by different hormone stimuli, abiotic stresses, or fungal pathogen treatments. Interestingly, two novel BnaMAPKKK genes, BnaMAPKKK18 and BnaMAPKKK19, which could elicit hypersensitive response (HR)-like cell death when transiently expressed in Nicotiana benthamiana leaves, were successfully identified. Moreover, it was found that BnaMAPKKK19 probably mediated cell death through BnaMKK9. Overall, the present work has laid the foundation for further characterization of this important MAPKKK gene family in canola. PMID:24604738

  18. Identification and functional analysis of mitogen-activated protein kinase kinase kinase (MAPKKK) genes in canola (Brassica napus L.).

    PubMed

    Sun, Yun; Wang, Chen; Yang, Bo; Wu, Feifei; Hao, Xueyu; Liang, Wanwan; Niu, Fangfang; Yan, Jingli; Zhang, Hanfeng; Wang, Boya; Deyholos, Michael K; Jiang, Yuan-Qing

    2014-05-01

    Mitogen-activated protein kinase (MAPK) signalling cascades, consisting of three types of reversibly phosphorylated kinases (MAPKKK, MAPKK, and MAPK), are involved in important processes including plant immunity and hormone responses. The MAPKKKs comprise the largest family in the MAPK cascades, yet only a few of these genes have been associated with physiological functions, even in the model plant Arabidopsis thaliana. Canola (Brassica napus L.) is one of the most important oilseed crops in China and worldwide. To explore MAPKKK functions in biotic and abiotic stress responses in canola, 66 MAPKKK genes were identified and 28 of them were cloned. Phylogenetic analysis of these canola MAPKKKs with homologous genes from representative species classified them into three groups (A-C), comprising four MAPKKKs, seven ZIKs, and 17 Raf genes. A further 15 interaction pairs between these MAPKKKs and the downstream BnaMKKs were identified through a yeast two-hybrid assay. The interactions were further validated through bimolecular fluorescence complementation (BiFC) analysis. In addition, by quantitative real-time reverse transcription-PCR, it was further observed that some of these BnaMAPKKK genes were regulated by different hormone stimuli, abiotic stresses, or fungal pathogen treatments. Interestingly, two novel BnaMAPKKK genes, BnaMAPKKK18 and BnaMAPKKK19, which could elicit hypersensitive response (HR)-like cell death when transiently expressed in Nicotiana benthamiana leaves, were successfully identified. Moreover, it was found that BnaMAPKKK19 probably mediated cell death through BnaMKK9. Overall, the present work has laid the foundation for further characterization of this important MAPKKK gene family in canola. PMID:24604738

  19. Variable intron/exon structure in the oligochaete lombricine kinase gene.

    PubMed

    Doumen, Chris

    2012-09-01

    Lombricine kinase is an annelid enzyme that belongs to the phosphagen kinase family of which creatine kinase and arginine kinase are the typical representatives. The enzymes play important roles in the cellular energy metabolism of animals. Biochemical, physiological and molecular information with respect to lombricine kinase is limited compared to other phosphagen kinases. This study presents data on the cDNA sequences of lombricine kinase from two smaller oligochaetes, Enchytraeus sp. and Stylaria sp. The deduced amino acid sequences are analyzed and compared with other selected phosphagen kinases. The intron/exon structure of the lombricine kinase gene was determined for these two species as well as two additional oligochaetes, Lumbriculus variegatus and Tubifex tubifex, and compared with available data for annelid phosphagen kinases. The data indicate the existence of a variable organization of the proposed 8-intron/9-exon gene structure. The results provide further insights in the evolution and position of these enzymes within the phosphagen kinase family. PMID:22705027

  20. 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

  1. 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

  2. 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

  3. Molecular characterization of the PK-LR gene in pyruvate kinase deficient Spanish patients. Red Cell Pathology Group of the Spanish Society of Haematology (AEHH).

    PubMed

    Zarza, R; Alvarez, R; Pujades, A; Nomdedeu, B; Carrera, A; Estella, J; Remacha, A; Sánchez, J M; Morey, M; Cortes, T; Pérez Lungmus, G; Bureo, E; Vives Corrons, J L

    1998-11-01

    The PK-LR gene has been studied in 12 unrelated patients with red cell pyruvate kinase deficiency and hereditary nonspherocytic haemolytic anaemia (CNSHA). The entire codifying region of the R-type PK gene and the flanking intronic regions were analysed by single-stranded conformation polymorphism (SSCP) followed by direct sequencing of abnormal DNA. 10 different mutations were identified in 22/24 alleles at risk. Eight of these were missense mutations that caused the following single amino acid changes: G514C (172Glu-Gln), G1010A (337Arg-Gln), G1015C (339Asp-Gln), T1070C (357Ile-Thr), C1223T (408Thr-Ile), G1291A (431Ala-Thr), C1456T (486Arg-Trp) and G1595A (532Arg-Gln). Two were nonsense mutations: G721T (241Glu-Stop) and C1675T (559Arg-Stop). 7/22 alleles demonstrated the same C1456 --> T mutation. The study of the polymorphic site at nucleotide (nt) 1705 performed in all cases disclosed a 1705 C/C mutation in 10 and a 1705 A/C mutation in three. This is the first report on the presence of several different L-type PK gene mutations within Spanish population. Furthermore, from the PK gene mutations found, six were unique and not previously described (1015C, 1070C, 1223T, 1291A, 1595A and 1675T) and one (C1456T) seems to be predominant in Spain. Interestingly, no case with the 1529A mutation commonly found in Northern European populations was present here. PMID:9827908

  4. 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. PMID:26948878

  5. 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

  6. 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. PMID:27344947

  7. Mutation hot spots in the canine herpesvirus thymidine kinase gene.

    PubMed

    Yamada, Shinya; Matsumoto, Yasunobu; Takashima, Yasuhiro; Otsuka, Haruki

    2005-08-01

    The guanine and cytosine content (GC-content) of alpha-herpesvirus genes are highly variable despite similar genome structures. It is known that drug resistant HSV, which has the genome with a high GC-content (approximately 70%), commonly includes frameshift mutations in homopolymer stretches of guanine (G) and cytosine (C) within the thymidine kinase (TK) gene. However, whether such mutation hotspots exist in the TK gene of canine herpesvirus (CHV) which has a low GC-content was unknown. In this study, we investigated mutations in the TK gene of CHV. CHV was passaged in the presence of iodo-deoxyuridine (IDU), and IDU-resistant clones were isolated. In all IDU-resistant virus clones, mutations in the TK gene were observed. The majority of these mutations were frameshift mutations of an adenine (A) insertion or deletion within either of 2 stretches of eight A's in the TK gene. It was demonstrated that CHV TK mutations frequently occur at a limited number of hot spots within long homopolymer nucleotide stretches. PMID:15965615

  8. Evolutionary Diversification of Plant Shikimate Kinase Gene Duplicates

    PubMed Central

    Fucile, Geoffrey; Falconer, Shannon; Christendat, Dinesh

    2008-01-01

    Shikimate kinase (SK; EC 2.7.1.71) catalyzes the fifth reaction of the shikimate pathway, which directs carbon from the central metabolism pool to a broad range of secondary metabolites involved in plant development, growth, and stress responses. In this study, we demonstrate the role of plant SK gene duplicate evolution in the diversification of metabolic regulation and the acquisition of novel and physiologically essential function. Phylogenetic analysis of plant SK homologs resolves an orthologous cluster of plant SKs and two functionally distinct orthologous clusters. These previously undescribed genes, shikimate kinase-like 1 (SKL1) and -2 (SKL2), do not encode SK activity, are present in all major plant lineages, and apparently evolved under positive selection following SK gene duplication over 400 MYA. This is supported by functional assays using recombinant SK, SKL1, and SKL2 from Arabidopsis thaliana (At) and evolutionary analyses of the diversification of SK-catalytic and -substrate binding sites based on theoretical structure models. AtSKL1 mutants yield albino and novel variegated phenotypes, which indicate SKL1 is required for chloroplast biogenesis. Extant SKL2 sequences show a strong genetic signature of positive selection, which is enriched in a protein–protein interaction module not found in other SK homologs. We also report the first kinetic characterization of plant SKs and show that gene expression diversification among the AtSK inparalogs is correlated with developmental processes and stress responses. This study examines the functional diversification of ancient and recent plant SK gene duplicates and highlights the utility of SKs as scaffolds for functional innovation. PMID:19057671

  9. PRMT5-Mediated Methylation of NF-κB p65 at Arg174 Is Required for Endothelial CXCL11 Gene Induction in Response to TNF-α and IFN-γ Costimulation

    PubMed Central

    Harris, Daniel P.; Chandrasekharan, Unnikrishnan M.; Bandyopadhyay, Smarajit; Willard, Belinda; DiCorleto, Paul E.

    2016-01-01

    Inflammatory agonists differentially activate gene expression of the chemokine family of proteins in endothelial cells (EC). TNF is a weak inducer of the chemokine CXCL11, while TNF and IFN-γ costimulation results in potent CXCL11 induction. The molecular mechanisms underlying TNF plus IFN-γ-mediated CXCL11 induction are not fully understood. We have previously reported that the protein arginine methyltransferase PRMT5 catalyzes symmetrical dimethylation of the NF-κB subunit p65 in EC at multiple arginine residues. Methylation of Arg30 and Arg35 on p65 is critical for TNF induction of CXCL10 in EC. Here we show that PRMT5-mediated methylation of p65 at Arg174 is required for induction of CXCL11 when EC are costimulated with TNF and IFN-γ. Knockdown of PRMT5 by RNAi reduced CXCL11 mRNA and protein levels in costimulated cells. Reconstitution of p65 Arg174Ala or Arg174Lys mutants into EC that were depleted of endogenous p65 blunted TNF plus IFN-γ-mediated CXCL11 induction. Mass spectrometric analyses showed that p65 Arg174 arginine methylation is enhanced by TNF plus IFN-γ costimulation, and is catalyzed by PRMT5. Chromatin immunoprecipitation assays (ChIP) demonstrated that PRMT5 is necessary for p65 association with the CXCL11 promoter in response to TNF plus IFN-γ. Further, reconstitution of p65 Arg174Lys mutant in EC abrogated this p65 association with the CXCL11 promoter. Finally, ChIP and Re-ChIP assays revealed that symmetrical dimethylarginine-containing proteins complexed with the CXCL11 promoter were diminished in p65 Arg174Lys-reconstituted EC stimulated with TNF and IFN-γ. In total, these results indicate that PRMT5-mediated p65 methylation at Arg174 is essential for TNF plus IFN-γ-mediated CXCL11 gene induction. We therefore suggest that the use of recently developed small molecule inhibitors of PRMT5 may present a therapeutic approach to moderating chronic inflammatory pathologies. PMID:26901772

  10. Functional Dissection of N-Acetylglutamate Synthase (ArgA) of Pseudomonas aeruginosa and Restoration of Its Ancestral N-Acetylglutamate Kinase Activity

    PubMed Central

    Sancho-Vaello, Enea; Fernández-Murga, María L.

    2012-01-01

    In many microorganisms, the first step of arginine biosynthesis is catalyzed by the classical N-acetylglutamate synthase (NAGS), an enzyme composed of N-terminal amino acid kinase (AAK) and C-terminal histone acetyltransferase (GNAT) domains that bind the feedback inhibitor arginine and the substrates, respectively. In NAGS, three AAK domain dimers are interlinked by their N-terminal helices, conforming a hexameric ring, whereas each GNAT domain sits on the AAK domain of an adjacent dimer. The arginine inhibition of Pseudomonas aeruginosa NAGS was strongly hampered, abolished, or even reverted to modest activation by changes in the length/sequence of the short linker connecting both domains, supporting a crucial role of this linker in arginine regulation. Linker cleavage or recombinant domain production allowed the isolation of each NAGS domain. The AAK domain was hexameric and inactive, whereas the GNAT domain was monomeric/dimeric and catalytically active although with ∼50-fold-increased and ∼3-fold-decreased Kmglutamate and kcat values, respectively, with arginine not influencing its activity. The deletion of N-terminal residues 1 to 12 dissociated NAGS into active dimers, catalyzing the reaction with substrate kinetics and arginine insensitivity identical to those for the GNAT domain. Therefore, the interaction between the AAK and GNAT domains from different dimers modulates GNAT domain activity, whereas the hexameric architecture appears to be essential for arginine inhibition. We proved the closeness of the AAK domains of NAGS and N-acetylglutamate kinase (NAGK), the enzyme that catalyzes the next arginine biosynthesis step, shedding light on the origin of classical NAGS, by showing that a double mutation (M26K L240K) in the isolated NAGS AAK domain elicited NAGK activity. PMID:22447897

  11. Functional dissection of N-acetylglutamate synthase (ArgA) of Pseudomonas aeruginosa and restoration of its ancestral N-acetylglutamate kinase activity.

    PubMed

    Sancho-Vaello, Enea; Fernández-Murga, María L; Rubio, Vicente

    2012-06-01

    In many microorganisms, the first step of arginine biosynthesis is catalyzed by the classical N-acetylglutamate synthase (NAGS), an enzyme composed of N-terminal amino acid kinase (AAK) and C-terminal histone acetyltransferase (GNAT) domains that bind the feedback inhibitor arginine and the substrates, respectively. In NAGS, three AAK domain dimers are interlinked by their N-terminal helices, conforming a hexameric ring, whereas each GNAT domain sits on the AAK domain of an adjacent dimer. The arginine inhibition of Pseudomonas aeruginosa NAGS was strongly hampered, abolished, or even reverted to modest activation by changes in the length/sequence of the short linker connecting both domains, supporting a crucial role of this linker in arginine regulation. Linker cleavage or recombinant domain production allowed the isolation of each NAGS domain. The AAK domain was hexameric and inactive, whereas the GNAT domain was monomeric/dimeric and catalytically active although with ∼50-fold-increased and ∼3-fold-decreased K(m)(glutamate) and k(cat) values, respectively, with arginine not influencing its activity. The deletion of N-terminal residues 1 to 12 dissociated NAGS into active dimers, catalyzing the reaction with substrate kinetics and arginine insensitivity identical to those for the GNAT domain. Therefore, the interaction between the AAK and GNAT domains from different dimers modulates GNAT domain activity, whereas the hexameric architecture appears to be essential for arginine inhibition. We proved the closeness of the AAK domains of NAGS and N-acetylglutamate kinase (NAGK), the enzyme that catalyzes the next arginine biosynthesis step, shedding light on the origin of classical NAGS, by showing that a double mutation (M26K L240K) in the isolated NAGS AAK domain elicited NAGK activity. PMID:22447897

  12. 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. PMID:19526298

  13. 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

  14. 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.

  15. 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.

  16. 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.

  17. Detection of a novel missense mutation in the mevalonate kinase gene in one Chinese family with DSAP

    PubMed Central

    Lu, Wen-Sheng; Zheng, Xiao-Dong; Yao, Xiu-Hua; Zhang, Lan-Fang; Hu, Bai; Lu, Yao-Juan

    2014-01-01

    Disseminated superficial actinic porokeratosis (DSAP) is the most common form of porokeratosis and a severe chronic autosomal dominant cutaneous disorder with high genetic heterogeneity. Recently, the mevalonate kinase (MVK) gene has been identified as a candidate gene responsible for DSAP and multiple mutations have been reported. Here, we report identification of a novel missense mutation in the MVK gene in a Chinese family with DSAP. A 50-year-old male was diagnosed as proband of DSAP based on the clinical and histological findings, which show numerous hyperpigmented macules by physical examination and cornoid lamella by skin biopsy. Similar skin symptoms were also observed in his father, who died many years ago. We prepared genomic DNA from the proband, unaffected individuals from his family members, as well as 100 unrelated healthy controls. PCR was then conducted using the above genomic DNA as template and the MVK gene-specific primers. The PCR product was subjected to direct sequencing and the sequence was compared to that of MVK gene within the NCBI database. We detected a heterozygous C to G transition at nucleotide 643 in exon 7 of MVK gene of the proband. This will result in an amino acid change at codon 215 (P.Arg215Gly.), which is from an arginine codon (CGA) to a Glycine codon (GGA). We did not detect any mutation in the unaffected family members or the 100 unrelated healthy controls, demonstrating that this is a novel missense mutation in MVK gene and therefore, contributes to the molecular diagnosis of DSAP. PMID:24551296

  18. Molecular Recognition of Lys and Arg Methylation.

    PubMed

    Beaver, Joshua E; Waters, Marcey L

    2016-03-18

    A network of reader proteins and enzymes precisely controls gene transcription through the dynamic addition, removal, and recognition of post-translational modifications (PTMs) of histone tails. Histone PTMs work in concert with this network to regulate gene transcription through the histone code, and the dysregulation of PTM maintenance is linked to a large number of diseases, including many types of cancer. A wealth of research aims to elucidate the functions of this code, but our understanding of the effects of PTMs, specifically the methylation of lysine (Lys) and arginine (Arg), is lacking. The development of new tools to study PTMs relies on a sophisticated understanding of the mechanisms that drive protein and small molecule recognition in water. In this review, we outline the physical organic concepts that drive the molecular recognition of Lys and Arg methylation by reader proteins and draw comparisons to the binding mechanisms of small molecule receptors for methylated Lys and Arg that have been developed recently. PMID:26759915

  19. Survival kinase genes present prognostic significance in glioblastoma

    PubMed Central

    Varghese, Robin T.; Liang, Yanping; Guan, Ting; Franck, Christopher T.; Kelly, Deborah F.; Sheng, Zhi

    2016-01-01

    Cancer biomarkers with a strong predictive power for diagnosis/prognosis and a potential to be therapeutic targets have not yet been fully established. Here we employed a loss-of-function screen in glioblastoma (GBM), an infiltrative brain tumor with a dismal prognosis, and identified 20 survival kinase genes (SKGs). Survival analyses using The Cancer Genome Atlas (TCGA) datasets revealed that the expression of CDCP1, CDKL5, CSNK1E, IRAK3, LATS2, PRKAA1, STK3, TBRG4, and ULK4 stratified GBM prognosis with or without temozolomide (TMZ) treatment as a covariate. For the first time, we found that GBM patients with a high level of NEK9 and PIK3CB had a greater chance of having recurrent tumors. The expression of CDCP1, IGF2R, IRAK3, LATS2, PIK3CB, ULK4, or VRK1 in primary GBM tumors was associated with recurrence-related prognosis. Notably, the level of PIK3CB in recurrent tumors was much higher than that in newly diagnosed ones. Congruent with these results, genes in the PI3K/AKT pathway showed a significantly strong correlation with recurrence rate, further highlighting the pivotal role of PIK3CB in the disease progression. Importantly, 17 SKGs together presented a novel GBM prognostic signature. SKGs identified herein are associated with recurrence rate and present prognostic significance in GBM, thereby becoming attractive therapeutic targets. PMID:26956052

  20. Survival kinase genes present prognostic significance in glioblastoma.

    PubMed

    Varghese, Robin T; Liang, Yanping; Guan, Ting; Franck, Christopher T; Kelly, Deborah F; Sheng, Zhi

    2016-04-12

    Cancer biomarkers with a strong predictive power for diagnosis/prognosis and a potential to be therapeutic targets have not yet been fully established. Here we employed a loss-of-function screen in glioblastoma (GBM), an infiltrative brain tumor with a dismal prognosis, and identified 20 survival kinase genes (SKGs). Survival analyses using The Cancer Genome Atlas (TCGA) datasets revealed that the expression of CDCP1, CDKL5, CSNK1E, IRAK3, LATS2, PRKAA1, STK3, TBRG4, and ULK4 stratified GBM prognosis with or without temozolomide (TMZ) treatment as a covariate. For the first time, we found that GBM patients with a high level of NEK9 and PIK3CB had a greater chance of having recurrent tumors. The expression of CDCP1, IGF2R, IRAK3, LATS2, PIK3CB, ULK4, or VRK1 in primary GBM tumors was associated with recurrence-related prognosis. Notably, the level of PIK3CB in recurrent tumors was much higher than that in newly diagnosed ones. Congruent with these results, genes in the PI3K/AKT pathway showed a significantly strong correlation with recurrence rate, further highlighting the pivotal role of PIK3CB in the disease progression. Importantly, 17 SKGs together presented a novel GBM prognostic signature. SKGs identified herein are associated with recurrence rate and present prognostic significance in GBM, thereby becoming attractive therapeutic targets. PMID:26956052

  1. Diversification of Lrk/Tak Kinase Gene Clusters is Associated with Subfunctionalization and Cultivar-specific Transcript Accumulation in Barley

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Lrk (Lr10 receptor-like kinase) and Tak (Triticum aestivum kinase) belong to the receptor-like kinase (RLK) super-gene 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 (Bacterial Artificial Chromosome) ...

  2. 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

  3. Global analysis of serine-threonine protein kinase genes in Neurospora crassa.

    PubMed

    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

    2011-11-01

    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

  4. Building a human kinase gene repository: Bioinformatics, molecular cloning, and functional validation

    PubMed Central

    Park, Jaehong; Hu, Yanhui; Murthy, T. V. S.; Vannberg, Fredrik; Shen, Binghua; Rolfs, Andreas; Hutti, Jessica E.; Cantley, Lewis C.; LaBaer, Joshua; Harlow, Ed; Brizuela, Leonardo

    2005-01-01

    Kinases catalyze the phosphorylation of proteins, lipids, sugars, nucleosides, and other important cellular metabolites and play key regulatory roles in all aspects of eukaryotic cell physiology. Here, we describe the mining of public databases to collect the sequence information of all identified human kinase genes and the cloning of the corresponding ORFs. We identified 663 genes, 511 encoding protein kinases, and 152 encoding nonprotein kinases. We describe the successful cloning and sequence verification of 270 of these genes. Subcloning of this gene set in mammalian expression vectors and their use in high-throughput cell-based screens allowed the validation of the clones at the level of expression and the identification of previously uncharacterized modulators of the survivin promoter. Moreover, expressions of the kinase genes in bacteria, followed by autophosphorylation assays, identified 21 protein kinases that showed autocatalytic activity. The work described here will facilitate the functional assaying of this important gene family in phenotypic screens and their use in biochemical and structural studies. PMID:15928075

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

    PubMed

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

    2006-01-01

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

  6. 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.

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

    PubMed Central

    Kwoh, T J; Engler, J A

    1984-01-01

    The entire DNA nucleotide sequence of a 3.0 kilobase pair Hind III fragment containing the chicken cytoplasmic thymidine kinase gene was determined. Oligonucleotide linker insertion mutations distributed throughout this gene and having known effects upon gene activity ( Kwoh , T.J., Zipser , D., and Wigler , M. 1983. J. Mol. Appl. Genet. 2, 191-200), were used to access regions of the Hind III fragment for sequencing reactions. The complete nucleotide sequence, together with the positions of the linker insertion mutations within the sequence, allows us to propose a structure for the chicken thymidine kinase gene. The protein coding sequence of the gene is divided into seven small segments (each less than 160 base pairs) by six small introns (each less than 230 base pairs). The proposed 244 amino acid polypeptide encoded by this gene bears strong homology to the vaccinia virus thymidine kinase. No homology with the thymidine kinases of the herpes simplex viruses was found. PMID:6328447

  8. Thymidine Kinase Suicide Gene-mediated Ganciclovir Ablation of Autologous Gene-modified Rhesus Hematopoiesis

    PubMed Central

    Barese, Cecilia N; Krouse, Allen E; Metzger, Mark E; King, Connor A; Traversari, Catia; Marini, Frank C; Donahue, Robert E; Dunbar, Cynthia E

    2012-01-01

    Despite the genotoxic complications encountered in clinical gene therapy trials for primary immunodeficiency diseases targeting hematopoietic cells with integrating vectors; this strategy holds promise for the cure of several monogenic blood, metabolic and neurodegenerative diseases. In this study, we asked whether the inclusion of a suicide gene in a standard retrovirus vector would allow elimination of vector-containing stem and progenitor cells and their progeny in vivo following transplantation, using our rhesus macaque transplantation model. Following stable engraftment with autologous CD34+ cells transduced with a retrovirus vector encoding a highly sensitive modified Herpes simplex virus thymidine kinase SR39, the administration of the antiviral prodrug ganciclovir (GCV) was effective in completely eliminating vector-containing cells in all hematopoietic lineages in vivo. The sustained absence of vector-containing cells over time, without additional GCV administration, suggests that the ablation of TkSR39 GCV-sensitive cells occurred in the most primitive hematopoietic long-term repopulating stem or progenitor cell compartment. These results are a proof-of-concept that the inclusion of a suicide gene in integrating vectors, in addition to a therapeutic gene, can provide a mechanism for later elimination of vector-containing cells, thereby increasing the safety of gene transfer. PMID:22910293

  9. LOCALIZATION OF THE MOUSE THYMIDINE KINASE GENE TO THE DISTAL PORTION OF CHROMOSOME 11

    EPA Science Inventory

    We report the regional mapping of the thymidine kinase (tk-1) gene in the mouse using two complementary analyses: 1) investigation of chromosome aberrations associated with tx-1 gene inactivation in the L5178Y TX+/-3.7.2c cell line and (2) in situ molecular hybridization of a clo...

  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. 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. PMID:26414948

  12. Utility of the Arg/Gln polymorphism of the factor VII (FVII) gene, serum lipid levels and body mass index in the prediction of the FVII:C and FVII:Ag in North Karelia; a cross-sectional and prospective study.

    PubMed

    Stengård, J H; Salomaa, V; Rasi, V; Vahtera, E; Ehnholm, C; Krusius, T; Perola, M; Vartiainen, E

    2001-09-01

    The arginine/glutamine (Arg/Gln) polymorphism of the factor VII (FVII) gene is associated with variation in coagulation activity (FVII:C) and antigen concentration (FVII:Ag) of the FVII protein. We estimated frequency distributions of the Arg and Gln alleles and respective genotypes in North Karelia, and evaluated the utility of this polymorphism, serum lipids, and body mass index (BMI) in the prediction of the distributions of FVII:C and FVII:Ag in a cross-sectional study and in a prospective cohort study. The sample comprised 203 males and 262 females (aged 45-64 years) who were seen twice, in 1992 and 1995. The Arg/Arg genotype and the Arg allele frequencies were among the highest reported so far (86 and 93% respectively, in men; and 89 and 94% respectively, in women). Intragenotypic means of both FVII:C and FVII:Ag were significantly higher in the Arg/Arg genotype than in the Arg/Gln genotype in both genders. Also, intragenotypic variances were different in different genotypes in females. Regression relationships between the FVII:C and FVII:Ag and serum triglyceride, and total cholesterol levels and BMI were positive in both genotypes in both genders, which has not been found in other populations. In prospective analyses, average changes in the FVII:C and FVII:Ag were genotype specific in both genders, as were also regression relationships between these changes and changes in triglyceride level in females (P = 0.065 for FVII:C and P = 0.061 for FVII:Ag). A consequence of these complex genetic architectures is that predictive utility of the Arg/Gln genotypes depends on population, gender, serum lipid levels, and BMI, and changes in these factors over time. PMID:11555697

  13. Calcium-Dependent Protein Kinase Genes in Corn Roots

    NASA Technical Reports Server (NTRS)

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

    1996-01-01

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

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

    SciTech Connect

    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 (ATR/sup 0/) 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 (/sup 125/I)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.

  15. MicroRNA-21 promotes phosphatase gene and protein kinase B/phosphatidylinositol 3-kinase expression in colorectal cancer

    PubMed Central

    Sheng, Wei-Zhong; Chen, Yu-Sheng; Tu, Chuan-Tao; He, Juan; Zhang, Bo; Gao, Wei-Dong

    2016-01-01

    AIM: To explore the regulatory mechanism of the target gene of microRNA-21 (miR-21), phosphatase gene (PTEN), and its downstream proteins, protein kinase B (AKT) and phosphatidylinositol 3-kinase (PI3K), in colorectal cancer (CRC) cells. METHODS: Quantitative real-time PCR (qRT-PCR) and Western blot were used to detect the expression levels of miR-21 and PTEN in HCT116, HT29, Colo32 and SW480 CRC cell lines. Also, the expression levels of PTEN mRNA and its downstream proteins AKT and PI3K in HCT116 cells after downregulating miR-21 were investigated. RESULTS: Comparing the miR-21 expression in CRC cells, the expression levels of miR-21 were highest in HCT116 cells, and the expression levels of miR-21 were lowest in SW480 cells. In comparing miR-21 and PTEN expression in CRC cells, we found that the protein expression levels of miR-21 and PTEN were inversely correlated (P < 0.05); when miR-21 expression was reduced, mRNA expression levels of PTEN did not significantly change (P > 0.05), but the expression levels of its protein significantly increased (P < 0.05). In comparing the levels of PTEN protein and downstream AKT and PI3K in HCT116 cells after downregulation of miR-21 expression, the levels of AKT and PI3K protein expression significantly decreased (P < 0.05). CONCLUSION: PTEN is one of the direct target genes of miR-21. Thus, phosphatase gene and its downstream AKT and PI3K expression levels can be regulated by regulating the expression levels of miR-21, which in turn regulates the development of CRC. PMID:27350731

  16. Investigation of the association between Rho/Rho-kinase gene polymorphisms and systemic sclerosis.

    PubMed

    Pehlivan, Yavuz; Yolbas, Servet; Cetin, Gozde Yıldırım; Alibaz-Oner, Fatma; Cagatay, Yonca; Yilmaz, Neslihan; Oztuzcu, Serdar; Donmez, Salim; Ozgen, Metin; Koca, Suleyman Serdar; Pamuk, Omer Nuri; Sayarlıoglu, Mehmet; Kisacik, Bunyamin; Direskeneli, Haner; Demiryurek, Abdullah Tuncay; Onat, Ahmet Mesut

    2016-03-01

    Systemic sclerosis (SSc) is a disease characterized by inflammation, vascular abnormalities and fibrosis. The role of Rho/Rho-kinase pathway was demonstrated in the pathogenesis of fibrosis, inflammation and vascular abnormalities. This study was aimed to investigate the relation between SSc and Rho/Rho-kinase gene polymorphisms. The study included 339 patients with SSc and 302 healthy subjects who were apparently healthy and at similar age and gender. Genotype distributions and allele frequencies were detected by using Chi-square test or Fisher's exact Chi-square test between groups, and the haplotype analysis was applied using online program (SHEsis). Significant association was found in a polymorphism in the ROCK1 gene (rs35996865), a polymorphism in ROCK2 gene (rs10178332), a polymorphism in RhoA gene (rs2177268) and two polymorphisms in RhoC gene (rs11102522 and rs11538960) with SSc disease (p < 0.0022). In this study, association between SSc disease and Rho/Rho-kinase gene polymorphisms was investigated for the first time; significant associations between ROCK1, ROCK2, RhoA and RhoC gene polymorphisms and SSc disease were demonstrated. The results strongly suggest that this SNP may be an important risk factor for development of SSc. However, further validation of these findings in an independent cohort is necessary. PMID:26615410

  17. TP53 codon 72 Arg/Arg polymorphism is associated with a higher risk for inflammatory bowel disease development

    PubMed Central

    Volodko, Natalia; Salla, Mohamed; Eksteen, Bertus; Fedorak, Richard N; Huynh, Hien Q; Baksh, Shairaz

    2015-01-01

    AIM: To investigate the association between tumor protein 53 (TP53) codon 72 polymorphisms and the risk for inflammatory bowel disease (IBD) development. METHODS: Numerous genetic and epigenetic drivers have been identified for IBD including the TP53 gene. Pathogenic mutations in TP53 gene have only been reported in 50% of colorectal cancer (CRC) patients. A single nucleotide polymorphism (SNP) in the TP53 gene resulting in the presence of either arginine (Arg) or proline (Pro) or both at codon 72 was shown to alter TP53 tumor-suppressor properties. This SNP has been investigated as a risk factor for numerous cancers, including CRC. In this study we analyzed TP53 codon 72 polymorphism distribution in 461 IBD, 181 primary sclerosing cholangitis patients and 62 healthy controls. Genotyping of TP53 was performed by sequencing and restriction fragment length polymorphism analysis of genomic DNA extracted from peripheral blood. RESULTS: The most frequent TP53 genotype in IBD patients was Arg/Arg occurring in 54%-64% of cases (and in only 32% of controls). Arg/Pro was the most prevalent genotype in controls (53%) and less common in patients (31%-40%). Pro/Pro frequency was not significantly different between controls and IBD patients. CONCLUSION: The data suggests that the TP53 codon 72 Arg/Arg genotype is associated with increased risk for IBD development. PMID:26420962

  18. Phosphoglycerate kinase gene from Zymomonas mobilis: cloning sequencing, and localization within the gap operon

    SciTech Connect

    Conway, T.; Ingram, L.O.

    1988-04-01

    The Zymomonas mobilis gene encoding phosphoglycerate kinase (EC 2.7.3.2), pgk, has been cloned into Escherichia coli and sequenced. It consists of 336 amino acids, including the N-terminal methionine, with a molecular weight of 47,384. This promoterless gene is located 225 base pairs downstream from the gap gene and is part of the gap operon. Previous studies have shown that the specific activities of glyceraldehyde-3-phosphate dehydrogenase and phosphoglycerate kinase do not change coordinately in Z. mobilis, although the two enzymes appear to be under the control of a common promoter. The translated amino acid sequence for the Z. mobilis phosphoglycerte kinase is less conserved than those of eucaryotic genes. A comparison of known sequences for phosphoglycerate kinase revealed a high degree of conservation of structure with 102 amino acid positions being retained by all. In general, the amino acid positions at the boundaries of ..beta..-sheet and ..cap alpha..-helical regions and those connecting these regions were more highly conserved than the amino acid positions within regions of secondary structure.

  19. Expression of complete chicken thymidine kinase gene inserted in a retrovirus vector.

    PubMed Central

    Bandyopadhyay, P K; Temin, H M

    1984-01-01

    The chicken thymidine kinase (tk) gene was inserted into spleen necrosis virus. Thymidine kinase activity was expressed even when the promoter and terminator sequences for tk RNA synthesis were retained. When the promoter was present in the same orientation as the promoter in the long terminal repeat of the virus, deletions occurred both in the virus and in the tk gene, and the thymidine kinase-transforming activity of the recovered virus was low. Splicing of apparent intervening sequences in the tk gene was also observed. When the orientation of the tk promoter was opposite to the promoter in the long terminal repeat, virus synthesis was diminished, whereas thymidine kinase activity was expressed at an elevated level compared with virus in which the promoter was in the same orientation. However, when the apparent tk promoter was deleted from virus with the tk gene in the opposite orientation, a high level of virus synthesis was observed, probably as a result of absence of interference of RNA synthesis from converging promoters. The intervening sequences in the virus in which the promoters were in opposite orientation were not spliced. Images PMID:6325895

  20. 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

  1. Mediator kinase inhibition further activates super-enhancer-associated genes in AML.

    PubMed

    Pelish, Henry E; Liau, Brian B; 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

    2015-10-01

    Super-enhancers (SEs), which are composed of large clusters of enhancers densely loaded with the Mediator complex, transcription factors and chromatin regulators, drive high expression of genes implicated in cell identity and disease, such as lineage-controlling transcription factors and oncogenes. BRD4 and CDK7 are positive regulators of SE-mediated transcription. By contrast, negative regulators of SE-associated genes have not been well described. Here we show that the 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 report that the natural product cortistatin A (CA) selectively inhibits Mediator kinases, has anti-leukaemic activity in vitro and in vivo, and disproportionately induces 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 transcription factors CEBPA, IRF8, IRF1 and ETV6 (refs 6-8). The BRD4 inhibitor I-BET151 downregulated these SE-associated genes, yet also has anti-leukaemic activity. Individually increasing or decreasing the expression of these transcription factors suppressed AML cell growth, providing evidence that leukaemia cells are sensitive to the 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

  2. Isolation of the human Xp21 glycerol kinase gene by positional cloning.

    PubMed

    Walker, A P; Muscatelli, F; Monaco, A P

    1993-02-01

    The gene for human glycerol kinase deficiency (GK) maps in Xp21.3 in a critical region of about 50-250 kb located distal to the Duchenne muscular dystrophy gene (DMD) by analysis of patient deletions and YAC contigs. We have used a genomic exon amplification strategy to isolate potential exons from two cosmids which mapped to this interval. The genomic exons were used to isolate six overlapping cDNA clones from human fetal liver which encode the X-linked glycerol kinase gene. The cDNA clones map to cosmids, YAC clones and deletions in patients which define the GK critical region and also hybridize to several autosomal fragments and one Xq fragment in genomic DNA. The GK gene is expressed most in human liver with three transcript sizes of 1.85, 2.7, and 3.7 kb. Sequence analysis of 1.5 kb of several overlapping liver cDNA clones predicted a protein with approximately 63% similarity to the E. coli and B. subtilis glycerol kinase genes. The liver cDNA clones have sequence identity with four genomic exons and the 3' untranslated region from an Xp21.3 cosmid thus indicating that this is the expressed GK gene which when deleted in patients gives rises to GK deficiency. PMID:8499898

  3. 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

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

    PubMed

    Hammond, R W; Zhao, Y

    2000-09-01

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

  5. 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.

  6. Nucleotide sequence of glycoprotein genes B, C, D, G, H and I, the thymidine kinase and protein kinase genes and gene homologue UL24 of an Australian isolate of canine herpesvirus.

    PubMed

    Reubel, Gerhard Herbert; Pekin, Jenny; Webb-Wagg, Kyleen; Hardy, Christopher Miles

    2002-10-01

    We report the complete nucleotide (nt) sequence of nine genes of an Australian isolate of canine herpesvirus (CHV). Four of them are located in the unique short (US) region: glycoprotein (g) genes gG, gD and gI, and the protein kinase gene. Five are in the unique long (UL) region: the thymidine kinase gene, gB, gC, gH, and gene homologue UL24. Partial sequence was determined for four genes, two in the UL region (UL21 and virion protein) and two in the US region (US2 and gE). A repeat sequence of 382 nt with unknown function was identified in the 615 nt intergenic region between gH and UL21. A total of 16.93 kb was sequenced and compared with sequences from CHV isolates from the USA, France, Japan and Australia. Only minor nt and/or amino acid (aa) differences were observed. PMID:12416682

  7. The Sch9 kinase is a chromatin-associated transcriptional activator of osmostress-responsive genes

    PubMed Central

    Pascual-Ahuir, Amparo; Proft, Markus

    2007-01-01

    The yeast Sch9 kinase has been implicated in the cellular adjustment to nutrient availability and in the regulation of aging. Here, we define a novel role for Sch9 in the transcriptional activation of osmostress inducible genes. Loss-of-function mutants sch9 are sensitive to hyperosmotic stress and show an impaired transcriptional response upon osmotic shock of several defense genes. We show that Sch9 is required for gene expression regulated by Sko1, a transcription factor, which is directly targeted by the Hog1 MAP kinase. Sch9 interacts in vitro with both Sko1 and Hog1. Additionally, Sch9 phosphorylates Sko1 in vitro. When artificially tethered to promoter DNA, Sch9 strongly activates transcription independently of osmotic stress. Using in vivo chromatin immunoprecipitation, we demonstrate that Sch9 is recruited to the GRE2 and CTT1 genes exclusively under osmostress conditions, and that this recruitment is dependent on Hog1 and Sko1. Furthermore, Sch9 is required for the proper recruitment of Hog1 at the same genes. Our data reveal the complexity of stress-induced transcription by the regulated association of signaling kinases to chromatin. PMID:17568771

  8. ADRB2 ARG16GLY POLYMORPHISM, LUNG FUNCTION, AND MORTALITY: RESULTS FROM THE ATHEROSCLEROSIS RISK IN COMMUNITIES STUDY

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Growing evidence suggests that the Arg16Arg genotype of the beta-2 adrenergic receptor gene may be associated with adverse effects of beta-agonist therapy. We sought to examine the association of beta-agonist use and the Arg16Gly polymorphism with lung function and mortality among participants in th...

  9. Familial amyloid polyneuropathy associated with TTRSer50Arg mutation in two Iberian families presenting a novel single base change in the mutant gene.

    PubMed

    Munar-Qués, Miguel; Masjuan, Jaime; Coelho, Teresa; Moreira, Paul; Viader-Farré, Carlos; Saraiva, María J M

    2007-06-01

    We present two families, from Spain and Portugal, with familial amyloid polyneuropathy (FAP) associated with the mutation TTRSer50Arg. This mutation was first described in two Japanese patients from independent families and later in a French-Italian patient and a Vietnamese family. The two families presented here, are the first to be diagnosed with this mutation in the Iberian Peninsula. In the patients of both families, FAP was very aggressive as they rapidly developed multiple symptoms with progressive deterioration; we emphasize the presence of severe orthostatic hypotension in the Spanish proband which confined him to a wheelchair. This proband was the first patient with this mutation to have undergone liver transplantation and results were encouraging. The mutation was detected in four patients and one disease-free relative by DNA sequencing of exon 3 and induced mutation restriction analysis. The most outstanding feature was the single base transversion A to C in codon 50 (CGT instead of AGT), whereas in both Japanese patients and the French-Italian patient it was T to G (AGG instead of AGT). To our knowledge only six FAP mutations with more than one single nucleotide mutation for the same codon have been reported to date. PMID:17577688

  10. Effect of various protein kinase inhibitors on the induction of milk protein gene expression by prolactin.

    PubMed

    Bayat-Sarmadi, M; Houdebine, L M

    1993-03-01

    Prolactin has many known functions and one of them is to induce the expression of milk protein gene expression in the mammary gland. Specific membrane receptors have been recently characterized but the transduction mechanism involved in the transfer of the prolactin signal to milk protein genes remains unknown. In the present work, it is shown that several protein kinase inhibitors block prolactin action on milk protein genes. Primary rabbit mammary cells were cultured for several days on floating collagen gel in a serum-free medium. Prolactin and the inhibitors of protein kinase were then added to the culture medium. After 1 day, the concentration of alpha s1-casein in the culture medium was measured using a specific radioimmunoassay. The concentration of several mRNAs in cell extracts was also evaluated using Northern blot analysis. alpha s1-Casein secretion and alpha s1-casein mRNA accumulation were induced by prolactin. This induction was blocked by staurosporine, sphingosine, quercetin, genistein and to some extent by o-hydroxyphenyl acetate, but not by H7, polymyxin B, benzylsuccinate and lavendustin A. The concentration of the mRNA coding for transferrin, which is abundantly secreted in rabbit milk independently of prolactin action, was only moderately altered by the inhibitors. The concentration of two house-keeping mRNAs, beta-actin and glyceraldehyde 3-phosphate dehydrogenase, was lowered only by genistein after 1 day but not after 4 h of culture. These data show for the first time that a Ser/Thre kinase, which is not kinase C, and possibly a tyrosine kinase is involved in the transduction of the prolactin message from the receptor to the milk protein genes. PMID:8472863

  11. High-Throughput Screening of Tyrosine Kinase Inhibitor Resistant Genes in CML.

    PubMed

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

    2016-01-01

    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

  12. Dysregulation of Protein Kinase Gene Expression in NK Cells from Chronic Fatigue Syndrome/Myalgic Encephalomyelitis Patients

    PubMed Central

    Chacko, Anu; Staines, Donald R.; Johnston, Samantha C.; Marshall-Gradisnik, Sonya M.

    2016-01-01

    BACKGROUND The etiology and pathomechanism of chronic fatigue syndrome/myalgic encephalomyelitis (CFS/ME) are unknown. However, natural killer (NK) cell dysfunction, in particular reduced NK cytotoxic activity, is a consistent finding in CFS/ME patients. Previous research has reported significant changes in intracellular mitogen-activated protein kinase pathways from isolated NK cells. The purpose of this present investigation was to examine whether protein kinase genes have a role in abnormal NK cell intracellular signaling in CFS/ME. METHOD Messenger RNA (mRNA) expression of 528 protein kinase genes in isolated NK cells was analyzed (nCounter GX Human Kinase Kit v2 (XT); NanoString Technologies) from moderate (n = 11; age, 54.9 ± 10.3 years) and severe (n = 12; age, 47.5 ± 8.0 years) CFS/ME patients (classified by the 2011 International Consensus Criteria) and nonfatigued controls (n = 11; age, 50.0 ± 12.3 years). RESULTS The expression of 92 protein kinase genes was significantly different in the severe CFS/ME group compared with nonfatigued controls. Among these, 37 genes were significantly upregulated and 55 genes were significantly downregulated in severe CFS/ME patients compared with nonfatigued controls. CONCLUSIONS In severe CFS/ME patients, dysfunction in protein kinase genes may contribute to impairments in NK cell intracellular signaling and effector function. Similar changes in protein kinase genes may be present in other cells, potentially contributing to the pathomechanism of this illness. PMID:27594784

  13. A new autoinhibited kinase conformation reveals a salt-bridge switch in kinase activation

    PubMed Central

    Wei, Qiang; Yang, Shaoyuan; Li, Dan; Zhang, Xiaoying; Zheng, Jimin; Jia, Zongchao

    2016-01-01

    In the structure of autoinhibited EphA2 tyrosine kinase reported herein, we have captured the entire activation segment, revealing a previously unknown role of the conserved Arg762 in kinase autoinhibition by interacting with the essential Mg2+-chelating Asp757. While it is well known that this Arg residue is involved in an electrostatic interaction with the phospho-residue of the activation loop to stabilize the active conformation, our structure determination revealed a new role for the Arg, acting as a switch between the autoinhibited and activated conformations. Mutation of Arg762 to Ala in EphA2 sensitized Mg2+ response, resulting in enhanced kinase catalytic activity and Mg2+ cooperativity. Furthermore, mutation of the corresponding Arg/Lys to Ala in PKA and p38MAPK also exhibited similar behavior. This new salt bridge-mediated switch may thus be an important mechanism of activation on a broader scope for kinases which utilize autophosphorylation. PMID:27324091

  14. Mitogen-Activated Protein Kinase Kinase 4 Gene Polymorphism and Cancer Risk

    PubMed Central

    Geng, Peiliang; Ou, Juanjuan; Xie, Ganfeng; Li, Jianjun; Zhao, Xiaoxin; Xiang, Lisha; Liao, Yunmei; Wang, Ning; Liang, Houjie

    2015-01-01

    Abstract A number of epidemiological studies have assessed the association of −1304T > G polymorphism in the MKK4 gene and risk of cancer, but the results lack of statistical power due to the limited subjects used in these studies. This study was devised to identify the genetic effects of the −1304T > G polymorphism on cancer risk in a large meta-analysis. Eligible studies were identified by searching both Chinese and English databases. General as well as subgroup analyses were performed for 8 independent case–control publications with a total of 4623 cases and 5256 cancer-free controls. Odds ratios (ORs) and 95% confidence intervals (CIs) were used to estimate the association. Overall, this meta-analysis showed that the association between the −1304T > G polymorphism and cancer risk was statistically significant (GG vs TT: OR = 0.63, 95% CI, 0.52–0.75; GG + TG vs TT: OR = 0.85, 95% CI, 0.79–0.91; GG vs TG + TT: OR = 0.67, 95% CI, 0.56–0.80; G vs T: OR = 0.82, 95% CI, 0.77–0.88; TG vs TT: OR = 0.86, 95% CI, 0.79–0.93). Our meta-analysis reveals that the presence of the −1304T > G polymorphism is likely to decrease risk of cancer. Future larger studies are necessary to validate the current finding. PMID:26554761

  15. Attenuation of serum inducibility of immediate early genes by oncoproteins in tyrosine kinase signaling pathways.

    PubMed Central

    Yu, C L; Prochownik, E V; Imperiale, M J; Jove, R

    1993-01-01

    Immediate early genes involved in controlling cell proliferation are rapidly and transiently induced following stimulation of susceptible cells with serum. To study how oncoproteins regulate immediate early genes, we examined serum inducibility of these genes in cells transformed by various oncoproteins. We found that induction of the immediate early gene, c-fos, by serum stimulation was markedly attenuated in four independent cell lines stably transformed by the v-Src tyrosine kinase. Cells chronically transformed by other oncoproteins implicated in tyrosine kinase signaling pathways, including v-Sis, v-Ras, and v-Raf, showed the same pattern of attenuation. In contrast, serum inducibility of c-fos was not attenuated in cells transformed by simian virus 40, which is thought to transform cells through a different pathway. Cell cycle analyses showed that proliferation of these transformed cell lines could be arrested effectively in 0.1% serum, demonstrating that the attenuation was not simply due to continuous cycling of transformed cells after serum deprivation. Moreover, serum inducibility of other immediate early genes, including c-jun, junB, egr-1, and NGFI-B, also was strikingly attenuated by these same oncoproteins. Nuclear run-on transcription assays established that this attenuation of serum inducibility occurred at the transcriptional level. Finally, flow cytometric analysis demonstrated that serum-starved v-Src-transformed cells were viable and able to progress into S phase of the cell cycle after serum stimulation, even though the induction of immediate early genes was greatly attenuated in these cells. Our results suggest that activation of immediate early genes is repressed by chronic stimulation of tyrosine kinase signaling pathways in transformed cells. Images PMID:8384301

  16. Genome-wide identification and expression analysis of calcium-dependent protein kinase and its closely related kinase genes in Capsicum annuum

    PubMed Central

    Cai, Hanyang; Cheng, Junbin; Yan, Yan; Xiao, Zhuoli; Li, Jiazhi; Mou, Shaoliang; Qiu, Ailian; Lai, Yan; Guan, Deyi; He, Shuilin

    2015-01-01

    As Ca2+ sensors and effectors, calcium-dependent protein kinases (CDPKs) play important roles in plant growth, development, and response to environmental cues. However, no CDPKs have been characterized in Capsicum annuum thus far. Herein, a genome wide comprehensive analysis of genes encoding CDPKs and CDPK-related protein kinases (CRKs) was performed in pepper, a total of 31 CDPK genes and five closely related kinase genes were identified, which were phylogenetically divided into four distinct subfamilies and unevenly distributed across nine chromosomes. Conserved sequence and exon-intron structures were found to be shared by pepper CDPKs within the same subfamily, and the expansion of the CDPK family in pepper was found to be due to segmental duplication events. Five CDPKs in the C. annuum variety CM334 were found to be mutated in the Chiltepin variety, and one CDPK present in CM334 was lost in Chiltepin. The majority of CDPK and CRK genes were expressed in different pepper tissues and developmental stages, and 10, 12, and 8 CDPK genes were transcriptionally modified by salt, heat, and Ralstonia solanacearum stresses, respectively. Furthermore, these genes were found to respond specifically to one stress as well as respond synergistically to two stresses or three stresses, suggesting that these CDPK genes might be involved in the specific or synergistic response of pepper to salt, heat, and R. solanacearum. Our results lay the foundation for future functional characterization of pepper CDPK and its closely related gene families. PMID:26442050

  17. Protein Kinase D1 Signaling in Angiogenic Gene Expression and VEGF-Mediated Angiogenesis

    PubMed Central

    Ren, Bin

    2016-01-01

    Protein kinase D 1 (PKD-1) is a signaling kinase important in fundamental cell functions including migration, proliferation, and differentiation. PKD-1 is also a key regulator of gene expression and angiogenesis that is essential for cardiovascular development and tumor progression. Further understanding molecular aspects of PKD-1 signaling in the regulation of angiogenesis may have translational implications in obesity, cardiovascular disease, and cancer. The author will summarize and provide the insights into molecular mechanisms by which PKD-1 regulates transcriptional expression of angiogenic genes, focusing on the transcriptional regulation of CD36 by PKD-1-FoxO1 signaling axis along with the potential implications of this axis in arterial differentiation and morphogenesis. He will also discuss a new concept of dynamic balance between proangiogenic and antiangiogenic signaling in determining angiogenic switch, and stress how PKD-1 signaling regulates VEGF signaling-mediated angiogenesis. PMID:27200349

  18. Protein Kinase D1 Signaling in Angiogenic Gene Expression and VEGF-Mediated Angiogenesis.

    PubMed

    Ren, Bin

    2016-01-01

    Protein kinase D 1 (PKD-1) is a signaling kinase important in fundamental cell functions including migration, proliferation, and differentiation. PKD-1 is also a key regulator of gene expression and angiogenesis that is essential for cardiovascular development and tumor progression. Further understanding molecular aspects of PKD-1 signaling in the regulation of angiogenesis may have translational implications in obesity, cardiovascular disease, and cancer. The author will summarize and provide the insights into molecular mechanisms by which PKD-1 regulates transcriptional expression of angiogenic genes, focusing on the transcriptional regulation of CD36 by PKD-1-FoxO1 signaling axis along with the potential implications of this axis in arterial differentiation and morphogenesis. He will also discuss a new concept of dynamic balance between proangiogenic and antiangiogenic signaling in determining angiogenic switch, and stress how PKD-1 signaling regulates VEGF signaling-mediated angiogenesis. PMID:27200349

  19. Expression Divergence of Duplicate Genes in the Protein Kinase Superfamily in Pacific Oyster.

    PubMed

    Gao, Dahai; Ko, Dennis C; Tian, Xinmin; Yang, Guang; Wang, Liuyang

    2015-01-01

    Gene duplication has been proposed to serve as the engine of evolutionary innovation. It is well recognized that eukaryotic genomes contain a large number of duplicated genes that evolve new functions or expression patterns. However, in mollusks, the evolutionary mechanisms underlying the divergence and the functional maintenance of duplicate genes remain little understood. In the present study, we performed a comprehensive analysis of duplicate genes in the protein kinase superfamily using whole genome and transcriptome data for the Pacific oyster. A total of 64 duplicated gene pairs were identified based on a phylogenetic approach and the reciprocal best BLAST method. By analyzing gene expression from RNA-seq data from 69 different developmental and stimuli-induced conditions (nine tissues, 38 developmental stages, eight dry treatments, seven heat treatments, and seven salty treatments), we found that expression patterns were significantly correlated for a number of duplicate gene pairs, suggesting the conservation of regulatory mechanisms following divergence. Our analysis also identified a subset of duplicate gene pairs with very high expression divergence, indicating that these gene pairs may have been subjected to transcriptional subfunctionalization or neofunctionalization after the initial duplication events. Further analysis revealed a significant correlation between expression and sequence divergence (as revealed by synonymous or nonsynonymous substitution rates) under certain conditions. Taken together, these results provide evidence for duplicate gene sequence and expression divergence in the Pacific oyster, accompanying its adaptation to harsh environments. Our results provide new insights into the evolution of duplicate genes and their expression levels in the Pacific oyster. PMID:26417197

  20. 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

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

    SciTech Connect

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

    1995-04-10

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

  2. Mouse thymidine kinase: the promoter sequence and the gene and pseudogene structures in normal cells and in thymidine kinase deficient mutants.

    PubMed Central

    Seiser, C; Knöfler, M; Rudelstorfer, I; Haas, R; Wintersberger, E

    1989-01-01

    The mouse genome carries one gene and two pseudogenes for cytoplasmic thymidine kinase. The overall structure of these genes was determined with the help of cosmids and lambda phage clones and the upstream sequence containing the promoter was determined. The data allow an allocation of bands seen in the complex patterns of genomic Southern blots obtained from the DNA of wild type cells and of thymidine kinase deficient mutants to the gene as well as to the two pseudogenes. The much used LTK cell line was found to lack the entire gene but to retain the pseudogenes. Two other TK cell lines had DNA patterns indistinguishable from the wild type. Whereas the LTK line did not produce any TKmRNA, the two other mutants had normal amounts of TKmRNA but no cytoplasmic TK activity. Images PMID:2911464

  3. Spermiogenesis initiation in Caenorhabditis elegans involves a casein kinase 1 encoded by the spe-6 gene.

    PubMed Central

    Muhlrad, Paul J; Ward, Samuel

    2002-01-01

    Immature spermatids from Caenorhabditis elegans are stimulated by an external activation signal to reorganize their membranes and cytoskeleton to form crawling spermatozoa. This rapid maturation, termed spermiogenesis, occurs without any new gene expression. To better understand this signal transduction pathway, we isolated suppressors of a mutation in the spe-27 gene, which is part of the pathway. The suppressors bypass the requirement for spe-27, as well as three other genes that act in this pathway, spe-8, spe-12, and spe-29. Eighteen of the suppressor mutations are new alleles of spe-6, a previously identified gene required for an early stage of spermatogenesis. The original spe-6 mutations are loss-of-function alleles that prevent major sperm protein (MSP) assembly in the fibrous bodies of spermatocytes and arrest development in meiosis. We have isolated the spe-6 gene and find that it encodes a predicted protein-serine/threonine kinase in the casein kinase 1 family. The suppressor mutations appear to be reduction-of-function alleles. We propose a model whereby SPE-6, in addition to its early role in spermatocyte development, inhibits spermiogenesis until the activation signal is received. The activation signal is transduced through SPE-8, SPE-12, SPE-27, and SPE-29 to relieve SPE-6 repression, thus triggering the formation of crawling spermatozoa. PMID:12019230

  4. MoARG1, MoARG5,6 and MoARG7 involved in arginine biosynthesis are essential for growth, conidiogenesis, sexual reproduction, and pathogenicity in Magnaporthe oryzae.

    PubMed

    Zhang, Yong; Shi, Huanbin; Liang, Shuang; Ning, Guoao; Xu, Nanchang; Lu, Jianping; Liu, Xiaohong; Lin, Fucheng

    2015-11-01

    Arginine is one of the most versatile amino acids in eukaryote cells, which plays important roles in a multitude of processes such as protein synthesis, nitrogen metabolism, nitric oxide (NO) and urea biosynthesis. The de novo arginine biosynthesis pathway is conserved among fungal kingdom, but poorly understood in plant pathogenic fungi. Here, we characterized the functions of three synthetic enzyme-encoding genes MoARG1, MoARG5,6, and MoARG7, which involved the seventh step, second-third step and fifth step of arginine biosynthesis in Magnaporthe oryzae, respectively. Deletion of MoARG1 or MoARG5,6, resulted in arginine auxotrophic mutants, which had a strict requirement for arginine on minimal medium (MM). Both ΔMoarg1 and ΔMoarg5,6 severely reduced in aerial hyphal growth, pigmentation, conidiogenesis, sexual reproduction and pathogenicity. Interestingly, like Saccharomyces cerevisiae, deletion of MoARG7 caused a leaky arginine auxotrophy, and attenuated pathogenicity. Limited appressorium-mediated penetration and restricted invasive hyphae growth in host cells are responsible for the severely attenuated pathogenicity of the Arg(-) mutants. Additionally, we monitored the NO generation during conidial germination and appressorial formation in both Arg(-) mutants and wild type, and demonstrated that NO generation may not occur via arginine-dependent pathway in M. oryzae. In summary, MoARG1, MoARG5,6, and MoARG7 are required for growth, conidiogenesis, sexual reproduction, and pathogenicity in M. oryzae. PMID:26505307

  5. Tissue-Specific and Developmentally Regulated Expression of a Cluster of Tandemly Arrayed Cell Wall-Associated Kinase-Like Kinase Genes in Arabidopsis1

    PubMed Central

    Verica, Joseph A.; Chae, Lee; Tong, Hongyun; Ingmire, Peter; He, Zheng-Hui

    2003-01-01

    The Arabidopsis cell wall-associated kinase (WAK) and WAK-like kinase (WAKL) family of receptor-like kinase genes encodes transmembrane proteins with a cytoplasmic serine/threonine kinase domain and an extracellular region containing epidermal growth factor-like repeats. Previous studies have suggested that some WAK members are involved in plant defense and heavy metal responses, whereas others are required for cell elongation and plant development. The WAK/WAKL gene family consists of 26 members in Arabidopsis and can be divided into four groups. Here, we describe the characterization of group 2 members that are composed of a cluster of seven tandemly arrayed WAKL genes. The predicted WAKL proteins are highly similar in their cytoplasmic region but are more divergent in their predicted extracellular ligand-binding region. WAKL7 encodes a truncated WAKL isoform that is predicted to be secreted from the cytoplasm. Ratios of nonsynonymous to synonymous substitutions suggest that the extracellular region is subject to diversifying selection. Comparison of the WAKL and WAK gene clusters suggests that they arose independently. Protein gel-blot and immunolocalization analyses suggest that WAKL6 is associated with the cell wall. Histochemical analyses of WAKL promoters fused with the β-glucuronidase reporter gene have shown that the expressions of WAKL members are developmentally regulated and tissue specific. Unlike WAK members whose expressions were found predominately in green tissues, WAKL genes are highly expressed in roots and flowers. The expression of WAKL5 and WAKL7 can be induced by wounding stress and by the salicylic acid analog 2,6-dichloroisonicotinic acid in an nonexpressor of pathogenesis-related gene 1-dependent manner, suggesting that they, like some WAK members, are wound inducible and can be defined as pathogenesis-related genes. PMID:14576286

  6. Transcriptional and posttranscriptional mechanisms regulate murine thymidine kinase gene expression in serum-stimulated cells.

    PubMed Central

    Lieberman, H B; Lin, P F; Yeh, D B; Ruddle, F H

    1988-01-01

    We previously isolated and characterized the structure of murine thymidine kinase (tk) genomic and cDNA sequences to begin a study designed to identify regions of the tk gene important for regulated expression during the transition of cells from G0 to a proliferating state. In this report, we describe the stable transfection of the cloned gene into L-M(TK-) cells and show that both thymidine kinase (TK) enzyme activity and DNA synthesis increase in parallel when transfectants in G0 arrest are stimulated by serum. To define promoter and regulatory regions more precisely, we have constructed a series of tk minigenes and have examined their expression in stable transfectants after serum stimulation. We have identified a 291-base-pair DNA fragment at the 5' end of the tk gene that has promoter function, and we have determined its sequence. In addition, we have found that DNA sequences which mediate serum-induced expression of TK are transcribed, since expression of the murine tk cDNA, fused to a promoter from either the murine tk gene, the simian virus 40 early region, or the herpes simplex virus tk gene, is stimulated by serum. Our constructs also reveal that the murine tk polyadenylation signal is not required for regulation, nor is most of the 3' untranslated region. RNA dot blot analysis indicates that murine cytoplasmic tk mRNA levels always parallel TK enzyme activity. Nuclear runon transcription assays show less than a 2-fold increase in transcription from the cloned tk gene in serum-stimulated transfectants, but an 11-fold increase in mouse L929 cells, which are inherently TK+. These results taken together suggest that the murine tk gene is controlled in serum-stimulated cells by a transcriptional mechanism influenced by DNA sequences that flank tk and also by a posttranscriptional system linked to gene sequences that are transcribed. Images PMID:3244356

  7. The sum of gains and losses of genes encoding the protein tyrosine kinase targets predicts response to multi-kinase inhibitor treatment: Characterization, validation, and prognostic value

    PubMed Central

    Jiang, Xiaojun; Pissaloux, Daniel; De La Fouchardiere, Christelle; Desseigne, Françoise; Wang, Qing; Attignon, Valery; Fondrevelle, Marie-Eve; De La Fouchardiere, Arnaud; Perol, Maurice; Cassier, Philippe; Seigne, Christelle; Perol, David; Coquard, Isabelle Ray; Meeus, Pierre; Fayette, Jerome; Flechon, Aude; Le Cesne, Axel; Penel, Nicolas; Tredan, Olivier; Blay, Jean-Yves

    2015-01-01

    Validated predictive biomarkers for multi-tyrosine kinase inhibitors (MTKI) efficacy are lacking. We hypothesized that interindividual response variability is partially dependent on somatic DNA copy number alterations (SCNAs), particularly those of genes encoding the protein tyrosines targeted by MTKI (called target genes). Genomic alterations were investigated in MTKI responsive and non responsive patients with different histological subtypes included in the ProfiLER protocol (NCT 01774409). From March 2013 to August 2014, 58 patients with advanced cancer treated with one of 7 MTKIs were included in the ProfiLER trial and split into one discovery cohort (n = 13), and 2 validation cohorts (n = 12 and 33). An analysis of the copy number alterations of kinase-coding genes for each of 7 MTKIs was conducted. A prediction algorithm (SUMSCAN) based on the presence of specific gene gains (Tumor Target Charge, TTC) and losses (Tumor Target Losses, TTL) was conceived and validated in 2 independent validation cohorts. MTKI sensitive tumors present a characteristic SCNA profile including a global gain profile, and specific gains for target genes while MTKI resistant tumors present the opposite. SUMSCAN favorable patients achieved longer progression-free and overall survival. This work shows that the copy number sum of kinase-coding genes enables the prediction of response of cancer patients to MTKI, opening a novel paradigm for the treatment selection of these patients. PMID:26317543

  8. The sum of gains and losses of genes encoding the protein tyrosine kinase targets predicts response to multi-kinase inhibitor treatment: Characterization, validation, and prognostic value.

    PubMed

    Jiang, Xiaojun; Pissaloux, Daniel; De La Fouchardiere, Christelle; Desseigne, Françoise; Wang, Qing; Attignon, Valery; Fondrevelle, Marie-Eve; De La Fouchardiere, Arnaud; Perol, Maurice; Cassier, Philippe; Seigne, Christelle; Perol, David; Ray-Coquard, Isabelle; Meeus, Pierre; Fayette, Jerome; Flechon, Aude; Le Cesne, Axel; Penel, Nicolas; Tredan, Olivier; Blay, Jean-Yves

    2015-09-22

    Validated predictive biomarkers for multi-tyrosine kinase inhibitors (MTKI) efficacy are lacking. We hypothesized that interindividual response variability is partially dependent on somatic DNA copy number alterations (SCNAs), particularly those of genes encoding the protein tyrosines targeted by MTKI (called target genes). Genomic alterations were investigated in MTKI responsive and non responsive patients with different histological subtypes included in the ProfiLER protocol (NCT 01774409). From March 2013 to August 2014, 58 patients with advanced cancer treated with one of 7 MTKIs were included in the ProfiLER trial and split into one discovery cohort (n = 13), and 2 validation cohorts (n = 12 and 33). An analysis of the copy number alterations of kinase-coding genes for each of 7 MTKIs was conducted. A prediction algorithm (SUMSCAN) based on the presence of specific gene gains (Tumor Target Charge, TTC) and losses (Tumor Target Losses, TTL) was conceived and validated in 2 independent validation cohorts. MTKI sensitive tumors present a characteristic SCNA profile including a global gain profile, and specific gains for target genes while MTKI resistant tumors present the opposite. SUMSCAN favorable patients achieved longer progression-free and overall survival. This work shows that the copy number sum of kinase-coding genes enables the prediction of response of cancer patients to MTKI, opening a novel paradigm for the treatment selection of these patients. PMID:26317543

  9. Human migration activities drive the fluctuation of ARGs: Case study of landfills in Nanjing, eastern China.

    PubMed

    Sun, Mingming; Ye, Mao; Schwab, Arthur P; Li, Xu; Wan, Jinzhong; Wei, Zhong; Wu, Jun; Friman, Ville-Petri; Liu, Kuan; Tian, Da; Liu, Manqiang; Li, Huixin; Hu, Feng; Jiang, Xin

    2016-09-01

    Landfills are perfect sites to study the effect of human migration on fluctuation of antibiotic resistance genes (ARGs) as they are the final destination of municipal waste. For example, large-scale human migration during the holidays is often accompanied by changes in waste dumping having potential effects on ARG abundance. Three landfills were selected to examine fluctuation in the abundance of fifteen ARGs and Intl1 genes for 14 months in Nanjing, eastern China. Mass human migration, the amount of dumped waste and temperature exerted the most significant effects on bimonthly fluctuations of ARG levels in landfill sites. As a middle-sized cosmopolitan city in China, millions of college students and workers migrate during holidays, contributing to the dramatic increases in waste production and fluctuation in ARG abundances. In line with this, mass migration explained most of the variation in waste dumping. The waste dumping also affected the bioaccessibility of mixed-compound pollutants that further positively impacted the level of ARGs. The influence of various bioaccessible compounds on ARG abundance followed the order: antibiotics>nutrients>metals>organic pollutants. Concentrations of bioaccessible compounds were more strongly correlated with ARG levels compared to total compound concentrations. Improved waste classification and management strategies could thus help to decrease the amount of bioaccessible pollutants leading to more effective control for urban ARG dissemination. PMID:27179703

  10. 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

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

    PubMed

    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

  12. Multiple Functions of Let-23, a Caenorhabditis Elegans Receptor Tyrosine Kinase Gene Required for Vulval Induction

    PubMed Central

    Aroian, R. V.; Sternberg, P. W.

    1991-01-01

    The let-23 gene, which encodes a putative tyrosine kinase of the epidermal growth factor (EGF) receptor subfamily, has multiple functions during Caenorhabditis elegans development. We show that let-23 function is required for vulval precursor cells (VPCs) to respond to the signal that induces vulval differentiation: a complete loss of let-23 function results in no induction. However, some let-23 mutations that genetically reduce but do not eliminate let-23 function result in VPCs apparently hypersensitive to inductive signal: as many as five of six VPCs can adopt vulval fates, in contrast to the three that normally do. These results suggest that the let-23 receptor tyrosine kinase controls two opposing pathways, one that stimulates vulval differentiation and another that negatively regulates vulval differentiation. Furthermore, analysis of 16 new let-23 mutations indicates that the let-23 kinase functions in at least five tissues. Since various let-23 mutant phenotypes can be obtained independently, the let-23 gene is likely to have tissue-specific functions. PMID:2071015

  13. 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.

  14. 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.

  15. Transcriptome Analysis of the ArgR Regulon in Pseudomonas aeruginosa

    PubMed Central

    Lu, Chung-Dar; Yang, Zhe; Li, Wei

    2004-01-01

    Arginine metabolism in pseudomonads with multiple catabolic pathways for its utilization as carbon and nitrogen sources is of particular interest as the model system to study control of metabolic integration. We performed transcriptome analyses to identify genes controlled by the arginine regulatory protein ArgR and to better understand arginine metabolic pathways of P. aeruginosa. We compared gene expression in wild-type strain PAO1 with that in argR mutant strain PAO501 grown in glutamate minimal medium in the presence and absence of arginine. Ten putative transcriptional units of 28 genes were inducible by ArgR and arginine, including all known ArgR-regulated operons under aerobic conditions. The newly identified genes include the putative adcAB operon, which encodes a catabolic arginine decarboxylase and an antiporter protein, and PA0328, which encodes a hypothetical fusion protein of a peptidase and a type IV autotransporter. Also identified as members of the arginine network are the following solute transport systems: PA1971 (braZ) for branched-chain amino acids permease; PA2042 for a putative sodium:serine symporter; PA3934, which belongs to the family of small oligopeptide transporters; and PA5152-5155, which encodes components of an ABC transporter for a putative opine uptake system. The effect of arginine on the expression of these genes was confirmed by lacZ fusion studies and by DNA binding studies with purified ArgR. Only five transcriptional units of nine genes were qualified as repressible by ArgR and arginine, with three operons (argF, carAB, and argG) in arginine biosynthesis and two operons (gltBD and gdhA) in glutamate biosynthesis. These results indicate that ArgR is important in control of arginine and glutamate metabolism and that arginine and ArgR may have a redundant effect in inducing the uptake systems of certain compounds. PMID:15175299

  16. Network analysis of neurotransmitter related human kinase genes: possible role of SRC, RAF1, PTK2B?

    PubMed

    Brys, Zoltan; Pluhar, Andras; Kis, Janos Tibor; Buda, Bela; Szabo, Attila

    2013-09-01

    Previous co-expression analysis of human kinase genes highlighted 119 genes in neurotransmitter-related activity (based on Go:Terms). Using a merged interactome dataset, we analyzed the network of these Neurotransmitter Related Human Kinase Genes. Using the full interactome dataset we extended the network and calculating degrees and closeness centralities we identified SRC, MAPK1, RAF1, PTK2B and AKT1 kinase genes as potentially relevant nodes which did not show relevant activity in the original experimental study. As AKT1 and MAPK1 have already been indicated in various neuronal functions, we hypothesize a potential direct or indirect role for SRC, RAF1, PTK2B genes in neurotransmission and in central nervous system signaling processes. PMID:24108181

  17. 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. PMID:27574806

  18. 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

  19. ARG2 impairs endothelial autophagy through regulation of MTOR and PRKAA/AMPK signaling in advanced atherosclerosis

    PubMed Central

    Xiong, Yuyan; Yepuri, Gautham; Forbiteh, Michael; Yu, Yi; Montani, Jean-Pierre; Yang, Zhihong; Ming, Xiu-Fen

    2015-01-01

    Impaired autophagy function and enhanced ARG2 (arginase 2)-MTOR (mechanistic target of rapamycin) crosstalk are implicated in vascular aging and atherosclerosis. We are interested in the role of ARG2 and the potential underlying mechanism(s) in modulation of endothelial autophagy. Using human nonsenescent “young” and replicative senescent endothelial cells as well as Apolipoprotein E-deficient (apoe−/−Arg2+/+) and Arg2-deficient apoe−/− (apoe−/−arg2−/−) mice fed a high-fat diet for 10 wk as the atherosclerotic animal model, we show here that overexpression of ARG2 in the young cells suppresses endothelial autophagy with concomitant enhanced expression of RICTOR, the essential component of the MTORC2 complex, leading to activation of the AKT-MTORC1-RPS6KB1/S6K1 (ribosomal protein S6 kinase, 70kDa, polypeptide 1) cascade and inhibition of PRKAA/AMPK (protein kinase, AMP-activated, α catalytic subunit). Expression of an inactive ARG2 mutant (H160F) had the same effect. Moreover, silencing RPS6KB1 or expression of a constitutively active PRKAA prevented autophagy suppression by ARG2 or H160F. In senescent cells, enhanced ARG2-RICTOR-AKT-MTORC1-RPS6KB1 and decreased PRKAA signaling and autophagy were observed, which was reversed by silencing ARG2 but not by arginase inhibitors. In line with the above observations, genetic ablation of Arg2 in apoe−/− mice reduced RPS6KB1, enhanced PRKAA signaling and endothelial autophagy in aortas, which was associated with reduced atherosclerosis lesion formation. Taken together, the results demonstrate that ARG2 impairs endothelial autophagy independently of the L-arginine ureahydrolase activity through activation of RPS6KB1 and inhibition of PRKAA, which is implicated in atherogenesis. PMID:25484082

  20. A c.1244G>A (p.Arg415Gln) mutation in SH3BP2 gene causes cherubism in a Turkish family: Report of a family with review of the literature

    PubMed Central

    Balta, Burhan; Hu, Ying; Reichenberger, Ernst J.; Etoz, Osman A.; Nazlim, Sinan; Bayrakdar, Ibrahim S.

    2014-01-01

    Objectives: The present study was aimed at advancing the understanding of the pathogenesis of cherubism by presenting a case study based on history, physical examination, typical radiological features, molecular and histopathological laboratory tests and a review of the literature. Study Design: This study began with a 7-year-old boy who was referred due to mandibular overgrowth. A panoramic radiograph revealed multilocular radiolucent lesions of the upper/lower jaws suggestive of cherubism. Overall, a total of four family members were tested for SH3BP2 mutations, namely two siblings and their parents. Both siblings had been clinically diagnosed with cherubism; however, the parents were clinically normal. Peripheral blood was collected from all participants and genomic DNA sequencing was carried out. Results: A missense mutation was found in the two affected siblings and their asymptomatic mother. The mutation was a 1244 G>A transversion which resulted in an amino acid substitution from arginine to glutamine (p.Arg415Gln) in exon 9. Conclusions: The present study emphasized the importance of further clinical and molecular investigation even when only a single case of cherubism is identified within a family. Genotype-phenotype association studies in individuals with cherubism are necessary to provide important insights into the molecular mechanisms associated with this disease. Key words:Cherubism, mandible, maxilla, SH3BP2, gene analysis, CBCT. PMID:24608212

  1. Phenotypic profiling of the human genome reveals gene products involved in plasma membrane targeting of SRC kinases

    PubMed Central

    Ritzerfeld, Julia; Remmele, Steffen; Wang, Tao; Temmerman, Koen; Brügger, Britta; Wegehingel, Sabine; Tournaviti, Stella; Strating, Jeroen R.P.M.; Wieland, Felix T.; Neumann, Beate; Ellenberg, Jan; Lawerenz, Chris; Hesser, Jürgen; Erfle, Holger; Pepperkok, Rainer; Nickel, Walter

    2011-01-01

    SRC proteins are non-receptor tyrosine kinases that play key roles in regulating signal transduction by a diverse set of cell surface receptors. They contain N-terminal SH4 domains that are modified by fatty acylation and are functioning as membrane anchors. Acylated SH4 domains are both necessary and sufficient to mediate specific targeting of SRC kinases to the inner leaflet of plasma membranes. Intracellular transport of SRC kinases to the plasma membrane depends on microdomains into which SRC kinases partition upon palmitoylation. In the present study, we established a live-cell imaging screening system to identify gene products involved in plasma membrane targeting of SRC kinases. Based on siRNA arrays and a human model cell line expressing two kinds of SH4 reporter molecules, we conducted a genome-wide analysis of SH4-dependent protein targeting using an automated microscopy platform. We identified and validated 54 gene products whose down-regulation causes intracellular retention of SH4 reporter molecules. To detect and quantify this phenotype, we developed a software-based image analysis tool. Among the identified gene products, we found factors involved in lipid metabolism, intracellular transport, and cellular signaling processes. Furthermore, we identified proteins that are either associated with SRC kinases or are related to various known functions of SRC kinases such as other kinases and phosphatases potentially involved in SRC-mediated signal transduction. Finally, we identified gene products whose function is less defined or entirely unknown. Our findings provide a major resource for future studies unraveling the molecular mechanisms that underlie proper targeting of SRC kinases to the inner leaflet of plasma membranes. PMID:21795383

  2. Expression Patterns of Protein Kinases Correlate with Gene Architecture and Evolutionary Rates

    PubMed Central

    Mariño-Ramírez, Leonardo; Johnson, Gibbes R.; Landsman, David; Spiridonov, Nikolay A.

    2008-01-01

    Background Protein kinase (PK) genes comprise the third largest superfamily that occupy ∼2% of the human genome. They encode regulatory enzymes that control a vast variety of cellular processes through phosphorylation of their protein substrates. Expression of PK genes is subject to complex transcriptional regulation which is not fully understood. Principal Findings Our comparative analysis demonstrates that genomic organization of regulatory PK genes differs from organization of other protein coding genes. PK genes occupy larger genomic loci, have longer introns, spacer regions, and encode larger proteins. The primary transcript length of PK genes, similar to other protein coding genes, inversely correlates with gene expression level and expression breadth, which is likely due to the necessity to reduce metabolic costs of transcription for abundant messages. On average, PK genes evolve slower than other protein coding genes. Breadth of PK expression negatively correlates with rate of non-synonymous substitutions in protein coding regions. This rate is lower for high expression and ubiquitous PKs, relative to low expression PKs, and correlates with divergence in untranslated regions. Conversely, rate of silent mutations is uniform in different PK groups, indicating that differing rates of non-synonymous substitutions reflect variations in selective pressure. Brain and testis employ a considerable number of tissue-specific PKs, indicating high complexity of phosphorylation-dependent regulatory network in these organs. There are considerable differences in genomic organization between PKs up-regulated in the testis and brain. PK genes up-regulated in the highly proliferative testicular tissue are fast evolving and small, with short introns and transcribed regions. In contrast, genes up-regulated in the minimally proliferative nervous tissue carry long introns, extended transcribed regions, and evolve slowly. Conclusions/Significance PK genomic architecture, the

  3. 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.

  4. 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

  5. Isolation and characterization of temperature-sensitive pantothenate kinase (coaA) mutants of Escherichia coli.

    PubMed Central

    Vallari, D S; Rock, C O

    1987-01-01

    Escherichia coli mutants conditionally defective in the conversion of pantothenate to coenzyme A were isolated and characterized. The gene was designated coaA and localized between argEH and rpoB near min 90 of the chromosome. The coaA15(Ts) mutation caused a temperature-sensitive growth phenotype and temperature-dependent inactivation of pantothenate kinase activity assayed both in vivo and in vitro. At 30 degrees C, coaA15(Ts) extracts contained less than 20% of the wild-type pantothenate kinase activity; the kinase had near normal kinetic constants for the substrates ATP and pantothenate and was inhibited by coenzyme A to the same degree as the wild-type enzyme. These data define the coaA gene as the structural gene for pantothenate kinase. PMID:2824448

  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. Dimeric 3-phosphoglycerate kinases from hyperthermophilic Archaea. Cloning, sequencing and expression of the 3-phosphoglycerate kinase gene of Pyrococcus woesei in Escherichia coli and characterization of the protein. Structural and functional comparison with the 3-phosphoglycerate kinase of Methanothermus fervidus.

    PubMed

    Hess, D; Krüger, K; Knappik, A; Palm, P; Hensel, R

    1995-10-01

    The gene coding for the 3-phosphoglycerate kinase (EC 2.7.2.3) of Pyrococcus woesei was cloned and sequenced. The gene sequence comprises 1230 bp coding for a polypeptide with the theoretical M(r) of 46,195. The deduced protein sequence exhibits a high similarity (46.1% and 46.6% identity) to the other known archaeal 3-phosphoglycerate kinases of Methanobacterium bryantii and Methanothermus fervidus [Fabry, S., Heppner, P., Dietmaier, W. & Hensel, R. (1990) Gene 91, 19-25]. By comparing the 3-phosphoglycerate kinase sequences of the mesophilic and the two thermophilic Archaea, trends in thermoadaptation were confirmed that could be deduced from comparisons of glyceraldehyde-3-phosphate dehydrogenase sequences from the same organisms [Zwickl, P., Fabry, S., Bogedain, C., Haas, A. & Hensel, R. (1990) J. Bacteriol. 172, 4329-4338]. With increasing temperature the average hydrophobicity and the portion of aromatic residues increases, whereas the chain flexibility as well as the content in chemically labile residues (Asn, Cys) decreases. To study the phenotypic properties of the 3-phosphoglycerate kinases from thermophilic Archaea in more detail, the 3-phosphoglycerate kinase genes from P. woesei and M. fervidus were expressed in Escherichia coli. Comparisons of kinetic and molecular properties of the enzymes from the original organisms and from E. coli indicate that the proteins expressed in the mesophilic host are folded correctly. Besides their higher thermostability according to their origin from hyperthermophilic organisms, both enzymes differ from their bacterial and eucaryotic homologues mainly in two respects. (a) The 3-phosphoglycerate kinases from P. woesei and M. fervidus are homomeric dimers in their native state contrary to all other known 3-phosphoglycerate kinases, which are monomers including the enzyme from the mesophilic Archaeum M. bryantii. (b) Monovalent cations are essential for the activity of both archaeal enzymes with K+ being significantly more

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

    PubMed Central

    Forbes, Sean C.; Bish, Lawrence T.; Ye, Fan; Spinazzola, Janelle; Baligand, Celine; Plant, Daniel; Vandenborne, Krista; Barton, Elisabeth R.; Sweeney, H. Lee; Walter, Glenn A.

    2014-01-01

    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 (31P-MRS). AK gene was injected into the gastrocnemius of the left hindlimb of C57Bl10 mice (age 5wk, male) using self-complementary AAV, type 2/8 with desmin promoter. Non-localized 31P-MRS data were acquired over nine months after injection using 11.1-T and 17.6-T Bruker Avance spectrometers. In addition, 31P 2-D 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 nine months (p<.05). Furthermore, PArg was primarily localized to the injected posterior hindimb region with the metabolite being 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 utilized to non-invasively monitor the transduction of genes for therapeutic interventions. PMID:24572791

  9. 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. PMID:24572791

  10. Identification of genes affecting expression of phosphoglycerate kinase on the surface of group B streptococcus.

    PubMed

    Boone, Tyler J; Tyrrell, Gregory J

    2012-04-01

    Group B streptococcal phosphoglycerate kinase (GBS-PGK), a glycolytic enzyme, has previously been identified on the surface of group B streptococcus (GBS). To identify genes involved in surface expression of GBS-PGK, we performed Tn917 mutagenesis followed by quantification of PGK expressed on the GBS surface. Tn917 mutagenesis identified 4 genes (sag0966, sag0979, sag0980, and sag1003) that when disrupted, alter expression of GBS-PGK on the bacterial surface. Three of the identified genes were localized to a region of the GBS genome containing genes (sag0973-sag0977) predicted to be involved in resistance to antimicrobial peptides. One mutant isolate, designated NCS13sag1003::Tn917, was found to have increased sensitivity to the antimicrobial peptides bacitracin and nisin. In addition, all of the mutant strains assayed were found to have decreased β-hemolysis. In conclusion, we have identified genes involved in surface expression of GBS-PGK. These genes also appear to be involved in antimicrobial peptide resistance and regulate expression of the β-hemolysin. PMID:22444251

  11. axl, a transforming gene isolated from primary human myeloid leukemia cells, encodes a novel receptor tyrosine kinase

    SciTech Connect

    O'Bryan, J.P.; Frye, R.A.; Cogswell, P.C.; Neubauer, A.; Kitch, B.; Prokop, C.; Earp, H.S.; Liu, E.T. ); Espinosa, R. III; Le Beau, M.M. )

    1991-10-01

    Using a sensitive transfection-tumorigenicity assay, the authors have isolated a novel transforming gene from the DNA of two patients with chronic myelogenous leukemia. Sequence analysis indicates that the product of this gene, axl, is a receptor tyrosine kinase. Overexpression of axl cDNA in NIH 3T3 cells induces neoplastic transformation with the concomitant appearance of a 140-kDa axl tyrosine-phosphorylated protein. Expression of axl cDNA in the baculovirus system results in the expression of the appropriate recombinant protein that is recognized by antophosphotyrosine antibodies, confirming that the axl protein is a tyrosine kinase. The juxtaposition of fibronectin type II and immunoglobulinlike repeats in the extracellular domain, as well as distinct amino acid sequences in the kinase domain, indicate that the axl protein represents a novel subclass of receptor tyrosine kinases.

  12. 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. PMID:25087164

  13. 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. PMID:26520101

  14. N- and C-terminal isoforms of Arg quantified by real-time PCR are specifically expressed in human normal and neoplastic cells, in neoplastic cell lines, and in HL-60 cell differentiation.

    PubMed

    Perego, Roberto A; Corizzato, Matteo; Bianchi, Cristina; Eroini, Barbara; Bosari, Silvano

    2005-04-01

    The human ABL2 (or ARG) gene codes for a nonreceptor tyrosine kinase is involved in translocation with the ETV6 gene in human leukemia and has an altered expression in several human carcinomas. Two isoforms of Arg with different N-termini (1A and 1B) have been described. The C-terminal domain of Arg contains two F-actin-binding sequences that perform a number of actions related to cell morphology and motility by interacting with actin filaments. We have identified different-sized specific cDNAs in hematopoietic, epithelial, nervous, and fibroblastic cells by means of the reverse transcription (RT)-polymerase chain reaction (PCR) analysis of human Arg mRNA. Some of these cDNAs showed an adjunctive alternative splice event involving the 63 bp sequence of exon II, thus leading to four cDNA types with different N-termini: 1A long and short, and 1B long and short. Other cDNAs lacked a 309 bp sequence in the last exon involving one of the C-terminal F-actin binding domains, thus giving rise to two cDNA types: C-termini long and short. Quantified by real-time PCR-quantitative RT-PCR-these Arg transcript isoforms have specific expression patterns not only in different normal and tumor cell types, but also during cell differentiation and growth arrest. These isoforms maintained the open reading frames, and eight putative proteins were predicted. The different C-termini isoforms seem to retain the same quantitative reciprocal ratio of their respective transcripts. The Arg protein isoforms with different C-terminal actin-binding domains and different N-termini might have specific cellular localizations/concentrations, and differently regulated catalytic activity with different implications in normal and neoplastic cells. PMID:15765532

  15. 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.

  16. 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.

  17. 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.

  18. 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

  19. Molecular Characterization and Expression Analysis of Creatine Kinase Muscle (CK-M) Gene in Horse

    PubMed Central

    Do, Kyong-Tak; Cho, Hyun-Woo; Badrinath, Narayanasamy; Park, Jeong-Woong; Choi, Jae-Young; Chung, Young-Hwa; Lee, Hak-Kyo; Song, Ki-Duk; Cho, Byung-Wook

    2015-01-01

    Since ancient days, domestic horses have been closely associated with human civilization. Today, horse racing is an important industry. Various genes involved in energy production and muscle contraction are differentially regulated during a race. Among them, creatine kinase (CK) is well known for its regulation of energy preservation in animal cells. CK is an iso-enzyme, encoded by different genes and expressed in skeletal muscle, heart, brain and leucocytes. We confirmed that the expression of CK-M significantly increased in the blood after a 30 minute exercise period, while no considerable change was observed in skeletal muscle. Analysis of various tissues showed an ubiquitous expression of the CK-M gene in the horse; CK-M mRNA expression was predominant in the skeletal muscle and the cardiac muscle compared to other tissues. An evolutionary study by synonymous and non-synonymous single nucleotide polymorphism ratio of CK-M gene revealed a positive selection that was conserved in the horse. More studies are warranted in order to develop the expression of CK-M gene as a biomarker in blood of thoroughbred horses. PMID:26580434

  20. Resequencing Analysis of the Candidate Tyrosine Kinase and RAS Pathway Gene Families in Multiple Myeloma

    PubMed Central

    Hucthagowder, Vishwanathan; Meyer, Rekha; Mullins, Chelsea; Nagarajan, Rakesh; DiPersio, John F.; Vij, Ravi; Tomasson, Michael H.; Kulkarni, Shashikant

    2012-01-01

    Multiple myeloma (MM) is an incurable, B-cell malignancy, characterized by the clonal proliferation and accumulation of malignant plasma cells in bone marrow. Despite recent advances in understanding of genomic aberrations, a comprehensive catalogue of clinically actionable mutations in MM is just beginning to emerge. The tyrosine kinase (TK) and RAS oncogenes, which encode important regulators of various signaling pathways, are among the most frequently altered gene families in cancer. To clarify the role of TK and RAS genes in pathogenesis of MM, we performed a systematic, targeted screening of mutations on prioritized RAS and TK genes, in CD138 sorted bone marrow specimens from 42 untreated patients. We identified a total of 24 mutations in KRAS, PIK3CA, INSR, LTK and MERTK genes. In particular, seven novel mutations in addition to known KRAS mutations were observed. Prediction analysis tools, PolyPhen and SIFT were used to assess the functional significance of these novel mutations. Our analysis predicted that these mutations may have a deleterious effect, resulting in functional alteration of proteins involved in the pathogenesis of myeloma. While further investigation is needed to determine the functional consequences of these proteins, mutational testing of the RAS and TK genes in larger myeloma cohorts might be also useful to establish the recurrent nature of these mutations. PMID:22939401

  1. Molecular Characterization and Expression Analysis of Creatine Kinase Muscle (CK-M) Gene in Horse.

    PubMed

    Do, Kyong-Tak; Cho, Hyun-Woo; Badrinath, Narayanasamy; Park, Jeong-Woong; Choi, Jae-Young; Chung, Young-Hwa; Lee, Hak-Kyo; Song, Ki-Duk; Cho, Byung-Wook

    2015-12-01

    Since ancient days, domestic horses have been closely associated with human civilization. Today, horse racing is an important industry. Various genes involved in energy production and muscle contraction are differentially regulated during a race. Among them, creatine kinase (CK) is well known for its regulation of energy preservation in animal cells. CK is an iso-enzyme, encoded by different genes and expressed in skeletal muscle, heart, brain and leucocytes. We confirmed that the expression of CK-M significantly increased in the blood after a 30 minute exercise period, while no considerable change was observed in skeletal muscle. Analysis of various tissues showed an ubiquitous expression of the CK-M gene in the horse; CK-M mRNA expression was predominant in the skeletal muscle and the cardiac muscle compared to other tissues. An evolutionary study by synonymous and non-synonymous single nucleotide polymorphism ratio of CK-M gene revealed a positive selection that was conserved in the horse. More studies are warranted in order to develop the expression of CK-M gene as a biomarker in blood of thoroughbred horses. PMID:26580434

  2. Sequence analysis of two genomic regions containing the KIT and the FMS receptor tyrosine kinase genes

    SciTech Connect

    Andre, C.; Hampe, A.; Lachaume, P.

    1997-01-15

    The KIT and FMS tyrosine kinase receptors, which are implicated in the control of cell growth and differentiation, stem through duplications from a common ancestor. We have conducted a detailed structural analysis of the two loci containing the KIT and FMS genes. The sequence of the {approximately}90-kb KIT locus reveals the position and size of the 21 introns and of the 5{prime} regulatory region of the KIT gene. The introns and the 3{prime}-untranslated parts of KIT and FMS have been analyzed in parallel. Comparison of the two sequences shows that, while introns of both genes have extensively diverged in size and sequence, this divergence is, at least in part, due to intron expansion through internal duplications, as suggested by the discrete extant analogies. Repetitive elements as well as exon predictions obtained using the GRAIL and GENEFINDER programs are described in detail. These programs led us to identify a novel gene, designated SMF, immediately downstream of FMS, in the opposite orientation. This finding emphasizes the gene-rich characteristic of this genomic region. 49 refs., 4 figs., 7 tabs.

  3. N-terminal stretch Arg2, Arg3, Arg4 and Arg5 of human lactoferrin is essential for binding to heparin, bacterial lipopolysaccharide, human lysozyme and DNA.

    PubMed Central

    van Berkel, P H; Geerts, M E; van Veen, H A; Mericskay, M; de Boer, H A; Nuijens, J H

    1997-01-01

    Human lactoferrin (hLF), a protein involved in host defence against infection and excessive inflammation, interacts with heparin, the lipid A moiety of bacterial lipopolysaccharide, human lysozyme (hLZ) and DNA. To determine which region of the molecule is important in these interactions, solid-phase ligand binding assays were performed with hLF from human milk (natural hLF) and N-terminally deleted hLF variants. Iron-saturated and natural hLF bound equally well to heparin, lipid A, hLZ and DNA. Natural hLF lacking the first two N-terminal amino acids (Gly1-Arg2) showed reactivities of one-half, two-thirds, one-third and one-third towards heparin, lipid A, hLZ and DNA respectively compared with N-terminally intact hLF. A lack of the first three residues (Gly1-Arg2-Arg3) decreased binding to the same ligands to one-eighth, one-quarter, one-twentieth and one-seventeenth respectively. No binding occurred with a mutant lacking the first five residues (Gly1-Arg2-Arg3-Arg4-Arg5). An anti-hLF monoclonal antibody (E11) that reacts to an N-lobe epitope including Arg5 completely blocked hLF-ligand interaction. These results show that the N-terminal stretch of four consecutive arginine residues, Arg2-Arg3-Arg4-Arg5, has a decisive role in the interaction of hLF with heparin, lipid A, hLZ and DNA. The role of limited N-terminal proteolysis of hLF in its anti-infective and anti-inflammatory properties is discussed. PMID:9359845

  4. Transcriptional cross-regulation between Gram-negative and gram-positive bacteria, demonstrated using ArgP-argO of Escherichia coli and LysG-lysE of Corynebacterium glutamicum.

    PubMed

    Marbaniang, Carmelita N; Gowrishankar, J

    2012-10-01

    The protein-gene pairs ArgP-argO of Escherichia coli and LysG-lysE of Corynebacterium glutamicum are orthologous, with the first member of each pair being a LysR-type transcriptional regulator and the second its target gene encoding a basic amino acid exporter. Whereas LysE is an exporter of arginine (Arg) and lysine (Lys) whose expression is induced by Arg, Lys, or histidine (His), ArgO exports Arg alone, and its expression is activated by Arg but not Lys or His. We have now reconstituted in E. coli the activation of lysE by LysG in the presence of its coeffectors and have shown that neither ArgP nor LysG can regulate expression of the noncognate orthologous target. Of several ArgP-dominant (ArgP(d)) variants that confer elevated Arg-independent argO expression, some (ArgP(d)-P274S, -S94L, and, to a lesser extent, -P108S) activated lysE expression in E. coli. However, the individual activating effects of LysG and ArgP(d) on lysE were mutually extinguished when both proteins were coexpressed in Arg- or His-supplemented cultures. In comparison with native ArgP, the active ArgP(d) variants exhibited higher affinity of binding to the lysE regulatory region and less DNA bending at both argO and lysE. We conclude that the transcription factor LysG from a Gram-positive bacterium, C. glutamicum, is able to engage appropriately with the RNA polymerase from a Gram-negative bacterium, E. coli, for activation of its cognate target lysE in vivo and that single-amino-acid-substitution variants of ArgP can also activate the distantly orthologous target lysE, but by a subtly different mechanism that renders them noninterchangeable with LysG. PMID:22904281

  5. Gene expression profiling and functional proteomic analysis reveal perturbed kinase-mediated signaling in genetic stroke susceptibility.

    PubMed

    Fornage, Myriam; Swank, Michael W; Boerwinkle, Eric; Doris, Peter A

    2003-09-29

    The stroke-prone spontaneously hypertensive rat (SHRSP) is a model of heritable hypertension-associated cerebrovascular injury. This study sought to compare SHRSP to the stroke-resistant SHR strain to identify genes and protein pathways whose expression and/or function was significantly altered between the strains prior to the onset of stroke. Cerebral cortex gene expression profiles from male SHRSPs and matched SHRs were examined by Affymetrix microarray analysis. mRNAs encoding the brain-derived neurotrophic factor receptor (TrkB) and multiple kinases of the MAPK/AKT signaling pathways, including JNK2, AKT2, and PI3K, were differentially expressed between SHRSP and SHR. Because these data suggest altered function in pathways involving MAP and AKT kinase activity, we performed Western blot using phosphorylation state-specific antibodies to characterize activity of MAP kinase and PI3K/AKT pathways. Changes in the levels of the phosphorylated forms of these kinases paralleled the changes in transcript levels observed between the strains. Two-dimensional gel electrophoresis and peptide fragment mass fingerprinting were used to identify altered protein substrates of these kinases. Protein profiling of kinase substrates further supported the notion of perturbed kinase-mediated signaling in SHRSP and identified adenylyl cyclase associated protein 2, TOAD-64, propionyl CoA carboxylase, APG-1, and valosin-containing protein as kinase targets whose phosphorylation state is altered between these strains. Altered gene and protein expression patterns in SHRSP are consistent with increased vulnerability of this strain to cerebrovascular injury. PMID:12902546

  6. Deletion of the phosphoinositide 3-Kinase p110(gamma) gene attenuates murine atherosclerosis

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Inflammatory cell activation by chemokines requires intracellular signaling through phosphoinositide 3-kinase (PI3-kinase) and the PI3-kinase-dependent protein serine/threonine kinase Akt. Atherosclerosis is a chronic inflammatory process driven by oxidatively modified (atherogenic) lipoproteins, ch...

  7. 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.

  8. 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

  9. 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

  10. 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.

  11. 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

  12. Generation of systemin signaling in tobacco by transformation with the tomato systemin receptor kinase gene

    PubMed Central

    Scheer, Justin M.; Pearce, Gregory; Ryan, Clarence A.

    2003-01-01

    The tomato systemin receptor, SR160, a plasma membrane-bound, leucine-rich repeat receptor kinase that signals systemic plant defense, and the brassinolide (BL) receptor, BRI1, that regulates developmental processes, have been shown recently to have identical amino acid sequences. We report herein that tobacco, a solanaceous species that does not express a systemin precursor gene nor responds to systemin, when transformed with the SR160 receptor gene, expresses the gene in suspension-cultured cells, evidenced by mRNA and protein analyses and photoaffinity-labeling experiments. Additionally, systemin induced an alkalinization response in the transgenic tobacco cells similar to that found in tomato cells, but not in WT cells. The gain in function in tobacco cells indicates that early steps of the systemin signaling pathway found in tomato are present in tobacco cells. A tomato line, cu-3, in which a mutation in the BRI1 gene has rendered the plant nonfunctional in BL signaling, exhibits a severely reduced response to systemin. In leaves of WT tomato plants, BL strongly and reversibly antagonized systemic signaling by systemin. The results suggest that the systemin-mediated systemic defense response may have evolved in some solanaceous species by co-opting the BRI1 receptor and associated components for defense signaling. PMID:12900501

  13. 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

  14. 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

  15. 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

  16. Molecular Effects of the CTG Repeats in Mutant Dystrophia Myotonica Protein Kinase Gene

    PubMed Central

    Llamusí, Beatriz; Artero, Ruben

    2008-01-01

    Myotonic Dystrophy type 1 (DM1) is a multi-system disorder characterized by muscle wasting, myotonia, cardiac conduction defects, cataracts, and neuropsychological dysfunction. DM1 is caused by expansion of a CTG repeat in the 3´untranslated region (UTR) of the Dystrophia Myotonica Protein Kinase (DMPK) gene. A body of work demonstrates that DMPK mRNAs containing abnormally expanded CUG repeats are toxic to several cell types. A core mechanism underlying symptoms of DM1 is that mutant DMPK RNA interferes with the developmentally regulated alternative splicing of defined pre-mRNAs. Expanded CUG repeats fold into ds(CUG) hairpins that sequester nuclear proteins including human Muscleblind-like (MBNL) and hnRNP H alternative splicing factors. DM1 cells activate CELF family member CUG-BP1 protein through hyperphosphorylation and stabilization in the cell nucleus. CUG-BP1 and MBNL1 proteins act antagonistically in exon selection in several pre-mRNA transcripts, thus MBNL1 sequestration and increase in nuclear activity of CUG-BP1 both act synergistically to missplice defined transcripts. Mutant DMPK-mediated effect on subcellular localization, and defective phosphorylation of cytoplasmic CUG-BP1, have additionally been linked to defective translation of p21 and MEF2A in DM1, possibly explaining delayed differentiation of DM1 muscle cells. Mutant DMPK transcripts bind and sequester transcription factors such as Specificity protein 1 leading to reduced transcription of selected genes. Recently, transcripts containing long hairpin structures of CUG repeats have been shown to be a Dicer ribonuclease target and Dicer-induced downregulation of the mutant DMPK transcripts triggers silencing effects on RNAs containing long complementary repeats. In summary, mutant DMPK transcripts alter gene transcription, alternative splicing, and translation of specific gene transcripts, and have the ability to trigger gene-specific silencing effects in DM1 cells. Therapies aimed at reversing

  17. The effects of knockdown of rho-associated kinase 1 and zipper-interacting protein kinase on gene expression and function in cultured human arterial smooth muscle cells.

    PubMed

    Deng, Jing-Ti; Wang, Xiu-Ling; Chen, Yong-Xiang; O'Brien, Edward R; Gui, Yu; Walsh, Michael P

    2015-01-01

    Rho-associated kinase (ROCK) and zipper-interacting protein kinase (ZIPK) have been implicated in diverse physiological functions. ROCK1 phosphorylates and activates ZIPK suggesting that at least some of these physiological functions may require both enzymes. To test the hypothesis that sequential activation of ROCK1 and ZIPK is commonly involved in regulatory pathways, we utilized siRNA to knock down ROCK1 and ZIPK in cultured human arterial smooth muscle cells (SMC). Microarray analysis using a whole-transcript expression chip identified changes in gene expression induced by ROCK1 and ZIPK knockdown. ROCK1 knockdown affected the expression of 553 genes, while ZIPK knockdown affected the expression of 390 genes. A high incidence of regulation of transcription regulator genes was observed in both knockdowns. Other affected groups included transporters, kinases, peptidases, transmembrane and G protein-coupled receptors, growth factors, phosphatases and ion channels. Only 76 differentially expressed genes were common to ROCK1 and ZIPK knockdown. Ingenuity Pathway Analysis identified five pathways shared between the two knockdowns. We focused on cytokine signaling pathways since ROCK1 knockdown up-regulated 5 and down-regulated 4 cytokine genes, in contrast to ZIPK knockdown, which affected the expression of only two cytokine genes (both down-regulated). IL-6 gene expression and secretion of IL-6 protein were up-regulated by ROCK1 knockdown, whereas ZIPK knockdown reduced IL-6 mRNA expression and IL-6 protein secretion and increased ROCK1 protein expression, suggesting that ROCK1 may inhibit IL-6 secretion. IL-1β mRNA and protein levels were increased in response to ROCK1 knockdown. Differences in the effects of ROCK1 and ZIPK knockdown on cell cycle regulatory genes suggested that ROCK1 and ZIPK regulate the cell cycle by different mechanisms. ROCK1, but not ZIPK knockdown reduced the viability and inhibited proliferation of vascular SMC. We conclude that ROCK1 and

  18. The Effects of Knockdown of Rho-Associated Kinase 1 and Zipper-Interacting Protein Kinase on Gene Expression and Function in Cultured Human Arterial Smooth Muscle Cells

    PubMed Central

    Deng, Jing-Ti; Wang, Xiu-Ling; Chen, Yong-Xiang; O’Brien, Edward R.; Gui, Yu; Walsh, Michael P.

    2015-01-01

    Rho-associated kinase (ROCK) and zipper-interacting protein kinase (ZIPK) have been implicated in diverse physiological functions. ROCK1 phosphorylates and activates ZIPK suggesting that at least some of these physiological functions may require both enzymes. To test the hypothesis that sequential activation of ROCK1 and ZIPK is commonly involved in regulatory pathways, we utilized siRNA to knock down ROCK1 and ZIPK in cultured human arterial smooth muscle cells (SMC). Microarray analysis using a whole-transcript expression chip identified changes in gene expression induced by ROCK1 and ZIPK knockdown. ROCK1 knockdown affected the expression of 553 genes, while ZIPK knockdown affected the expression of 390 genes. A high incidence of regulation of transcription regulator genes was observed in both knockdowns. Other affected groups included transporters, kinases, peptidases, transmembrane and G protein-coupled receptors, growth factors, phosphatases and ion channels. Only 76 differentially expressed genes were common to ROCK1 and ZIPK knockdown. Ingenuity Pathway Analysis identified five pathways shared between the two knockdowns. We focused on cytokine signaling pathways since ROCK1 knockdown up-regulated 5 and down-regulated 4 cytokine genes, in contrast to ZIPK knockdown, which affected the expression of only two cytokine genes (both down-regulated). IL-6 gene expression and secretion of IL-6 protein were up-regulated by ROCK1 knockdown, whereas ZIPK knockdown reduced IL-6 mRNA expression and IL-6 protein secretion and increased ROCK1 protein expression, suggesting that ROCK1 may inhibit IL-6 secretion. IL-1β mRNA and protein levels were increased in response to ROCK1 knockdown. Differences in the effects of ROCK1 and ZIPK knockdown on cell cycle regulatory genes suggested that ROCK1 and ZIPK regulate the cell cycle by different mechanisms. ROCK1, but not ZIPK knockdown reduced the viability and inhibited proliferation of vascular SMC. We conclude that ROCK1 and

  19. Calspermin gene transcription is regulated by two cyclic AMP response elements contained in an alternative promoter in the calmodulin kinase IV gene.

    PubMed Central

    Sun, Z; Sassone-Corsi, P; Means, A R

    1995-01-01

    The transcript for the high-affinity Ca2+/calmodulin-binding protein calspermin is generated from the gene encoding Ca2+/calmodulin-dependent protein kinase IV only in postmeiotic germ cells during spermatogenesis. We demonstrate that this testis-specific calspermin transcript can be produced in heterologous cells by utilization of a promoter located in an intron of the calmodulin (CaM) kinase IV gene. Critical motifs within this promoter are two cyclic AMP response element (CRE)-like sequences located about -70 and -50 bp upstream of the transcriptional initiation site. Both CRE motifs are footprinted by the authentic testis-specific transcriptional activator CREM tau or by CREM tau present in adult testis nuclear extract. Whereas a 2.1-kb DNA fragment containing the calspermin promoter is inactive when transfected into NIH 3T3 cells, activity can be restored by cotransfection of CREM tau and protein kinase A or CaM kinase IV but not CaM kinase II alpha. Restoration of activity is greatly reduced by mutation of the two CRE motifs. Since CRE-like motifs have been identified in many genes uniquely expressed in postmeiotic germ cells, which contain abundant CREM tau protein, we suggest that CREM tau may function as one transcription factor responsible for the expression of postmeiotic germ cell-specific genes. PMID:7799965

  20. Molecular cloning and chromosomal mapping of the mouse cyclin-dependent kinase 5 gene

    SciTech Connect

    Ohshima, Toshio; Nagle, J.W.; Brady, R.O.; Kozak, C.A.

    1995-08-10

    Cyclin-dependent kinase 5 (Cdk5) is predominantly expressed in neurons. In vitro, Cdk5 purified from the nervous tissue phosphorylates both high-molecular-weight neurofilament and microtubule-associated tau. The mouse gene encoding Cdk5 (Cdk5) was found to be 5 kb in length and divided into 12 exons. All of the exon-intron junctions matched the expected consensus sequence with the exception of the splice junction for intron 9, which has AT and AC dinucleotides instead of the usual GT and AG bordering sequence. In the 5{prime}-flanking region of mouse Cdk5, several putative promoter elements were present, including AP1, Sp1, PuF, and TATA motifs. A metal regulatory element was also identified at position -207 to -201. Nucleotide sequence analysis of mouse Cdk5 showed high identity to the homologues of other vertebrate species, indicating that this kinase is highly conserved during evolution. Mouse Cdk5 was mapped to the centromeric region of mouse chromosome 5. 20 refs., 2 figs., 1 tab.

  1. Two antagonistic clock-regulated histidine kinases time the activation of circadian gene expression

    PubMed Central

    Gutu, Andrian; O’Shea, Erin K.

    2013-01-01

    Summary The cyanobacterial circadian pacemaker consists of a three-protein clock – KaiA, KaiB and KaiC – that generates oscillations in the phosphorylation state of KaiC. Here we investigate how temporal information encoded in KaiC phosphorylation is transduced to RpaA, a transcription factor required for circadian gene expression. We show that phosphorylation of RpaA is regulated by two antagonistic histidine kinases, SasA and CikA, which are sequentially activated at distinct times by the Kai clock complex. SasA acts as a kinase toward RpaA, whereas CikA, previously implicated in clock input, acts as a phosphatase that dephosphorylates RpaA. CikA and SasA cooperate to generate an oscillation of RpaA activity that is distinct from that generated by either enzyme alone and offset from the rhythm of KaiC phosphorylation. Our observations reveal how circadian clocks can precisely control the timing of output pathways via the concerted action of two oppositely acting enzymes. PMID:23541768

  2. 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

  3. The herpes simplex virus 1 protein kinase encoded by the US3 gene mediates posttranslational modification of the phosphoprotein encoded by the UL34 gene.

    PubMed Central

    Purves, F C; Spector, D; Roizman, B

    1991-01-01

    Earlier studies have shown that a herpes simplex virus 1 (HSV-1) open reading frame, US3, encodes a novel protein kinase and have characterized the cognate amino acid sequence which is phosphorylated by this enzyme. This report identifies an apparently essential viral phosphoprotein whose posttranslational processing involves the viral protein kinase. Analyses of viral proteins phosphorylated in the course of productive infection revealed a phosphoprotein whose mobility was viral protein kinase and serotype dependent. Thus, the corresponding HSV-1 and HSV-2 phosphoproteins differ in their electrophoretic mobilities, and the phosphoprotein specified by the HSV-1 mutant deleted in US3 (R7041) differs from that of the corresponding HSV-1 and HSV-2 proteins. Analyses of HSV-1 x HSV-2 recombinants mapped the phosphoprotein between 0.42 and 0.47 map units on the prototype HSV-1 DNA map. Within this region, the UL34 open reading frame was predicted to encode a protein of appropriate molecular weight which would also contain the consensus target site for phosphorylation by the viral protein kinase as previously defined with synthetic peptides. Replacement of the native UL34 gene with a UL34 gene tagged with a 17-amino-acid epitope from the alpha 4 protein identified this gene as encoding the phosphoprotein. Finally, mutagenesis of the predicted phosphorylation site on UL34 in the viral genome, and specifically the substitution of threonine or serine with alanine in the product of the UL34 gene, yielded phosphoproteins whose electrophoretic mobilities could not be differentiated from that of the US3- mutant. We conclude that the posttranslational processing of the UL34 gene product to its wild-type phenotype requires the participation of the viral protein kinase. While the viral protein kinase is not essential for viral replication in cells in culture, the UL34 gene product itself may not be dispensable. Images PMID:1656069

  4. Constitutive modulation of Raf-1 protein kinase is associated with differential gene expression of several known and unknown genes.

    PubMed Central

    Patel, S.; Wang, F. H.; Whiteside, T. L.; Kasid, U.

    1997-01-01

    BACKGROUND: Raf-1, a cytoplasmic serine/threonine protein kinase, plays an important role in mitogen- and damage-responsive cellular signal transduction pathways. Consistent with this notion is the fact that constitutive modulation of expression and/or activity of Raf-1 protein kinase modifies cell growth, proliferation, and cell survival. Although these effects are controlled at least in part by transcriptional mechanisms, the role of Raf-1 in the regulation of specific gene expression is unclear. MATERIALS AND METHODS: Differential display of mRNA was used to identify the genes differentially expressed in human head and neck squamous carcinoma cells (PCI-06A) transfected with either the antisense c-raf-1 cDNA (PCI-06A-Raf(AS)), or a portion of cDNA coding for the kinase domain of Raf-1 (PCI-06A-Raf(K)). The differentially expressed fragments were cloned and sequenced, and they were used as probes to compare the expression patterns in parent transfectants by Northern blot analysis. In addition, expression patterns of the novel genes were examined in normal tissues and cancer cell lines. RESULTS: Six differentially expressed cDNA fragments were identified and sequenced. Northern blot analysis revealed that four of these fragments representing human alpha 1-antichymotrypsin (alpha 1-ACT), mitochondrial cytochrome c oxidase subunit II (COX-II), and two as-yet unidentified cDNAs (KAS-110 and KAS-111) were relatively overexpressed in PCI-06A-Raf(AS) transfectants compared with PCI-06A-Raf(K) transfectants. The other two cDNA fragments representing human elongation factor-1 alpha (HEF-1 alpha) and ornithine decarboxylase antizyme (OAz) were overexpressed in PCI-06A-Raf(K) transfectants compared with PCI-06A-Raf(AS) transfectants. The KAS-110 (114 bp) and KAS-111 (202 bp) cDNAs did not show significant matches with sequences in the GenEMBL, TIGR, and HGS DNA databases, and these may represent novel genes. The KAS-110 and KAS-111 transcripts, approximately 0.9 kb and

  5. 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.

  6. Myelodysplastic syndrome without ring sideroblasts and with Janus kinase 2 gene mutation: An unusual case report

    PubMed Central

    Ornellas, Maria Helena; De França Silva, Monique; Solza, Cristiana; De Lucena Gonçalves, Stella Beatriz Sampaio; Silva De Almeida, Liliane; De Paula Ayres-Silva, Jackline; Seixas, Taís Leite; Bastos, Elenice Ferreira; Liehr, Thomas; Alves, Gilda

    2016-01-01

    Myelodysplastic syndrome (MDS) cases comprise a heterogeneous group of hematological disorders that are characterized by impaired hematopoiesis, with cytopenias of different grades and risk of developing acute myeloid leukemia. MDS may rarely be associated with thrombocytosis. In such cases, myelodysplasia and myeloproliferative disorders may overlap, making correct diagnosis difficult. We herein describe a case of MDS with thrombocytosis, Janus kinase 2 gene mutation-positive and Perls' staining-negative, which was initially classified as essential thrombocythemia (ET). This case highlights that MDS may be misdiagnosed as ET and inappropriate treatment may be initiated. Therefore, it is crucial to carefully combine all available data on morphology and immunophenotyping, and to perform the necessary molecular, cytogenetic and molecular cytogenetic analyses, in order to correctly diagnose this disease.

  7. 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.

  8. 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

  9. Treatment of experimental human mesothelioma using adenovirus transfer of the herpes simplex thymidine kinase gene.

    PubMed Central

    Smythe, W R; Hwang, H C; Elshami, A A; Amin, K M; Eck, S L; Davidson, B L; Wilson, J M; Kaiser, L R; Albelda, S M

    1995-01-01

    OBJECTIVE: The authors demonstrate the ability of an adenovirus vector expressing the herpes simplex thymidine kinase (HSVtk) gene to treat human malignant mesothelioma growing within the peritoneal cavity of severe combined immunodeficient (SCID) mice. BACKGROUND DATA: Introduction of the HSVtk gene into tumor cells renders them sensitive to the antiviral drug ganciclovir (GCV). This approach has been used previously to treat experimental brain tumors. Although malignant mesothelioma is refractory to current therapies, its localized nature and the accessibility of the pleural space make it a potential target for a similar type of in vivo gene therapy using adenovirus. METHODS: An adenovirus containing the HSVtk gene (Ad.RSVtk) was used to transduce mesothelioma cells in vitro. These cells were then injected into the flanks of SCID mice. Ad.RSVtk was also injected directly into the peritoneal cavity of SCID mice with established human mesothelioma tumors. Mice were subsequently treated for 7 days with GCV at a dose of 5 mg/kg. RESULTS: Mesothelioma cells transduced in vitro with Ad.RSVtk formed nodules when injected in the subcutaneous tissue. These tumors could be eliminated by the administration of GCV, even when as few as 10% of cells were transduced to express HSVtk (bystander effect). Administration of Ad.RSVtk into the peritoneal space of animals with established multifocal human mesothelioma followed by GCV therapy resulted in the eradication of macroscopic tumor in 90% of animals and microscopic tumor in 80% of animals when evaluated after 30 days. The median survival of animals treated with Ad.RSVtk/GCV was significantly longer than that of control animals treated with similar protocols. CONCLUSION: These results indicate that an adenoviral vector containing the HSVtk gene is effective in treating established malignant mesothelioma in an in vivo setting and raise the possibility of using adenovirus transfer of HSVtk for clinical trials in mesothelioma and

  10. Plant thymidine kinase 1: a novel efficient suicide gene for malignant glioma therapy.

    PubMed

    Khan, Zahidul; Knecht, Wolfgang; Willer, Mette; Rozpedowska, Elzbieta; Kristoffersen, Peter; Clausen, Anders Ranegaard; Munch-Petersen, Birgitte; Almqvist, Per M; Gojkovic, Zoran; Piskur, Jure; Ekström, Tomas J

    2010-06-01

    The prognosis for malignant gliomas remains poor, and new treatments are urgently needed. Targeted suicide gene therapy exploits the enzymatic conversion of a prodrug, such as a nucleoside analog, into a cytotoxic compound. Although this therapeutic strategy has been considered a promising regimen for central nervous system (CNS) tumors, several obstacles have been encountered such as inefficient gene transfer to the tumor cells, limited prodrug penetration into the CNS, and inefficient enzymatic activity of the suicide gene. We report here the cloning and successful application of a novel thymidine kinase 1 (TK1) from the tomato plant, with favorable characteristics in vitro and in vivo. This enzyme (toTK1) is highly specific for the nucleoside analog prodrug zidovudine (azidothymidine, AZT), which is known to penetrate the blood-brain barrier. An important feature of toTK1 is that it efficiently phosphorylates its substrate AZT not only to AZT monophosphate, but also to AZT diphosphate, with excellent kinetics. The efficiency of the toTK1/AZT system was confirmed when toTK1-transduced human glioblastoma (GBM) cells displayed a 500-fold increased sensitivity to AZT compared with wild-type cells. In addition, when neural progenitor cells were used as delivery vectors for toTK1 in intracranial GBM xenografts in nude rats, substantial attenuation of tumor growth was achieved in animals exposed to AZT, and survival of the animals was significantly improved compared with controls. The novel toTK1/AZT suicide gene therapy system in combination with stem cell-mediated gene delivery promises new treatment of malignant gliomas. PMID:20154339

  11. Activation of proglucagon gene transcription by protein kinase-A in a novel mouse enteroendocrine cell line.

    PubMed

    Drucker, D J; Jin, T; Asa, S L; Young, T A; Brubaker, P L

    1994-12-01

    The gene encoding proglucagon is expressed predominantly in the pancreas and intestine. The physiological importance of glucagon secreted from the islets of Langerhans has engendered considerable interest in the molecular control of proglucagon gene transcription in the endocrine pancreas. In contrast, little is known about the molecular control of proglucagon gene expression in the intestine. The recent demonstration that glucagon-like peptide-1 (GLP-1) secreted from the intestine is a potent regulator of insulin secretion and glucose homeostasis has stimulated renewed interest in the factors that control GLP-1 synthesis in the intestinal L-cell. To develop a model for the analysis of intestinal proglucagon gene expression, we have targeted expression of a proglucagon gene-simian virus-40 large T-antigen fusion gene to enteroendocrine cells in transgenic mice. These mice develop intestinal tumors that were used to derive a novel cell line, designated GLUTag, that expresses the proglucagon gene and secretes immunoreactive GLP-1 in vitro. GLUTag cells demonstrate morphological characteristics of enteroendocrine cells by electron microscopy and are plurihormonal, as shown by immunocytochemistry and RNA analyses. GLUTag cells express the proglucagon and cholecystokinin genes, consistent with the pattern of lineage-specific enteroendocrine differentiation described for mouse intestine. Proglucagon gene expression was induced by activators of the protein kinase-A pathway, and a combination of messenger RNA half-life and nuclear run-on experiments demonstrated that the protein kinase-A-induction is mediated by an increase in proglucagon gene transcription. In contrast, activators of protein kinase-C stimulated secretion, but not biosynthesis of the PGDPs in GLUTag cell cultures. Analysis of proglucagon processing in GLUTag cells demonstrated the liberation of glucagon, oxyntomodulin, glicentin, and multiple forms of GLP-1. These observations provide evidence for the

  12. 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

  13. 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...

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

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

  15. Organization and post-transcriptional processing of focal adhesion kinase gene

    PubMed Central

    Corsi, Jean-Marc; Rouer, Evelyne; Girault, Jean-Antoine; Enslen, Hervé

    2006-01-01

    Background Focal adhesion kinase (FAK) is a non-receptor tyrosine kinase critical for processes ranging from embryo development to cancer progression. Although isoforms with specific molecular and functional properties have been characterized in rodents and chicken, the organization of FAK gene throughout phylogeny and its potential to generate multiple isoforms are not well understood. Here, we study the phylogeny of FAK, the organization of its gene, and its post-transcriptional processing in rodents and human. Results A single orthologue of FAK and the related PYK2 was found in non-vertebrate species. Gene duplication probably occurred in deuterostomes after the echinoderma embranchment, leading to the evolution of PYK2 with distinct properties. The amino acid sequence of FAK and PYK2 is conserved in their functional domains but not in their linker regions, with the absence of autophosphorylation site in C. elegans. Comparison of mouse and human FAK genes revealed the existence of multiple combinations of conserved and non-conserved 5'-untranslated exons in FAK transcripts suggesting a complex regulation of their expression. Four alternatively spliced coding exons (13, 14, 16, and 31), previously described in rodents, are highly conserved in vertebrates. Cis-regulatory elements known to regulate alternative splicing were found in conserved alternative exons of FAK or in the flanking introns. In contrast, other reported human variant exons were restricted to Homo sapiens, and, in some cases, other primates. Several of these non-conserved exons may correspond to transposable elements. The inclusion of conserved alternative exons was examined by RT-PCR in mouse and human brain during development. Inclusion of exons 14 and 16 peaked at the end of embryonic life, whereas inclusion of exon 13 increased steadily until adulthood. Study of various tissues showed that inclusion of these exons also occurred, independently from each other, in a tissue-specific fashion

  16. Functional reconstitution of receptors for bradykinin and des argZ-bradykinin from pulmonary artery membranes

    SciTech Connect

    Cahill, M.C.; Polgar, P.; Dickey, B.F.; Fishman, J.B.

    1987-05-01

    Bradykinin (BK) is a vasoactive peptide which mediates a number of vascular functions, including activation of prostaglandin biosynthesis and modulation of vasomotor tone. BK and its kinase II metabolite, des argZ-BK, have been reported to activate the B2 and B1 receptors, respectively. The authors prepared membranes from the bovine pulmonary artery and solubilized membrane proteins using the zwitterionic detergent CHAPS (3-((3-cholamidopropyl) dimethylammonio)-1-propanesulfonate). The solubilized proteins were reconstituted into liposomes via a gel filtration method. The vesicles specifically bound both TH-BK and TH-des argZ-BK, although the latter bound with significantly lower affinity. The binding of TH-BK was inhibited 65% by guanosine 5'-0-thiotriphosphate S while the binding of TH-des argZ-BK was unaffected. This suggests that the receptor for BK was associated with a guanine-nucleotide binding protein whereas the receptor for des argZ-BK was not. Since des argZ-BK has recently been reported to be considerably less potent than BK at activating the turnover of phosphatidylinositol, the authors data suggest that this is due to the des argZ-BK receptor not being coupled to a G-protein. Further work towards characterizing these receptors is now underway.

  17. 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.

  18. 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

  19. IL-4 regulates specific Arg-1(+) macrophage sFlt-1-mediated inhibition of angiogenesis.

    PubMed

    Wu, Wei-Kang; Georgiadis, Anastasios; Copland, David A; Liyanage, Sidath; Luhmann, Ulrich F O; Robbie, Scott J; Liu, Jian; Wu, Jiahui; Bainbridge, James W; Bates, David O; Ali, Robin R; Nicholson, Lindsay B; Dick, Andrew D

    2015-08-01

    One of the main drivers for neovascularization in age-related macular degeneration is activation of innate immunity in the presence of macrophages. Here, we demonstrate that T helper cell type 2 cytokines and, in particular, IL-4 condition human and murine monocyte phenotype toward Arg-1(+), and their subsequent behavior limits angiogenesis by increasing soluble fms-like tyrosine kinase 1 (sFlt-1) gene expression. We document that T helper cell type 2 cytokine-conditioned murine macrophages neutralize vascular endothelial growth factor-mediated endothelial cell proliferation (human umbilical vein endothelial cell and choroidal vasculature) in a sFlt-1-dependent manner. We demonstrate that in vivo intravitreal administration of IL-4 attenuates laser-induced choroidal neovascularization (L-CNV) due to specific IL-4 conditioning of macrophages. IL-4 induces the expression of sFlt-1 by resident CD11b(+) retinal microglia and infiltrating myeloid cells but not from retinal pigment epithelium. IL-4-induced suppression of L-CNV is not prevented when sFlt-1 expression is attenuated in retinal pigment epithelium. IL-4-mediated suppression of L-CNV was abrogated in IL-4R-deficient mice and in bone marrow chimeras reconstituted with myeloid cells that had undergone lentiviral-mediated shRNA silencing of sFlt-1, demonstrating the critical role of this cell population. Together, these data establish how lL-4 directly drives macrophage sFlt-1 production expressing an Arg-1(+) phenotype and support the therapeutic potential of targeted IL-4 conditioning within the tissue to regulate disease conditions such as neovascular age-related macular degeneration. PMID:26079814

  20. Identification and Characterization of the Cyclin-Dependent Kinases Gene Family in Silkworm, Bombyx mori.

    PubMed

    Li, Yinü; Jiang, Feng; Shi, Xiaofeng; Liu, Xingjian; Yang, Huipeng; Zhang, Zhifang

    2016-01-01

    Cyclin-dependent protein kinases (CDKs) play key roles at different checkpoint regulations of the eukaryotic cell cycle. However, only few studies of lepidoptera CDK family proteins have been reported so far. In this study, we performed the cDNA sequencing of 10 members of the CDK family in Bombyx mori. Gene structure analysis suggested that CDK12 and CDC2L1 owned two and three isoforms, respectively. Phylogenetic analysis showed that CDK genes in different species were highly conserved, implying that they evolved independently even before the split between vertebrates and invertebrates. We found that the expression levels of BmCDKs in 13 tissues of fifth-instar day 3 larvae were different: CDK1, CDK7, and CDK9 had a high level of expression, whereas CDK4 was low-level expressed and was detected only in the testes and fat body cells. Similar expression profiles of BmCDKs during embryo development were obtained. Among the variants of CDK12, CDK12 transcript variant A had the highest expression, and the expression of CDC2L1 transcript variant A was the highest among the variants of CDC2L1. It was shown from the RNAi experiments that the silencing of CDK1, CDK10, CDK12, and CDC2L1 could influence the cells from G0/G1 to S phase transition. PMID:26544066

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

    SciTech Connect

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

    1982-04-01

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

  2. A phase I clinical trial of thymidine kinase-based gene therapy in advanced hepatocellular carcinoma.

    PubMed

    Sangro, B; Mazzolini, G; Ruiz, M; Ruiz, J; Quiroga, J; Herrero, I; Qian, C; Benito, A; Larrache, J; Olagüe, C; Boan, J; Peñuelas, I; Sádaba, B; Prieto, J

    2010-12-01

    The aim of this phase I clinical trial was to assess the feasibility and safety of intratumoral administration of a first-generation adenoviral vector encoding herpes simplex virus thymidine kinase (HSV-TK) gene (Ad.TK) followed by systemic ganciclovir to patients with advanced hepatocellular carcinoma (HCC). Secondarily, we have analyzed its antitumor effect. Ten patients were enrolled in five dose-level cohorts that received from 10¹⁰ to 2 × 10¹² viral particles (vp). Ad.TK was injected intratumorally and patients received up to three doses at 30-day intervals. Positron emission tomography was used to monitor TK gene expression. Ad.TK injection was feasible in 100% of cases. Treatment was well tolerated and dose-limiting toxicity was not achieved. Cumulative toxicity was not observed. Hepatic toxicity was absent even in cirrhotic patients. Fever, flu-like syndrome, pain at the injection site and pancytopenia were the most common side effects. No partial responses were observed and 60% of patients showed tumor stabilization of the injected lesion. Importantly, two patients who received the highest dose showed signs of intratumoral necrosis by imaging procedures. One of them achieved a sustained stabilization and survived for 26 months. In conclusion, Ad.TK can be safely administered by intratumoral injection to patients with HCC up to 2 × 10¹² vp per patient. PMID:20689572

  3. 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.

  4. Cloning and characterization of the gene encoding Halobacterium halobium adenylate kinase.

    PubMed

    Song, S; Inouye, S; Kawai, M; Fukami-Kobayashi, K; Gõ, M; Nakazawa, A

    1996-10-10

    The gene (AK) encoding adenylate kinase (AK) of Halobacterium halobium was cloned. AK consisted of 648 bp and coded for 216 amino acids (aa). S1 mapping and primer extension experiments indicated that the transcription start point (tsp) was located immediately upstream from the start codon. The TAT-like promoter sequence was found at a position 20-24 bp upstream from tsp. The most striking property of the enzyme was a putative Zn finger-like structure with four cysteines. It might contribute to the structural stability of the molecule in high-salt conditions. Phylogenetic analysis indicated two lineages of the AK family, the short and long types which diverged a long time ago, possibly before the separation of prokaryotes and eukaryotes. Although the H. halobium AK belongs to the long-type AK lineage, it is located in an intermediary position between the two lineages of the phylogenetic tree, indicating early divergence of the gene along the long-type lineage. PMID:8917077

  5. 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

  6. 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

  7. Identification of a Bohle iridovirus thymidine kinase gene and demonstration of activity using vaccinia virus.

    PubMed

    Coupar, B E H; Goldie, S G; Hyatt, A D; Pallister, J A

    2005-09-01

    In recent years interest in the family Iridoviridae has been renewed by the identification of a number of viruses, particularly from the genus Ranavirus, associated with disease in a range of poikilotherms. Ranaviruses have been isolated from amphibian, piscine and reptilian species. Here we describe an open reading frame (ORF) identified in the genome of Bohle iridovirus (BIV) which contains a nucleotide binding motif conserved within the thymidine kinase (TK) genes of iridoviruses from other genera (lymphocystis disease virus, LCDV, type species of the genus Lymphocystivirus; Chilo iridescent virus, CIV, type species of the genus Iridovirus). The ability of this putative gene to express a functional TK was confirmed by rescue of a TK negative mutant vaccinia virus in the presence of selective media, when expression was controlled by a vaccinia virus promoter. The sequence of the BIV TK was compared with the homologous sequences from epizootic haematopoietic necrosis virus (EHNV), a virus associated with disease in fish, from Wamena iridovirus (WIV) associated with systemic disease in green pythons, and from frog virus 3 (FV3) the ranavirus type species. Comparisons between these sequences and those available from other ranaviruses, other iridoviruses, other DNA viruses and cellular TKs are presented. PMID:15883656

  8. The TMK1 gene from Arabidopsis codes for a protein with structural and biochemical characteristics of a receptor protein kinase.

    PubMed Central

    Chang, C; Schaller, G E; Patterson, S E; Kwok, S F; Meyerowitz, E M; Bleecker, A B

    1992-01-01

    Genomic and cDNA clones that code for a protein with structural and biochemical properties similar to the receptor protein kinases from animals were obtained from Arabidopsis. Structural features of the predicted polypeptide include an amino-terminal membrane targeting signal sequence, a region containing blocks of leucine-rich repeat elements, a single putative membrane spanning domain, and a characteristic serine/threonine-specific protein kinase domain. The gene coding for this receptor-like transmembrane kinase was designated TMK1. Portions of the TMK1 gene were expressed in Escherichia coli, and antibodies were raised against the recombinant polypeptides. These antibodies immunodecorated a 120-kD polypeptide present in crude extracts and membrane preparations. The immunodetectable band was present in extracts from leaf, stem, root, and floral tissues. The kinase domain of TMK1 was expressed as a fusion protein in E. coli, and the purified fusion protein was found capable of autophosphorylation on serine and threonine residues. The possible role of the TMK1 gene product in transmembrane signaling is discussed. PMID:1332795

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

    PubMed 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

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

    PubMed

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

    2016-01-01

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

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

    PubMed

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

    1999-08-01

    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. PMID:10436217

  12. 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.

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

    PubMed

    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

    2016-03-01

    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

  14. The yes-related cellular gene lyn encodes a possible tyrosine kinase similar to p56lck.

    PubMed Central

    Yamanashi, Y; Fukushige, S; Semba, K; Sukegawa, J; Miyajima, N; Matsubara, K; Yamamoto, T; Toyoshima, K

    1987-01-01

    With v-yes DNA as the probe, a human cDNA library made from placental RNA was screened under relaxed conditions, and DNA clones derived from a novel genetic locus, termed lyn, were obtained. Nucleotide sequencing revealed that lyn could encode a novel tyrosine kinase that was very similar to mouse T-lymphocyte-specific tyrosine kinase p56lck and the v-yes protein as well as to the gene products of v-fgr and v-src. Northern hybridization analysis revealed that a 3.2-kilobase lyn mRNA was expressed in a variety of tissues of the human fetus. The pattern of lyn mRNA expression was different from those of related genes, such as yes and syn. Hybridization analysis of DNA from sorted chromosomes showed that the lyn gene is located on human chromosome 8 q13-qter. Images PMID:3561390

  15. 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. PMID:26320869

  16. Epidermal Growth Factor Receptor Tyrosine Kinase Defines Critical Prognostic Genes of Stage I Lung Adenocarcinoma

    PubMed Central

    Nagasaki, Masao; Shimamura, Teppei; Imoto, Seiya; Saito, Ayumu; Ueno, Kazuko; Hatanaka, Yousuke; Yoshida, Ryo; Higuchi, Tomoyuki; Nomura, Masaharu; Beer, David G.; Yokota, Jun; Miyano, Satoru; Gotoh, Noriko

    2012-01-01

    Purpose To identify stage I lung adenocarcinoma patients with a poor prognosis who will benefit from adjuvant therapy. Patients and Methods Whole gene expression profiles were obtained at 19 time points over a 48-hour time course from human primary lung epithelial cells that were stimulated with epidermal growth factor (EGF) in the presence or absence of a clinically used EGF receptor tyrosine kinase (RTK)-specific inhibitor, gefitinib. The data were subjected to a mathematical simulation using the State Space Model (SSM). “Gefitinib-sensitive” genes, the expressional dynamics of which were altered by addition of gefitinib, were identified. A risk scoring model was constructed to classify high- or low-risk patients based on expression signatures of 139 gefitinib-sensitive genes in lung cancer using a training data set of 253 lung adenocarcinomas of North American cohort. The predictive ability of the risk scoring model was examined in independent cohorts of surgical specimens of lung cancer. Results The risk scoring model enabled the identification of high-risk stage IA and IB cases in another North American cohort for overall survival (OS) with a hazard ratio (HR) of 7.16 (P = 0.029) and 3.26 (P = 0.0072), respectively. It also enabled the identification of high-risk stage I cases without bronchioalveolar carcinoma (BAC) histology in a Japanese cohort for OS and recurrence-free survival (RFS) with HRs of 8.79 (P = 0.001) and 3.72 (P = 0.0049), respectively. Conclusion The set of 139 gefitinib-sensitive genes includes many genes known to be involved in biological aspects of cancer phenotypes, but not known to be involved in EGF signaling. The present result strongly re-emphasizes that EGF signaling status in cancer cells underlies an aggressive phenotype of cancer cells, which is useful for the selection of early-stage lung adenocarcinoma patients with a poor prognosis. Trial Registration The Gene Expression Omnibus (GEO) GSE31210 PMID:23028479

  17. Inhibition of human insulin gene transcription and MafA transcriptional activity by the dual leucine zipper kinase

    PubMed Central

    Stahnke, Marie-Jeannette; Dickel, Corinna; Schröder, Sabine; Kaiser, Diana; Blume, Roland; Stein, Roland; Pouponnot, Celio; Oetjen, Elke

    2016-01-01

    Insulin biosynthesis is an essential β-cell function and inappropriate insulin secretion and biosynthesis contribute to the pathogenesis of diabetes mellitus type 2. Previous studies showed that the dual leucine zipper kinase (DLK) induces β-cell apoptosis. Since β-cell dysfunction precedes β-cell loss, in the present study the effect of DLK on insulin gene transcription was investigated in the HIT-T15 β-cell line. Downregulation of endogenous DLK increased whereas overexpression of DLK decreased human insulin gene transcription. 5′- and 3′-deletion human insulin promoter analyses resulted in the identification of a DLK responsive element that mapped to the DNA binding-site for the β-cell specific transcription factor MafA. Overexpression of DLK wild-type but not its kinase-dead mutant inhibited MafA transcriptional activity conferred by its transactivation domain. Furthermore, in the non-β-cell line JEG DLK inhibited MafA overexpression-induced human insulin promoter activity. Overexpression of MafA and DLK or its kinase-dead mutant into JEG cells revealed that DLK but not its mutant reduced MafA protein content. Inhibition of the down-stream DLK kinase c-Jun N-terminal kinase (JNK) by SP600125 attenuated DLK-induced MafA loss. Furthermore, mutation of the serine 65 to alanine, shown to confer MafA protein stability, increased MafA-dependent insulin gene transcription and prevented DLK-induced MafA loss in JEG cells. These data suggest that DLK by activating JNK triggers the phosphorylation and degradation of MafA thereby attenuating insulin gene transcription. Given the importance of MafA for β-cell function, the inhibition of DLK might preserve β-cell function and ultimately retard the development of diabetes mellitus type 2. PMID:24726898

  18. Inhibition of human insulin gene transcription and MafA transcriptional activity by the dual leucine zipper kinase.

    PubMed

    Stahnke, Marie-Jeannette; Dickel, Corinna; Schröder, Sabine; Kaiser, Diana; Blume, Roland; Stein, Roland; Pouponnot, Celio; Oetjen, Elke

    2014-09-01

    Insulin biosynthesis is an essential β-cell function and inappropriate insulin secretion and biosynthesis contribute to the pathogenesis of diabetes mellitus type 2. Previous studies showed that the dual leucine zipper kinase (DLK) induces β-cell apoptosis. Since β-cell dysfunction precedes β-cell loss, in the present study the effect of DLK on insulin gene transcription was investigated in the HIT-T15 β-cell line. Downregulation of endogenous DLK increased whereas overexpression of DLK decreased human insulin gene transcription. 5'- and 3'-deletion human insulin promoter analyses resulted in the identification of a DLK responsive element that mapped to the DNA binding-site for the β-cell specific transcription factor MafA. Overexpression of DLK wild-type but not its kinase-dead mutant inhibited MafA transcriptional activity conferred by its transactivation domain. Furthermore, in the non-β-cell line JEG DLK inhibited MafA overexpression-induced human insulin promoter activity. Overexpression of MafA and DLK or its kinase-dead mutant into JEG cells revealed that DLK but not its mutant reduced MafA protein content. Inhibition of the down-stream DLK kinase c-Jun N-terminal kinase (JNK) by SP600125 attenuated DLK-induced MafA loss. Furthermore, mutation of the serine 65 to alanine, shown to confer MafA protein stability, increased MafA-dependent insulin gene transcription and prevented DLK-induced MafA loss in JEG cells. These data suggest that DLK by activating JNK triggers the phosphorylation and degradation of MafA thereby attenuating insulin gene transcription. Given the importance of MafA for β-cell function, the inhibition of DLK might preserve β-cell function and ultimately retard the development of diabetes mellitus type 2. PMID:24726898

  19. Nucleotide sequence of the phosphoglycerate kinase gene from the extreme thermophile Thermus thermophilus. Comparison of the deduced amino acid sequence with that of the mesophilic yeast phosphoglycerate kinase.

    PubMed Central

    Bowen, D; Littlechild, J A; Fothergill, J E; Watson, H C; Hall, L

    1988-01-01

    Using oligonucleotide probes derived from amino acid sequencing information, the structural gene for phosphoglycerate kinase from the extreme thermophile, Thermus thermophilus, was cloned in Escherichia coli and its complete nucleotide sequence determined. The gene consists of an open reading frame corresponding to a protein of 390 amino acid residues (calculated Mr 41,791) with an extreme bias for G or C (93.1%) in the codon third base position. Comparison of the deduced amino acid sequence with that of the corresponding mesophilic yeast enzyme indicated a number of significant differences. These are discussed in terms of the unusual codon bias and their possible role in enhanced protein thermal stability. Images Fig. 1. PMID:3052437

  20. 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

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

    PubMed

    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

  2. Induction by ionizing radiation of the gadd45 gene in cultured human cells: lack of mediation by protein kinase C.

    PubMed Central

    Papathanasiou, M A; Kerr, N C; Robbins, J H; McBride, O W; Alamo, I; Barrett, S F; Hickson, I D; Fornace, A J

    1991-01-01

    The effect of ionizing radiation on the expression of two DNA-damage-inducible genes, designated gadd45 and gadd153, was examined in cultured human cells. These genes have previously been shown to be strongly and coordinately induced by UV radiation and alkylating agents in human and hamster cells. We found that the gadd45 but not the gadd153 gene is strongly induced by X rays in human cells. The level of gadd45 mRNA increased rapidly after X rays at doses as low as 2 Gy. After 20 Gy of X rays, gadd45 induction, as measured by increased amounts of mRNA, was similar to that produced by the most effective dose of the alkylating agent methyl methanesulfonate. No induction was seen after treatment of either human or hamster cells with 12-O-tetradecanoylphorbol-13-acetate, a known activator of protein kinase C (PKC). Therefore, gadd45 represents the only known mammalian X-ray-responsive gene whose induction is not mediated by PKC. However, induction was blocked by the protein kinase inhibitor H7, indicating that induction is mediated by some other kinase(s). Sequence analysis of human and hamster cDNA clones demonstrated that this gene has been highly conserved and encodes a novel 165-amino-acid polypeptide which is 96% identical in the two species. This gene was localized to the short arm of human chromosome 1 between p12 and p34. When induction in lymphoblast lines from four normal individuals was compared with that in lines from four patients with ataxia telangiectasia, induction by X rays of gadd45 mRNA was less in the cell lines from this cancer-prone radiosensitive disorder. Our results provide evidence for the existence of an X-ray stress response in human cells which is independent of PKC and which is abnormal in taxia telangiectasia. Images PMID:1990262

  3. Structure-function relationship of arginyl-tRNA synthetase from Escherichia coli: isolation and characterization of the argS mutation MA5002.

    PubMed Central

    Eriani, G; Dirheimer, G; Gangloff, J

    1990-01-01

    The Escherichia coli K12 argS MA5002 mutant appears to have a functionally altered arginyl-tRNA synthetase (ArgRS). The gene coding for this enzyme was isolated from E. coli genomic DNA using the PCR procedure and inserted into a pUC18 multicopy vector. Sequencing revealed that it differs from the wildtype ArgRS structural gene only by one mutation: a replacement of a C by an A residue which results in substitution of an arginine by a serine at position 134, located two residues downstream from the HVGH consensus sequence. As compared to the genomic enzyme level, this recombinant vector, containing the mutated gene, produces in E. coli JM103, about 100 times as much modified ArgRS. This enzyme was obtained nearly pure after only two chromatographic steps; it exhibits a 4-6 times as low activity and a 5 times as high Km value for ATP as the wildtype enzyme in the aminoacylation and ATP-PPi reactions; Km values for arginine and tRNAArg remained unaltered. The position of this mutation and its effect on enzymatic properties suggest the implication of arginine 134 in ATP binding as well as in the activation catalytic process. Images PMID:2183195

  4. 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

  5. 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

  6. The human gene for neurotrophic tyrosine kinase receptor type 2 (NTRK2) is located on chromosome 9 but is not the familial dysautonomia gene

    SciTech Connect

    Slaugenhaupt, S.A. |; Liebert, C.B.; Lucente, D.E.

    1995-02-10

    The neurotrophic tyrosine kinase receptor type 2 (NTRK2) gene is a member of the trk family of tyrosine protein kinases, which encode receptors for the nerve growth factor-related proteins known as neurotrophins. The neurotrophins and their receptors have long been considered candidate genes for familial dysautonomia (FD), a hereditary sensory neuropathy resulting from the congenital loss of both sensory and autonomic neurons. The DYS gene has recently been mapped to human chromosome 9q31-q33, and therefore we set out to determine the chromosomal localization of the candidate gene NTRK2. A mouse trkB probe was hybridized to both somatic cell hybrids containing human chromosome 9 and a human chromosome 9 flow-sorted cosmid library. The human homologue of trkB, NTRK2, was assigned to chromosome 9. To localize the NTRK2 gene further, a dinucleotide repeat polymorphism was identified within a cosmid that contains NTRK2 exon sequences. This marker was genotyped in the CEPH reference pedigrees and places the NTRK2 gene near D9S1 on the proximal long arm of human chromosome 9. The NTRK2 gene is located approximately 22 cm proximal to DYS and shows several recombinants in disease families. Therefore, the NTRK2 gene can now be excluded as a candidate gene for familial dysautonomia. 18 refs., 1 fig.

  7. Elicitor- and A23187-induced expression of WCK-1, a gene encoding mitogen-activated protein kinase in wheat.

    PubMed

    Takezawa, D

    1999-08-01

    Wheat cultured cells were used to study the role of Ca2+ in regulating protein kinases during the induction of defense-related genes by fungal elicitor treatments. Manipulation of intracellular Ca2+ concentrations by treatment with calcium ionophore A23187 in the presence of high extracellular Ca2+ resulted in the induction of mRNA expression of WCK-1, a gene encoding mitogen-activated protein (MAP) kinase. The induction of WCK-1 mRNA by A23187 did not occur when extracellular Ca2+ was chelated by 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA). The WCK-1 mRNA was also induced by Typhula ishikariensis-derived elicitors, suggesting a possible involvement of WCK-1 in the plant defense response against pathogens. BAPTA and a calcium channel blocker, La3+, inhibited the elicitor-induced expression of the WCK-1 mRNA. A recombinant fusion protein of WCK-1 (GST-WCK-1) autophosphorylated at the Tyr residue and exhibited an autophosphorylation-dependent protein kinase activity towards myelin basic protein. Alteration of Tyr-196 in the conserved 'TEY' motif in GST-WCK-1 to Phe by site-directed mutagenesis abolished the autophosphorylation. The GST-WCK-1 protein was activated by elicitor-treated wheat cell extracts but not by the control extract. These results suggest that fungal elicitors activate WCK-1, a specific MAP kinase in wheat. Furthermore, the results suggest a possible involvement of Ca2+ in enhancing the MAP kinase signaling cascade in plants by controlling the levels of the MAP kinase transcripts. PMID:10527417

  8. Evidence for an arginine exporter encoded by yggA (argO) that is regulated by the LysR-type transcriptional regulator ArgP in Escherichia coli.

    PubMed

    Nandineni, Madhusudan R; Gowrishankar, J

    2004-06-01

    The anonymous open reading frame yggA of Escherichia coli was identified in this study as a gene that is under the transcriptional control of argP (previously called iciA), which encodes a LysR-type transcriptional regulator protein. Strains with null mutations in either yggA or argP were supersensitive to the arginine analog canavanine, and yggA-lac expression in vivo exhibited argP(+)-dependent induction by arginine. Lysine supplementation phenocopied the argP null mutation in that it virtually abolished yggA expression, even in the argP+ strain. The dipeptides arginylalanine and lysylalanine behaved much like arginine and lysine, respectively, to induce and to turn off yggA transcription. Dominant missense mutations in argP (argPd) that conferred canavanine resistance and rendered yggA-lac expression constitutive were obtained. The protein deduced to be encoded by yggA shares similarity with a basic amino acid exporter (LysE) of Corynebacterium glutamicum, and we obtained evidence for increased arginine efflux from E. coli strains with either the argPd mutation or multicopy yggA+. The null yggA mutation abolished the increased arginine efflux from the argPd strain. Our results suggest that yggA encodes an ArgP-regulated arginine exporter, and we have accordingly renamed it argO (for "arginine outward transport"). We propose that the physiological function of argO may be either to prevent the accumulation to toxic levels of canavanine (which is a plant-derived antimetabolite) or arginine or to maintain an appropriate balance between the intracellular lysine and arginine concentrations. PMID:15150242

  9. Transcription factor AtbZIP60 regulates expression of Ca2+ -dependent protein kinase genes in transgenic cells.

    PubMed

    Tang, Wei; Page, Michael

    2013-03-01

    The Arabidopsis thaliana bZIP60 (AtbZIP60) transcription factor regulates stress signaling. However, its molecular mechanism remains to be elucidated. In this investigation, cell suspension cultures of two different plant species rice (Oryza sativa L.) and white pine (Pinus strobes L.) were transformed using Agrobacterium tumefaciens strain LBA4404 harboring pBI-AtZIP60. Integration of the AtbZIP60 gene into the genome of rice and white pine has been confirmed by polymerase chain reaction (PCR), southern blotting, and northern blotting analyses. Six transgenic cell lines from O. sativa and three transgenic cell lines from P. strobus were used to analyze the salt, drought, and cold tolerance conferred by the overexpression of the AtbZIP60 gene. Our results demonstrated that expression of the AtbZIP60 gene enhanced salt, drought, and cold tolerance in rice and white pine transgenic cell lines. In rice, transcription factor AtbZIP60 increased expression of Ca(2+)-dependent protein kinase genes OsCPK6, OsCPK9, OsCPK10, OsCPK19, OsCPK25, and OsCPK26 under treatment of salt, drought, and cold. These results demonstrated that overexpression of the AtbZIP60 gene in transgenic cell lines improved salt, drought, and cold stress tolerances by regulating expression of Ca(2+)-dependent protein kinase genes. Overexpression of the AtbZIP60 gene could be an alternative choice for engineering plant abiotic stress tolerance. PMID:23275191

  10. 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. PMID:27106120

  11. In silico analyses identify gene-sets, associated with clinical outcome in ovarian cancer: role of mitotic kinases

    PubMed Central

    Ocaña, Alberto; Pérez-Peña, Javier; Alcaraz-Sanabria, Ana; Sánchez-Corrales, Verónica; Nieto-Jiménez, Cristina; Templeton, Arnoud J.; Seruga, Bostjan; Pandiella, Atanasio; Amir, Eitan

    2016-01-01

    Introduction Accurate assessment of prognosis in early stage ovarian cancer is challenging resulting in suboptimal selection of patients for adjuvant therapy. The identification of predictive markers for cytotoxic chemotherapy is therefore highly desirable. Protein kinases are important components in oncogenic transformation and those relating to cell cycle and mitosis control may allow for identification of high-risk early stage ovarian tumors. Methods Genes with differential expression in ovarian surface epithelia (OSE) and ovarian cancer epithelial cells (CEPIs) were identified from public datasets and analyzed with dChip software. Progression-free (PFS) and overall survival (OS) associated with these genes in stage I/II and late stage ovarian cancer was explored using the Kaplan Meier Plotter online tool. Results Of 2925 transcripts associated with modified expression in CEPIs compared to OSE, 66 genes coded for upregulated protein kinases. Expression of 9 of these genes (CDC28, CHK1, NIMA, Aurora kinase A, Aurora kinase B, BUB1, BUB1βB, CDKN2A and TTK) was associated with worse PFS (HR:3.40, log rank p<0.001). The combined analyses of CHK1, CDKN2A, AURKA, AURKB, TTK and NEK2 showed the highest magnitude of association with PFS (HR:4.62, log rank p<0.001). Expression of AURKB predicted detrimental OS in stage I/II ovarian cancer better than all other combinations Conclusion Genes linked to cell cycle control are associated with worse outcome in early stage ovarian cancer. Incorporation of these biomarkers in clinical studies may help in the identification of patients at high risk of relapse for whom optimizing adjuvant therapeutic strategies is needed. PMID:26992217

  12. 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

  13. Identification of the human pim-1 gene product as a 33-kilodalton cytoplasmic protein with tyrosine kinase activity

    SciTech Connect

    Telerman, A.; Amson, R.; Zakut-Houri, R.; Givol, D.

    1988-04-01

    The human pim-1 gene was recently identified as a new putative oncogene located on chromosome 6p21, a region showing karyotypic abnormalities in particular leukemias. In the present work the authors characterized the pim protein product. In vitro translation of positively selected poly(A)/sup +/ mRNA indicates that this gene encodes a 33-kilodalton protein. Anti-pim antibodies were raised against a fused TrpE-pim protein induced in a bacterial expression vector. This antibody immunoprecipitated a 33-kilodalton protein from in vivo (/sup 35/S)methionine-labeled K562 and KCl myelogenous origin cell lines. This protein was localized to the cytoplasm, and in vivo labeling as well as in vitro kinase assay suggests that it is a phosphoprotein with tyrosine kinase activity. This was further confirmed by performing autophosphorylation directly on a p33/sup pim/-containing gel band cut out after sodium dodecyl sulfate-polyacrylamide gel electrphoresis. The results imply that the tyrosine kinase activity of pim can be recovered after boiling the pim-1 protein in sample buffer: a feature not described yet for this class of protein. These results suggest that pim-1 is a new member of the subgroup of oncogenes encoding tyrosine kinases.

  14. Mutagenesis of charged residues in a conserved sequence in the 2-kinase domain of 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase.

    PubMed Central

    Bertrand, L; Vertommen, D; Feytmans, E; Di Pietro, A; Rider, M H; Hue, L

    1997-01-01

    Arg-136, Glu-137, Arg-138 and Arg-139 are conserved in all sequences of the 2-kinase domain of 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase. Their role was studied by site-directed mutagenesis. All the mutations had little, if any, effect on fructose-2,6-bisphosphatase activity. Mutations of Arg-136 and Glu-137 into Ala caused only minor modifications of phosphofructo-2-kinase activity. In contrast, mutation of Arg138 into Ala increased 280-fold the Km for fructose 6-phosphate of phosphofructo-2-kinase. Mutation of Arg-139 into Ala resulted in decreases in phosphofructo-2-kinase Vmax/Km for MgATP and fructose 6-phosphate 600-fold and 5000-fold respectively. Mutation of Arg-139 into Lys and Gln increased the Km of phosphofructo-2-kinase for MgATP (20-fold and 25-fold respectively) and for fructose 6-phosphate (8-fold and 13-fold), and the IC50 for MgADP (30-fold and 50-fold) and for magnesium citrate (7-fold and 25-fold). However, these two mutations did not affect nucleotide binding, as measured by quenching of intrinsic fluorescence. The changes in kinetic properties induced by mutations could not be attributed to structural changes. It is proposed that Arg-138 is involved in fructose 6-phosphate binding and that Arg-139 is probably involved in the stabilization of the transition state and so participates in catalysis. PMID:9032444

  15. 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

  16. Human creatine kinase genes on chromosomes 15 and 19, and proximity of the gene for the muscle form to the genes for apolipoprotein C2 and excision repair.

    PubMed Central

    Stallings, R L; Olson, E; Strauss, A W; Thompson, L H; Bachinski, L L; Siciliano, M J

    1988-01-01

    The human chromosomal assignments of genes of the creatine kinase (CK) family--loci for brain (CKBB), muscle (CKMM), and mitochondrial (CKMT) forms--were studied by Southern filter hybridization analysis of DNAs isolated from a human x rodent somatic cell hybrid clone panel. Probes for the 3'-noncoding sequences of human CKBB and CKMM hybridized concordantly only to DNAs from somatic cell hybrids containing chromosomes 14 and 19, respectively. Thus the earlier assignment of the gene coding for the CKBB isozyme to chromosome 14 was confirmed by molecular means, as was the provisional assignment of CKMM to the long arm of chromosome 19. A probe containing canine sequences for CKMM cross-hybridized with human sequences on chromosomes 14 and 19, a result consistent with the assignments of CKBB and CKMM. A probe containing human sequences for CKMT enabled the provisional assignment of CKMT to human chromosome 15. Independent hybrids with portions of the long arm of chromosome 19 missing indicated the order of genes on the long arm of chromosome 19 as being cen-GPI-(TGFB, CYP1)-[CKMM, (APOC2-ERCC1)]-(CGB, FTL). The unexpectedly more distal location of APOC2 among the genes on the long arm--and APOC2's close association with CKMM--is discussed with respect to the close linkage relationship of APOC2 to myotonic muscular dystrophy. Images Figure 1 PMID:3400641

  17. 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. PMID:25617765

  18. Sphingosine kinase-1 is a hypoxia-regulated gene that stimulates migration of human endothelial cells

    SciTech Connect

    Schwalm, Stephanie; Doell, Frauke; Roemer, Isolde; Bubnova, Svetlana

    2008-04-18

    Sphingosine kinases (SK) catalyze the production of sphingosine-1-phosphate which in turn regulates cell responses such as proliferation and migration. Here, we show that exposure of the human endothelial cell line EA.hy 926 to hypoxia stimulates a increased SK-1, but not SK-2, mRNA, protein expression, and activity. This effect was due to stimulated SK-1 promoter activity which contains two putative hypoxia-inducible factor-responsive-elements (HRE). By deletion of one of the two HREs, hypoxia-induced promoter activation was abrogated. Furthermore, hypoxia upregulated the expression of HIF-1{alpha} and HIF-2{alpha}, and both contributed to SK-1 gene transcription as shown by selective depletion of HIF-1{alpha} or HIF-2{alpha} by siRNA. The hypoxia-stimulated SK-1 upregulation was functionally coupled to increased migration since the selective depletion of SK-1, but not of SK-2, by siRNAs abolished the migratory response. In summary, these data show that hypoxia upregulates SK-1 activity and results in an accelerated migratory capacity of endothelial cells. SK-1 may thus serve as an attractive therapeutic target to treat diseases associated with increased endothelial migration and angiogenesis such as cancer growth and progression.

  19. PRKAR1A gene analysis and protein kinase A activity in endometrial tumors.

    PubMed

    Tsigginou, A; Bimpaki, E; Nesterova, M; Horvath, A; Boikos, S; Lyssikatos, C; Papageorgiou, C; Dimitrakakis, C; Rodolakis, A; Stratakis, C A; Antsaklis, A

    2012-08-01

    PRKAR1A codes for the type 1a regulatory subunit (RIα) of the cAMP-dependent protein kinase A (PKA), an enzyme with an important role in cell cycle regulation and proliferation. PKA dysregulation has been found in various tumors, and PRKAR1A-inactivating mutations have been reported in mostly endocrine neoplasias. In this study, we investigated PKA activity and the PRKAR1A gene in normal and tumor endometrium. Specimens were collected from 31 patients with endometrial cancer. We used as controls 41 samples of endometrium that were collected from surrounding normal tissues or from women undergoing gynecological operations for other reasons. In all samples, we sequenced the PRKAR1A-coding sequence and studied PKA subunit expression; we also determined PKA activity and cAMP binding. PRKAR1A mutations were not found. However, PKA regulatory subunit protein levels, both RIα and those of regulatory subunit type 2b (RIIβ), were lower in tumor samples; cAMP binding was also lower in tumors compared with normal endometrium (P<0.01). Free PKA activity was higher in tumor samples compared with that of control tissue (P<0.01). There are significant PKA enzymatic abnormalities in tumors of the endometrium compared with surrounding normal tissue; as these were not due to PRKAR1A mutations, other mechanisms affecting PKA function ought to be explored. PMID:22461635

  20. Epigenetic deregulation of the anaplastic lymphoma kinase gene modulates mesenchymal characteristics of oral squamous cell carcinomas

    PubMed Central

    Huang, Tim Hui-Ming

    2013-01-01

    DNA hypermethylation of promoter CpG islands is associated with epigenetic silencing of tumor suppressor genes in oral squamous cell carcinomas (OSCCs). We used a methyl-CpG-binding domain protein capture method coupled with next-generation sequencing (MBDCap-seq) to survey global DNA methylation patterns in OSCCs with and without nodal metastasis and normal mucosa (total n = 58). Of 1462 differentially methylated CpG islands identified in OSCCs relative to normal controls, MBDCap-seq profiling uncovered 359 loci linked to lymph node metastasis. Interactive network analysis revealed a subset of these loci (n = 23), including the anaplastic lymphoma kinase (ALK) gene, are potential regulators and effectors of invasiveness and metastatic progression. Promoter methylation of ALK was preferentially observed in OSCCs without node metastasis, whereas relatively lower methylation levels were present in metastatic tumors, implicating an active state of ALK transcription in the latter group. The OSCC cell line, SCC4, displayed reduced ALK expression that corresponded to extensive promoter CpG island methylation. SCC4 treatment with demethylating agents induced ALK expression and increased invasion and migration characteristics. Inhibition of ALK activity in OSCC cells with high ALK expression (CAL27, HSC3 and SCC25), decreased cell growth and resulted in changes in invasive potential and mesenchymal marker expression that were cell-line dependent. Although ALK is susceptible to epigenetic silencing during oral tumorigenesis, overwriting this default state may be necessary for modulating invasive processes involved in nodal metastases. Given the complex response of OSCC cells to ALK inhibition, future studies are required to assess the feasibility of targeting ALK to treat invasive OSCCs. PMID:23568951

  1. Aberrant Expression of Anaplastic Lymphoma Kinase in Ovarian Carcinoma Independent of Gene Rearrangement.

    PubMed

    Tang, Shaoxian; Yang, Fei; Du, Xiang; Lu, Yongming; Zhang, Ling; Zhou, Xiaoyan

    2016-07-01

    Ovarian carcinoma is the leading cause of death from gynecologic malignancies. The oncogenic role of anaplastic lymphoma kinase (ALK) is well characterized in many hematopoietic and solid tumors. ALK expression in ovarian carcinoma has been reported but the exact status of ALK protein and its association with clinicopathologic features requires further investigation. ALK expression was determined by immunohistochemistry in 110 primary ovarian carcinomas, including 85 cases of serous carcinoma and 25 cases of mucinous carcinoma. Fluorescence in situ hybridization (FISH) and real-time reverse transcription polymerase chain reaction (RT-PCR) were used for evaluating ALK translocation in ALK-positive ovarian carcinomas. Among 110 ovarian carcinomas, 23 (20.9%) cases were ALK positive by immunohistochemistry. All ALK-positive cases were ovarian high-grade serous carcinoma. ALK expression was detected in 23/85 (27.1%) ovarian serous carcinoma and 0/25 (0%) in ovarian mucinous carcinoma. None of the 23 ALK IHC-positive cases harbored ALK gene translocations by FISH or RT-PCR. ALK protein expression was associated with patient age, tumor stage, and histologic type. Specifically, the probability of ALK protein expression was significantly higher in high-grade serous carcinomas in older patients (above 50 y) with advanced disease (FIGO stage III and IV) compared with the low-grade serous and mucinous carcinomas in younger patients with relatively early disease. In conclusion, aberrant ALK expression is observed in ovarian serous carcinoma but not in mucinous carcinoma, is independent of gene translocation, and might be associated with progression and prognosis. PMID:27271776

  2. 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.

  3. 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

  4. Complete nucleotide sequence of a gene conferring polymyxin B resistance on yeast: similarity of the predicted polypeptide to protein kinases.

    PubMed

    Boguslawski, G; Polazzi, J O

    1987-08-01

    Polymyxin B is an antibiotic that kills sensitive cells by disrupting their membranes. We have cloned a wild-type yeast gene that, when present on a high-copy-number plasmid, renders the cells resistant to the drug. The nucleotide sequence of this gene is presented. A single open reading frame within the sequence has the potential to encode a polypeptide (molecular mass of 77.5 kDa) that shows strong homologies to polypeptides of the protein kinase family. The gene, PBS2, located on chromosome X, is not allelic to the previously described PBS1 gene (where PBS signifies polymyxin B sensitivity). Although pbs1 mutations confer resistance to high levels of polymyxin B, double mutants, pbs1 pbs2, are not resistant to the drug, indicating that PBS2 is essential for pbs1 activity. Models based on the proposed protein kinase activity of the PBS2 gene product are presented to explain the interaction between PBS1 and PBS2 gene products involved in conferring polymyxin B resistance on yeast cells. PMID:3039511

  5. A MAP kinase gene, BMK1, is required for conidiation and pathogenicity in the rice leaf spot pathogen Bipolaris oryzae.

    PubMed

    Moriwaki, Akihiro; Kihara, Junichi; Mori, Chie; Arase, Sakae

    2007-01-01

    We isolated and characterized BMK1, a gene encoding a mitogen-activated protein kinase (MAPK), from the rice leaf spot pathogen Bipolaris oryzae. The deduced amino acid sequence showed significant homology with Fus3/Kss1 MAPK homologues from other phytopathogenic fungi. The BMK1 disruptants showed impaired hyphal growth, no conidial production, and loss of virulence against rice leaves, indicating that the BMK1 is essential for conidiation and pathogenicity in B. oryzae. PMID:16546358

  6. Chromatinized protein kinase C-θ directly regulates inducible genes in epithelial to mesenchymal transition and breast cancer stem cells.

    PubMed

    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; Rao, Sudha

    2014-08-01

    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

  7. 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

  8. Chromosomal location of the Syk and ZAP-70 tyrosine kinase genes in mice and humans

    SciTech Connect

    Ku, G.; Malissen, B.; Mattei, M.G.

    1994-12-31

    Several protein tyrosine kinases (PTKs), which may be grouped into two structurally different families, have been implicated in antigen receptor proximal signaling. Blk, Fyn, Lck, Lyn, and Yes belong to the Src-family kinases, whereas the spleen tyrosine kinase (Syk) and the CD3-{zeta}-associated PTK (ZAP-70) define a new one, the Syk family. These kinases differ from the Src-family kinases in that they are non-myristylated cytoplasmic polypeptides composed of two N-terminal Src homology-2 (SH2) domains and a C-terminal catalytic domain. ZAP-70 appears to be expressed exclusively in T cells and NK cells, whereas Syk is preferentially expressed in B cells, T cells, and myeloid cells. 15 refs., 2 figs.

  9. Role of the Cys154Arg Substitution in Ribosomal Protein L3 in Oxazolidinone Resistance in Mycobacterium tuberculosis.

    PubMed

    Makafe, Gaëlle Guiewi; Cao, Yuanyuan; Tan, Yaoju; Julius, Mugweru; Liu, Zhiyong; Wang, Changwei; Njire, Moses M; Cai, Xingshan; Liu, Tianzhou; Wang, Bangxing; Pang, Wei; Tan, Shouyong; Zhang, Buchang; Yew, Wing Wai; Lamichhane, Gyanu; Guo, Jintao; Zhang, Tianyu

    2016-05-01

    We expressed the wild-type rplC and mutated rplC (Cys154Arg) genes, respectively, in Mycobacterium tuberculosis H37Ra and H37Rv in an attempt to delineate the role of rplC (Cys154Arg) regarding oxazolidinone resistance. An increase of the MICs of linezolid (LZD) and sutezolid (PNU-100480, PNU) against the recombinant mycobacteria with overexpressed rplC mutation (Cys154Arg) was found, suggesting the rplC gene is a determinant of bacillary susceptibilities to LZD and PNU. PMID:26953211

  10. 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.

  11. Molecular characterization of a Cyrtochilum loxense Somatic Embryogenesis Receptor-like Kinase (SERK) gene expressed during somatic embryogenesis.

    PubMed

    Cueva, Augusta; Concia, Lorenzo; Cella, Rino

    2012-06-01

    Somatic embryogenesis is crucial for the propagation of endangered Ecuadorian orchid species, among them Cyrtochilum loxense, in view of the fact that their number in nature or in collections is quite reduced. One of the genes expressed during somatic and zygotic embryogenesis is Somatic Embryogenesis Receptor-like Kinase (SERK). Despite the development of somatic embryogenesis protocols for orchids, no SERK genes have been isolated from this family. This is the first report on the isolation of a full-length orchid SERK sequence, namely that of Cyrtochilum loxense (ClSERK). The identity of ClSERK was inferred by the presence of all domains typical of SERK proteins: a signal peptide, a leucine zipper domain, five LRRs, a serine proline-rich domain, a transmembrane domain, a kinase domain, and the C-terminal region. We have observed that the ClSERK gene is highly expressed in embryogenic calluses generated from protocorms at the time of appearance of embryonic morphological features. At later stages when embryos become well visible on calluses, ClSERK gene expression decreases. Compared to early stages of embryo formation on calluses, the expression detected in leaf tissue is far lower, thus suggesting a role of this gene during development. PMID:22350407

  12. Cloning and organization of seven arginine biosynthesis genes from Neisseria gonorrhoeae.

    PubMed Central

    Picard, F J; Dillon, J R

    1989-01-01

    A genomic library for Neisseria gonorrhoeae, constructed in the lambda cloning vector EMBL4, was screened for clones carrying arginine biosynthesis genes by complementation of Escherichia coli mutants. Clones complementing defects in argA, argB, argE, argG, argIF, carA, and carB were isolated. An E. coli defective in the acetylornithine deacetylase gene (argE) was complemented by the ornithine acetyltransferase gene (argJ) from N. gonorrhoeae. This heterologous complementation is reported for the first time. The carAB operon from E. coli hybridized with the gonococcal clones that carried carA or carB genes under conditions of high stringency, detecting 80% or greater similarity and showing that the nucleotide sequence of the carbamoylphosphate synthetase genes is very similar in these two organisms. Under these conditions for hybridization, the gonococcal clones carrying argB or argF genes did not hybridize with plasmids containing the corresponding E. coli gene. Cocomplementation experiments established gene linkage between carA and carB. Clones complementing a gene defect in argE were also able to complement an argA mutation. This suggests that the enzyme ornithine acetyltransferase from N. gonorrhoeae (encoded by argJ) may be able to complement both argA and argE mutations in E. coli. The arginine biosynthesis genes in N. gonorrhoeae appear to be scattered as in members of the family Pseudomonadaceae. Images PMID:2493452

  13. Molecular cloning and expression of the human deoxythymidylate kinase gene in yeast.

    PubMed Central

    Su, J Y; Sclafani, R A

    1991-01-01

    (Deoxy)thymidylate (dTMP) kinase is an enzyme which phosphorylates dTMP to dTDP in the presence of ATP and magnesium. This enzyme is important in cellular DNA synthesis because the synthesis of dTTP, either via the de novo pathway or through the exogenous supply of thymidine, requires the activity of this enzyme. It has been suggested that the activities of the enzymes involved in DNA precursor biosynthesis, such as thymidine kinase, thymidylate synthase, thymidylate kinase, and dihydrofolate reductase, are subjected to cell cycle regulation. Here we describe the cloning of a human dTMP kinase cDNA by functional complementation of a yeast dTMP kinase temperature-sensitive mutant at the non-permissive temperature. The nucleotide sequence of the cloned human cDNA is predicted to encode a 24 KD protein that shows considerable homology with the yeast and vaccinia virus dTMP kinase enzymes. The human enzyme activity has been investigated by expressing it in yeast. In this work, we demonstrate that the cloned human cDNA, when expressed in yeast, produces dTMP kinase activity. Images PMID:2017365

  14. Multiple Promoters in the WNK1 Gene: One Controls Expression of a Kidney-Specific Kinase-Defective Isoform

    PubMed Central

    Delaloy, Celine; Lu, Jingyu; Houot, Anne-Marie; Disse-Nicodeme, Sandra; Gasc, Jean-Marie; Corvol, Pierre; Jeunemaitre, Xavier

    2003-01-01

    WNK1 is a serine-threonine kinase, the expression of which is affected in pseudohypoaldosteronism type II, a Mendelian form of arterial hypertension. We characterized human WNK1 transcripts to determine the molecular mechanisms governing WNK1 expression. We report the presence of two promoters generating two WNK1 isoforms with a complete kinase domain. Further variations are achieved by the use of two polyadenylation sites and tissue-specific splicing. We also determined the structure of a kidney-specific isoform regulated by a third promoter and starting at a novel exon. This transcript is kinase defective and has a predominant expression in the kidney compared to the other WNK1 isoforms, with, furthermore, a highly restricted expression profile in the distal convoluted tubule. We confirmed that the ubiquitous and kidney-specific promoters are functional in several cells lines and identified core promoters and regulatory elements. In particular, a strong enhancer element upstream from the kidney-specific exon seems specific to renal epithelial cells. Thus, control of human WNK1 gene expression of kinase-active or -deficient isoforms is mediated predominantly through the use of multiple transcription initiation sites and tissue-specific regulatory elements. PMID:14645531

  15. 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.

  16. Cell biological characterization of a multidomain adaptor protein, ArgBP2, in epithelial NMuMG cells, and identification of a novel short isoform.

    PubMed

    Murase, Kana; Ito, Hidenori; Kanoh, Hiroyuki; Sudo, Kaori; Iwamoto, Ikuko; Morishita, Rika; Soubeyran, Philippe; Seishima, Mariko; Nagata, Koh-Ichi

    2012-12-01

    ArgBP2 is a member of the SoHo (sorbin-homology) family of adaptor proteins believed to play roles in cell adhesion, cytoskeletal organization, and signaling. We show here a novel splicing isoform of ArgBP2, i.e., ArgBP2™, composed of only three SH3 (src-homology 3) domains and structurally similar to vinexinß. We then characterized the biochemical and cell biological properties of ArgBP2 to compare these with vinexin. Similar to vinexin, ArgBP2 was enriched at focal adhesions in REF52 fibroblast cells and induced anchorage-dependent extracellular signal-regulated kinase activation in NIH3T3 fibroblast cells. In epithelial NMuMG cells, immunofluorescence analyses revealed localization of ArgBP2 at tight junctions (TJs), whereas vinexin was distributed in cytoplasm as well as cell-cell boundaries. During TJ formation, recruitment of ZO-1 to TJs was followed by ArgBP2. Based on mutation analyses, a second SH3 domain was found to be important for ArgBP2 localization to the cell-cell contact sites. These data suggest some role of ArgBP2 in NMuMG cells at TJs that may be distinct from the function of vinexin. PMID:22431180

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

    PubMed Central

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

    2015-01-01

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

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

    PubMed

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

    2015-01-01

    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. PMID:26634459

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

    PubMed

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

    2015-10-01

    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

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

    PubMed

    Del Coso, Juan; 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

  1. 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

  2. An efficient method for organic acetylation and use of DL-phosphinothricin as a negative selection agent in argE transgenic rice.

    PubMed

    Byeon, Ji-Hui; Cho, Joon-Hyeong

    2013-11-01

    We present an efficient method for the production of N-acetyl-L-phosphinothricin (N-AcPt) from commercial DL-phosphinothricin (DL-PPT) by organic acetylation for use as a negative selection agent (NSA) that induces cell death in argE transgenic rice. DL-PPT was efficiently converted into N-AcPt with tetrahydrofuran (THF) and acetic anhydride (Ac2O). Chemical changes were confirmed using NMR and ATR-FTIR analyses. DL-PPT was toxic but N-AcPt did not show cytotoxic effects on leaf discs or seed germination of wild-type rice. Conversely, in argE-hpt transgenic rice, non-toxic N-AcPt showed the negative selection (NS) effect by inducing cell destruction in leaf discs and restricting seed germination. For inducing NS, ≥0.1 mg ml(-1) and ≥0.5 mg ml(-1) of N-AcPt were effective in leaf and seed assays, respectively. Further, the NS effect occurred faster in the leaf assay compared with the seed germination assay, again indicating the leaf assay was a more sensitive indicator of N-AcPt as an NSA to argE transgenic rice than the seed germination assay. This negative selection approach could be useful for the development of selectable marker free transgenic plants in the economically important monocot species and its commercialization for multiple gene transformation. PMID:24141117

  3. 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

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

    PubMed

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

    2013-01-01

    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. PMID:24149340

  5. 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.

  6. Targeting Abl Kinases to Regulate Vascular Leak During Sepsis and ARDS

    PubMed Central

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

    2015-01-01

    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 (ARDS), due to the formation of gaps between endothelial cells (EC). Although these disorders are leading causes of mortality in the ICU, no medical therapies exist to restore EC barrier function. Recent evidence highlights a key role for the Abl family of non-receptor 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 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. Additionally, both c-Abl and Arg are activated by hyperoxia and contribute to oxidant-induced EC injury. These numerous roles of Abl kinases in EC 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. PMID:25814671

  7. Protein Kinase Cδ Blocks Immediate-Early Gene Expression in Senescent Cells by Inactivating Serum Response Factor

    PubMed Central

    Wheaton, Keith; Riabowol, Karl

    2004-01-01

    Fibroblasts lose the ability to replicate in response to growth factors and become unable to express growth-associated immediate-early genes, including c-fos and egr-1, as they become senescent. The serum response factor (SRF), a major transcriptional activator of immediate-early gene promoters, loses the ability to bind to the serum response element (SRE) and becomes hyperphosphorylated in senescent cells. We identify protein kinase C delta (PKCδ) as the kinase responsible for inactivation of SRF both in vitro and endogenously in senescent cells. This is due to a higher level of PKCδ activity as cells age, production of the PKCδ catalytic fragment, and its nuclear localization in senescent but not in low-passage-number cells. The phosphorylation of T160 of SRF by PKCδ in vitro and in vivo led to loss of SRF DNA binding activity. Both the PKCδ inhibitor rottlerin and ectopic expression of a dominant negative form of PKCδ independently restored SRE-dependent transcription and immediate-early gene expression in senescent cells. Modulation of PKCδ activity in vivo with rottlerin or bistratene A altered senescent- and young-cell morphology, respectively. These observations support the idea that the coordinate transcriptional inhibition of several growth-associated genes by PKCδ contributes to the senescent phenotype. PMID:15282327

  8. Characterization of the cyclin-dependent kinase 6 gene in Apis cerana cerana in response to multiple environmental stresses.

    PubMed

    Luo, Lu; Kangb, Mingjiang; Liu, Kai; Guo, Xingqi; Xu, Baohua

    2012-01-01

    Cyclin-dependent kinases (CDKs) are serine/threonine kinases that play critical roles in the cell cycle regulation. Herein, we describe the identification of a CDK gene from Apis cerana cerana, named AccCDK6. The full-length cDNA is 1778 bp long, including an ORF of 1380 bp that encodes a polypeptide of 459 amino acid residues. Multiple sequence alignment analysis showed that the predicted AccCDK6 sequence shares a high similarity with CDK6 genes of other species, and this protein may share an evolutionary predecessor with Drosophila CDK4. The expression patterns of the gene were also analysed, and the transcript was detected throughout the larval, pupal, and adult developmental stages. Furthermore, the expression level of the mRNA of the gene in adult workers was influenced by H2O2, ultraviolet (UV) light, temperature (42 degrees C), HgCl2, and pyriproxyfen. These results indicate that AccCDK6 responds to multiple environmental stresses and may also participate in intracellular reactions of reactive oxygen species (ROS) and development processes in honey-bees. PMID:22888541

  9. The apoptosis associated tyrosine kinase gene is frequently hypermethylated in human cancer and is regulated by epigenetic mechanisms

    PubMed Central

    Haag, Tanja; Herkt, Christina E.; Walesch, Sara K.; Richter, Antje M.; Dammann, Reinhard H.

    2014-01-01

    Epigenetic gene inactivation through promoter hypermethylation is an important aberration involved in the silencing of tumor-associated genes in cancer. Here we identified the apoptosis associated tyrosine kinase (AATK) as an epigenetically downregulated tumor related gene. We analyzed the epigenetic regulation of AATK in several human cancer cell lines and normal tissues by methylation and expression analysis. Hypermethylation of AATK was also analyzed in 25 primary lung tumors, 30 breast cancers and 24 matching breast tissues. In normal tissues the AATK CpG island promoter was unmethylated and AATK was expressed. Hypermethylation of AATK occurred frequently in 13 out of 14 (93%) human cancer cell lines. Methylation was reversed by 5-aza-2′-deoxycytidine treatment leading to re-expression of AATK in cancer cell lines. Aberrant methylation of AATK was also revealed in primary lung (40%) and breast (53%) cancers, but was found to be significantly less methylated in matching normal breast tissues (17%; p<0.01). In addition, we observed that AATK is epigenetically reactivated through the chromatin regulator CTCF. We further show that overexpression of Aatk significantly suppresses colony formation in cancer cell lines. Our findings suggest that the apoptosis associated tyrosine kinase is frequently inactivated in human cancers and acts as a tumor suppressive gene. PMID:25352953

  10. Transcriptional regulation of the cyclin-dependent kinase inhibitor 1A (p21) gene by NFI in proliferating human cells

    PubMed Central

    Ouellet, Stéphane; Vigneault, François; Lessard, Maryse; Leclerc, Steeve; Drouin, Régen; Guérin, Sylvain L.

    2006-01-01

    The cyclin-dependent kinase inhibitor 1A (CDKN1A), also known as p21 (WAF1/CIP1) modulates cell cycle, apoptosis, senescence and differentiation via specific protein–protein interactions with the cyclins, cyclin-dependent kinase (Cdk), and many others. Expression of the p21 gene is mainly regulated at the transcriptional level. By conducting both ligation-mediated PCR (LMPCR) and chromatin immunoprecipitation (ChIP) in vivo, we identified a functional target site for the transcription factor, nuclear factor I (NFI), in the basal promoter from the p21 gene. Transfection of recombinant constructs bearing mutations in the p21 NFI site demonstrated that NFI acts as a repressor of p21 gene expression in various types of cultured cells. Inhibition of NFI in human skin fibroblasts through RNAi considerably increased p21 promoter activity suggesting that NFI is a key repressor of p21 transcription. Over-expression of each of the four NFI isoforms in HCT116 cells established that each of them contribute to various extend to the repression of the p21 gene. Most of all, over-expression of NFI-B in doxorubicin, growth-arrested HCT116 increased the proportion of cells in the S-phase of the cell cycle whereas NFI-A and NFI-X reduced it, thereby establishing a role for NFI in the cell cycle dependent expression of p21. PMID:17130157

  11. Loss of cyclin-dependent kinase inhibitor genes and chromosome 9 karyotypic abnormalities in human bladder cancer cell lines.

    PubMed Central

    Southgate, J.; Proffitt, J.; Roberts, P.; Smith, B.; Selby, P.

    1995-01-01

    Loss of cell cycle control through the structural or functional aberration of checkpoint genes and their products is a potentially important process in carcinogenesis. In this study, a panel of well-characterised established human bladder cancer cell lines was screened by the polymerase chain reaction for homozygous loss of the cyclin-dependent kinase inhibitor genes p15, p16 and p27. The results demonstrate that, whereas there was no genetic loss of p27, homozygous deletion of both p15 and p16 genes occurred in seven of 13 (54%) independent bladder cell lines tested. Differential loss of either the p15 or p16 gene was not seen. The p15 and p16 genes are known to be juxtaposed on chromosome 9p21 at the locus of a putative tumour-suppressor gene involved in the initiation of bladder cancer. Cytogenetic analysis of the cell lines revealed karyotypes ranging from near diploid to near pentaploid with complex rearrangements of some chromosomes and a high prevalence of chromosome 9p rearrangements, although all cell lines contained at least one cytogenetically normal 9p21 region. These observations support a role for p15/p16 gene inactivation in bladder carcinogenesis and/or the promotion of cell growth in vitro and lend support to the hypothesis that homozygous deletion centred on 9p21 is a mechanism by which both p15 and p16 genes are co-inactivated. Images Figure 1 Figure 2 Figure 3 PMID:7577470

  12. Intracellular calcium-release and protein kinase C-activation stimulate sonic hedgehog gene expression during gastric acid secretion

    PubMed Central

    El-Zaatari, Mohamad; Zavros, Yana; Tessier, Art; Waghray, Meghna; Lentz, Steve; Gumucio, Deborah; Todisco, Andrea; Merchant, Juanita L.

    2010-01-01

    Introduction Hypochlorhydria during Helicobacter pylori infection inhibits gastric Shh expression. We investigated whether acid-secretory mechanisms regulate Shh gene expression through Ca2+i-dependent protein kinase C (PKC) or cAMP-dependent protein kinase A (PKA)-activation. Method We blocked Hedgehog signaling by transgenically overexpressing a secreted form of the Hedgehog interacting protein-1 (sHip-1), a natural inhibitor of hedgehog ligands, which induced hypochlorhydria. Gadolinium, EGTA+BAPTA, PKC-overexpressing adenoviruses, and PKC-inhibitors were used to modulate Ca2+i-release, PKC-activity and Shh gene expression in primary gastric cell, organ, and AGS cell line cultures. PKA hyperactivity was induced in the H+/K+-β-cholera-toxin overexpressing mice (Ctox). Results Mice that expressed sHip-1 had lower levels of gastric acid (hypochlorhydria), reduced production of somatostatin, and increased gastrin gene expression. Hypochlorhydria in these mice repressed Shh gene expression, similar to the levels obtained with omeprazole treatment of wild-type mice. However, Shh expression was also repressed in the hyperchlorhydric Ctox model with elevated cAMP, suggesting that the regulation of Shh was not solely acid-dependent, but pertained to specific acid-stimulatory signaling pathways. Based on previous reports that Ca2+i-release also stimulates acid secretion in parietal cells, we showed that gadolinium-, thapsigargin- and carbachol-mediated release of Ca2+i induced Shh expression. Ca2+-chelation with BAPTA+EGTA reduced Shh expression. Overexpression of PKC-α, -β and -δ (but not PKC-ε) induced Shh gene expression. In addition, phorbol esters induced a Shh-regulated reporter gene. Conclusion Secretagogues that stimulate gastric acid secretion induce Shh gene expression through increased Ca2+i-release and PKC activation. Shh might be the ligand transducing changes in gastric acidity to the regulation of G-cell secretion of gastrin. PMID:20816837

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

    PubMed

    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

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

    PubMed Central

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

    2016-01-01

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

  15. 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.

  16. 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.

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

    NASA Astrophysics Data System (ADS)

    Panicali, Dennis; Paoletti, Enzo

    1982-08-01

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

  18. A single-nucleotide polymorphism in serine-threonine kinase 11, the gene encoding liver kinase B1, is a risk factor for multiple sclerosis.

    PubMed

    Boullerne, Anne I; Skias, Demetrios; Hartman, Elizabeth M; Testai, Fernando D; Kalinin, Sergey; Polak, Paul E; Feinstein, Douglas L

    2015-01-01

    We identified a family in which five siblings were diagnosed with multiple sclerosis (MS) or clinically isolated syndrome. Several women in the maternal lineage have comorbidities typically associated with Peutz Jeghers Syndrome, a rare autosomal-dominant disease caused by mutations in the serine-threonine-kinase 11 (STK11) gene, which encodes liver kinase B1. Sequence analysis of DNA from one sibling identified a single-nucleotide polymorphism (SNP) within STK11 intron 5. This SNP (dbSNP ID: rs9282860) was identified by TaqMan polymerase chain reaction (PCR) assays in DNA samples available from two other siblings. Further screening was carried out in samples from 654 relapsing-remitting MS patients, 100 primary progressive MS patients, and 661 controls. The STK11-SNP has increased frequency in all female patients versus controls (odds ratio = 1.66, 95% CI = 1.05, 2.64, p = .032). The STK11-SNP was not associated with disease duration or onset; however, it was significantly associated with reduced severity (assessed by MS severity scores), with the lowest scores in patients who also harbored the HLA-DRB1*1501 allele. In vitro studies showed that peripheral blood mononuclear cells from members of the family were more sensitive to the mitochondrial inhibitor metformin than cells from MS patients with the major STK11 allele. The increased association of SNP rs9282860 in women with MS defines this variant as a genetic risk factor. The lower disease severity observed in the context of HLA-DRB1*1501 combined with limited in vitro studies raises the provocative possibility that cells harboring the STK11-SNP could be targeted by drugs which increase metabolic stress. PMID:25694554

  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. 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.

  1. Role of Plc1p in regulation of Mcm1p-dependent genes

    PubMed Central

    Guzinska, Katarzyna; Varghese, Roger; Vancura, Ales

    2009-01-01

    In budding yeast, phosphoinositide-specific phospholipase C (Plc1p encoded by PLC1 gene) and several inositol polyphosphate kinases represent a nuclear pathway for synthesis of inositol polyphosphates (InsPs) that are involved in several aspects of DNA and RNA metabolism, including transcriptional regulation. Plc1p-produced InsP3 is phosphorylated by Ipk2p/Arg82p to yield InsP4/InsP5. Ipk2p/Arg82p is also a component of ArgR-Mcm1p complex that regulates transcription of genes involved in arginine metabolism. The role of Ipk2p/Arg82p in this complex is to stabilize the essential MADS box protein Mcm1p. Consequently, ipk2Δ cells display reduced level of Mcm1p and attenuated expression of Mcm1p-dependent genes. Since plc1Δ cells display aberrant expression of several groups of genes, including genes involved in stress response, the objective of this study was to determine whether Plc1p also affects expression of Mcm1p-dependent genes. We report here that not only ipk2Δ, but also plc1Δ cells display decreased expression of Mcm1p-dependent genes. However, Plc1p is not involved in stabilization of Mcm1p and affects transcription of Mcm1p-dependent genes by a different mechanism, probably involving regulation of chromatin remodeling complexes. PMID:19459978

  2. 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.

  3. 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).

  4. Tumor accumulation of protein kinase-responsive gene carrier/DNA polyplex stabilized by alkanethiol for intravenous injection.

    PubMed

    Li, Kai; Sato, Hikari; Kim, Chan Woo; Nakamura, Yuta; Zhao, Guo Xi; Funamoto, Daiki; Nobori, Takanobu; Kishimura, Akihiro; Mori, Takeshi; Katayama, Yoshiki

    2015-01-01

    We synthesized polymeric gene carriers consisting of poly-L-lysine (PLL) main chain modified both with substrate peptide for protein kinase Cα (PKCα) and alkanethiol (pentadecanethiol). Due to the grafted substrate peptide, the polyplex prepared from these carriers is expected to show gene expression triggered by the phosphorylation of the peptide by intracellular PKCα. The modified alkanethiol on the main chain stabilized the polyplex both via disulfide crosslinking and hydrophobic interaction. The polyplex found to show gene expression in vitro when the alkanethiol content in the main chain was enough low (4-mol%-modification of PLL's ε-amine group) to minimize cytotoxic effect. Even though the content of alkanethiol is low, the polyplex had significant stability in a model serum solution and showed longer blood circulation in vivo. The polyplex clearly accumulated in tumor after intravenous injection. PMID:26011738

  5. Involvement of a Magnaporthe grisea serine/threonine kinase gene, MgATG1, in appressorium turgor and pathogenesis.

    PubMed

    Liu, Xiao-Hong; Lu, Jian-Ping; Zhang, Lei; Dong, Bo; Min, Hang; Lin, Fu-Cheng

    2007-06-01

    We isolated an MgATG1 gene encoding a serine/threonine protein kinase from the rice blast fungus Magnaporthe grisea. In the DeltaMgatg1 mutant, in which the MgATG1 gene had been deleted, autophagy was blocked; the mutant also showed fewer lipid droplets in its conidia, lower turgor pressure of the appressorium, and such defects in morphogenesis as delayed initiation and slower germination of conidia. As a result of lower turgor pressure of the appressorium, the DeltaMgatg1 mutant lost its ability to penetrate and infect the two host plants, namely, rice and barley. However, normal values of the parameters and infective abilities were restored on reintroducing an intact copy of the MgATG1 gene into the mutant. Autophagy is thus necessary for turnover of organic matter during the formation of conidia and appressoria and for normal development and pathogenicity in M. grisea. PMID:17416896

  6. Restricted expression of herpes simplex virus lytic genes during establishment of latent infection by thymidine kinase-negative mutant viruses.

    PubMed Central

    Kosz-Vnenchak, M; Coen, D M; Knipe, D M

    1990-01-01

    Infection of cells by herpes simplex virus (HSV) can lead to either lytic, productive infection or nonlytic, latent infection. The factors influencing this infection pathway decision are largely unknown. Thymidine kinase-negative mutant viruses can establish latent infection in neurons of mouse trigeminal ganglia but do not replicate productively in these cells. We show that during the early stages of establishment of latency by these mutants, expression of viral lytic genes is drastically reduced or undetectable as assayed by in situ hybridization. Thus, establishment of latent infection by HSV can occur despite severely restricted levels of lytic gene expression. This suggests that the block to productive replication during establishment of latent infection by HSV occurs before or early during the expression of alpha genes. Images PMID:2170678

  7. Differential gene expression for glutamic acid decarboxylase and type II calcium-calmodulin-dependent protein kinase in basal ganglia, thalamus, and hypothalamus of the monkey

    SciTech Connect

    Benson, D.L.; Isackson, P.J.; Hendry, S.H.; Jones, E.G. )

    1991-06-01

    In situ hybridization histochemistry, using cRNA probes, revealed a complementarity in the distributions of cells in the basal ganglia, basal nucleus of Meynert, thalamus, hypothalamus, and rostral part of the midbrain that showed gene expression for glutamic acid decarboxylase (GAD) or the alpha-subunit of type II calcium-calmodulin-dependent protein kinase (CAM II kinase-alpha). Cells in certain nuclei such as the thalamic reticular nucleus, globus pallidus, and pars reticulata of the substantia nigra show GAD gene expression only; others in nuclei such as the basal nucleus of Meynert, medial mamillary nuclei, and ventromedial hypothalamic nuclei show CAM II kinase-alpha gene expression only. A few nuclei, for example, the pars compacta of the substantia nigra and the greater part of the subthalamic nucleus, display gene expression for neither GAD nor CAM II kinase-alpha. In other nuclei, notably those of the dorsal thalamus, and possibly in the striatum, GAD- and CAM II kinase-expressing cells appear to form two separate populations that, in most thalamic nuclei, together account for the total cell population. In situ hybridization reveals large amounts of CAM II kinase-alpha mRNA in the neuropil of most nuclei containing CAM II kinase-alpha-positive cells, suggesting its association with dendritic polyribosomes. The message may thus be translated at those sites, close to the synapses with which the protein is associated. The in situ hybridization results, coupled with those from immunocytochemical staining for CAM II kinase-alpha protein, indicate that CAM II kinase-alpha is commonly found in certain non-GABAergic afferent fiber systems but is not necessarily present in the postsynaptic cells on which they terminate. It appears to be absent from most GABAergic fiber systems but can be present in the cells on which they terminate.

  8. 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.

  9. Genetic Variations of Kinase Inserts Domain Receptor (KDR) Gene Are Associated with the Risk of Astrocytomas.

    PubMed

    Gao, Yufei; Ma, Piyong; He, Yichun; Liu, Yan; Jiang, Yang

    2016-05-01

    Astrocytomas is one of the most common central nervous system (CNS) tumors with high mortality rate. Kinase insert domain receptor (KDR) is involved in the regulation of tumor angiogenesis, migration, and vascular permeability. The aim of the study was to explore the relationship between KDR polymorphisms and risk of astrocytomas. Blood samples were collected from 157 astrocytomas patients and 160 healthy controls. Three tag-SNPs (rs2071559C/T, rs2305948T/C, and rs1870377A/T) were identified from the International HapMap Project Databases and genotyped using the method of polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP). We evaluated the astrocytomas risk caused by individual SNPs and haplotype using odds ratios (ORs) and their 95 % confidence intervals (CIs). In the overall individual SNP analysis, the C allele of rs2071559 was correlated with an increased risk of astrocytomas. However, individuals with mutant allele A and genotype TA + AA of rs1870377 showed a protective effect against astrocytomas. Subgroup analysis based on WHO tumor grade revealed that the C allele of rs2071559 had more influence with the risk of astrocytomas in the grade III-IV (OR = 1.91) subgroup than the grade I-II (OR = 1.47) group. Genotype TT of rs2305948 was found to be significantly associated with susceptibility of astrocytomas only in the grade III-IV subgroup. The protective effect of rs1870377 did not reveal significant difference between the grade III-IV and grade I-II subgroups. Meanwhile, stratified analysis demonstrated that mutation of rs2071559 and rs2305948 could elevate the risk of astrocytomas more significantly in the subgroup of smokers than the nonsmokers. Interestingly, the protective effect of rs1870377 was more obvious in the nonsmokers than the smokers. Additionally, haplotype-specific analysis showed that haplotype CCT and CTT were related with an increased risk of astrocytomas. We found that individual with variants of rs

  10. Molecular Characterization and Comparative Sequence Analysis of Defense-Related Gene, Oryza rufipogon Receptor-Like Protein Kinase 1

    PubMed Central

    Law, Yee-Song; Gudimella, Ranganath; Song, Beng-Kah; Ratnam, Wickneswari; Harikrishna, Jennifer Ann

    2012-01-01

    Many of the plant leucine rich repeat receptor-like kinases (LRR-RLKs) have been found to regulate signaling during plant defense processes. In this study, we selected and sequenced an LRR-RLK gene, designated as Oryza rufipogon receptor-like protein kinase 1 (OrufRPK1), located within yield QTL yld1.1 from the wild rice Oryza rufipogon (accession IRGC105491). A 2055 bp coding region and two exons were identified. Southern blotting determined OrufRPK1 to be a single copy gene. Sequence comparison with cultivated rice orthologs (OsI219RPK1, OsI9311RPK1 and OsJNipponRPK1, respectively derived from O. sativa ssp. indica cv. MR219, O. sativa ssp. indica cv. 9311 and O. sativa ssp. japonica cv. Nipponbare) revealed the presence of 12 single nucleotide polymorphisms (SNPs) with five non-synonymous substitutions, and 23 insertion/deletion sites. The biological role of the OrufRPK1 as a defense related LRR-RLK is proposed on the basis of cDNA sequence characterization, domain subfamily classification, structural prediction of extra cellular domains, cluster analysis and comparative gene expression. PMID:22942769

  11. 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

  12. Relationship between bacterial virulence and nucleotide metabolism: a mutation in the adenylate kinase gene renders Yersinia pestis avirulent.

    PubMed Central

    Munier-Lehmann, Hélène; Chenal-Francisque, Viviane; Ionescu, Mihaela; Chrisova, Petya; Foulon, Jeannine; Carniel, Elisabeth; Bârzu, Octavian

    2003-01-01

    Nucleoside monophosphate kinases (NMPKs) are essential catalysts for bacterial growth and multiplication. These enzymes display high primary sequence identities among members of the family Enterobacteriaceae. Yersinia pestis, the causative agent of plague, belongs to this family. However, it was previously shown that its thymidylate kinase (TMPKyp) exhibits biochemical properties significantly different from those of its Escherichia coli counterpart [Chenal-Francisque, Tourneux, Carniel, Christova, Li de la Sierra, Barzu and Gilles (1999) Eur. J. Biochem. 265, 112-119]. In this work, the adenylate kinase (AK) of Y. pestis (AKyp) was characterized. As with TMPKyp, AKyp displayed a lower thermodynamic stability than other studied AKs. Two mutations in AK (Ser129Phe and Pro87Ser), previously shown to induce a thermosensitive growth defect in E. coli, were introduced into AKyp. The recombinant variants had a lower stability than wild-type AKyp and a higher susceptibility to proteolytic digestion. When the Pro87Ser substitution was introduced into the chromosomal adk gene of Y. pestis, growth of the mutant strain was altered at the non-permissive temperature of 37 degree C. In virulence testings, less than 50 colony forming units (CFU) of wild-type Y. pestis killed 100% of the mice upon subcutaneous infection, whereas bacterial loads as high as 1.5 x 10(4) CFU of the adk mutant were unable to kill any animals. PMID:12879903

  13. Mutations in the DDR2 kinase gene identify a novel therapeutic target in squamous cell lung cancer

    PubMed Central

    Hammerman, Peter S; Sos, Martin L; Ramos, Alex H; Xu, Chunxiao; Dutt, Amit; Zhou, Wenjun; Brace, Lear E; Woods, Brittany A; Lin, Wenchu; Zhang, Jianming; Deng, Xianming; Lim, Sang Min; Heynck, Stefanie; Peifer, Martin; Simard, Jeffrey R; Lawrence, Michael S; Onofrio, Robert C; Salvesen, Helga B; Seidel, Danila; Zander, Thomas; Heuckmann, Johannes M; Soltermann, Alex; Moch, Holger; Koker, Mirjam; Leenders, Frauke; Gabler, Franziska; Querings, Silvia; Ansén, Sascha; Brambilla, Elisabeth; Brambilla, Christian; Lorimier, Philippe; Brustugun, Odd Terje; Helland, Åslaug; Petersen, Iver; Clement, Joachim H; Groen, Harry; Timens, Wim; Sietsma, Hannie; Stoelben, Erich; Wolf, Jürgen; Beer, David G; Tsao, Ming Sound; Hanna, Megan; Hatton, Charles; Eck, Michael J; Janne, Pasi A; Johnson, Bruce E; Winckler, Wendy; Greulich, Heidi; Bass, Adam J; Cho, Jeonghee; Rauh, Daniel; Gray, Nathanael S; Wong, Kwok-Kin; Haura, Eric B; Thomas, Roman K; Meyerson, Matthew

    2011-01-01

    While genomically targeted therapies have improved outcomes for patients with lung adenocarcinoma, little is known about the genomic alterations which drive squamous cell lung cancer. Sanger sequencing of the tyrosine kinome identified mutations in the DDR2 kinase gene in 3.8% of squamous cell lung cancers and cell lines. Squamous lung cancer cell lines harboring DDR2 mutations were selectively killed by knock-down of DDR2 by RNAi or by treatment with the multi-targeted kinase inhibitor dasatinib. Tumors established from a DDR2 mutant cell line were sensitive to dasatinib in xenograft models. Expression of mutated DDR2 led to cellular transformation which was blocked by dasatinib. A squamous cell lung cancer patient with a response to dasatinib and erlotinib treatment harbored a DDR2 kinase domain mutation. These data suggest that gain-of-function mutations in DDR2 are important oncogenic events and are amenable to therapy with dasatinib. As dasatinib is already approved for use, these findings could be rapidly translated into clinical trials. PMID:22328973

  14. Basal expression of the cystic fibrosis transmembrane conductance regulator gene is dependent on protein kinase A activity.

    PubMed Central

    McDonald, R A; Matthews, R P; Idzerda, R L; McKnight, G S

    1995-01-01

    The cystic fibrosis transmembrane conductance regulator (CFTR) functions as a Cl- channel that becomes activated after phosphorylation by cAMP-dependent protein kinase (PKA). We demonstrate that PKA also plays a crucial role in maintaining basal expression of the CFTR gene in the human colon carcinoma cell line T84. Inhibition of PKA activity by expression of a dominant-negative regulatory subunit or treatment with the PKA-selective inhibitor N-[2-(p-bromocinnamylamino)ethyl]-5-isoquinolinesulfonamide (H-89) caused a complete suppression of CFTR gene expression without affecting other constitutively active genes. Basal expression of a 2.2-kb region of the CFTR promoter linked to a luciferase reporter gene (CFTR-luc) exhibited the same dependence on PKA. The ability of cAMP to induce CFTR over basal levels is cell-type specific. In T84 cells, both the endogenous CFTR gene and CFTR-luc exhibited only a modest inducibility (approximately 2-fold), whereas in the human choriocarcinoma cell line JEG-3, CFTR-luc could be induced at least 4-fold. A variant cAMP-response element is present at position -48 to -41 in the CFTR promoter, and mutation of this sequence blocks basal expression. We conclude that cAMP, acting through PKA, is an essential regulator of basal CFTR gene expression and may mediate an induction of CFTR in responsive cell types. Images Fig. 1 Fig. 3 PMID:7543684

  15. Delineation of gastric cancer subtypes by co-regulated expression of receptor tyrosine kinases and chemosensitivity genes.

    PubMed

    Li, Shu-Chun; Ma, Rong; Wu, Jian-Zhong; Xiao, Xia; Wu, Wei; Li, Gang; Chen, Bo; Sharma, Ashok; Bai, Shan; Dun, Bo-Ying; She, Jin-Xiong; Tang, Jin-Hai

    2015-01-01

    Chemotherapy plays a key role in improving disease-free survival and overall survival of gastric cancer (GC); however, response rates are variable and a non-negligible proportion of patients undergo toxic and costly chemotherapeutic regimens without a survival benefit. Several studies have shown the existence of GC subtypes which may predict survival and respond differently to chemotherapy. It is also known that the expression level of chemotherapy-related and target therapy-related genes correlates with response to specific antitumor drugs. Nevertheless, these genes have not been considered jointly to define GC subtypes. In this study, we evaluated seven genes known to influence chemotherapeutic response (ERCC1, BRCA1, RRM1, TUBB3, STMN1, TYMS and TOP2A) and five receptor tyrosine kinases (RTKs) (EGFR, ERBB2, PDGFRB, VEGFR1 and VEGFR2). We demonstrate significant heterogeneity of gene expression among GC patients and identified four GC subtypes using the expression profiles of eight genes in two co-regulation groups: chemosensitivity (BRCA1, STMN1, TYMS and TOP2A) and RTKs (EGFR, PDGFRB, VEGFR1 and VEGFR2). The results are of immediate translational value regarding GC diagnostics and therapeutics, as many of these genes are curently widely used in relevant clinical testing. PMID:26396673

  16. New splicing mutation in the choline kinase beta (CHKB) gene causing a muscular dystrophy detected by whole-exome sequencing.

    PubMed

    Oliveira, Jorge; Negrão, Luís; Fineza, Isabel; Taipa, Ricardo; Melo-Pires, Manuel; Fortuna, Ana Maria; Gonçalves, Ana Rita; Froufe, Hugo; Egas, Conceição; Santos, Rosário; Sousa, Mário

    2015-06-01

    Muscular dystrophies (MDs) are a group of hereditary muscle disorders that include two particularly heterogeneous subgroups: limb-girdle MD and congenital MD, linked to 52 different genes (seven common to both subgroups). Massive parallel sequencing technology may avoid the usual stepwise gene-by-gene analysis. We report the whole-exome sequencing (WES) analysis of a patient with childhood-onset progressive MD, also presenting mental retardation and dilated cardiomyopathy. Conventional sequencing had excluded eight candidate genes. WES of the trio (patient and parents) was performed using the ion proton sequencing system. Data analysis resorted to filtering steps using the GEMINI software revealed a novel silent variant in the choline kinase beta (CHKB) gene. Inspection of sequence alignments ultimately identified the causal variant (CHKB:c.1031+3G>C). This splice site mutation was confirmed using Sanger sequencing and its effect was further evaluated with gene expression analysis. On reassessment of the muscle biopsy, typical abnormal mitochondrial oxidative changes were observed. Mutations in CHKB have been shown to cause phosphatidylcholine deficiency in myofibers, causing a rare form of CMD (only 21 patients reported). Notwithstanding interpretative difficulties that need to be overcome before the integration of WES in the diagnostic workflow, this work corroborates its utility in solving cases from highly heterogeneous groups of diseases, in which conventional diagnostic approaches fail to provide a definitive diagnosis. PMID:25740612

  17. Analysis of Noncanonical Calcium-Dependent Protein Kinases in Toxoplasma gondii by Targeted Gene Deletion Using CRISPR/Cas9.

    PubMed

    Long, Shaojun; Wang, Qiuling; Sibley, L David

    2016-05-01

    Calcium-dependent protein kinases (CDPKs) are expanded in apicomplexan parasites, especially in Toxoplasma gondii where 14 separate genes encoding these enzymes are found. Although previous studies have shown that several CDPKs play a role in controlling invasion, egress, and cell division in T. gondii, the roles of most of these genes are unexplored. Here we developed a more efficient method for gene disruption using CRISPR (clustered regularly interspaced short palindromic repeats)/Cas9 (CRISPR-associated protein 9) that was modified to completely delete large, multiexonic genes from the genome and to allow serial replacement by recycling of the selectable marker using Cre-loxP. Using this system, we generated a total of 24 mutants in type 1 and 2 genetic backgrounds to ascertain the functions of noncanonical CDPKs. Remarkably, although we were able to confirm the essentiality of CDPK1 and CDPK7, the majority of CDPKs had no discernible phenotype for growth in vitro or infection in the mouse model. The exception to this was CDPK6, loss of which leads to reduced plaquing, fitness defect in a competition assay, and reduced tissue cyst formation in chronically infected mice. Our findings highlight the utility of CRISPR/Cas9 for rapid serial gene deletion and also suggest that additional models are needed to reveal the functions of many genes in T. gondii. PMID:26755159

  18. Delineation of gastric cancer subtypes by co-regulated expression of receptor tyrosine kinases and chemosensitivity genes

    PubMed Central

    Li, Shu-Chun; Ma, Rong; Wu, Jian-Zhong; Xiao, Xia; Wu, Wei; Li, Gang; Chen, Bo; Sharma, Ashok; Bai, Shan; Dun, Bo-Ying; She, Jin-Xiong; Tang, Jin-Hai

    2015-01-01

    Chemotherapy plays a key role in improving disease-free survival and overall survival of gastric cancer (GC); however, response rates are variable and a non-negligible proportion of patients undergo toxic and costly chemotherapeutic regimens without a survival benefit. Several studies have shown the existence of GC subtypes which may predict survival and respond differently to chemotherapy. It is also known that the expression level of chemotherapy-related and target therapy-related genes correlates with response to specific antitumor drugs. Nevertheless, these genes have not been considered jointly to define GC subtypes. In this study, we evaluated seven genes known to influence chemotherapeutic response (ERCC1, BRCA1, RRM1, TUBB3, STMN1, TYMS and TOP2A) and five receptor tyrosine kinases (RTKs) (EGFR, ERBB2, PDGFRB, VEGFR1 and VEGFR2). We demonstrate significant heterogeneity of gene expression among GC patients and identified four GC subtypes using the expression profiles of eight genes in two co-regulation groups: chemosensitivity (BRCA1, STMN1, TYMS and TOP2A) and RTKs (EGFR, PDGFRB, VEGFR1 and VEGFR2). The results are of immediate translational value regarding GC diagnostics and therapeutics, as many of these genes are curently widely used in relevant clinical testing. PMID:26396673

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

    SciTech Connect

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

    1993-03-01

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

  20. A cotton fiber associated cyclin-dependent kinase A gene: Characterization and chromosomal location

    Technology Transfer Automated Retrieval System (TEKTRAN)

    A cotton fiber cell normally originates and elongates as a single ovular epidermal cell. The cessation of fiber cell division and ensuing elongation imply that the cell cycle is differentially regulated in fiber cells. Cyclin-dependent kinases (CDKs) play a central role in the regulation of cell cy...

  1. Activation of c-fos gene expression by a kinase-deficient epidermal growth factor receptor.

    PubMed Central

    Eldredge, E R; Korf, G M; Christensen, T A; Connolly, D C; Getz, M J; Maihle, N J

    1994-01-01

    The intrinsic tyrosine kinase activity of the epidermal growth factor receptor (EGFR) has been shown to be responsible for many of the pleiotropic intracellular effects resulting from ligand stimulation [W.S. Chen, C.S. Lazar, M. Poenie, R.Y. Tsien, G.N. Gill, and M.G. Rosenfeld, Nature (London) 328:820-823, 1987; A.M. Honegger, D. Szapary, A. Schmidt, R. Lyall, E. Van Obberghen, T.J. Dull, A. Ulrich, and J. Schlessinger, Mol. Cell. Biol. 7:4568-4571, 1987]. Recently, however, it has been shown that addition of ligand to cells expressing kinase-defective EGFR mutants can result in the phosphorylation of mitogen-activated protein kinase (R. Campos-González and J.R. Glenney, Jr., J. Biol. Chem. 267:14535-14538, 1992; E. Selva, D.L. Raden, and R.J. Davis, J. Biol. Chem. 268:2250-2254, 1993), as well as stimulation of DNA synthesis (K.J. Coker, J.V. Staros, and C.A. Guyer, Proc. Natl. Acad. Sci. USA 91:6967-6971, 1994). Moreover, mitogen-activated protein kinase has been shown to phosphorylate the transcription factor p62TCF in vitro, leading to enhanced ternary complex formation between p62TCF, p67SRF, and the c-fos serum response element (SRE) [H. Gille, A.D. Sharrocks, and P.E. Shaw, Nature (London) 358:414-417, 1992]. On the basis of these observations, we have investigated the possibility that the intrinsic tyrosine kinase activity of the EGFR may not be necessary for transcriptional activation mediated via p62TCF. Here, we demonstrate that a kinase-defective EGFR mutant can signal ligand-induced expression of c-fos protein and that a significant component of this induction appears to be mediated at the transcriptional level. Investigation of transcriptional activation mediated by the c-fos SRE shows that this response is impaired by mutations in the SRE which eliminate binding of p62(TCF). These data indicate that information inherent in the structure of the EGFR can be accessed by ligand stimulation independent of the receptor's catalytic kinase function

  2. Transcriptomics-based screen for genes induced by flagellin and repressed by pathogen effectors identifies a cell wall-associated kinase involved in plant immunity

    PubMed Central

    2013-01-01

    Background Microbe-associated molecular patterns, such as those present in bacterial flagellin, are powerful inducers of the innate immune response in plants. Successful pathogens deliver virulence proteins, termed effectors, into the plant cell where they can interfere with the immune response and promote disease. Engineering the plant immune system to enhance disease resistance requires a thorough understanding of its components. Results We describe a high-throughput screen, using RNA sequencing and virus-induced gene silencing, to identify tomato genes whose expression is enhanced by the flagellin microbe-associated molecular pattern flgII-28, but reduced by activities of the Pseudomonas syringae pv. tomato (Pst) type III effectors AvrPto and AvrPtoB. Gene ontology terms for this category of Flagellin-induced repressed by effectors (FIRE) genes showed enrichment for genes encoding certain subfamilies of protein kinases and transcription factors. At least 25 of the FIRE genes have been implicated previously in plant immunity. Of the 92 protein kinase-encoding FIRE genes, 33 were subjected to virus-induced gene silencing and their involvement in pattern-triggered immunity was tested with a leaf-based assay. Silencing of one FIRE gene, which encodes the cell wall-associated kinase SlWAK1, compromised the plant immune response resulting in increased growth of Pst and enhanced disease symptoms. Conclusions Our transcriptomic approach identifies FIRE genes that represent a pathogen-defined core set of immune-related genes. The analysis of this set of candidate genes led to the discovery of a cell wall-associated kinase that participates in plant defense. The FIRE genes will be useful for further elucidation of the plant immune system. PMID:24359686

  3. Identification of a receptor-like protein kinase gene rapidly induced by abscisic acid, dehydration, high salt, and cold treatments in Arabidopsis thaliana.

    PubMed Central

    Hong, S W; Jon, J H; Kwak, J M; Nam, H G

    1997-01-01

    A cDNA clone for a receptor-like protein kinase gene (RPK1) was isolated from Arabidopsis thaliana. The clone is 1952 bp long with 1623 bp of an open reading frame encoding a peptide of 540 amino acids. The deduced peptide (RPK1) contains four distinctive domains characteristic of receptor kinases: (a) a putative amino-terminal signal sequence domain; (b) a domain with five extracellular leucine-rich repeat sequences; (c) a membrane-spanning domain; and (d) a cytoplasmic protein kinase domain that contains all of the 11 subdomains conserved among protein kinases. The RPK1 gene is expressed in flowers, stems, leaves, and roots. Expression of the RPK1 gene is induced within 1 h after treatment with abscisic acid (ABA). The gene is also rapidly induced by several environmental stresses such as dehydration, high salt, and low temperature, suggesting that the gene is involved in a general stress response. The dehydration-induced expression is not impaired in aba-1, abi1-1, abi2-1, and abi3-1 mutants, suggesting that the dehydration-induced expression of the RPK1 gene is ABA-independent. A possible role of this gene in the signal transduction pathway of ABA and the environmental stresses is discussed. PMID:9112773

  4. Proline-Rich Tyrosine Kinase 2 Mediates Gonadotropin-Releasing Hormone Signaling to a Specific Extracellularly Regulated Kinase-Sensitive Transcriptional Locus in the Luteinizing Hormone β-Subunit Gene

    PubMed Central

    Maudsley, Stuart; Naor, Zvi; Bonfil, David; Davidson, Lindsay; Karali, Dimitra; Pawson, Adam J.; Larder, Rachel; Pope, Caroline; Nelson, Nancy; Millar, Robert P.; Brown, Pamela

    2007-01-01

    G protein-coupled receptor regulation of gene transcription primarily occurs through the phosphorylation of transcription factors by MAPKs. This requires transduction of an activating signal via scaffold proteins that can ultimately determine the outcome by binding signaling kinases and adapter proteins with effects on the target transcription factor and locus of activation. By investigating these mechanisms, we have elucidated how pituitary gonadotrope cells decode an input GnRH signal into coherent transcriptional output from the LH β-subunit gene promoter. We show that GnRH activates c-Src and multiple members of the MAPK family, c-Jun NH2-terminal kinase 1/2, p38MAPK, and ERK1/2. Using dominant-negative point mutations and chemical inhibitors, we identified that calcium-dependent proline-rich tyrosine kinase 2 specifically acts as a scaffold for a focal adhesion/cytoskeleton-dependent complex comprised of c-Src, Grb2, and mSos that translocates an ERK-activating signal to the nucleus. The locus of action of ERK was specifically mapped to early growth response-1 (Egr-1) DNA binding sites within the LH β-subunit gene proximal promoter, which was also activated by p38MAPK, but not c-Jun NH2-terminal kinase 1/2. Egr-1 was confirmed as the transcription factor target of ERK and p38MAPK by blockade of protein expression, transcriptional activity, and DNA binding. We have identified a novel GnRH-activated proline-rich tyrosine kinase 2-dependent ERK-mediated signal transduction pathway that specifically regulates Egr-1 activation of the LH β-subunit proximal gene promoter, and thus provide insight into the molecular mechanisms required for differential regulation of gonadotropin gene expression. PMID:17327421

  5. Identification, expression and interaction analyses of calcium-dependent protein kinase (CPK) genes in canola (Brassica napus L.)

    PubMed Central

    2014-01-01

    Background Canola (Brassica napus L.) is one of the most important oil-producing crops in China and worldwide. The yield and quality of canola is frequently threatened by environmental stresses including drought, cold and high salinity. Calcium is a well-known ubiquitous intracellular secondary messenger in plants. Calcium-dependent protein kinases (CPKs) are Ser/Thr protein kinases found only in plants and some protozoans. CPKs are Ca2+ sensors that have both Ca2+ sensing function and kinase activity within a single protein and play crucial roles in plant development and responses to various environmental stresses. Results In this study, we mined the available expressed sequence tags (ESTs) of B. napus and identified a total of 25 CPK genes, among which cDNA sequences of 23 genes were successfully cloned from a double haploid cultivar of canola. Phylogenetic analysis demonstrated that they could be clustered into four subgroups. The subcellular localization of five selected BnaCPKs was determined using green fluorescence protein (GFP) as the reporter. Furthermore, the expression levels of 21 BnaCPK genes in response to salt, drought, cold, heat, abscisic acid (ABA), low potassium (LK) and oxidative stress were studied by quantitative RT-PCR and were found to respond to multiple stimuli, suggesting that canola CPKs may be convergence points of different signaling pathways. We also identified and cloned five and eight Clade A basic leucine zipper (bZIP) and protein phosphatase type 2C (PP2C) genes from canola and, using yeast two-hybrid and bimolecular fluorescence complementation (BiFC), determined the interaction between individual BnaCPKs and BnabZIPs or BnaPP2Cs (Clade A). We identified novel, interesting interaction partners for some of the BnaCPK proteins. Conclusion We present the sequences and characterization of CPK gene family members in canola for the first time. This work provides a foundation for further crop improvement and improved understanding of

  6. Cloning and characterization of a maize SnRK2 protein kinase gene confers enhanced salt tolerance in transgenic Arabidopsis.

    PubMed

    Ying, Sheng; Zhang, Deng-Feng; Li, Hui-Yong; Liu, Ying-Hui; Shi, Yun-Su; Song, Yan-Chun; Wang, Tian-Yu; Li, Yu

    2011-09-01

    SnRK2 (sucrose non-fermenting 1-related protein kinases 2) represents a unique family of protein kinase in regulating signaling transduction in plants. Although the regulatory mechanisms of SnRK2 have been well demonstrated in Arabidopsis thaliana, their functions in maize are still unknown. In our study, we cloned an SnRK2 gene from maize, ZmSAPK8, which encoded a putative homolog of the rice SAPK8 protein. ZmSAPK8 had two copies in the maize genome and harbored eight introns in its coding region. We demonstrated that ZmSAPK8 expressed differentially in various organs of maize plants and was up-regulated by high-salinity and drought treatment. A green fluorescent protein (GFP)-tagged ZmSAPK8 showed subcellular localization in the cell membrane, cytoplasm and nucleus. In vitro kinase assays indicated that ZmSAPK8 preferred Mn(2+) to Mg(2+) as cofactor for phosphorylation, and Ser-182 and Thr-183 in activation loop was important for its activity. Heterologous overexpression of ZmSAPK8 in Arabidopsis could significantly strengthen tolerance to salt stress. Under salt treatment, ZmSAPK8-overexpressed transgenic plants exhibited higher germination rate and proline content, low electrolyte leakage and higher survival rate than wild type. Further analysis indicated that transgenic plants showed increased transcription of the stress-related genes, RD29A, RD29B, RAB18, ABI1, DREB2A and P5CS1, under high-salinity conditions. The results demonstrated that ZmSAPK8 was involved in diverse stress signal transduction. Moreover, no obvious adverse effects on growth and development in the ZmSAPK8-overexpressed transgenic plants implied that ZmSAPK8 was potentially useful in transgenic breeding to improve salt tolerance in crops. PMID:21638061

  7. 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

  8. 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. PMID:27330719

  9. A gene encoding a potential adenosine 5'-phosphosulphate kinase is necessary for timely development of Myxococcus xanthus.

    PubMed

    Wang, Daoyong; Xu, Shihui; Song, Dan; Knight, Stefan; Mao, Xiaohua

    2016-04-01

    A Myxococcus xanthus gene, MXAN3487, was identified by transposon mutagenesis to be required for the expression of mcuABC, an operon coding for part of the chaperone-usher (CU) system in this bacterium. The MXAN3487 protein displays sequence and structural homology to adenosine 5'-phosphosulphate (APS) kinase family members and contains putative motifs for ATP and APS binding. Although the MXAN3487 locus is not linked to other sulphate assimilation genes, its protein product may have APS kinase activity in vivo and the importance of the ATP-binding site for activity was demonstrated. Expression of MXAN3487 was not affected by sulphate availability, suggesting that MXAN3487 may not function in a reductive sulphate assimilation pathway. Deletion of MXAN3487 significantly delayed fruiting body formation and the production of McuA, a spore coat protein secreted by the M. xanthus Mcu CU system. Based on these observations and data from our previous studies, we propose that MXAN3487 may phosphorylate molecules structurally related to APS, generating metabolites necessary for M. xanthus development, and that MXAN3487 exerts a positive effect on the mcuABC operon whose expression is morphogenesis dependent. PMID:26860640

  10. Aberrant promoter hypermethylation of the death-associated protein kinase gene is early and frequent in murine lung tumors induced by cigarette smoke and tobacco carcinogens.

    PubMed

    Pulling, Leah C; Vuillemenot, Brian R; Hutt, Julie A; Devereux, Theodora R; Belinsky, Steven A

    2004-06-01

    Loss of expression of the death-associated protein (DAP)-kinase gene by aberrant promoter methylation may play an important role in cancer development and progression. The purpose of this investigation was to determine the commonality for inactivation of the DAP-kinase gene in adenocarcinomas induced in mice by chronic exposure to mainstream cigarette smoke, the tobacco carcinogens 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) and vinyl carbamate, and the occupational carcinogen methylene chloride. The timing for inactivation was also determined in alveolar hyperplasias that arise in lung cancer induced in the A/J mouse by NNK. The DAP-kinase gene was not expressed in three of five NNK-induced lung tumor-derived cell lines or in a spontaneously arising lung tumor-derived cell line. Treatment with 5-aza-2'-deoxycytidine restored expression; dense methylation throughout the DAP-kinase CpG island detected by bisulfite sequencing supported methylation as the inactivating event in these cell lines. Methylation-specific PCR detected inactivation of the DAP-kinase gene in 43% of tumors associated with cigarette smoke, a frequency similar to those reported in human non-small cell lung cancer. In addition, DAP-kinase methylation was detected in 52%, 60%, and 50% of tumors associated with NNK, vinyl carbamate, and methylene chloride, respectively. Methylation was observed at similar prevalence in both NNK-induced hyperplasias and adenocarcinomas (46% versus 52%), suggesting that inactivation of this gene is one pathway for tumor development in the mouse lung. Bisulfite sequencing of both premalignant and malignant lesions revealed dense methylation, substantiating that this gene is functionally inactivated at the earliest histological stages of adenocarcinoma development. This study is the first to use a murine model of cigarette smoke-induced lung cancer and demonstrate commonality for inactivation by promoter hypermethylation of a gene implicated in the development

  11. Different regulatory sequences control creatine kinase-M gene expression in directly injected skeletal and cardiac muscle.

    PubMed Central

    Vincent, C K; Gualberto, A; Patel, C V; Walsh, K

    1993-01-01

    Regulatory sequences of the M isozyme of the creatine kinase (MCK) gene have been extensively mapped in skeletal muscle, but little is known about the sequences that control cardiac-specific expression. The promoter and enhancer sequences required for MCK gene expression were assayed by the direct injection of plasmid DNA constructs into adult rat cardiac and skeletal muscle. A 700-nucleotide fragment containing the enhancer and promoter of the rabbit MCK gene activated the expression of a downstream reporter gene in both muscle tissues. Deletion of the enhancer significantly decreased expression in skeletal muscle but had no detectable effect on expression in cardiac muscle. Further deletions revealed a CArG sequence motif at position -179 within the promoter that was essential for cardiac-specific expression. The CArG element of the MCK promoter bound to the recombinant serum response factor and YY1, transcription factors which control expression from structurally similar elements in the skeletal actin and c-fos promoters. MCK-CArG-binding activities that were similar or identical to serum response factor and YY1 were also detected in extracts from adult cardiac muscle. These data suggest that the MCK gene is controlled by different regulatory programs in adult cardiac and skeletal muscle. Images PMID:8423791

  12. Ultrasound Microbubble-Mediated Delivery of Integrin-Linked Kinase Gene Improves Endothelial Progenitor Cells Dysfunction in Pre-Eclampsia

    PubMed Central

    Cui, Kai; Yan, Ting; Luo, Qingqing; Zheng, Yanfang; Liu, Xiaoxia; Huang, Xiaoyu

    2014-01-01

    Pre-eclampsia (PE) is a specific vascular complication in pregnancy whose precise mechanism is still unclear. We hypothesized that endothelial progenitor cells (EPCs), the precursor of endothelial cells, might be impaired in patients with PE and hold a great promise for the treatment of PE. In the present study, we analyzed the EPCs number and expression of integrin-linked kinase (ILK) in PE patients. We confirmed that both EPCs number and ILK expression were diminished in PE patients. Next, we transfected EPCs with ILK gene using ultrasonic microbubble technique (UMT) for the first time, as UMT is a novel type of gene transfer technology showing promising applications in stem cells apart from EPCs. To further investigate the transfection efficiency of UMT, RT-PCR analysis and western blot were used to examine the messenger RNA (mRNA) and protein level of ILK. After transfection of the ILK gene, EPCs function was tested to illustrate the role of ILK in cell proliferation, apoptosis, migration, and secretion. The results of the in vitro study suggested that UMT, a novel gene delivery system, could be considered a potent physical method for EPCs transfection. Moreover, the growth and angiogenetic properties of EPCs are enhanced by introducing ILK. This study may afford a new trend for EPCs transfection and gene therapy in PE. PMID:24564279

  13. Toward Understanding the Functional Role of Ss-riok-1, a RIO Protein Kinase-Encoding Gene of Strongyloides stercoralis

    PubMed Central

    Yuan, Wang; Lok, James B.; Stoltzfus, Jonathan D.; Gasser, Robin B.; Fang, Fang; Lei, Wei-Qiang; Fang, Rui; Zhou, Yan-Qin; Zhao, Jun-Long; Hu, Min

    2014-01-01

    Background Some studies of Saccharomyces cerevisiae and mammals have shown that RIO protein kinases (RIOKs) are involved in ribosome biogenesis, cell cycle progression and development. However, there is a paucity of information on their functions in parasitic nematodes. We aimed to investigate the function of RIOK-1 encoding gene from Strongyloides stercoralis, a nematode parasitizing humans and dogs. Methodology/Principal Findings The RIOK-1 protein-encoding gene Ss-riok-1 was characterized from S. stercoralis. The full-length cDNA, gDNA and putative promoter region of Ss-riok-1 were isolated and sequenced. The cDNA comprises 1,828 bp, including a 377 bp 5′-UTR, a 17 bp 3′-UTR and a 1,434 bp ORF encoding a protein of 477 amino acids containing a RIOK-1 signature motif. The genomic sequence of the Ss-riok-1 coding region is 1,636 bp in length and has three exons and two introns. The putative promoter region comprises 4,280 bp and contains conserved promoter elements, including four CAAT boxes, 12 GATA boxes, eight E-boxes (CANNTG) and 38 TATA boxes. The Ss-riok-1 gene is transcribed throughout all developmental stages with the highest transcript abundance in the infective third-stage larva (iL3). Recombinant Ss-RIOK-1 is an active kinase, capable of both phosphorylation and auto-phosphorylation. Patterns of transcriptional reporter expression in transgenic S. stercoralis larvae indicated that Ss-RIOK-1 is expressed in neurons of the head, body and tail as well as in pharynx and hypodermis. Conclusions/Significance The characterization of the molecular and the temporal and spatial expression patterns of the encoding gene provide first clues as to functions of RIOKs in the biological processes of parasitic nematodes. PMID:25101874

  14. Differential Roles of Two Homologous Cyclin-Dependent Kinase Inhibitor Genes in Regulating Cell Cycle and Innate Immunity in Arabidopsis.

    PubMed

    Hamdoun, Safae; Zhang, Chong; Gill, Manroop; Kumar, Narender; Churchman, Michelle; Larkin, John C; Kwon, Ashley; Lu, Hua

    2016-01-01

    Precise cell-cycle control is critical for plant development and responses to pathogen invasion. Two homologous cyclin-dependent kinase inhibitor genes, SIAMESE (SIM) and SIM-RELATED 1 (SMR1), were recently shown to regulate Arabidopsis (Arabidopsis thaliana) defense based on phenotypes conferred by a sim smr1 double mutant. However, whether these two genes play differential roles in cell-cycle and defense control is unknown. In this report, we show that while acting synergistically to promote endoreplication, SIM and SMR1 play different roles in affecting the ploidy of trichome and leaf cells, respectively. In addition, we found that the smr1-1 mutant, but not sim-1, was more susceptible to a virulent Pseudomonas syringae strain, and this susceptibility could be rescued by activating salicylic acid (SA)-mediated defense. Consistent with these results, smr1-1 partially suppressed the dwarfism, high SA levels, and cell death phenotypes in acd6-1, a mutant used to gauge the change of defense levels. Thus, SMR1 functions partly through SA in defense control. The differential roles of SIM and SMR1 are due to differences in temporal and spatial expression of these two genes in Arabidopsis tissues and in response to P. syringae infection. In addition, flow-cytometry analysis of plants with altered SA signaling revealed that SA is necessary, but not sufficient, to change cell-cycle progression. We further found that a mutant with three CYCD3 genes disrupted also compromised disease resistance to P. syringae. Together, this study reveals differential roles of two homologous cyclin-dependent kinase inhibitors in regulating cell-cycle progression and innate immunity in Arabidopsis and provides insights into the importance of cell-cycle control during host-pathogen interactions. PMID:26561564

  15. Arginine kinase: differentiation of gene expression and protein activity in the red imported fire ant, Solenopsis invicta.

    PubMed

    Wang, Haichuan; Zhang, Lan; Zhang, Lee; Lin, Qin; Liu, Nannan

    2009-02-01

    Arginine kinase (AK), a primary enzyme in cell metabolism and adenosine 5'-triphosphate (ATP)-consuming processes, plays an important role in cellular energy metabolism and maintaining constant ATP levels in invertebrate cells. In order to identify genes that are differentially expressed between larvae and adults, queens and workers, and female alates (winged) and queens (wingless), AK cDNA was obtained from the red imported fire ant. The cDNA sequence of the gene has open reading frames of 1065 nucleotides, encoding a protein of 355 amino acid residues that includes the substrate recognition region, the signature sequence pattern of ATP:guanidino kinases, and an "actinin-type" actin binding domain. Northern blot analysis and protein activity analysis demonstrated that the expression of the AK gene and its protein activity were developmentally, caste specifically, and tissue specifically regulated in red imported fire ants with a descending order of worker> alate (winged adult) female> alate (winged adult) male> larvae> worker pupae approximately alate pupae. These results suggest a different demand for energy-consumption and production in the different castes of the red imported fire ant, which may be linked to their different missions and physiological activities in the colonies. The highest level of the AK gene expression and activity was identified in head tissue of both female alates and workers and thorax tissue of workers, followed by thorax tissue of female alates and abdomen tissue of male alates, suggesting the main tissues or cells in these body parts, such as brain, neurons and muscles, which have been identified as the major tissues and/or cells that display high and variable rates of energy turnover in other organisms, play a key role in energy production and its utilization in the fire ant. In contrast, in the male alate, the highest AK expression and activity were found in the abdomen, suggesting that here energy demand may relate to sperm formation

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

    PubMed

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

    2014-08-01

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

  17. Sphingosine 1-phosphate stimulation of the p42/p44 mitogen-activated protein kinase pathway in airway smooth muscle. Role of endothelial differentiation gene 1, c-Src tyrosine kinase and phosphoinositide 3-kinase.

    PubMed Central

    Rakhit, S; Conway, A M; Tate, R; Bower, T; Pyne, N J; Pyne, S

    1999-01-01

    We report here that cultured airway smooth muscle cells contain transcripts of endothelial differentiation gene 1 (EDG-1), a prototypical orphan Gi-coupled receptor whose natural ligand is sphingosine 1-phosphate (S1P). This is consistent with data that showed that S1P activated both c-Src and p42/p44 mitogen-activated protein kinase (p42/p44 MAPK) in a pertussis toxin (PTX)-sensitive manner in these cells. An essential role for c-Src was confirmed by using the c-Src inhibitor, PP1, which markedly decreased p42/p44 MAPK activation. We have also shown that phosphoinositide 3-kinase (PI-3K) inhibitors (wortmannin and LY294002) decreased p42/p44 MAPK activation. An essential role for PI-3K was supported by experiments that showed that PI-3K activity was increased in Grb-2 immunoprecipitates from S1P-stimulated cells. Significantly, Grb-2 associated PI-3K activity was decreased by pretreatment of cells with PTX. Finally, we have shown that the co-stimulation of cells with platelet-derived growth factor (PDGF) and S1P (which failed to stimulate DNA synthesis) elicited a larger p42/p44 MAPK activation over a 30 min stimulation compared with each agonist alone. This was associated with a S1P-dependent increase in PDGF-stimulated DNA synthesis. These results demonstrate that S1P activates c-Src and Grb-2-PI-3K (intermediates in the p42/p44 MAPK cascade) via a PTX-sensitive mechanism. This action of S1P is consistent with the stimulation of EDG-1 receptors. S1P might also function as a co-mitogen with PDGF, producing a more robust activation of a common permissive signal transduction pathway linked to DNA synthesis. PMID:10051434

  18. A phosphatidylinositol (PI) kinase gene family in Dictyostelium discoideum: biological roles of putative mammalian p110 and yeast Vps34p PI 3-kinase homologs during growth and development.

    PubMed Central

    Zhou, K; Takegawa, K; Emr, S D; Firtel, R A

    1995-01-01

    Three groups of phosphatidylinositol (PI) kinases convert PI into PI(3)phosphate, PI(4)phosphate, PI(4,5) bisphosphate, and PI(3,4,5)trisphosphate. These phosphoinositides have been shown to function in vesicle-mediated protein sorting, and they serve as second-messenger signaling molecules for regulating cell growth. To further elucidate the mechanism of regulation and function of phosphoinositides, we cloned genes encoding five putative PI kinases from Dictyostelium discoideum. Database analysis indicates that D. discoideum PIK1 (DdPIK1), -2, and -3 are most closely related to the mammalian p110 PI 3-kinase, DdPIK5 is closest to the yeast Vps34p PI 3-kinase, and DdPIK4 is most homologous to PI 4-kinases. Together with other known PI kinases, a superfamily of PI kinase genes has been defined, with all of the encoded proteins sharing a common highly conserved catalytic core domain. DdPIK1, -2, and -3 may have redundant functions because disruption of any single gene had no effect on D. discoideum growth or development. However, strains in which both of the two most highly related genes, DdPIK1 and DdPIK2, were disrupted showed both growth and developmental defects, while double knockouts of DdPIK1 and DdPIK3 and DdPIK2 and DdPIK3 appear to be lethal. The delta Ddpik1 delta Ddpik2 null cells were smaller than wild-type cells and grew slowly both in association with bacteria and in axenic medium when attached to petri plates but were unable to grow in suspension in axenic medium. When delta Ddpik1 delta Ddpik2 null cells were plated for multicellular development, they formed aggregates having multiple tips and produced abnormal fruiting bodies. Antisense expression of DdPIK5 (a putative homolog of the Saccharomyces cerevisiae VPS34) led to a defect in the growth of D. discoideum cells on bacterial lawns and abnormal development. DdPIK5 complemented the temperature-sensitive growth defect of a Schizosaccharomyces pombe delta Svps34 mutant strain, suggesting DdPIK5

  19. Nuclear localization of lymphocyte-specific protein tyrosine kinase (Lck) and its role in regulating LIM domain only 2 (Lmo2) gene

    SciTech Connect

    Venkitachalam, Srividya; Chueh, Fu-Yu; Yu, Chao-Lan

    2012-01-20

    Highlights: Black-Right-Pointing-Pointer Lmo2 expression is elevated in Lck-transformed cells. Black-Right-Pointing-Pointer Both endogenous and exogenous Lck localize in the nucleus. Black-Right-Pointing-Pointer Nuclear Lck is active in Lck-transformed cells. Black-Right-Pointing-Pointer Lck binds to the promoter region of Lmo2 gene in vivo. Black-Right-Pointing-Pointer In contrast to JAK2, Lck does not increase histone H3 phosphorylation on Tyr 41. -- Abstract: LIM domain only protein 2 (Lmo2) is a transcription factor that plays a critical role in the development of T-acute lymphoblastic leukemia (T-ALL). A previous report established a link between Lmo2 expression and the nuclear presence of oncogenic Janus kinase 2 (JAK2), a non-receptor protein tyrosine kinase. The oncogenic JAK2 kinase phosphorylates histone H3 on Tyr 41 that leads to the relief of Lmo2 promoter repression and subsequent gene expression. Similar to JAK2, constitutive activation of lymphocyte-specific protein tyrosine kinase (Lck) has been implicated in lymphoid malignancies. However, it is not known whether oncogenic Lck regulates Lmo2 expression through a similar mechanism. We show here that Lmo2 expression is significantly elevated in T cell leukemia LSTRA overexpressing active Lck kinase and in HEK 293 cells expressing oncogenic Y505FLck kinase. Nuclear localization of active Lck kinase was confirmed in both Lck-transformed cells by subcellular fractionation and immunofluorescence microscopy. More importantly, in contrast to oncogenic JAK2, oncogenic Lck kinase does not result in significant increase in histone H3 phosphorylation on Tyr 41. Instead, chromatin immunoprecipitation experiment shows that oncogenic Y505FLck kinase binds to the Lmo2 promoter in vivo. This result raises the possibility that oncogenic Lck may activate Lmo2 promoter through direct interaction.

  20. Metallothionein gene expression is regulated by serum factors and activators of protein kinase C.

    PubMed Central

    Imbra, R J; Karin, M

    1987-01-01

    The exact physiological role of metallothionein (MT) is not clear. It has been suggested that these low-molecular-weight, highly inducible, heavy-metal-binding proteins serve in the regulation of intracellular Zn metabolism. Among the Zn-requiring systems are several enzymes involved in DNA replication and repair. Therefore, during periods of active DNA synthesis there is likely to be an increased demand for Zn, which could be met by elevated MT synthesis. For that reason, we examined whether stimulation of cellular proliferation leads to increased expression of MT. We report here that treatment of cultured mammalian cells with serum growth factors and activators of protein kinase C, all of which are known to have growth stimulatory activity, led to induction of MT mRNA. One of the required steps in the signal transduction pathways triggered by these agents, ending in MT induction, appears to be the activation of protein kinase C. Images PMID:3600629

  1. Selective anticancer activity of a hexapeptide with sequence homology to a non-kinase domain of Cyclin Dependent Kinase 4

    PubMed Central

    2011-01-01

    Background Cyclin-dependent kinases 2, 4 and 6 (Cdk2, Cdk4, Cdk6) are closely structurally homologous proteins which are classically understood to control the transition from the G1 to the S-phases of the cell cycle by combining with their appropriate cyclin D or cyclin E partners to form kinase-active holoenzymes. Deregulation of Cdk4 is widespread in human cancer, CDK4 gene knockout is highly protective against chemical and oncogene-mediated epithelial carcinogenesis, despite the continued presence of CDK2 and CDK6; and overexpresssion of Cdk4 promotes skin carcinogenesis. Surprisingly, however, Cdk4 kinase inhibitors have not yet fulfilled their expectation as 'blockbuster' anticancer agents. Resistance to inhibition of Cdk4 kinase in some cases could potentially be due to a non-kinase activity, as recently reported with epidermal growth factor receptor. Results A search for a potential functional site of non-kinase activity present in Cdk4 but not Cdk2 or Cdk6 revealed a previously-unidentified loop on the outside of the C'-terminal non-kinase domain of Cdk4, containing a central amino-acid sequence, Pro-Arg-Gly-Pro-Arg-Pro (PRGPRP). An isolated hexapeptide with this sequence and its cyclic amphiphilic congeners are selectively lethal at high doses to a wide range of human cancer cell lines whilst sparing normal diploid keratinocytes and fibroblasts. Treated cancer cells do not exhibit the wide variability of dose response typically seen with other anticancer agents. Cancer cell killing by PRGPRP, in a cyclic amphiphilic cassette, requires cells to be in cycle but does not perturb cell cycle distribution and is accompanied by altered relative Cdk4/Cdk1 expression and selective decrease in ATP levels. Morphological features of apoptosis are absent and cancer cell death does not appear to involve autophagy. Conclusion These findings suggest a potential new paradigm for the development of broad-spectrum cancer specific therapeutics with a companion diagnostic

  2. Selection variability for Arg48His in alcohol dehydrogenase ADH1B among Asian populations.

    PubMed

    Evsyukov, Alexey; Ivanov, Denis

    2013-08-01

    The variant His at codon 48 of the alcohol dehydrogenase gene (ADH1B) results in more efficient ethanol metabolism than with the "typical" codon 48Arg. In this study we introduced selection properties of Arg48His genotypes of ADH1B and estimated fitness in four ethnic-geographical clusters in Asia. Population genetics models were employed that derive observed gene frequencies from fitness relationships among genotypes, to infer the selection pattern of polymorphisms in an indirect manner. The data were analyzed using the model of "complete stationary distribution" by Wright that takes into account random genetic drift, pressure of migrations, mutations, and selection as influential factors of gene frequency. We found that the different population groups showed some variation in the types of selection for Arg48His. Han Chinese from eastern and southeastern China and the Japanese and Korean populations showed stabilizing selection, while the groups from Central Asian and Indochina showed divergent selection. However, all the groups demonstrated a strong positive selection for Arg48His. PMID:25019189

  3. GSIV serine/threonine kinase can induce apoptotic cell death via p53 and pro-apoptotic gene Bax upregulation in fish cells.

    PubMed

    Reshi, Latif; Wu, Horng-Cherng; Wu, Jen-Leih; Wang, Hao-Ven; Hong, Jiann-Ruey

    2016-04-01

    Previous studies have shown that GSIV induces apoptotic cell death through upregulation of the pro-apoptotic genes Bax and Bak in Grouper fin cells (GF-1 cells). However, the role of viral genome-encoded protein(s) in this death process remains unknown. In this study, we demonstrated that the Giant seaperch iridovirus (GSIV) genome encoded a serine/threonine kinase (ST kinase) protein, and induced apoptotic cell death via a p53-mediated Bax upregulation approach and a downregulation of Bcl-2 in fish cells. The ST kinase expression profile was identified through Western blot analyses, which indicated that expression started at day 1 h post-infection (PI), increased up to day 3, and then decreased by day 5 PI. This profile indicated the role of ST kinase expression during the early and middle phases of viral replication. We then cloned the ST kinase gene and tested its function in fish cells. The ST kinase was transiently expressed and used to investigate possible novel protein functions. The transient expression of ST kinase in GF-1 cells resulted in apoptotic cell features, as revealed with Terminal deoxynucleotidyl transferase biotin-dUTP nick-end labeling (TUNEL) assays and Hoechst 33258 staining at 24 h (37 %) and 48 h post-transfection (PT) (49 %). Then, through studies on the mechanism of cell death, we found that ST kinase overexpression could upregulate the anti-stress gene p53 and the pro-apoptotic gene Bax at 48 h PT. Interestingly, this upregulation of p53 and Bax also correlated to alterations in the mitochondria function that induced loss of mitochondrial membrane potential (MMP) and activated the initiator caspase-9 and the effector caspase-3 in the downstream. Moreover, when the p53-dependent transcriptional downstream gene was blocked by a specific transcriptional inhibitor, it was found that pifithrin-α not only reduced Bax expression, but also averted cell death in GF-1 cells during the ST kinase overexpression. Taken altogether, these

  4. Src Subfamily Kinases Regulate Nuclear Export and Degradation of Transcription Factor Nrf2 to Switch Off Nrf2-mediated Antioxidant Activation of Cytoprotective Gene Expression*

    PubMed Central

    Niture, Suryakant K.; Jain, Abhinav K.; Shelton, Phillip M.; Jaiswal, Anil K.

    2011-01-01

    Nrf2 (NF-E2-related factor 2) is a nuclear transcription factor that in response to chemical and radiation stress regulates coordinated induction of a battery of cytoprotective gene expressions leading to cellular protection. In this study, we investigated the role of Src kinases in the regulation of Nrf2 and downstream signaling. siRNA-mediated inhibition of Fyn, Src, Yes, and Fgr, but not Lyn, in mouse hepatoma Hepa-1 cells, led to nuclear accumulation of Nrf2 and up-regulation of Nrf2 downstream gene expression. Mouse embryonic fibroblasts with combined deficiency of Fyn/Src/Yes/Fgr supported results from siRNA. In addition, steady-state overexpression of Fyn, Src, and Yes phosphorylated Nrf2Tyr568 that triggered nuclear export and degradation of Nrf2 and down-regulation of Nrf2 downstream gene expression. Exposure of cells to antioxidant, oxidant, or UV radiation increased nuclear import of Fyn, Src, and Yes kinases, which phosphorylated Nrf2Tyr568 resulting in nuclear export and degradation of Nrf2. Further analysis revealed that stress-activated GSK3β acted upstream to the Src kinases and phosphorylated the Src kinases, leading to their nuclear localization and Nrf2 phosphorylation. The overexpression of Src kinases in Hepa-1 cells led to decreased Nrf2, increased apoptosis, and decreased cell survival. Mouse embryonic fibroblasts deficient in Src kinases showed nuclear accumulation of Nrf2, induction of Nrf2 and downstream gene expression, reduced apoptosis, and increased cell survival. The studies together demonstrate that Src kinases play a critical role in nuclear export and degradation of Nrf2, thereby providing a negative feedback mechanism to switch off Nrf2 activation and restore normal cellular homeostasis. PMID:21690096

  5. Turn-on of Inactive Genes by Promoter Recruitment in ESCHERICHIA COLI: Inverted Repeats Resulting in Artificial Divergent Operons

    PubMed Central

    Charlier, Daniel; Severne, Yvonne; Zafarullah, Muhamad; Glansdorff, Nicolas

    1983-01-01

    We have characterized two rearrangements consisting of inverted repeats of the argE gene. The promoters (p) of argE and of argCBH face each other over an internal operator. The rearrangements were obained as reactivations of argE in a strain harboring an argEp deletion on a λdarg prophage. In both cases the repeat included argE and argCBHp on either side of a unique sequence; the result is a divergent operon in which each copy of argCBHp reads into the adjacent argE repeat. In one case, the pair of repeats adjoins the silent parental gene, forming a triplication (← → ←). The other rearrangement consists of a single argE palindrome, but the whole prophage is rearranged into an inverted repeat, analogous to certain λdv's. Both structures could be explained by breakage of a replication fork passing argE and by inaccurate rejoining of strands. The λdv-like rearrangement would result from breakage at both replication forks of a phage or prophage replicating during transient release of immunity. The triplication would imply breaking of a chromosomal replication fork, formation of a cyclic intermediate by recombination between the daughter duplex molecules and reinsertion into the parental argE gene. Formation of a triplication by replication errors involving appropriate strand switchings and branch migrations can not be excluded however. PMID:6227522

  6. Gene-Environment Interactions Target Mitogen-activated Protein 3 Kinase 1 (MAP3K1) Signaling in Eyelid Morphogenesis*

    PubMed Central

    Mongan, Maureen; Meng, Qinghang; Wang, Jingjing; Kao, Winston W.-Y.; Puga, Alvaro; Xia, Ying

    2015-01-01

    Gene-environment interactions determine the biological outcomes through mechanisms that are poorly understood. Mouse embryonic eyelid closure is a well defined model to study the genetic control of developmental programs. Using this model, we investigated how exposure to dioxin-like environmental pollutants modifies the genetic risk of developmental abnormalities. Our studies reveal that mitogen-activated protein 3 kinase 1 (MAP3K1) signaling is a focal point of gene-environment cross-talk. Dioxin exposure, acting through the aryl hydrocarbon receptor (AHR), blocked eyelid closure in genetic mutants in which MAP3K1 signaling was attenuated but did not disturb this developmental program in either wild type or mutant mice with attenuated epidermal growth factor receptor or WNT signaling. Exposure also markedly inhibited c-Jun phosphorylation in Map3k1+/− embryonic eyelid epithelium, suggesting that dioxin-induced AHR pathways can synergize with gene mutations to inhibit MAP3K1 signaling. Our studies uncover a novel mechanism through which the dioxin-AHR axis interacts with the MAP3K1 signaling pathways during fetal development and provide strong empirical evidence that specific gene alterations can increase the risk of developmental abnormalities driven by environmental pollutant exposure. PMID:26109068

  7. Genome-wide survey and expression analysis of the calcium-dependent protein kinase gene family in cassava.

    PubMed

    Hu, Wei; Hou, Xiaowan; Xia, Zhiqiang; Yan, Yan; Wei, Yunxie; Wang, Lianzhe; Zou, Meiling; Lu, Cheng; Wang, Wenquan; Peng, Ming

    2016-02-01

    Calcium-dependent protein kinases (CPKs) play important roles in regulating plant tolerance to abiotic stress and signal transduction; however, no data are currently available regarding the CPK family in cassava. Herein, we identified 27 CPK genes from cassava based on our previous genome sequencing data. Phylogenetic analysis showed that cassava CPKs could be clustered into three groups, which was further supported by gene structure and conserved protein motif analyses. Global expression analysis suggested that MeCPK genes showed distinct expression patterns in different tissues between wild subspecies and cultivated varieties, indicating their involvement in the functional diversity of different varieties. Transcriptomics, interaction networks, and co-expression assays revealed a broad transcriptional response of cassava CPKs and CPK-mediated networks to drought stress and their differential expression profiles in different varieties, implying their contribution to drought stress tolerance in cassava. Expression analysis of eight MeCPK genes suggested a comprehensive response to osmotic stress, salt, cold, abscisic acid, and H2O2, which indicated that cassava CPKs might be convergence points for different signaling pathways. This study provides a basis for crop improvements and understanding of abiotic stress responses and signal transduction mediated by CPKs in cassava. PMID:26272723

  8. Gene-Environment Interactions Target Mitogen-activated Protein 3 Kinase 1 (MAP3K1) Signaling in Eyelid Morphogenesis.

    PubMed

    Mongan, Maureen; Meng, Qinghang; Wang, Jingjing; Kao, Winston W-Y; Puga, Alvaro; Xia, Ying

    2015-08-01

    Gene-environment interactions determine the biological outcomes through mechanisms that are poorly understood. Mouse embryonic eyelid closure is a well defined model to study the genetic control of developmental programs. Using this model, we investigated how exposure to dioxin-like environmental pollutants modifies the genetic risk of developmental abnormalities. Our studies reveal that mitogen-activated protein 3 kinase 1 (MAP3K1) signaling is a focal point of gene-environment cross-talk. Dioxin exposure, acting through the aryl hydrocarbon receptor (AHR), blocked eyelid closure in genetic mutants in which MAP3K1 signaling was attenuated but did not disturb this developmental program in either wild type or mutant mice with attenuated epidermal growth factor receptor or WNT signaling. Exposure also markedly inhibited c-Jun phosphorylation in Map3k1(+/-) embryonic eyelid epithelium, suggesting that dioxin-induced AHR pathways can synergize with gene mutations to inhibit MAP3K1 signaling. Our studies uncover a novel mechanism through which the dioxin-AHR axis interacts with the MAP3K1 signaling pathways during fetal development and provide strong empirical evidence that specific gene alterations can increase the risk of developmental abnormalities driven by environmental pollutant exposure. PMID:26109068

  9. Polymorphisms in the Tyrosine Kinase 2 and Interferon Regulatory Factor 5 Genes Are Associated with Systemic Lupus Erythematosus

    PubMed Central

    Sigurdsson, Snaevar; Nordmark, Gunnel; Göring, Harald H. H.; Lindroos, Katarina; Wiman, Ann-Christin; Sturfelt, Gunnar; Jönsen, Andreas; Rantapää-Dahlqvist, Solbritt; Möller, Bozena; Kere, Juha; Koskenmies, Sari; Widén, Elisabeth; Eloranta, Maija-Leena; Julkunen, Heikki; Kristjansdottir, Helga; Steinsson, Kristjan; Alm, Gunnar; Rönnblom, Lars; Syvänen, Ann-Christine

    2005-01-01

    Systemic lupus erythematosus (SLE) is a complex systemic autoimmune disease caused by both genetic and environmental factors. Genome scans in families with SLE point to multiple potential chromosomal regions that harbor SLE susceptibility genes, and association studies in different populations have suggested several susceptibility alleles for SLE. Increased production of type I interferon (IFN) and expression of IFN-inducible genes is commonly observed in SLE and may be pivotal in the molecular pathogenesis of the disease. We analyzed 44 single-nucleotide polymorphisms (SNPs) in 13 genes from the type I IFN pathway in 679 Swedish, Finnish, and Icelandic patients with SLE, in 798 unaffected family members, and in 438 unrelated control individuals for joint linkage and association with SLE. In two of the genes—the tyrosine kinase 2 (TYK2) and IFN regulatory factor 5 (IRF5) genes—we identified SNPs that displayed strong signals in joint analysis of linkage and association (unadjusted P<10-7) with SLE. TYK2 binds to the type I IFN receptor complex and IRF5 is a regulator of type I IFN gene expression. Thus, our results support a disease mechanism in SLE that involves key components of the type I IFN system. PMID:15657875

  10. Overexpression of GbRLK, a putative receptor-like kinase gene, improved cotton tolerance to Verticillium wilt

    PubMed Central

    Jun, Zhao; Zhang, Zhiyuan; Gao, Yulong; Zhou, Lei; Fang, Lei; Chen, Xiangdong; Ning, Zhiyuan; Chen, Tianzi; Guo, Wangzhen; Zhang, Tianzhen

    2015-01-01

    Verticillium dahliae is a causative fungal pathogen and only a few genes have been identified that exhibit critical roles in disease resistance and few has shown positive effects on the resistance to Verticillium wilt in transgenic cotton. We cloned a receptor-like kinase gene (GbRLK) induced by Verticillium dahliae (VD) in the disease-resistant cotton Gossypium barbadense cv. Hai7124. Northern blotting revealed that the GbRLK was induced by VD at 96 h after inoculation. The functional GbRLK is from D subgenome since a single base deletion results in a frameshift or dysfunctional homologue in the A subgenome in tetraploid cotton. To verify the function of GbRLK, we developed the overexpression transgenic GbRLK cotton and Arabidopsis lines, and found that they all showed the higher resistance to Verticillium in the greenhouse and field trial. The results of the expression profile using transgenic and non-transgenic Arabidopsis thaliana revealed that the GbRLK regulated expressions of a series genes associated with biotic and abiotic stresses. Therefore, we propose that the increased resistance to Verticillium dahliae infection in transgnic plants could result from reduction in the damage of water loss and regulation of defense gene expression. PMID:26446555

  11. The role of immunosuppression in the efficacy of cancer gene therapy using adenovirus transfer of the herpes simplex thymidine kinase gene.

    PubMed Central

    Elshami, A A; Kucharczuk, J C; Sterman, D H; Smythe, W R; Hwang, H C; Amin, K M; Litzky, L A; Albelda, S M; Kaiser, L R

    1995-01-01

    OBJECTIVE: To determine whether the immune system limits or improves the therapeutic efficacy of an adenovirus vector expressing the herpes simplex thymidine kinase (HSVtk) gene in a subcutaneous tumor model. BACKGROUND DATA: Enhanced immune reactions against tumors may be therapeutically useful. However, recent studies with adenoviral vectors show that immune responses limit the efficacy and persistence of gene expression. The effect of the immune response on cancer gene therapy with HSVtk gene delivery by an adenovirus vector followed by treatment with ganciclovir is unclear. METHODS: After adenoviral transduction of a Fischer rat syngeneic mesothelioma cell line with the HSVtk gene in vitro, subcutaneous flank tumors were established. The ability of the HSVtk/ganciclovir system to inhibit tumor growth was compared among normal Fischer rats, immunodeficient nude rats, and Fischer rats immunosuppressed with cyclosporin. RESULTS: HSVtk/ganciclovir therapy was more effective in nude rats and immunosuppressed Fischer rats than in immunocompetent Fischer rats. CONCLUSION: These results indicate that the immune response against adenovirally transduced cells limits the efficacy of the HSVtk/ganciclovir system and that immunosuppression appears to be a useful adjunct. These findings have important implications for clinical trials using currently available adenovirus vectors as well as for future vector design. PMID:7677460

  12. Rearranged Anaplastic Lymphoma Kinase (ALK) Gene in Adult-Onset Papillary Thyroid Cancer Amongst Atomic Bomb Survivors

    PubMed Central

    Mukai, Mayumi; Takahashi, Keiko; Hayashi, Yuzo; Nakachi, Kei; Kusunoki, Yoichiro

    2012-01-01

    Background We previously noted that among atomic bomb survivors (ABS), the relative frequency of cases of adult papillary thyroid cancer (PTC) with chromosomal rearrangements (mainly RET/PTC) was significantly greater in those with relatively higher radiation exposure than those with lower radiation exposure. In contrast, the frequency of PTC cases with point mutations (mainly BRAFV600E) was significantly lower in patients with relatively higher radiation exposure than those with lower radiation exposure. We also found that among ABS, the frequency of PTC cases with no detectable gene alterations in RET, neurotrophic tyrosine kinase receptor 1 (NTRK1), BRAF, or RAS was significantly higher in patients with relatively higher radiation exposure than those with lower radiation exposure. However, in ABS with PTC, the relationship between the presence of the anaplastic lymphoma kinase (ALK) gene fused with other gene partners and radiation exposure has received little study. In this study, we tested the hypothesis that the relative frequency of rearranged ALK in ABS with PTC, and with no detectable gene alterations in RET, NTRK1, BRAF, or RAS, would be greater in those having relatively higher radiation exposures. Methods The 105 subjects in the study were drawn from the Life Span Study cohort of ABS of Hiroshima and Nagasaki who were diagnosed with PTC between 1956 and 1993. Seventy-nine were exposed (>0 mGy), and 26 were not exposed to A-bomb radiation. In the 25 ABS with PTC, and with no detectable gene alterations in RET, NTRK1, BRAF, or RAS, we examined archival, formalin-fixed, paraffin-embedded PTC specimens for rearrangement of ALK using reverse transcription–polymerase chain reaction and 5′ rapid amplification of cDNA ends (5′ RACE). Results We found rearranged ALK in 10 of 19 radiation-exposed PTC cases, but none among 6 patients with PTC with no radiation exposure. In addition, solid/trabecular-like architecture in PTC was closely associated with ALK

  13. Living with high putrescine: expression of ornithine and arginine biosynthetic pathway genes in high and low putrescine producing poplar cells.

    PubMed

    Page, Andrew F; Minocha, Rakesh; Minocha, Subhash C

    2012-01-01

    Arginine (Arg) and ornithine (Orn), both derived from glutamate (Glu), are the primary substrates for polyamine (PA) biosynthesis, and also play important roles as substrates and intermediates of overall N metabolism in plants. Their cellular homeostasis is subject to multiple levels of regulation. Using reverse transcription quantitative PCR (RT-qPCR), we studied changes in the expression of all genes of the Orn/Arg biosynthetic pathway in response to up-regulation [via transgenic expression of mouse Orn decarboxylase (mODC)] of PA biosynthesis in poplar (Populus nigra × maximowiczii) cells grown in culture. Cloning and sequencing of poplar genes involved in the Orn/Arg biosynthetic pathway showed that they have high homology with similar genes in other plants. The expression of the genes of Orn, Arg and PA biosynthetic pathway fell into two hierarchical clusters; expression of one did not change in response to high putrescine, while members of the other cluster showed a shift in expression pattern during the 7-day culture cycle. Gene expression of branch point enzymes (N-acetyl-Glu synthase, Orn aminotransferase, Arg decarboxylase, and spermidine synthase) in the sub-pathways, constituted a separate cluster from those involved in intermediary reactions of the pathway (N-acetyl-Glu kinase, N-acetyl-Glu-5-P reductase, N-acetyl-Orn aminotransferase, N (2)-acetylOrn:N-acetyl-Glu acetyltransferase, N (2)-acetyl-Orn deacetylase, Orn transcarbamylase, argininosuccinate synthase, carbamoylphosphate synthetase, argininosuccinate lyase, S-adenosylmethionine decarboxylase, spermine synthase). We postulate that expression of all genes of the Glu-Orn-Arg pathway is constitutively coordinated and is not influenced by the increase in flux rate through this pathway in response to increased utilization of Orn by mODC; thus the pathway involves mostly biochemical regulation rather than changes in gene expression. We further suggest that Orn itself plays a major role in the

  14. Calcitonin gene-related peptide activated ATP-sensitive K+ currents in rabbit arterial smooth muscle via protein kinase A.

    PubMed Central

    Quayle, J M; Bonev, A D; Brayden, J E; Nelson, M T

    1994-01-01

    1. Whole-cell K+ currents activated by calcitonin gene-related peptide (CGRP) in smooth muscle cells enzymatically isolated from rabbit mesenteric arteries were measured in the conventional and perforated configurations of the patch clamp technique. The signal transduction pathway from CGRP receptors to activation of potassium currents was investigated. 2. CGRP (10 nM) activated a whole-cell current that was blocked by glibenclamide (10 microM), an inhibitor of ATP-sensitive K+ channels. Elevating intracellular ATP reduced glibenclamide-sensitive currents. CGRP increased the glibenclamide-sensitive currents by 3- to 6-fold in cells dialysed with 0.1 mM ATP, 3.0 mM ATP or in intact cells. The reversal potential of the glibenclamide-sensitive current in the presence of CGRP shifted with the potassium equilibrium potential, while its current-voltage relationship exhibited little voltage dependence. 3. Forskolin (10 microM), an adenylyl cyclase activator, Sp-cAMPS (500 microM) and the catalytic subunit of protein kinase A increased glibenclamide-sensitive K+ currents 2.1-, 3.3- and 8.2-fold, respectively. 4. Nitric oxide and nitroprusside did not activate glibenclamide-sensitive K+ currents. 5. Dialysis of the cell's interior with inhibitors of protein kinase A (synthetic peptide inhibitor, 4.6 microM or H-8, 100 microM) completely blocked activation of K+ currents by CGRP. 6. Our results suggest the following signal transduction scheme for activation of K+ currents by CGRP in arterial smooth muscle: (1) CGRP stimulates adenylyl cyclase, which leads to an elevation of cAMP; (2) cAMP activates protein kinase A, which opens ATP-sensitive K+ channels. PMID:8189394

  15. Cloning, expression and characterization of a gene encoding mitogen activated protein kinase 2 (MPK2) from Tetrahymena thermophila.

    PubMed

    Arslanyolu, Muhittin; Yıldız, Mehmet Taha

    2014-08-01

    Environmental effects and mitogens determine cell phenotype in eukaryotes mainly through MAPK pathways. However, MAPK signaling pathways in T. thermophila have not been studied comprehensively. This study aims to express recombinant MPK2, a MAPK from T. thermophila, in E. coli to characterize its kinase activity. MPK2 was cloned by RT-PCR using degenerate oligonucleotide primers and RACE method. The full-length cDNA of the MPK2 gene is 1705bp that includes 1281bp ORF coding for a putative protein of 426 amino acids having a mass of 50.2kDa. The putative MPK2 protein contains all eleven conserved subdomains that are characteristics of serine/threonine protein kinases, and a TDY motif, which is a putative dual phosphorylation site common in Protista. MPK2 displays highest 48% overall identity to human ERK5 (MAPK7). The expression vector pGEX4T-1-MPK2 was constructed by inserting the coding region of MPK2 cDNA into pGEX4T-1 after introducing the nine point mutations, and then transformed into E. coli BL21(DE3). Autophosphorylation of 76kDa GST-MPK2 at tyrosine residues was confirmed not only by Western blot using anti-phosphotyrosine monoclonal antibody but also by in vitro kinase assay. GST-MPK2 was also able to phosphorylate the artificial substrate myelin basic protein. This study concludes that the free-living unicellular protist T. thermophila MPK2 has commonly conserved MAPK enzyme features, possibly involved in the regulation of cell survival responding to abiotic or biotic stressors, and the production and movement of haploid gametic nuclei between pairs during conjugation. PMID:24858074

  16. Distribution of OCA2∗481Thr and OCA2∗615Arg, associated with hypopigmentation, in several additional populations.

    PubMed

    Yuasa, Isao; Harihara, Shinji; Jin, Feng; Nishimukai, Hiroaki; Fujihara, Junko; Fukumori, Yasuo; Takeshita, Haruo; Umetsu, Kazuo; Saitou, Naruya

    2011-07-01

    Two mutants, OCA2∗481Thr (c.1441G>A, p.Ala481Thr) and OCA2∗615Arg (c.1844A>G, p.His615Arg), in the OCA2 (oculocutaneous albinism type II) gene are associated with hypopigmentation in East Asians. Here, these two alleles were studied to assess the frequencies in five different populations. In addition, the allele frequency of OCA2∗615Arg was investigated in seven populations. Among a total of 24 global populations investigated, Oroqens in Heihe showed the highest frequency for OCA2∗481Thr (0.519), and among 26 populations, Han Chinese in Changsha showed the highest frequency for OCA2∗615Arg (0.673). This study confirmed that these two East Asian-specific alleles are characteristic of northern and central-southern East Asian populations. PMID:21565543

  17. Gene Expression Profile of Calcium/Calmodulin-Dependent Protein Kinase IIα in Rat's Hippocampus during Morphine Withdrawal

    PubMed Central

    Ahmadi, Shamseddin; Amiri, Shahin; Rafieenia, Fatemeh; Rostamzadeh, Jalal

    2013-01-01

    Introduction Calcium/calmodulin-dependent protein kinase II (CaMKII) which is highly expressed in the hippocampus is known to play a pivotal role in reward-related memories and morphine dependence. Methods In the present study, repeated morphine injections once daily for 7 days was done to induce morphine tolerance in male Wistar rats, after which gene expression profile of α-isoform of CaMKII (CaMKIIα) in the hippocampus was evaluated upon discontinuation of morphine injection over 21 days of morphine withdrawal. Control groups received saline for 7 consecutive days. For gene expression study, rats’ brains were removed and the hippocampus was dissected in separate groups on days 1, 3, 7, 14, and 21 since discontinuation of of morphine injection. A semi-quantitative RT-PCR method was used to evaluate the gene expression profile. Results Tolerance to morphine was verified by a significant decrease in morphine analgesia in a hotplate test on day 8 (one day after the final repeated morphine injections). Results showed that gene expression of CaMKIIα at mRNA level on day 1, 3, 7, 14 and 21 of morphine withdrawal was significantly altered as compared to the saline control group. Post hoc Tukey's test revealed a significantly enhanced CaMKIIα gene expression on day 14. Discussion It can be concluded that CaMKIIα gene expression during repeated injections of morphine is increased and this increase continues up to 14 days of withdrawal then settles at a new set point. Therefore, the strong morphine reward-related memory in morphine abstinent animals may, at least partly be attributed to, the up-regulation of CaMKIIα in the hippocampus over 14 days of morphine withdrawal. PMID:25337341

  18. Genome of Epinotia aporema granulovirus (EpapGV), a polyorganotropic fast killing betabaculovirus with a novel thymidylate kinase gene

    PubMed Central

    2012-01-01

    Background Epinotia aporema (Lepidoptera: Tortricidae) is an important pest of legume crops in South America. Epinotia aporema granulovirus (EpapGV) is a baculovirus that causes a polyorganotropic infection in the host larva. Its high pathogenicity and host specificity make EpapGV an excellent candidate to be used as a biological control agent. Results The genome of Epinotia aporema granulovirus (EpapGV) was sequenced and analyzed. Its circular double-stranded DNA genome is 119,082 bp in length and codes for 133 putative genes. It contains the 31 baculovirus core genes and a set of 19 genes that are GV exclusive. Seventeen ORFs were unique to EpapGV in comparison with other baculoviruses. Of these, 16 found no homologues in GenBank, and one encoded a thymidylate kinase. Analysis of nucleotide sequence repeats revealed the presence of 16 homologous regions (hrs) interspersed throughout the genome. Each hr was characterized by the presence of 1 to 3 clustered imperfect palindromes which are similar to previously described palindromes of tortricid-specific GVs. Also, one of the hrs (hr4) has flanking sequences suggestive of a putative non-hr ori. Interestingly, two more complex hrs were found in opposite loci, dividing the circular dsDNA genome in two halves. Gene synteny maps showed the great colinearity of sequenced GVs, being EpapGV the most dissimilar as it has a 20 kb-long gene block inversion. Phylogenetic study performed with 31 core genes of 58 baculoviral genomes suggests that EpapGV is the baculovirus isolate closest to the putative common ancestor of tortricid specific betabaculoviruses. Conclusions This study, along with previous characterization of EpapGV infection, is useful for the better understanding of the pathology caused by this virus and its potential utilization as a bioinsecticide. PMID:23051685

  19. A Genomewide Overexpression Screen Identifies Genes Involved in the Phosphatidylinositol 3-Kinase Pathway in the Human Protozoan Parasite Entamoeba histolytica

    PubMed Central

    Koushik, Amrita B.; Welter, Brenda H.; Rock, Michelle L.

    2014-01-01

    Entamoeba histolytica is a protozoan parasite that causes amoebic dysentery and liver abscess. E. histolytica relies on motility, phagocytosis, host cell adhesion, and proteolysis of extracellular matrix for virulence. In eukaryotic cells, these processes are mediated in part by phosphatidylinositol 3-kinase (PI3K) signaling. Thus, PI3K may be critical for virulence. We utilized a functional genomics approach to identify genes whose products may operate in the PI3K pathway in E. histolytica. We treated a population of trophozoites that were overexpressing genes from a cDNA library with a near-lethal dose of the PI3K inhibitor wortmannin. This screen was based on the rationale that survivors would be overexpressing gene products that directly or indirectly function in the PI3K pathway. We sequenced the overexpressed genes in survivors and identified a cDNA encoding a Rap GTPase, a protein previously shown to participate in the PI3K pathway. This supports the validity of our approach. Genes encoding a coactosin-like protein, EhCoactosin, and a serine-rich E. histolytica protein (SREHP) were also identified. Cells overexpressing EhCoactosin or SREHP were also less sensitive to a second PI3K inhibitor, LY294002. This corroborates the link between these proteins and PI3K. Finally, a mutant cell line with an increased level of phosphatidylinositol (3,4,5)-triphosphate, the product of PI3K activity, exhibited increased expression of SREHP and EhCoactosin. This further supports the functional connection between these proteins and PI3K in E. histolytica. To our knowledge, this is the first forward-genetics screen adapted to reveal genes participating in a signal transduction pathway in this pathogen. PMID:24442890

  20. Cloning of a gene (SR-A1), encoding for a new member of the human Ser/Arg-rich family of pre-mRNA splicing factors: overexpression in aggressive ovarian cancer

    PubMed Central

    Scorilas, A; Kyriakopoulou, L; Katsaros, D; Diamandis, E P

    2001-01-01

    By using the positional cloning gene approach, we were able to identify a novel gene encoding for a serine/arginine-rich protein, which appears to be the human homologue of the rat A1 gene. We named this new gene SR-A1. Members of the SR family of proteins have been shown to interact with the C-terminal domain (CTD) of the large subunit of RNA polymerase II and participate in pre-mRNA splicing. We have localized the SR-A1 gene between the known genes IRF3 and RRAS on chromosome 19q13.3. The novel gene spans 16.7 kb of genomic sequence and it is formed of 11 exons and 10 intervening introns. The SR-A1 protein is composed of 1312 amino acids, with a molecular mass of 139.3 kDa and a theoretical isoelectric point of 9.31. The SR-A1 protein contains an SR-rich domain as well as a CTD-binding domain present only in a subset of SR-proteins. Through interactions with the pre-mRNA and the CTD domain of the Polymerase II, SR proteins have been shown to regulate alternative splicing. The SR-A1 gene is expressed in all tissues tested, with highest levels found in fetal brain and fetal liver. Our data suggest that this gene is overexpressed in a subset of ovarian cancers which are clinically more aggressive. Studies with the steroid hormone receptor-positive breast and prostate carcinoma cell lines ZR-75-1, BT-474 and LNCaP, respectively, suggest that SR-A1 is constitutively expressed. Furthermore, the mRNA of the SR-A1 gene in these cell lines appears to increase by estrogens, androgens and glucocorticoids, and to a lesser extend by progestins. © 2001 Cancer Research Campaign http://www.bjcancer.com PMID:11461075

  1. A polymorphism in the protein kinase C gene PRKCB is associated with α2-adrenoceptor-mediated vasoconstriction

    PubMed Central

    Ruohonen, Saku; Valve, Laura; Muszkat, Mordechai; Sofowora, Gbenga G.; Kurnik, Daniel; Stein, C. Michael; Perola, Markus; Scheinin, Mika; Snapir, Amir

    2013-01-01

    Objectives α2-Adrenoceptors (α2-AR) mediate both constriction and dilatation of blood vessels. There is substantial inter-individual variability in dorsal hand vein (DHV) constriction responses to α2-AR agonist activation. Genetic factors appear to contribute significantly to this variation. The present study was designed to identify genetic factors contributing to the inter-individual variability in α2-AR-mediated vascular constriction induced by the selective α2-AR agonist dexmedetomidine. Methods DHV constriction responses to local infusion of dexmedetomidine were assessed by measuring changes in vein diameter with a linear variable differential transformer. The outcome variable was log-transformed dexmedetomidine ED50 for constriction. A genome-wide association study (GWAS) of 433,378 single nucleotide polymorphisms (SNPs) was performed for the sensitivity of DHV responses in 64 healthy Finnish subjects. 20 SNPs were selected based on the GWAS results and their associations with the ED50 of dexmedetomidine were tested in an independent North American study population of 68 healthy individuals. Results In both study populations (GWAS and replication samples), the SNP rs9922316 in the gene for protein kinase C type β was consistently associated with dexmedetomidine ED50 for dorsal hand vein constriction (unadjusted p = 0.00016 for the combined population). Conclusions Genetic variation in protein kinase C type β may contribute to the inter-individual variation in dorsal hand vein constriction responses to α2-AR activation by the agonist dexmedetomidine. PMID:23337848

  2. Gene expression analysis of microtubule affinity-regulating kinase 2 in non-small cell lung cancer

    PubMed Central

    Marshall, Erin A.; Ng, Kevin W.; Anderson, Christine; Hubaux, Roland; Thu, Kelsie L.; Lam, Wan L.; Martinez, Victor D.

    2015-01-01

    Lung cancer is the leading cause of cancer death worldwide, and has a five-year survival rate of 18% [1]. MARK2 is a serine/threonine-protein kinase, and is a key component in the phosphorylation of microtubule-associated proteins [2], [3]. A recent study published by Hubaux et al. found that microtubule affinity-regulating kinase 2 (MARK2) showed highly frequent DNA and RNA level disruption in lung cancer cell lines and independent non-small cell lung cancer (NSCLC) cohorts [4]. These alterations result in the acquisition of oncogenic properties in cell lines, such as increased viability and anchorage-independent growth. Furthermore, a microarray-based transcriptome analysis of three short hairpin RNA (shRNA)-mediated MARK2 knockdown lung adenocarcinoma cell lines (GEO#: GSE57966) revealed an association between MARK2 gene expression and cell cycle activation and DNA damage response. Here, we present a detailed description of transcriptome analysis to support the described role of MARK2 in promoting a malignant phenotype. PMID:26697357

  3. Oncoprotein kinase

    DOEpatents

    Karin, Michael; Hibi, Masahiko; Lin, Anning

    2001-02-27

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

  4. Naturally Occurring Mutations in the MPS1 Gene Predispose Cells to Kinase Inhibitor Drug Resistance.

    PubMed

    Gurden, Mark D; Westwood, Isaac M; Faisal, Amir; Naud, Sébastien; Cheung, Kwai-Ming J; McAndrew, Craig; Wood, Amy; Schmitt, Jessica; Boxall, Kathy; Mak, Grace; Workman, Paul; Burke, Rosemary; Hoelder, Swen; Blagg, Julian; Van Montfort, Rob L M; Linardopoulos, Spiros

    2015-08-15

    Acquired resistance to therapy is perhaps the greatest challenge to effective clinical management of cancer. With several inhibitors of the mitotic checkpoint kinase MPS1 in preclinical development, we sought to investigate how resistance against these inhibitors may arise so that mitigation or bypass strategies could be addressed as early as possible. Toward this end, we modeled acquired resistance to the MPS1 inhibitors AZ3146, NMS-P715, and CCT251455, identifying five point mutations in the kinase domain of MPS1 that confer resistance against multiple inhibitors. Structural studies showed how the MPS1 mutants conferred resistance by causing steric hindrance to inhibitor binding. Notably, we show that these mutations occur in nontreated cancer cell lines and primary tumor specimens, and that they also preexist in normal lymphoblast and breast tissues. In a parallel piece of work, we also show that the EGFR p.T790M mutation, the most common mutation conferring resistance to the EGFR inhibitor gefitinib, also preexists in cancer cells and normal tissue. Our results therefore suggest that mutations conferring resistance to targeted therapy occur naturally in normal and malignant cells and these mutations do not arise as a result of the increased mutagenic plasticity of cancer cells. PMID:26202014

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

    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, are virulent on most of the known race-specific resistance genes. We report here the map-based cloning of the gene Yr36 (WKS1), ...

  6. The mammalian AMP-activated protein kinase complex mediates glucose regulation of gene expression in the yeast Saccharomyces cerevisiae.

    PubMed

    Ye, Tian; Bendrioua, Loubna; Carmena, David; García-Salcedo, Raúl; Dahl, Peter; Carling, David; Hohmann, Stefan

    2014-06-01

    The AMP-activated protein kinase (AMPK) controls energy homeostasis in eukaryotic cells. Here we expressed hetero-trimeric mammalian AMPK complexes in a Saccharomyces cerevisiae mutant lacking all five genes encoding yeast AMPK/SNF1 components. Certain mammalian complexes complemented the growth defect of the yeast mutant on non-fermentable carbon sources. Phosphorylation of the AMPK α1-subunit was glucose-regulated, albeit not by the Glc7-Reg1/2 phosphatase, which performs this function on yeast AMPK/SNF1. AMPK could take over SNF1 function in glucose derepression. While indirectly acting anti-diabetic drugs had no effect on AMPK in yeast, compound 991 stimulated α1-subunit phosphorylation. Our results demonstrate a remarkable functional conservation of AMPK and that glucose regulation of AMPK may not be mediated by regulatory features of a specific phosphatase. PMID:24815694

  7. Deletion of the thymidine kinase gene induces complete attenuation of the Georgia isolate of African swine fever virus.

    PubMed

    Sanford, B; Holinka, L G; O'Donnell, V; Krug, P W; Carlson, J; Alfano, M; Carrillo, C; Wu, Ping; Lowe, Andre; Risatti, G R; Gladue, D P; Borca, M V

    2016-02-01

    African swine fever virus (ASFV) is the etiological agent of a contagious and often lethal viral disease of domestic pigs. There are no vaccines to control Africa swine fever (ASF). Experimental vaccines have been developed using genetically modified live attenuated ASFVs obtained by specifically deleting virus genes involved in virulence, including the thymidine kinase (TK) gene. TK has been shown to be involved in the virulence of several viruses, including ASFV. Here we report the construction of a recombinant virus (ASFV-G/V-ΔTK) obtained by deleting the TK gene in a virulent strain of ASFV Georgia adapted to replicate in Vero cells (ASFV-G/VP30). ASFV-G/P-ΔTK demonstrated decreased replication both in primary swine macrophage cell cultures and in Vero cells compared with ASFV-G/VP30. In vivo, intramuscular administration of up to 10(6) TCID50 of ASFV-G/V-ΔTK does not result in ASF disease. However, these animals are not protected when challenged with the virulent parental Georgia strain. PMID:26656424

  8. Intron sequences of arginine kinase in an intertidal snail suggest an ecotype-specific selective sweep and a gene duplication

    PubMed Central

    Kemppainen, P; Lindskog, T; Butlin, R; Johannesson, K

    2011-01-01

    Many species with restricted gene flow repeatedly respond similarly to local selection pressures. To fully understand the genetic mechanisms behind this process, the phylogeographic history of the species (inferred from neutral markers) as well as the loci under selection need to be known. Here we sequenced an intron in the arginine kinase gene (Ark), which shows strong clinal variation between two locally adapted ecotypes of the flat periwinkle, Littorina fabalis. The ‘small-sheltered' ecotype was almost fixed for one haplotype, H1, in populations on both sides of the North Sea, unlike the ‘large-moderately exposed ecotype', which segregated for ten different haplotypes. This contrasts with neutral markers, where the two ecotypes are equally variable. H1 could have been driven to high frequency in an ancestral population and then repeatedly spread to sheltered habitats due to local selection pressures with the colonization of both sides of the North Sea, after the last glacial maximum (∼18 000 years ago). An alternative explanation is that a positively selected mutation, in or linked to Ark, arose after the range expansion and secondarily spread through sheltered populations throughout the distribution range, causing this ecotype to evolve in a concerted fashion. Also, we were able to sequence up to four haplotypes consistently from some individuals, suggesting a gene duplication in Ark. PMID:20877396

  9. Ultrasound-targeted microbubble destruction mediated herpes simplex virus-thymidine kinase gene treats hepatoma in mice

    PubMed Central

    2010-01-01

    Objective The purpose of the study was to explore the anti-tumor effect of ultrasound -targeted microbubble destruction mediated herpes simplex virus thymidine kinase (HSV-TK) suicide gene system on mice hepatoma. Methods Forty mice were randomly divided into four groups after the models of subcutaneous transplantation tumors were estabilished: (1) PBS; (2) HSV-TK (3) HSV-TK+ ultrasound (HSV-TK+US); (4) HSV-TK+ultrasound+microbubbles (HSV-TK+US+MB). The TK protein expression in liver cancer was detected by western-blot. Applying TUNEL staining detected tumor cell apoptosis. At last, the inhibition rates and survival time of the animals were compared among all groups. Results The TK protein expression of HSV-TK+MB+US group in tumor-bearing mice tissues were significantly higher than those in other groups. The tumor inhibitory effect of ultrasound-targeted microbubble destruction mediated HSV-TK on mice transplantable tumor was significantly higher than those in other groups (p < 0.05), and can significantly improve the survival time of tumor-bearing mice. Conclusion Ultrasound-targeted microbubble destruction can effectively transfect HSV-TK gene into target tissues and play a significant inhibition effect on tumors, which provides a new strategy for gene therapy in liver cancer. PMID:21176239

  10. Intron sequences of arginine kinase in an intertidal snail suggest an ecotype-specific selective sweep and a gene duplication.

    PubMed

    Kemppainen, P; Lindskog, T; Butlin, R; Johannesson, K

    2011-05-01

    Many species with restricted gene flow repeatedly respond similarly to local selection pressures. To fully understand the genetic mechanisms behind this process, the phylogeographic history of the species (inferred from neutral markers) as well as the loci under selection need to be known. Here we sequenced an intron in the arginine kinase gene (Ark), which shows strong clinal variation between two locally adapted ecotypes of the flat periwinkle, Littorina fabalis. The 'small-sheltered' ecotype was almost fixed for one haplotype, H1, in populations on both sides of the North Sea, unlike the 'large-moderately exposed ecotype', which segregated for ten different haplotypes. This contrasts with neutral markers, where the two ecotypes are equally variable. H1 could have been driven to high frequency in an ancestral population and then repeatedly spread to sheltered habitats due to local selection pressures with the colonization of both sides of the North Sea, after the last glacial maximum (~18 000 years ago). An alternative explanation is that a positively selected mutation, in or linked to Ark, arose after the range expansion and secondarily spread through sheltered populations throughout the distribution range, causing this ecotype to evolve in a concerted fashion. Also, we were able to sequence up to four haplotypes consistently from some individuals, suggesting a gene duplication in Ark. PMID:20877396

  11. Growth properties and vaccine efficacy of recombinant pseudorabies virus defective in glycoprotein E and thymidine kinase genes.

    PubMed

    Wu, Ching-Ying; Liao, Chih-Ming; Chi, Jiun-Ni; Chien, Maw-Sheng; Huang, Chienjin

    2016-07-10

    Pseudorabies virus (PRV) is an alphaherpesvirus that causes pseudorabies (PR), an economically important viral disease of pigs. Marker vaccines were widely used in PR prevention and eradication programs. The purpose of this study was to construct a novel recombinant virus with deletions at defined regions in the glycoprotein E (gE) and thymine kinase (TK) genes by homologous recombination. This study also evaluated the safety and efficacy of the virus for a live attenuated marker vaccine. No significant difference was observed in virus replication between gE gene-deleted (gE(-)), gE/TK double gene-deleted (gE(-)TK(-)), and wild-type PRV by growth curve analysis. However, gE(-)TK(-) PRV was completely attenuated in mice. To evaluate the immunogenicity of gE(-)TK(-) PRV, four 12-week-old specific-pathogen-free pigs per group were immunized intramuscularly with viral titers of 1×10(4), 1×10(5), or 1×10(6) TCID50, followed by intranasal challenge infection with virulent PRV (1×10(8) TCID50) at 3 weeks post vaccination. The gE(-)TK(-) PRV-vaccinated pigs displayed no general adverse effects after immunization and had protective immune responses after PRV challenge. Thus, gE(-)TK(-) PRV was safe and efficacious and might be a potential candidate for a live attenuated marker vaccine against PRV. PMID:27164258

  12. Nucleotide sequence variation within the human tyrosine kinase B neurotrophin receptor gene: association with antisocial alcohol dependence.

    PubMed

    Xu, K; Anderson, T R; Neyer, K M; Lamparella, N; Jenkins, G; Zhou, Z; Yuan, Q; Virkkunen, M; Lipsky, R H

    2007-12-01

    To identify sequence variants in genes that may have roles in neuronal responses to alcohol, we resequenced the 5' region of tyrosine kinase B neurotrophin receptor gene (NTRK2) and determined linkage disequilibrium (LD) values, haplotype structure, and performed association analyses using 43 single nucleotide polymorphisms (SNPs) covering the entire NTRK2 region in a Finnish Caucasian sample of 229 alcohol-dependent subjects with antisocial personality disorder (ASPD) and 287 healthy controls. Individually, three SNPs were associated with alcohol dependence and alcohol abuse (AD) (P-value from 0.0019 to 0.0059, significance level was set at Pgene in addiction in a Caucasian population with AD and a subtype of ASPD. PMID:17200667

  13. RNA interference suppression of the receptor tyrosine kinase Torso gene impaired pupation and adult emergence in Leptinotarsa decemlineata.

    PubMed

    Zhu, Tao-Tao; Meng, Qing-Wei; Guo, Wen-Chao; Li, Guo-Qing

    2015-12-01

    In Drosophila melanogaster prothoracic gland (PG) cells, Torso mediates prothoracicotropic hormone (PTTH)-triggered mitogen activated protein kinase (MAPK) pathway (consisting of four core components Ras, Raf, MEK and ERK) to stimulate ecdysteroidogenesis. In this study, LdTorso, LdRas, LdRaf and LdERK were cloned in Leptinotarsa decemlineata. The four genes were highly or moderately expressed in the larval prothoracic glands. At the first- to third-instar stages, their expression levels were higher just before and right after the molt, and were lower in the mid instars. At the fourth-instar stage, their transcript levels were higher before prepupal stage. RNA interference-mediated knockdown of LdTorso delayed larval development, increased pupal weight, and impaired pupation and adult emergence. Moreover, knockdown of LdTorso decreased the mRNA levels of LdRas, LdRaf and LdERK, repressed the transcription of two ecdysteroidogenesis genes (LdPHM and LdDIB), lowered 20E titer, and downregulated the expression of several 20E-response genes (LdEcR, LdUSP, LdHR3 and LdFTZ-F1). Furthermore, silencing of LdTorso induced the expression of a JH biosynthesis gene LdJHAMT, increased JH titer, and activated the transcription of a JH early-inducible gene LdKr-h1. Thus, our results suggest that Torso transduces PTTH-triggered MAPK signal to regulate ecdysteroidogenesis in the PGs in a non-drosophiline insect. PMID:26518287

  14. Decreased in vivo virulence and altered gene expression by a Brucella melitensis light-sensing histidine kinase mutant

    PubMed Central

    Gourley, Christopher R.; Petersen, Erik; Harms, Jerome; Splitter, Gary

    2015-01-01

    Brucella species utilize diverse virulence factors. Previously, Brucella abortus light-sensing histidine kinase was identified as important for cellular infection. Here, we demonstrate that a Brucella melitensisLOV-HK (BM-LOV-HK) mutant strain has strikingly different gene expression than wild type. General stress response genes including the alternative sigma factor rpoE1 and its anti-anti-sigma factor phyR were downregulated, while flagellar, quorum sensing (QS), and type IV secretion system genes were upregulated in the ΔBM-LOV-HK strain vs. wild type. Contextually, expression results agree with other studies of transcriptional regulators involving ΔrpoE1, ΔphyR, ΔvjbR, and ΔblxR (ΔbabR) Brucella strains. Additionally, deletion of BM-LOV-HK decreases virulence in mice. During C57BL/6 mouse infection, the ΔBM-LOV-HK strain had 2 logs less CFUs in the spleen 3 days postinfection, but similar levels 6 days post infection compared to wild type. Infection of IRF-1−/− mice more specifically define ΔBM-LOV-HK strain attenuation with fewer bacteria in spleens and significantly increased survival of mutant vs. wild-type infected IRF-1−/− mice. Upregulation of flagella, QS, and VirB genes, along with downregulation of rpoE1 and related sigma factor, rpoH2 (BMEI0280) suggest that BM-LOV-HK modulates both QS and general stress response regulatory components to control Brucella gene expression on a global level. PMID:25132657

  15. Up-regulated uridine kinase gene identified by RLCS in the ventral horn after crush injury to rat sciatic nerves.

    PubMed

    Yuh, I; Yaoi, T; Watanabe, S; Okajima, S; Hirasawa, Y; Fushiki, S

    1999-12-01

    Rat sciatic nerve crush injury is one of the models commonly employed for studying the mechanisms of nerve regeneration. In this study, we analyzed the temporal change of gene expression after injury in this model, to elucidate the molecular mechanisms involved in nerve regeneration. First, a cDNA analysis method, Restriction Landmark cDNA Scanning (RLCS), was applied to cells in the ventral horn of the spinal cord during a 7-day period after the crush injury. A total of 1991 cDNA species were detected as spots on gels, and 37 of these were shown to change after the injury. Temporally changed patterns were classified into three categories: the continuously up-regulated type (10 species), the transiently up-regulated type (22 species), and the down-regulated type (5 species). These complex patterns of gene expression demonstrated after the injury suggest that precise regulation in molecular pathways is required for accomplishing nerve regeneration. Secondly, the rat homologue of uridine kinase gene was identified as one of the up-regulated genes. Northern blot analysis on rat ventral horn tissue and brain revealed that the UK gene had three transcripts with different sizes (4.3, 1. 4, and 1.35 kb, respectively). All of the transcripts, especially the 4.3 kb one, were up-regulated mainly in a bimodal fashion during the 28-day period after the injury. The RLCS method that we employed in the present study shows promise as a means to fully analyze molecular changes in nerve regeneration in detail. PMID:10581173

  16. Mitogen-activated protein kinase kinase 5 (MKK5)-mediated signalling cascade regulates expression of iron superoxide dismutase gene in Arabidopsis under salinity stress

    PubMed Central

    Xing, Yu; Chen, Wei-hua; Jia, Wensuo; Zhang, Jianhua

    2015-01-01

    Superoxide dismutases (SODs) are involved in plant adaptive responses to biotic and abiotic stresses but the upstream signalling process that modulates their expression is not clear. Expression of two iron SODs, FSD2 and FSD3, was significantly increased in Arabidopsis in response to NaCl treatment but blocked in transgenic MKK5-RNAi plant, mkk5. Using an assay system for transient expression in protoplasts, it was found that mitogen-activated protein kinase kinase 5 (MKK5) was also activated in response to salt stress. Overexpression of MKK5 in wild-type plants enhanced their tolerance to salt treatments, while mkk5 mutant exhibited hypersensitivity to salt stress in germination on salt-containing media. Moreover, another kinase, MPK6, was also involved in the MKK5-mediated iron superoxide dismutase (FSD) signalling pathway in salt stress. The kinase activity of MPK6 was totally turned off in mkk5, whereas the activity of MPK3 was only partially blocked. MKK5 interacted with the MEKK1 protein that was also involved in the salt-induced FSD signalling pathway. These data suggest that salt-induced FSD2 and FSD3 expressions are influenced by MEKK1 via MKK5–MPK6-coupled signalling. This MAP kinase cascade (MEKK1, MKK5, and MPK6) mediates the salt-induced expression of iron superoxide dismutases. PMID:26136265

  17. PRKCE gene encoding protein kinase C-epsilon-Dual roles at sarcomeres and mitochondria in cardiomyocytes.

    PubMed

    Scruggs, Sarah B; Wang, Ding; Ping, Peipei

    2016-09-15

    Protein kinase C-epsilon (PKCε) is an isoform of a large PKC family of enzymes that has a variety of functions in different cell types. Here we discuss two major roles of PKCε in cardiac muscle cells; specifically, its role in regulating cardiac muscle contraction via targeting the sarcomeric proteins, as well as modulating cardiac cell energy production and metabolism by targeting cardiac mitochondria. The importance of PKCε action is described within the context of intracellular localization, as substrate selectivity and specificity is achieved through spatiotemporal targeting of PKCε. Accordingly, the role of PKCε in regulating myocardial function in physiological and pathological states has been documented in both cardioprotection and cardiac hypertrophy. PMID:27312950

  18. Regulation of Lhb and Egr1 Gene Expression by GNRH Pulses in Rat Pituitaries Is Both c-Jun N-Terminal Kinase (JNK)- and Extracellular Signal-Regulated Kinase (ERK)-Dependent1

    PubMed Central

    Burger, Laura L.; Haisenleder, Daniel J.; Aylor, Kevin W.; Marshall, John C.

    2009-01-01

    Pulsatile GNRH regulates the gonadotropin subunit genes in a differential manner, with faster frequencies favoring Lhb gene expression and slower frequencies favoring Fshb. Early growth response 1 (EGR1) is critical for Lhb gene transcription. We examined GNRH regulation of EGR1 and its two corepressors, Ngfi-A-binding proteins 1 and 2 (NAB1 and NAB2), both in vivo and in cultured rat pituitary cells. In rats, fast GNRH pulses (every 30 min) stably induced Egr1 primary transcript (PT) and mRNA 2-fold (P < 0.05) for 1–24 h. In contrast, slow GNRH pulses (every 240 min) increased Egr1 PT at 24 h (6-fold; P < 0.05) but increased Egr1 mRNA 4- to 5-fold between 4 and 24 h. Both GNRH pulse frequencies increased EGR1 protein 3- to 4-fold. In cultured rat pituitary cells, GNRH pulses (every 60 min) increased Egr1 (PT, 2.5- to 3-fold; mRNA, 1.5- to 2-fold; P < 0.05). GNRH pulses had little effect on Nab1/2 PT/mRNAs either in vivo or in vitro. We also examined specific intracellular signaling cascades activated by GNRH. Inhibitors of mitogen-activated protein kinase 8/9 (MAPK8/9 [also known as JNK]; SP600125) and MAP Kinase Kinase 1 (MAP2K1 [also known as MEK1]; PD98059) either blunted or totally suppressed the GNRH induction of Lhb PT and Egr1 PT/mRNA, whereas the MAPK14 (also known as p38) inhibitor SB203580 did not. In summary, pulsatile GNRH stimulates Egr1 gene expression and protein in vivo but not in a frequency-dependent manner. Additionally, GNRH-induced Egr1 gene expression is mediated by MAPK8/9 and MAPK1/3, and both are critical for Lhb gene transcription. PMID:19710510

  19. KEA: kinase enrichment analysis

    PubMed Central

    Lachmann, Alexander; Ma'ayan, Avi

    2009-01-01

    Motivation: Multivariate experiments applied to mammalian cells often produce lists of proteins/genes altered under treatment versus control conditions. Such lists can be projected onto prior knowledge of kinase–substrate interactions to infer the list of kinases associated with a specific protein list. By computing how the proportion of kinases, associated with a specific list of proteins/genes, deviates from an expected distribution, we can rank kinases and kinase families based on the likelihood that these kinases are functionally associated with regulating the cell under specific experimental conditions. Such analysis can assist in producing hypotheses that can explain how the kinome is involved in the maintenance of different cellular states and can be manipulated to modulate cells towards a desired phenotype. Summary: Kinase enrichment analysis (KEA) is a web-based tool with an underlying database providing users with the ability to link lists of mammalian proteins/genes with the kinases that phosphorylate them. The system draws from several available kinase–substrate databases to compute kinase enrichment probability based on the distribution of kinase–substrate proportions in the background kinase–substrate database compared with kinases found to be associated with an input list of genes/proteins. Availability: The KEA system is freely available at http://amp.pharm.mssm.edu/lib/kea.jsp Contact: avi.maayan@mssm.edu PMID:19176546

  20. Choline availability modulates human neuroblastoma cell proliferation and alters the methylation of the promoter region of the cyclin-dependent kinase inhibitor 3 gene.

    PubMed

    Niculescu, Mihai D; Yamamuro, Yutaka; Zeisel, Steven H

    2004-06-01

    Choline is an important methyl donor and a component of membrane phospholipids. In this study, we tested the hypothesis that choline availability can modulate cell proliferation and the methylation of genes that regulate cell cycling. In several other model systems, hypomethylation of cytosine bases that are followed by a guanosine (CpG) sites in the promoter region of a gene is associated with increased gene expression. We found that in choline-deficient IMR-32 neuroblastoma cells, the promoter of the cyclin-dependent kinase inhibitor 3 gene (CDKN3) was hypomethylated. This change was associated with increased expression of CDKN3 and increased levels of its gene product, kinase-associated phosphatase (KAP), which inhibits the G(1)/S transition of the cell cycle by dephosphorylating cyclin-dependent kinases. Choline deficiency also reduced global DNA methylation. The percentage of cells that accumulated bromodeoxyuridine (proportional to cell proliferation) was 1.8 times lower in the choline-deficient cells than in the control cells. Phosphorylated retinoblastoma (p110) levels were 3 times lower in the choline-deficient cells than in control cells. These findings suggest that the mechanism whereby choline deficiency inhibits cell proliferation involves hypomethylation of key genes regulating cell cycling. This may be a mechanism for our previously reported observation that stem cell proliferation in hippocampus neuroepithelium is decreased in choline-deficient rat and mouse fetuses. PMID:15147518

  1. Choline availability modulates human neuroblastoma cell proliferation and alters the methylation of the promoter region of the cyclin-dependent kinase inhibitor 3 gene

    PubMed Central

    Niculescu, Mihai D.; Yamamuro, Yutaka; Zeisel, Steven H.

    2006-01-01

    Choline is an important methyl donor and a component of membrane phospholipids. In this study, we tested the hypothesis that choline availability can modulate cell proliferation and the methylation of genes that regulate cell cycling. In several other model systems, hypomethylation of cytosine bases that are followed by a guanosine (CpG) sites in the promoter region of a gene is associated with increased gene expression. We found that in choline-deficient IMR-32 neuroblastoma cells, the promoter of the cyclin-dependent kinase inhibitor 3 gene (CDKN3) was hypomethylated. This change was associated with increased expression of CDKN3 and increased levels of its gene product, kinase-associated phosphatase (KAP), which inhibits the G1/S transition of the cell cycle by dephosphorylating cyclin-dependent kinases. Choline deficiency also reduced global DNA methylation. The percentage of cells that accumulated bromodeoxyuridine (proportional to cell proliferation) was 1.8 times lower in the choline-deficient cells than in the control cells. Phosphorylated retinoblastoma (p110) levels were 3 times lower in the choline-deficient cells than in control cells. These findings suggest that the mechanism whereby choline deficiency inhibits cell proliferation involves hypomethylation of key genes regulating cell cycling. This may be a mechanism for our previously reported observation that stem cell proliferation in hippocampus neuroepithelium is decreased in choline-deficient rat and mouse fetuses. PMID:15147518

  2. Characterization and expression analysis of SOMATIC EMBRYOGENESIS RECEPTOR KINASE (SERK) genes in sexual and apomictic Paspalum notatum.

    PubMed

    Podio, Maricel; Felitti, Silvina Andrea; Siena, Lorena Adelina; Delgado, Luciana; Mancini, Micaela; Seijo, José Guillermo; González, Ana María; Pessino, Silvina Claudia; Ortiz, Juan Pablo A

    2014-03-01

    The SOMATIC EMBRYOGENESIS RECEPTOR-LIKE KINASE (SERK) gene plays a fundamental role in somatic embryogenesis of angiosperms, and is associated with apomixis in Poa pratensis. The objective of this work was to isolate, characterize and analyze the expression patterns of SERK genes in apomictic and sexual genotypes of Paspalum notatum. A conserved 200-bp gene fragment was amplified from genomic DNA with heterologous primers, and used to initiate a chromosomal walking strategy for cloning the complete sequence. This procedure allowed the isolation of two members of the P. notatum SERK family; PnSERK1, which is similar to PpSERK1, and PnSERK2, which is similar to ZmSERK2 and AtSERK1. Phylogenetic analyses indicated that PnSERK1 and PnSERK2 represent paralogous sequences. Southern-blot hybridization indicated the presence of at least three copies of SERK genes in the species. qRT-PCR analyses revealed that PnSERK2 was expressed at significantly higher levels than PnSERK1 in roots, leaves, reproductive tissues and embryogenic calli. Moreover, in situ hybridization experiments revealed that PnSERK2 displayed a spatially and chronologically altered expression pattern in reproductive organs of the apomictic genotype with respect to the sexual one. PnSERK2 is expressed in nucellar cells of the apomictic genotype at meiosis, but only in the megaspore mother cell in the sexual genotype. Therefore, apomixis onset in P. notatum seems to be correlated with the expression of PnSERK2 in nucellar tissue. PMID:24146222

  3. Expression of sphingosine kinase gene in the interactions between human gastric carcinoma cell and vascular endothelial cell

    PubMed Central

    Ren, Juan; Dong, Lei; Xu, Cang-Bao; Pan, Bo-Rong

    2002-01-01

    AIM: To study the interactions between human gastric carcinoma cell (HGCC) and human vascular endothelial cell (HVEC), and if the expression of sphingosine kinase (SPK) gene was involved in these interactions. METHODS: The specific inhibitor to SPK, dimethyl sphingosine (DMS), was added acting on HGCC and HVEC, then the cell proliferation was measured by MTT. The conditioned mediums (CMs) of HGCC and HVEC were prepared. The CM of one kind of cell was added to the other kind of cell, and the cell proliferation was measured by MTT. After the action of CM, the cellular expression of SPK gene in mRNA level was detected with in situ hybridization (ISH). RESULTS: DMS could almost completely inhibit the proliferation of HGCC and HVEC. The growth inhibitory rates could amount to 97.21%, 83.42%, respectively (P < 0.01). The CM of HGCC could stimulate the growth of HVEC (2.70 ± 0.01, P < 0.01) while the CM of HVEC could inhibit the growth of HGCC (52.97% ± 0.01%, P < 0.01). There was no significant change in the mRNA level of SPK gene in one kind of cell after the action of the CM of the other kind of cell. CONCLUSION: SPK plays a key role in regulating the proliferation of HGCC and HVEC. There exist complicated interactions between HGCC and HVEC. HGCC can significantly stimulate the growth of HVEC while HVEC can significantly inhibit the growth of HGCC. The expression of SPK gene is not involved in the interactions. PMID:12174364

  4. Aluminum-induced gene expression and protein localization of a cell wall-associated receptor kinase in Arabidopsis.

    PubMed

    Sivaguru, Mayandi; Ezaki, Bunichi; He, Zheng-Hui; Tong, Hongyun; Osawa, Hiroki; Baluska, Frantisek; Volkmann, Dieter; Matsumoto, Hideaki

    2003-08-01

    Here, we report the aluminum (Al)-induced organ-specific expression of a WAK1 (cell wall-associated receptor kinase 1) gene and cell type-specific localization of WAK proteins in Arabidopsis. WAK1-specific reverse transcriptase-polymerase chain reaction analysis revealed an Al-induced WAK1 gene expression in roots. Short- and long-term analysis of gene expression in root fractions showed a typical "on" and "off" pattern with a first peak at 3 h of Al exposure followed by a sharp decline at 6 h and a complete disappearance after 9 h of Al exposure, suggesting the WAK1 is a further representative of Al-induced early genes. In shoots, upon root Al exposure, an increased but stable WAK1 expression was observed. Using confocal microscopy, we visualized Al-induced closure of leaf stomata, consistent with previous suggestions that the Al stress primarily experienced in roots associated with the transfer of root-shoot signals. Elevated levels of WAK protein in root cells were observed through western blots after 6 h of Al exposure, indicating a lag time between the Al-induced WAK transcription and translation. WAK proteins are localized abundantly to peripheries of cortex cells within the elongation zone of the root apex. In these root cells, disintegration of cortical microtubules was observed after Al treatment but not after the Al analog lanthanum treatments. Tip-growing control root hairs, stem stomata, and leaf stomatal pores are characterized with high amounts of WAKs, suggesting WAKs are accumulating at plasma membrane domains, which suffer from mechanical stress and lack dense arrays of supporting cortical microtubules. Further, transgenic plants overexpressing WAK1 showed an enhanced Al tolerance in terms of root growth when compared with the wild-type plants, making the WAK1 one of the important candidates for plant defense against Al toxicity. PMID:12913180

  5. Variation in the Phosphoinositide 3-Kinase Gamma Gene Affects Plasma HDL-Cholesterol without Modification of Metabolic or Inflammatory Markers

    PubMed Central

    Kächele, Martin; Hennige, Anita M.; Machann, Jürgen; Hieronimus, Anja; Lamprinou, Apostolia; Machicao, Fausto; Schick, Fritz; Fritsche, Andreas; Stefan, Norbert; Nürnberg, Bernd; Häring, Hans-Ulrich; Staiger, Harald

    2015-01-01

    Objective Phosphoinositide 3-kinase γ (PI3Kγ) is a G-protein-coupled receptor-activated lipid kinase mainly expressed in leukocytes and cells of the cardiovascular system. PI3Kγ plays an important signaling role in inflammatory processes. Since subclinical inflammation is a hallmark of atherosclerosis, obesity-related insulin resistance, and pancreatic β-cell failure, we asked whether common genetic variation in the PI3Kγ gene (PIK3CG) contributes to body fat content/distribution, serum adipokine/cytokine concentrations, alterations in plasma lipid profiles, insulin sensitivity, insulin release, and glucose homeostasis. Study Design Using a tagging single nucleotide polymorphism (SNP) approach, we analyzed genotype-phenotype associations in 2,068 German subjects genotyped for 10 PIK3CG SNPs and characterized by oral glucose tolerance tests. In subgroups, data from hyperinsulinaemic-euglycaemic clamps, magnetic resonance spectroscopy of the liver, whole-body magnetic resonance imaging, and intravenous glucose tolerance tests were available, and peripheral blood mononuclear cells (PBMCs) were used for gene expression analysis. Results After appropriate adjustment, none of the PIK3CG tagging SNPs was significantly associated with body fat content/distribution, adipokine/cytokine concentrations, insulin sensitivity, insulin secretion, or blood glucose concentrations (p>0.0127, all; Bonferroni-corrected α-level: 0.0051). However, six non-linked SNPs displayed at least nominal associations with plasma HDL-cholesterol concentrations, two of them (rs4288294 and rs116697954) reaching the level of study-wide significance (p = 0.0003 and p = 0.0004, respectively). More precisely, rs4288294 and rs116697954 influenced HDL2-, but not HDL3-, cholesterol. With respect to the SNPs’ in vivo functionality, rs4288294 was significantly associated with PIK3CG mRNA expression in PBMCs. Conclusions We could demonstrate that common genetic variation in the PIK3CG locus, possibly

  6. Functional characterization of sucrose non-fermenting 1 protein kinase complex genes in the Ascomycete Fusarium graminearum.

    PubMed

    Yu, Jungheon; Son, Hokyoung; Park, Ae Ran; Lee, Seung-Ho; Choi, Gyung Ja; Kim, Jin-Cheol; Lee, Yin-Won

    2014-02-01

    Sucrose non-fermenting 1 (SNF1) protein kinase complex is a heterotrimer that functions in energy homeostasis in eukaryotes by regulating transcription of glucose-repressible genes. Our previous study revealed that SNF1 of the homothallic ascomycete fungus Fusarium graminearum plays important roles in vegetative growth, sexual development, and virulence. In this study, we further identified the components of the SNF1 complex in F. graminearum and characterized their functions. We found that the SNF1 complex in F. graminearum consists of one alpha subunit (FgSNF1), one beta subunit (FgGAL83), and one gamma subunit (FgSNF4). Deletion of Fggal83 and Fgsnf4 resulted in alleviated phenotype changes in vegetative growth and sexual development as compared to those of the Fgsnf1 deletion mutant. However, all of the single, double, and triple deletion mutants among Fgsnf1, Fggal83, and Fgsnf4 had similar levels of decreased virulence. In addition, there was no synergistic effect of the mutant (single, double, or triple deletions of SNF1 complex component genes) phenotypes except for sucrose utilization. In this study, we revealed that FgSNF1 is mainly required for SNF1 complex functions, and the other two SNF1 complex components have adjunctive roles with FgSNF1 in sexual development and vegetative growth but have a major role in virulence in F. graminearum. PMID:24057127

  7. Regulation of gene expression by the small GTPase Rho through the ERK6 (p38γ) MAP kinase pathway

    PubMed Central

    Marinissen, Maria Julia; Chiariello, Mario; Gutkind, J. Silvio

    2001-01-01

    Small GTP-binding proteins of the Rho-family, Rho, Rac, and Cdc42, have been traditionally linked to the regulation of the cellular actin-based cytoskeleton. Rac and Cdc42 can also control the activity of JNK, thus acting in a molecular pathway transmitting extracellular signals to the nucleus. Interestingly, Rho can also regulate gene expression, albeit by a not fully understood mechanism. Here, we found that activated RhoA can stimulate c-jun expression and the activity of the c-jun promoter. As the complexity of the signaling pathways controlling the expression of c-jun has begun to be unraveled, this finding provided a unique opportunity to elucidate the biochemical routes whereby RhoA regulates nuclear events. We found that RhoA can initiate a linear kinase cascade leading to the activation of ERK6 (p38γ), a recently identified member of the p38 family of MAPKs. Furthermore, we present evidence that RhoA, PKN, MKK3/MKK6, and ERK6 (p38γ) are components of a novel signal transduction pathway involved in the regulation of gene expression and cellular transformation. PMID:11238375

  8. Never in mitosis gene A-related kinase 6 promotes cell proliferation of hepatocellular carcinoma via cyclin B modulation

    PubMed Central

    ZHANG, BIAO; ZHANG, HAI; WANG, DONG; HAN, SHENG; WANG, KE; YAO, AIHUA; LI, XIANGCHENG

    2014-01-01

    Never in mitosis gene A-related kinase (Nek) 6 is a recently identified Nek that is required for mitotic cell cycle progression; however, the role and mechanism of Nek6 activity during hepatocarcinogenesis is not well known. The aim of this study was to investigate the potential roles and internal mechanism of Nek6 in hepatocellular carcinoma (HCC) development. In the present study, Nek6 was found to be overexpressed in HCC samples and cell lines by florescent real-time quantitative polymerase chain reaction, immunohistochemistry and western blot analysis. Furthermore, it was evidenced to contribute to oncogenesis and progression. The ectopic overexpression of Nek6 promoted cell proliferation and colony formation, whereas gene silencing of Nek6 inhibited these phenotypes, as documented in Huh7, PLC/PRF/5, Hep3B and HepG2 HCC cell lines. Mechanistic analyses indicated that Nek6 regulates the transcription of cyclin B through cdc2 activation, and promotes the accumulation of G0/G1-phase cells. In conclusion, the findings of the current study suggested that Nek6 contributes to the oncogenic potential of HCC, and may present as a potential therapeutic target in this disease. PMID:25120679

  9. The FvMK1 mitogen-activated protein kinase gene regulates conidiation, pathogenesis, and fumonisin production in Fusarium verticillioides.

    PubMed

    Zhang, Yueping; Choi, Yoon-E; Zou, Xuexiao; Xu, Jin-Rong

    2011-02-01

    Fusarium verticillioides is one of the most important fungal pathogens to cause destructive diseases of maize worldwide. Fumonisins produced by the fungus are harmful to human and animal health. To date, our understanding of the molecular mechanisms associated with pathogenicity and fumonisin biosynthesis in F. verticillioides is limited. Because MAP kinase pathways have been implicated in regulating diverse processes important for plant infection in phytopathogenic fungi, in this study we identified and functionally characterized the FvMK1 gene in F. verticillioides. FvMK1 is orthologous to FMK1 in F. oxysporum and GPMK1 in F. graminearum. The Fvmk1 deletion mutant was reduced in vegetative growth and production of microconidia. However, it was normal in sexual reproduction and increased in the production of macroconidia. In infection assays with developing corn kernels, the Fvmk1 mutant was non-pathogenic and failed to colonize through wounding sites. It also failed to cause stalk rot symptoms beyond the inoculation sites on corn stalks, indicating that FvMK1 is essential for plant infection. Furthermore, the Fvmk1 mutant was significantly reduced in fumonisin production and expression levels of FUM1 and FUM8, two genes involved in fumonisin biosynthesis. The defects of the Fvmk1 mutant were fully complemented by re-introducing the wild type FvMK1 allele. These results demonstrate that FvMK1 plays critical roles in the regulation of vegetative growth, asexual reproduction, fumonisin biosynthesis, and pathogenicity. PMID:20887797

  10. Genes, enzymes and regulation of arginine biosynthesis in plants.

    PubMed

    Slocum, Robert D

    2005-08-01

    Arabidopsis genes encoding enzymes for each of the eight steps in L-arginine (Arg) synthesis were identified, based upon sequence homologies with orthologs from other organisms. Except for N-acetylglutamate synthase (NAGS; EC 2.3.1.1), which is encoded by two genes, all remaining enzymes are encoded by single genes. Targeting predictions for these enzymes, based upon their deduced sequences, and subcellular fractionation studies, suggest that most enzymes of Arg synthesis reside within the plastid. Synthesis of the L-ornthine (Orn) intermediate in this pathway from L-glutamate occurs as a series of acetylated intermediates, as in most other organisms. An N-acetylornithine:glutamate acetyltransferase (NAOGAcT; EC 2.3.1.35) facilitates recycling of the acetyl moiety during Orn formation (cyclic pathway). A putative N-acetylornithine deacetylase (NAOD; EC 3.5.1.16), which participates in the "linear" pathway for Orn synthesis in some organisms, was also identified. Previous biochemical studies have indicated that allosteric regulation of the first and, especially, the second steps in Orn synthesis (NAGS; N-acetylglutamate kinase (NAGK), EC 2.7.2.8) by the Arg end-product are the major sites of metabolic control of the pathway in organisms using the cyclic pathway. Gene expression profiling for pathway enzymes further suggests that NAGS, NAGK, NAOGAcT and NAOD are coordinately regulated in response to changes in Arg demand during plant growth and development. Synthesis of Arg from Orn is further coordinated with pyrimidine nucleotide synthesis, at the level of allocation of the common carbamoyl-P intermediate. PMID:16122935

  11. Deletion of the gene Pip4k2c, a novel phosphatidylinositol kinase, results in hyperactivation of the immune system.

    PubMed

    Shim, Hyeseok; Wu, Chuan; Ramsamooj, Shivan; Bosch, Kaitlyn N; Chen, Zuojia; Emerling, Brooke M; Yun, Jihye; Liu, Hui; Choo-Wing, Rayman; Yang, Zhiwei; Wulf, Gerburg M; Kuchroo, Vijay Kumar; Cantley, Lewis C

    2016-07-01

    Type 2 phosphatidylinositol-5-phosphate 4-kinase (PI5P4K) converts phosphatidylinositol-5-phosphate to phosphatidylinositol-4,5-bisphosphate. Mammals have three enzymes PI5P4Kα, PI5P4Kβ, and PI5P4Kγ, and these enzymes have been implicated in metabolic control, growth control, and a variety of stress responses. Here, we show that mice with germline deletion of type 2 phosphatidylinositol-5-phosphate 4-kinase gamma (Pip4k2c), the gene encoding PI5P4Kγ, appear normal in regard to growth and viability but have increased inflammation and T-cell activation as they age. Immune cell infiltrates increased in Pip4k2c(-/-) mouse tissues. Also, there was an increase in proinflammatory cytokines, including IFNγ, interleukin 12, and interleukin 2 in plasma of Pip4k2c(-/-) mice. Pip4k2c(-/-) mice had an increase in T-helper-cell populations and a decrease in regulatory T-cell populations with increased proliferation of T cells. Interestingly, mammalian target of rapamycin complex 1 (mTORC1) signaling was hyperactivated in several tissues from Pip4k2c(-/-) mice and treating Pip4k2c(-/-) mice with rapamycin reduced the inflammatory phenotype, resulting in a decrease in mTORC1 signaling in tissues and a decrease in proinflammatory cytokines in plasma. These results indicate that PI5P4Kγ plays a role in the regulation of the immune system via mTORC1 signaling. PMID:27313209

  12. A Dominant Negative Mutant of Cyclin-Dependent Kinase A Reduces Endoreduplication but Not Cell Size or Gene Expression in Maize Endosperm

    PubMed Central

    Leiva-Neto, João T.; Grafi, Gideon; Sabelli, Paolo A.; Dante, Ricardo A.; Woo, Young-min; Maddock, Sheila; Gordon-Kamm, William J.; Larkins, Brian A.

    2004-01-01

    Cells in maize (Zea mays) endosperm undergo multiple cycles of endoreduplication, with some attaining DNA contents as high as 96C and 192C. Genome amplification begins around 10 d after pollination, coincident with cell enlargement and the onset of starch and storage protein accumulation. Although the role of endoreduplication is unclear, it is thought to provide a mechanism that increases cell size and enhances gene expression. To investigate this process, we reduced endoreduplication in transgenic maize endosperm by ectopically expressing a gene encoding a dominant negative mutant form of cyclin-dependent kinase A. This gene was regulated by the 27-kD γ-zein promoter, which restricted synthesis of the defective enzyme to the endoreduplication rather than the mitotic phase of endosperm development. Overexpression of a wild-type cyclin-dependent kinase A increased enzyme activity but had no effect on endoreduplication. By contrast, ectopic expression of the defective enzyme lowered kinase activity and reduced by half the mean C-value and total DNA content of endosperm nuclei. The lower level of endoreduplication did not affect cell size and only slightly reduced starch and storage protein accumulation. There was little difference in the level of endosperm gene expression with high and low levels of endoreduplication, suggesting that this process may not enhance transcription of genes associated with starch and storage protein synthesis. PMID:15208390

  13. Activation of ATP binding for the autophosphorylation of DosS, a Mycobacterium tuberculosis histidine kinase lacking an ATP lid motif.

    PubMed

    Cho, Ha Yeon; Lee, Young-Hoon; Bae, Young-Seuk; Kim, Eungbin; Kang, Beom Sik

    2013-05-01

    The sensor histidine kinases of Mycobacterium tuberculosis, DosS and DosT, are responsible for sensing hypoxic conditions and consist of sensor and kinase cores responsible for accepting signals and phosphorylation activity, respectively. The kinase core contains a dimerization and histidine phosphate-accepting (DHp) domain and an ATP binding domain (ABD). The 13 histidine kinase genes of M. tuberculosis can be grouped based on the presence or absence of the ATP lid motif and F box (elements known to play roles in ATP binding) in their ABDs; DosS and DosT have ABDs lacking both these elements, and the crystal structures of their ABDs indicated that they were unsuitable for ATP binding, as a short loop covers the putative ATP binding site. Although the ABD alone cannot bind ATP, the kinase core is functional in autophosphorylation. Appropriate spatial arrangement of the ABD and DHp domain within the kinase core is required for both autophosphorylation and ATP binding. An ionic interaction between Arg(440) in the DHp domain and Glu(537) in the short loop of the ABD is available and may open the ATP binding site, by repositioning the short loop away from the site. Mutations at Arg(440) and Glu(537) reduce autophosphorylation activity. Unlike other histidine kinases containing an ATP lid, which protects bound ATP, DosS is unable to accept ATP until the ABD is properly positioned relative to the histidine; this may prevent unexpected ATP reactions. ATP binding can, therefore, function as a control mechanism for histidine kinase activity. PMID:23486471

  14. Transcriptome and proteome analysis of tyrosine kinase inhibitor treated canine mast cell tumour cells identifies potentially kit signaling-dependent genes

    PubMed Central

    2012-01-01

    Background Canine mast cell tumour proliferation depends to a large extent on the activity of KIT, a tyrosine kinase receptor. Inhibitors of the KIT tyrosine kinase have recently been introduced and successfully applied as a therapeutic agent for this tumour type. However, little is known on the downstream target genes of this signaling pathway and molecular changes after inhibition. Results Transcriptome analysis of the canine mast cell tumour cell line C2 treated for up to 72 hours with the tyrosine kinase inhibitor masitinib identified significant changes in the expression levels of approximately 3500 genes or 16% of the canine genome. Approximately 40% of these genes had increased mRNA expression levels including genes associated with the pro-proliferative pathways of B- and T-cell receptors, chemokine receptors, steroid hormone receptors and EPO-, RAS and MAP kinase signaling. Proteome analysis of C2 cells treated for 72 hours identified 24 proteins with changed expression levels, most of which being involved in gene transcription, e.g. EIA3, EIA4, TARDBP, protein folding, e.g. HSP90, UCHL3, PDIA3 and protection from oxidative stress, GSTT3, SELENBP1. Conclusions Transcriptome and proteome analysis of neoplastic canine mast cells treated with masitinib confirmed the strong important and complex role of KIT in these cells. Approximately 16% of the total canine genome and thus the majority of the active genes were significantly transcriptionally regulated. Most of these changes were associated with reduced proliferation and metabolism of treated cells. Interestingly, several pro-proliferative pathways were up-regulated, which may represent attempts of masitinib treated cells to activate alternative pro-proliferative pathways. These pathways may contain hypothetical targets for a combination therapy with masitinib to further improve its therapeutic effect. PMID:22747577

  15. Gene expression in bovine oocytes and cumulus cells after meiotic inhibition with the cyclin-dependent kinase inhibitor butyrolactone I.

    PubMed

    Leal, C L V; Mamo, S; Fair, T; Lonergan, P

    2012-08-01

    The aim of this study was to determine the effect of temporary inhibition of meiosis using the cyclin-dependent kinase inhibitor butyrolactone I (BLI) on gene expression in bovine oocytes and cumulus cells. Immature bovine cumulus-oocyte complexes (COCs) were assigned to groups: (i) Control COCs collected immediately after recovery from the ovary or (ii) after in vitro maturation (IVM) for 24 h, (iii) Inhibited COCs collected 24 h after incubation with 100 μm BLI or (iv) after meiotic inhibition for 24 h followed by IVM for a further 22 h. For mRNA relative abundance analysis, pools of 10 denuded oocytes and respective cumulus cells were collected. Transcripts related to cell cycle regulation and oocyte competence were evaluated in oocytes and cumulus cells by quantitative real-time PCR (qPCR). Most of the examined transcripts were downregulated (p < 0.05) after IVM in control and inhibited oocytes (19 of 35). Nine transcripts remained stable (p > 0.05) after IVM in control oocytes; only INHBA did not show this pattern in inhibited oocytes. Seven genes were upregulated after IVM in control oocytes (p < 0.05), and only PLAT, RBP1 and INHBB were not upregulated in inhibited oocytes after IVM. In cumulus cells, six genes were upregulated (p < 0.05) after IVM and eight were downregulated (p < 0.05). Cells from inhibited oocytes showed the same pattern of expression regarding maturation profile, but were affected by the temporary meiosis inhibition of the oocyte when the same maturation stages were compared between inhibited and control groups. In conclusion, changes in transcript abundance in oocytes and cumulus cells during maturation in vitro were mostly mirrored after meiotic inhibition followed by maturation. PMID:22034924

  16. Transcriptional induction of pim-1 protein kinase gene expression by interferon gamma and posttranscriptional effects on costimulation with steel factor.

    PubMed

    Yip-Schneider, M T; Horie, M; Broxmeyer, H E

    1995-06-15

    Steel factor (SLF) synergizes with interferon gamma (IFN gamma) to stimulate proliferation of the human factor-dependent cell line MO7e. We examined the effects of IFN gamma and SLF treatment, alone or in combination, on the expression of a 33-kD cytoplasmic protein serine/threonine kinase designated pim-1 whose expression has been closely associated with proliferation induced by related myeloid cytokines. IFN gamma alone, but not SLF, stimulated expression of pim-1 RNA and protein in MO7e cells; compared with IFN gamma alone, costimulation with IFN gamma and SLF resulted in a twofold to threefold increase in pim-1 message and protein expression, correlating with synergistic effects on cell proliferation. Both IFN gamma and IFN gamma/SLF induced pim-1 mRNA in the absence of de novo protein synthesis. Nuclear run-on assays showed that, although IFN gamma alone increased the rate of pim-1 gene transcription, costimulation with IFN gamma and SLF did not further potentiate this effect; however, the stability of pim-1 message was significantly enhanced in the presence of both cytokines. An IFN gamma-responsive element within the 5' flanking region of the pim-1 gene that could confer IFN gamma responsiveness on a heterologous promoter was identified. This sequence, designated PMGAS, formed a specific complex with Stat (signal transducers and activators of transcription) 1 alpha (the p91 subunit of the transcription factor ISGF3 [interferon-stimulated gene factor 3]) in IFN gamma-treated cell extracts, suggesting that the transcriptional effects of IFN gamma on pim-1 expression may be mediated by Stat 1 alpha. PMID:7540064

  17. Characterization and mapping of the human rhodopsin kinase gene and screening of the gene for mutations in patients with retinitis pigmentosa

    SciTech Connect

    Khani, S.C.; Lin, D.; Magovcevic, I.

    1994-09-01

    Rhodopsin kinase (RK) is a cytosolic enzyme in rod photoreceptors that initiates the deactivation of the phototransductions cascade by phosphorylating photoactivated rhodopsin. Although the cDNA sequence of bovine RK has been determined previously, no human cDNA or genomic sequence has thus far been available for genetic studies. In order to investigate the possible role of this candidate gene in retinitis pigmentosa (RP) and allied diseases, we have isolated and characterized human cDNA and genomic clones derived from the RK locus. The coding sequence of the human gene is 1692 nucleotides in length and is split into seven exons. The human and the bovine sequence show 84% identity at the nucleotide level and 92% identity at the amino acid level. Thus far, the intronic sequences flanking each exon except for one have been determined. We have also mapped the human RK gene to chromosome 13q34 using fluorescence in situ hybridization. To our knowledge, no RP gene has as yet been linked to this region. However, since the substrate for RK (rhodopsin) and other members of the phototransduction cascade have been implicated in the pathogenesis of RP, it is conceivable that defects in RK can also cause some forms of this disease. We are evaluating this possibility by screening DNA from 173 patients with autosomal recessive RP and 190 patients with autosomal dominant RP. So far, we have found 11 patients with variant bands. In one patient with autosomal dominant RP we discovered the missense change Ser536Leu. Cosegregation studies and further sequencing of the variant bands are currently underway.

  18. Tyrosine kinase inhibitors for epidermal growth factor receptor gene mutation-positive non-small cell lung cancers: an update for recent advances in therapeutics.

    PubMed

    Chung, Clement

    2016-06-01

    The presence of activating gene mutations in the epidermal growth factor receptor of non-small cell lung cancer patients is predictive (improved progression-free survival and improved response rate) when treated with small molecule tyrosine kinase inhibitors such as gefitinib, erlotinib and afatinib. The two most common mutations that account for greater than 85% of all EGFR gene mutations are in-frame deletions in exon 19 (LREA deletions) and substitution in exon 21 (L858R). Exon 18 mutations occur much less frequently at about 4% of all EGFR gene mutations. Together, exon 19 deletion and exon 21 L858R gene substitution are present in about 10% of Caucasian patients and 20-40% of Asian patients with non-small cell lung cancer. T790M gene mutation at exon 20 is associated with acquired resistance to epidermal growth factor receptor tyrosine kinase inhibitors. Early studies showed that activating EGFR gene mutations are most common in patients with adenocarcinoma histology, women, never smokers and those of Asian ethnicity. A recent multi-center phase III trial suggested that frontline epidermal growth factor receptor tyrosine kinase inhibitor therapy with afatinib is associated with improved progression-free survival compared to chemotherapy regardless of race. Moreover, guidelines now suggest EGFR gene mutation testing should be conducted in all patients with lung adenocarcinoma or mixed lung cancers with an adenocarcinoma component, regardless of characteristics such as smoking status, gender or race. The success of targeted therapies in non-small cell lung cancer patients has changed the treatment paradigm in metastatic non-small cell lung cancer. However, despite a durable response of greater than a year, resistance to epidermal growth factor receptor tyrosine kinase inhibitors inevitably occurs. This mini-review describes the clinically relevant EGFR gene mutations and the efficacy/toxicity of small molecule epidermal growth factor receptor tyrosine kinase

  19. Expression of Arg-Gingipain RgpB Is Required for Correct Glycosylation and Stability of Monomeric Arg-Gingipain RgpA from Porphyromonas gingivalis W50

    PubMed Central

    Rangarajan, Minnie; Hashim, Ahmed; Aduse-Opoku, Joseph; Paramonov, Nikolay; Hounsell, Elizabeth F.; Curtis, Michael A.

    2005-01-01

    Arg-gingipains are extracellular cysteine proteases produced by the gram-negative periodontal pathogen Porphyromonas gingivalis and are encoded by rgpA and rgpB. Three Arg-gingipains, heterodimeric high-molecular-mass Arg-gingipain HRgpA comprising the α-catalytic chain and the β-adhesin chain, the monomeric soluble Arg-gingipain comprising only the α-catalytic chain (RgpAcat), and the monomeric membrane-type heavily glycosylated Arg-gingipain comprising the α-catalytic chain (mt-RgPAcat), are derived from rgpA. The monomeric enzymes contain between 14 and 30% carbohydrate by weight. rgpB encodes two monomeric enzymes, RgpB and mt-RgpB. Earlier work indicated that rgpB is involved in the glycosylation process, since inactivation of rgpB results in the loss of not only RgpB and mt-RgpB but also mt-RgpAcat. This work aims to confirm the role of RgpB in the posttranslational modification of RgpAcat and the effect of aberrant glycosylation on the properties of this enzyme. Two-dimensional gel electrophoresis of cellular proteins from W50 and an inactivated rgpB strain (D7) showed few differences, suggesting that loss of RgpB has a specific effect on RgpA maturation. Inactivation of genes immediately upstream and downstream of rgpB had no effect on rgpA-derived enzymes, suggesting that the phenotype of the rgpB mutant is not due to a polar effect on transcription at this locus. Matrix-assisted laser desorption ionization-time of flight analysis of purified RgpAcat from W50 and D7 strains gave identical peptide mass fingerprints, suggesting that they have identical polypeptide chains. However, RgpAcat from D7 strain had a higher isoelectric point and a dramatic decrease in thermostability and did not cross-react with a monoclonal antibody which recognizes a glycan epitope on the parent strain enzyme. Although it had the same total sugar content as the parent strain enzyme, there were significant differences in the monosaccharide composition and linking sugars. These

  20. Lethal Netherton syndrome due to homozygous p.Arg371X mutation in SPINK5.

    PubMed

    Diociaiuti, Andrea; Castiglia, Daniele; Fortugno, Paola; Bartuli, Andrea; Pascucci, Monica; Zambruno, Giovanna; El Hachem, May

    2013-01-01

    Here we report a lethal case of Netherton syndrome presenting with neurologic complications, hypernatremic dehydration, failure to thrive, and episodes of sepsis. Molecular analysis of the serine protease inhibitor Kazal-type 5 gene identified a homozygous mutation (c.1111C>T, p.Arg371X). This case highlights the importance of early diagnosis to start appropriate care in a timely fashion and prevent disease complications. PMID:23331056

  1. Chromosome mapping of the human arrestin (SAG), {beta}-arrestin 2 (ARRB2), and {beta}-adrenergic receptor kinase 2 (ADRBK2) genes

    SciTech Connect

    Calabrese, G.; Sallese, M.; Stornaiuolo, A.

    1994-09-01

    Two types of proteins play a major role in determining homologous desensitization of G-coupled receptors: {beta}-adrenergic receptor kinase ({beta}ARK), which phosphorylates the agonist-occupied receptor and its functional cofactor, {beta}-arrestin. Both {beta}ARK and {beta}-arrestin are members of multigene families. The family of G-protein-coupled receptor kinases includes rhodopsin kinase, {beta}ARK1, {beta}ARK2, IT11-A (GRK4), GRK5, and GRK6. The arrestin/{beta}-arrestin gene family includes arrestin (also known as S-antigen), {beta}-arrestin 1, and {beta}-arrestin 2. Here we report the chromosome mapping of the human genes for arrestin (SAG), {beta}arrestin 2 (ARRB2), and {beta}ARK2 (ADRBK2) by fluorescence in situ hybridization (FISH). FISH results confirmed the assignment of the gene coding for arrestin (SAG) to chromosome 2 and allowed us to refine its localization to band q37. The gene coding for {beta}-arrestin 2 (ARRB2) was mapped to chromosome 17p13 and that coding for {beta}ARK2 (ADRBK2) to chromosome 22q11. 17 refs., 1 fig.

  2. Generation of a High Number of Healthy Erythroid Cells from Gene-Edited Pyruvate Kinase Deficiency Patient-Specific Induced Pluripotent Stem Cells

    PubMed Central

    Garate, Zita; Quintana-Bustamante, Oscar; Crane, Ana M.; Olivier, Emmanuel; Poirot, Laurent; Galetto, Roman; Kosinski, Penelope; Hill, Collin; Kung, Charles; Agirre, Xabi; Orman, Israel; Cerrato, Laura; Alberquilla, Omaira; Rodriguez-Fornes, Fatima; Fusaki, Noemi; Garcia-Sanchez, Felix; Maia, Tabita M.; Ribeiro, Maria L.; Sevilla, Julian; Prosper, Felipe; Jin, Shengfang; Mountford, Joanne; Guenechea, Guillermo; Gouble, Agnes; Bueren, Juan A.; Davis, Brian R.; Segovia, Jose C.

    2015-01-01

    Summary Pyruvate kinase deficiency (PKD) is a rare erythroid metabolic disease caused by mutations in the PKLR gene. Erythrocytes from PKD patients show an energetic imbalance causing chronic non-spherocytic hemolytic anemia, as pyruvate kinase defects impair ATP production in erythrocytes. We generated PKD induced pluripotent stem cells (PKDiPSCs) from peripheral blood mononuclear cells (PB-MNCs) of PKD patients by non-integrative Sendai viral vectors. PKDiPSCs were gene edited to integrate a partial codon-optimized R-type pyruvate kinase cDNA in the second intron of the PKLR gene by TALEN-mediated homologous recombination (HR). Notably, we found allele specificity of HR led by the presence of a single-nucleotide polymorphism. High numbers of erythroid cells derived from gene-edited PKDiPSCs showed correction of the energetic imbalance, providing an approach to correct metabolic erythroid diseases and demonstrating the practicality of this approach to generate the large cell numbers required for comprehensive biochemical and metabolic erythroid analyses. PMID:26549847

  3. Generation of a High Number of Healthy Erythroid Cells from Gene-Edited Pyruvate Kinase Deficiency Patient-Specific Induced Pluripotent Stem Cells.

    PubMed

    Garate, Zita; Quintana-Bustamante, Oscar; Crane, Ana M; Olivier, Emmanuel; Poirot, Laurent; Galetto, Roman; Kosinski, Penelope; Hill, Collin; Kung, Charles; Agirre, Xabi; Orman, Israel; Cerrato, Laura; Alberquilla, Omaira; Rodriguez-Fornes, Fatima; Fusaki, Noemi; Garcia-Sanchez, Felix; Maia, Tabita M; Ribeiro, Maria L; Sevilla, Julian; Prosper, Felipe; Jin, Shengfang; Mountford, Joanne; Guenechea, Guillermo; Gouble, Agnes; Bueren, Juan A; Davis, Brian R; Segovia, Jose C

    2015-12-01

    Pyruvate kinase deficiency (PKD) is a rare erythroid metabolic disease caused by mutations in the PKLR gene. Erythrocytes from PKD patients show an energetic imbalance causing chronic non-spherocytic hemolytic anemia, as pyruvate kinase defects impair ATP production in erythrocytes. We generated PKD induced pluripotent stem cells (PKDiPSCs) from peripheral blood mononuclear cells (PB-MNCs) of PKD patients by non-integrative Sendai viral vectors. PKDiPSCs were gene edited to integrate a partial codon-optimized R-type pyruvate kinase cDNA in the second intron of the PKLR gene by TALEN-mediated homologous recombination (HR). Notably, we found allele specificity of HR led by the presence of a single-nucleotide polymorphism. High numbers of erythroid cells derived from gene-edited PKDiPSCs showed correction of the energetic imbalance, providing an approach to correct metabolic erythroid diseases and demonstrating the practicality of this approach to generate the large cell numbers required for comprehensive biochemical and metabolic erythroid analyses. PMID:26549847

  4. Molecular evolution and nucleotide diversity of nuclear plastid phosphoglycerate kinase (PGK) gene in Triticeae (Poaceae).

    PubMed

    Adderley, Shawn; Sun, Genlou

    2014-01-01

    Levels of nucleotide divergence provide key evidence in the evolution of polyploids. The nucleotide diversity of 226 sequences of pgk1 gene in Triticeae species was characterized. Phylogenetic analyses based on the pgk1 gene were carried out to determine the diploid origin of polyploids within the tribe in relation to their A(u), B, D, St, Ns, P, and H haplomes. Sequences from the Ns genome represented the highest nucleotide diversity values for both polyploid and diploid species with π=0.03343 and θ=0.03536 for polyploid Ns genome sequences and π=0.03886 and θ=0.03886 for diploid Psathyrostachys sequences, while Triticum urartu represented the lowest diversity among diploid species at π=0.0011 and θ=0.0011. Nucleotide variation of diploid Aegilops speltoides (π=0.2441, presumed the B genome donor of Triticum species) is five times higher than that (π=0.00483) of B genome in polyploid species. Significant negative Tajima's D values for the St, A(u), and D genomes along with high rates of polymorphisms and low sequence diversity were observed. Origins of the A(u), B, and D genomes were linked to T. urartu, A. speltoides, and A. tauschii, respectively. Putative St genome donor was Pseudoroegneria, while Ns and P donors were Psathyrostachys and Agropyron. H genome diploid donor is Hordeum. PMID:24120623

  5. E sub 1 BF is an essential RNA polymerase I transcription factor with an intrinsic protein kinase activity that can modulate rRNA gene transcription

    SciTech Connect

    Ji Zhang; Huifeng Niu; Jacob, S.T. )

    1991-10-01

    The authors previously described the purification and characterization of E{sub 1}BF, a rat rRNA gene core promoter-binding factor that consists of two polypeptides of 89 and 79 kDa. When this factor was incubated in the absence of any exogenous protein kinase under conditions optimal for protein phosphorylation, the 79-kDa polypeptide of E{sub 1}BF was selectively phosphorylated. The labeled phosphate could be removed from the E{sub 1}BF polypeptide by treatment with calf intestinal alkaline phosphatase or potato acid phosphatase. Elution of the protein from the E{sub 1}BF-promoter complex formed in an electrophoretic mobility-shift assay followed by incubation of the concentrated eluent with ({gamma}-{sup 32}P)ATP resulted in the selective labeling o the 79-kDa band. The E{sub 1}BF-associated protein kinase did not phosphorylate casein or histone H1. These data demonstrate that (1) polymerase I promoter-binding factor E{sub 1}BF contains an intrinsic substrate-specific protein kinase and (2) E{sub 1}BF is an essential polymerase I transcription factor that can modulate rRNA gene transcription by protein phosphorylation. Further, these studies have provided a direct means to identify a protein kinase or any other enzyme that can interact with a specific DNA sequence.

  6. The effects of various kinase and phosphatase inhibitors on the transmission of the prolactin and extracellular matrix signals to rabbit alpha S1-casein and transferrin genes.

    PubMed

    Bayat-Sarmadi, M; Puissant, C; Houdebine, L M

    1995-07-01

    In all species, milk protein genes are specifically expressed in the mammary gland under the control of lactogenic hormones and extracellular matrix. In rabbit, casein gene expression is induced by prolactin alone and this induction is amplified by extracellular matrix. Transferrin gene expression is induced by extracellular matrix in the absence of hormones. The transduction mechanisms of prolactin and extracellular matrix to milk protein genes is only partly known. The present study has been undertaken to determine if protein kinases and phosphatases are involved in these mechanisms. Rabbit primary mammary cells were cultured in three different conditions (i) directly on floating collagen I, (ii) on plastic after a trypsinization to remove endogenous extracellular matrix, and (iii) on floating collagen I after a trypsinization to restore a functional extracellular matrix. In these culture conditions, prolactin and several protein kinase and phosphatase inhibitors were added to the medium. The expression of alpha S1-casein and transferrin genes was evaluated using Northern blotting analysis. In cells cultured directly on collagen I, staurosporine, quercetin and 6-dimethylaminopurine strongly inhibited prolactin action of alpha S1-casein gene whereas herbimycin A was only partly inhibitory. An erbstatin analogue, tyrosine phosphate, 1(5 isoquinolylsulphonyl) 2-methylpiperazine and GF 109 203 X did not alter prolactin action. The inhibitors which inhibited prolactin action when cells were directly cultured on collagen I were also those which prevented the induction of alpha S1-casein gene expression when cells were cultured on plastic in the absence of extracellular matrix. The induction of transferrin gene by the extracellular matrix was inhibited slightly by quercetin. Okadaic acid, phenylarsine oxide and sodium pervanadate which inhibit Ser/Thr and Tyr phosphatase inhibitors were unable to mimic prolactin action on alpha S1-casein gene expression. On the contrary

  7. Isolation, characterization and expression of cyclin and cyclin-dependent kinase genes in Jerusalem artichoke (Helianthus tuberosus L.).

    PubMed

    Freeman, Donna; Riou-Khamlichi, Catherine; Oakenfull, E Ann; Murray, James A H

    2003-01-01

    Tuber explants of Jerusalem artichoke (Helianthus tuberosus L.) are a model system for cell-cycle re-entry from a quiescent state, involving the activation of division of tuber parenchyma cells in response to exogenous auxin. To enable molecular studies of this system, two cyclin (Heltu;CYCD1;1 and Heltu; CYCD3;1) and two cyclin-dependent kinase (Heltu; CDKA;1 and Heltu;CDKB1;1) genes have been isolated from a Jerusalem artichoke cDNA library and their expression demonstrated during the activation of cell division. It was found that CDKA;1 transcripts are present in quiescent tubers, whereas CYCD1;1, CYCD3;1 and CDKB1;1 transcripts are induced during cell-cycle re-entry as well as during bud growth of whole tubers. Both CYCD1;1 and CYCD3;1 transcripts appear shortly before, or coincident with, the onset of S phase. PMID:12493857

  8. [Cloning and expression analysis of 4- (cytidine-5-diphospho) -2-C-methyl-D-erythritol kinase gene in Tripterygium wilfordii].

    PubMed

    Tong, Yu-ru; Su, Ping; Zhao, Yu-jun; Zhang, Meng; Wang, Xiu-juan; Hu, Tian-yuan; Gao, Wei; Huang, Lu-qi

    2015-11-01

    4-(Cytidine-5-diphospho) -2-C-methyl-D-erythritol kinase is a key enzyme in the biosynthesis pathway of terpenoids. According to the transcriptome database, the specific primers were designed and used in PCR. The bioinformatic analysis of the sequenced TwCMK gene was performed in several bioinformatics software. The Real-time fluorescence quantification polymerase chain reaction (RT-qPCR) were used to detect the expression levels of TwCMK from T. wilfordii after elicitor MeJA supplied. The results showed that the full length of TwCMK cDNA was 1 732 bp encoding 387 amino acids. The theoretical isoelectric point of the putative TwCMK protein was 5.79 and the molecular weight was about 42.85 kDa. MeJA stimulated the rising of TwCMK expression in suspension cell and signally impacted at 24 h. The research provides a basis for further study on the regulation of terpenoid secondary metabolism and biological synthesis. PMID:27071250

  9. Role of polyphosphate kinase gene (ppk) for survival of Vibrio cholerae O1 in surface water of Bangladesh.

    PubMed

    Jahid, Iqbal Kabir; Hasan, Md Mahmud; Abdul Matin, Mohammad; Mahmud, Zahid Hayat; Neogi, Sucharit Basu; Uddin, Md Hafiz; Islam, Md Sirajul

    2013-11-15

    Polyphosphate provides a substitute for ATP and energy source when phosphorus is a limiting resource in nature. The present study focuses on the role ofpolyphosphate for the survival of Vibrio cholerae in the aquatic habitats as an autochthonous bacterium. The survival advantages of polyphosphate of V. cholerae O1 having (wild type) and lacking (mutant) polyphosphate kinase (ppk) gene in surface water and with Anabaena variabilis were compared by cultural, Direct Fluorescent Antibody (DFA) and polymerase chain reaction methods in natural water microcosms. The microcosm's water was prepared by filtering and physicochemical parameters were also investigated by standard methods. The results revealed that both fresh and saline water, the wild type strain enhanced survival in cultural conditioned than ppk mutant strain. However, Fluorescent Antibody Direct Viable Counts (FADVC) and Polymerase Chain Reaction (PCR) results noted both strains have the equal survival strategy in viable but nonculturable state (VNC). In conclusion, it could be hypothesized that the polyphosphate inclusion body might keep cultivable and survivable at low phosphate natural environment of the aquatic bacterium. PMID:24511696

  10. Identification of PRKDC (Protein Kinase, DNA-Activated, Catalytic Polypeptide) as an essential gene for colorectal cancer (CRCs) cells.

    PubMed

    Sun, Shangfeng; Cheng, Shuguang; Zhu, Yunxiao; Zhang, Peng; Liu, Ning; Xu, Tong; Sun, Chao; Lv, Yanfeng

    2016-06-10

    Oncogene and non-oncogene addictions describe the phenomenon that tumor cells become reliant on certain genes for maintenance of malignancy. Reversal of these mutations profoundly affects tumor growth and survival, providing a fundamental rationale for development of targeted cancer therapy. However, inadequate knowledge on cancer signaling networks and lack of potential drug targets limited its clinical application. A screen was conducted using a custom small interfering RNA (siRNA) library in colorectal cancer (CRC). Transient knockdown followed by cell proliferation assays were performed to validate the essentiality of PRKDC (Protein Kinase, DNA-Activated, Catalytic Polypeptide) in CRC. Western blot analysis was performed to examine the mechanism by which PRKDC confers selective survival advantage in CRC cells. Inducible knockdown and overexpression cell lines were introduced into nude mice to assess PRKDC dependency of CRC cells in vivo. PRKDC expression level in patient samples and overall survival of patients with low or high PRKDC expression were analyzed. Transient knockdown of PRKDC reduced cell proliferation/survival in HCT116 and DLD1, but not FHC cells. PRKDC down-regulation induced apoptosis partially through inhibiting AKT activation, and sensitized HCT116 cells to chemotherapeutic agents interfering with DNA replication. Inducible knockdown of PRKDC inhibited tumor growth in vivo. PRKDC was up-regulated in cancerous tissues compared with normal tissues. Patients with high PRKDC expression showed poorer overall survival. PRKDC is an essential gene required for CRC cell proliferation/survival, which may represent as a potential prognostic biomarker and an ideal therapeutic target for CRC. PMID:26992638

  11. TaCIPK29, a CBL-Interacting Protein Kinase Gene from Wheat, Confers Salt Stress Tolerance in Transgenic Tobacco

    PubMed Central

    Zhou, Shiyi; Zhang, Fan; Han, Jiapeng; Chen, Lihong; Li, Yin; Feng, Jialu; Fang, Bin; Luo, Qingchen; Li, Shasha; Liu, Yunyi; Yang, Guangxiao; He, Guangyuan

    2013-01-01

    Calcineurin B-like protein-interacting protein kinases (CIPKs) have been found to be responsive to abiotic stress. However, their precise functions and the related molecular mechanisms in abiotic stress tolerance are not completely understood, especially in wheat. In the present study, TaCIPK29 was identified as a new member of CIPK gene family in wheat. TaCIPK29 transcript increased after NaCl, cold, methyl viologen (MV), abscisic acid (ABA) and ethylene treatments. Over-expression of TaCIPK29 in tobacco resulted in increased salt tolerance, which was demonstrated by higher germination rates, longer root lengths and better growth status of transgenic tobacco plants compared to controls when both were treated with salt stress. Physiological measurements indicated that transgenic tobacco seedlings retained high K+/Na+ ratios and Ca2+ content by up-regulating some transporter genes expression and also possessed lower H2O2 levels and reduced membrane injury by increasing the expression and activities of catalase (CAT) and peroxidase (POD) under salt stress. Moreover, transgenic lines conferred tolerance to oxidative stress by increasing the activity and expression of CAT. Finally, TaCIPK29 was located throughout cells and it preferentially interacted with TaCBL2, TaCBL3, NtCBL2, NtCBL3 and NtCAT1. Taken together, our results showed that TaCIPK29 functions as a positive factor under salt stress and is involved in regulating cations and reactive oxygen species (ROS) homeostasis. PMID:23922838

  12. MET gene amplification or EGFR mutation activate MET in lung cancers untreated with EGFR tyrosine kinase inhibitors

    PubMed Central

    Kubo, Takafumi; Yamamoto, Hiromasa; Lockwood, William W.; Valencia, Ilse; Soh, Junichi; Peyton, Michael; Jida, Masaru; Otani, Hiroki; Fujii, Tetsuya; Ouchida, Mamoru; Takigawa, Nagio; Kiura, Katsuyuki; Shimizu, Kenji; Date, Hiroshi; Minna, John D.; Varella-Garcia, Marileila; Lam, Wan L.; Gazdar, Adi F.; Toyooka, Shinichi

    2009-01-01

    We analyzed MET protein and copy number in NSCLC with or without EGFR mutations untreated with EGFR tyrosine kinase inhibitors (TKIs). MET copy number was examined in 28 NSCLC and 4 human bronchial epithelial cell lines (HBEC) and 100 primary tumors using quantitative real-time PCR. Positive results were confirmed by array comparative genomic hybridization and fluorescence in-situ hybridization. Total and phospho-MET protein expression was determined in 24 NSCLC and 2 HBEC cell lines using Western blot. EGFR mutations were examined for exon 19 deletions, T790M, and L858R. Knockdown of EGFR with siRNA was performed to examine the relation between EGFR and MET activation. High-level MET amplification was observed in 3 of 28 NSCLC cell lines and in 2 of 100 primary lung tumors that had not been treated with EGFR-TKIs. MET protein was highly expressed and phosphorylated in all the 3 cell lines with high MET amplification. In contrast, 6 NSCLC cell lines showed phospho-MET among 21 NSCLC cell lines without MET amplification (p = 0.042). Furthermore, those 6 cell lines harboring phospho-MET expression without MET amplification were all EGFR mutant (p = 0.0039). siRNA-mediated knockdown of EGFR abolished phospho-MET expression in examined 3 EGFR mutant cell lines of which MET gene copy number was not amplified. By contrast, phospho-MET expression in 2 cell lines with amplified MET gene was not down-regulated by knockdown of EGFR. Our results indicated that MET amplification was present in untreated NSCLC and EGFR mutation or MET amplification activated MET protein in NSCLC. PMID:19117057

  13. Abnormal dentin structure in two novel gene mutations [COL1A1, Arg134Cys] and [ADAMTS2, Trp795-to-ter] causing rare type I collagen disorders.

    PubMed

    De Coster, P J; Cornelissen, M; De Paepe, A; Martens, L C; Vral, A

    2007-02-01

    Histological and ultrastructural observations of dentin of two patients affected with rare types of type I collagen disorders are presented. In the first case, a homozygous nonsense mutation in ADAMTS2 (substitution of a codon for tryptophan by a stopcodon) causes type VIIC Ehlers-Danlos syndrome (EDS) with multiple tooth agenesis and focal dysplastic dentin defects. In the second case, a missense mutation in COL1A1 (substitution of arginine by cysteine) results in a type I EDS phenotype with clinically normal-appearing dentition. Tooth samples are investigated by using light microscopy (LM), transmission electron microscopy (TEM) and immunostaining for types I and III collagen, and tenascin. These are compared with samples from patients with types III and IV osteogenesis imperfecta (OI) in association with dentinogenesis imperfecta (DI), showing a consistently abnormal appearance of the dentin in all specimens, with variations being primarily those of degree of change. Similarities in histological changes include the alternating presence of normal and severe pathological areas in primary and secondary dentin, the latter being characterized by large canal-like structures in atubular areas. Ultrastructural evidence of pathological dentinogenesis include abnormal distribution, size and organization of collagen fibers, which may also be found in clinically unaffected teeth. The histological and ultrastructural changes seen can be explained on the basis of odontoblast dysfunction which may be secondary to the collagen defect, interfering with different levels of odontoblast cell function and intercellular communication. These observations on (ultra)structural dentin defects associated with the two novel gene mutations are the first ever reported. PMID:17118335

  14. Protein Kinases and Addiction

    PubMed Central

    Lee, Anna M.; Messing, Robert O.

    2011-01-01

    Although drugs of abuse have different chemical structures and interact with different protein targets, all appear to usurp common neuronal systems that regulate reward and motivation. Addiction is a complex disease that is thought to involve drug-induced changes in synaptic plasticity due to alterations in cell signaling, gene transcription, and protein synthesis. Recent evidence suggests that drugs of abuse interact with and change a common network of signaling pathways that include a subset of specific protein kinases. The best studied of these kinases are reviewed here and include extracellular signal-regulated kinase, cAMP-dependent protein kinase, cyclin-dependent protein kinase 5, protein kinase C, calcium/calmodulin-dependent protein kinase II, and Fyn tyrosine kinase. These kinases have been implicated in various aspects of drug addiction including acute drug effects, drug self-administration, withdrawal, reinforcement, sensitization, and tolerance. Identifying protein kinase substrates and signaling pathways that contribute to the addicted state may provide novel approaches for new pharma-cotherapies to treat drug addiction. PMID:18991950

  15. Draft Genome Sequence of Methylobacterium sp. Strain ARG-1 Isolated from the White-Rot Fungus Armillaria gallica

    PubMed Central

    Collins, Caitlin; Kowalski, Caitlin; Zebrowski, Jessica; Tulchinskaya, Yevgeniya; Tai, Albert K.; James-Pederson, Magdalena

    2016-01-01

    Methylobacterium sp. strain ARG-1 was isolated from a cell culture of hyphal tips of the white-rot fungus Armillaria gallica. We describe here the sequencing, assembly, and annotation of its genome, confirming the presence of genes involved in methylotrophy. This is the first genome announcement of a strain of Methylobacterium associated with A. gallica. PMID:27257212

  16. Exon junction complex subunits are required to splice Drosophila MAP kinase, a large heterochromatic gene

    PubMed Central

    Roignant, Jean-Yves; Treisman, Jessica E.

    2010-01-01

    Summary The exon junction complex (EJC) is assembled on spliced mRNAs upstream of exon-exon junctions, and can regulate their subsequent translation, localization, or degradation. We isolated mutations in Drosophila mago nashi (mago), which encodes a core EJC subunit, based on their unexpectedly specific effects on photoreceptor differentiation. Loss of Mago prevents Epidermal growth factor receptor signaling, due to a large reduction in MAPK mRNA levels. MAPK expression also requires the EJC subunits Y14 and eIF4AIII, and EJC-associated splicing factors. Mago depletion does not affect the transcription or stability of MAPK mRNA, but alters its splicing pattern. MAPK expression from an exogenous promoter requires Mago only when the template includes introns. MAPK is the primary functional target of mago in eye development; in cultured cells, Mago knockdown disproportionately affects other large genes located in heterochromatin. These data support a nuclear role for EJC components in splicing a specific subset of introns. PMID:20946982

  17. Seven, eight and nine-membered anticodon loop mutants of tRNA(2Arg) which cause +1 frameshifting. Tolerance of DHU arm and other secondary mutations.

    PubMed

    Tuohy, T M; Thompson, S; Gesteland, R F; Atkins, J F

    1992-12-20

    The mutant tRNA(2Arg) encoded by the genetically-selected frameshift suppressor, sufT621, inserts arginine and causes a +1 reading-frame shift at the proline codon, CCG(U). There is an extra base, G36.1, in argV beta, one of the four identical genes for tRNA(2Arg) in the position between bases 36 and 37, corresponding to the 3' side of the anticodon. The new four-base anticodon, predicted from DNA sequencing to be 3' GGCA 5', is complementary to the four-base codon CCGU. Quadruplet translocation promoted by mutant argV does not require perfect complementarity between the codon and the anticodon since synthetic genes encoding derivatives of tRNA(2Arg) and tRNA(1Pro), with four-base anticodons complementary to three out of the four bases of CCGU, were also shown to be capable of frameshifting. Two other mutants of argV, inferred to have normal-size, seven-base anticodon loops, were also found to be capable of four-base-decoding demonstrating that quadruplet translocation promoted by mutant argV does not require an enlarged anticodon loop. Other alleles of argV, predicted to have nine bases in the anticodon loop, were also found to cause frameshifting. The DNA sequence of two of these showed in addition, either a deletion of G24, or a ten-base duplication in the region corresponding to the TFC arm. A general finding is that mutations in the DHU arm of tRNA(2Arg) are compatible with, and in one case necessary for, frameshifting. PMID:1474576

  18. Quantitative 1H nuclear magnetic resonance diffusion spectroscopy of BT4C rat glioma during thymidine kinase-mediated gene therapy in vivo: identification of apoptotic response.

    PubMed

    Hakumäki, J M; Poptani, H; Puumalainen, A M; Loimas, S; Paljärvi, L A; Ylä-Herttuala, S; Kauppinen, R A

    1998-09-01

    We have investigated the effects of thymidine kinase-mediated gene therapy in a malignant rat BT4C glioma by using 1H nuclear magnetic resonance spectroscopy in vivo. Ganciclovir has been successfully used in thymidine kinase gene therapy as treatment for various experimental malignancies. The cell damaging effect seems to be mediated by apoptosis, optimally leading to eradication of tumor tissue. In this study, we show that ganciclovir treatment of tumors transfected with the herpes simplex thymidine kinase gene causes profound changes in water, metabolites, and macromolecules observable by diffusion spectroscopy. During treatment, a 50% reduction from 0.14 +/- 0.01 x 10(-9) m2/s in the apparent diffusion coefficient of choline-containing compounds can be observed, concomitant with a 219% increase in the apparent diffusion coefficient of the rapidly diffusing water component. These changes are associated with an increase in the relative fraction of this water component from 87 to 94%. The apparent diffusion coefficients of the slowly diffusing water component and macromolecules remain unaltered. The results imply a reduction in cell size and number, a significant increase in intracellular viscosity, and a possible reduction in the hydrodynamic radii of macromolecular components, which are ascribed as biophysical signatures for apoptotic cell death. PMID:9731486

  19. 5-Aminoimidazole-4-carboxyamide-1-β-D-ribofranoside stimulates the rat enhancer of split- and hairy-related protein-2 gene via atypical protein kinase C lambda.

    PubMed

    Komatsu, Yoshiko; Yanagisawa, Yuki; Moriizumi, Maya; Tsuchiya, Yuuki; Yokouchi, Honami; Otsuka, Hatsumi; Aoyagi, Mizuki; Tsukada, Akiko; Kanai, Yukiko; Haneishi, Ayumi; Takagi, Katsuhiro; Asano, Kosuke; Ono, Moe; Tanaka, Takashi; Tomita, Koji; Yamada, Kazuya

    2016-04-01

    The 5'-AMP-activated protein kinase (AMPK) functions as a cellular energy sensor. 5-Aminoimidazole-4-carboxyamide-1-β-D-ribofranoside (AICAR) is a chemical activator of AMPK. In the liver, AICAR suppresses expression of thephosphoenolpyruvate carboxykinase(PEPCK) gene. The rat enhancer of split- and hairy-related protein-2 (SHARP-2) is an insulin-inducible transcriptional repressor and its target is thePEPCKgene. In this study, we examined an issue of whether theSHARP-2gene expression is regulated by AICAR via the AMPK. AICAR increased the level of SHARP-2 mRNA in H4IIE cells. Whereas an AMPK inhibitor, compound-C, had no effects on the AICAR-induction, inhibitors for both phosphoinositide 3-kinase (PI 3-K) and protein kinase C (PKC) completely diminished the effects of AICAR. Western blot analyses showed that AICAR rapidly activated atypical PKC lambda (aPKCλ). In addition, when a dominant negative form of aPKCλ was expressed, the induction of SHARP-2 mRNA level by AICAR was inhibited. Calcium ion is not required for the activation of aPKCλ. A calcium ion-chelating reagent had no effects on the AICAR-induction. Furthermore, the AICAR-induction was inhibited by treatment with an RNA polymerase inhibitor or a protein synthesis inhibitor. Thus, we conclude that the AICAR-induction of theSHARP-2gene is mediated at transcription level by a PI 3-K/aPKCλ pathway. PMID:26590300

  20. Structural analysis of APOB variants, p.(Arg3527Gln), p.(Arg1164Thr) and p.(Gln4494del), causing Familial Hypercholesterolaemia provides novel insights into variant pathogenicity

    PubMed Central

    Fernández-Higuero, J. A.; Etxebarria, A.; Benito-Vicente, A.; Alves, A. C.; Arrondo, J. L. R.; Ostolaza, H.; Bourbon, M.; Martin, C.

    2015-01-01

    Familial hypercholesterolaemia (FH) is an inherited autosomal dominant disorder resulting from defects in the low-density lipoprotein receptor (LDLR), in the apolipoprotein B (APOB) or in the proprotein convertase subtilisin/kexin type 9 (PCSK9) genes. In the majority of the cases FH is caused by mutations occurring within LDLR, while only few mutations in APOB and PCSK9 have been proved to cause disease. p.(Arg3527Gln) was the first mutation in APOB being identified and characterized. Recently two novel pathogenic APOB variants have been described: p.(Arg1164Thr) and p.(Gln4494del) showing impaired LDLR binding capacity, and diminished LDL uptake. The objective of this work was to analyse the structure of p.(Arg1164Thr) and p.(Gln4494del) variants to gain insight into their pathogenicity. Secondary structure of the human ApoB100 has been investigated by infrared spectroscopy (IR) and LDL particle size both by dynamic light scattering (DLS) and electron microscopy. The results show differences in secondary structure and/or in particle size of p.(Arg1164Thr) and p.(Gln4494del) variants compared with wild type. We conclude that these changes underlie the defective binding and uptake of p.(Arg1164Thr) and p.(Gln4494del) variants. Our study reveals that structural studies on pathogenic variants of APOB may provide very useful information to understand their role in FH disease. PMID:26643808

  1. Structures of Arg- and Gln-type bacterial cysteine dioxygenase homologs: Arg- and Gln-type Bacterial CDO Homologs

    DOE PAGESBeta

    Driggers, Camden M.; Hartman, Steven J.; Karplus, P. Andrew

    2015-01-01

    In some bacteria, cysteine is converted to cysteine sulfinic acid by cysteine dioxygenases (CDO) that are only ~15–30% identical in sequence to mammalian CDOs. Among bacterial proteins having this range of sequence similarity to mammalian CDO are some that conserve an active site Arg residue (“Arg-type” enzymes) and some having a Gln substituted for this Arg (“Gln-type” enzymes). Here, we describe a structure from each of these enzyme types by analyzing structures originally solved by structural genomics groups but not published: a Bacillus subtilis “Arg-type” enzyme that has cysteine dioxygenase activity (BsCDO), and a Ralstonia eutropha “Gln-type” CDO homolog ofmore » uncharacterized activity (ReCDOhom). The BsCDO active site is well conserved with mammalian CDO, and a cysteine complex captured in the active site confirms that the cysteine binding mode is also similar. The ReCDOhom structure reveals a new active site Arg residue that is hydrogen bonding to an iron-bound diatomic molecule we have interpreted as dioxygen. Notably, the Arg position is not compatible with the mode of Cys binding seen in both rat CDO and BsCDO. As sequence alignments show that this newly discovered active site Arg is well conserved among “Gln-type” CDO enzymes, we conclude that the “Gln-type” CDO homologs are not authentic CDOs but will have substrate specificity more similar to 3-mercaptopropionate dioxygenases.« less

  2. Structures of Arg- and Gln-type bacterial cysteine dioxygenase homologs: Arg- and Gln-type Bacterial CDO Homologs

    SciTech Connect

    Driggers, Camden M.; Hartman, Steven J.; Karplus, P. Andrew

    2015-01-01

    In some bacteria, cysteine is converted to cysteine sulfinic acid by cysteine dioxygenases (CDO) that are only ~15–30% identical in sequence to mammalian CDOs. Among bacterial proteins having this range of sequence similarity to mammalian CDO are some that conserve an active site Arg residue (“Arg-type” enzymes) and some having a Gln substituted for this Arg (“Gln-type” enzymes). Here, we describe a structure from each of these enzyme types by analyzing structures originally solved by structural genomics groups but not published: a Bacillus subtilis “Arg-type” enzyme that has cysteine dioxygenase activity (BsCDO), and a Ralstonia eutropha “Gln-type” CDO homolog of uncharacterized activity (ReCDOhom). The BsCDO active site is well conserved with mammalian CDO, and a cysteine complex captured in the active site confirms that the cysteine binding mode is also similar. The ReCDOhom structure reveals a new active site Arg residue that is hydrogen bonding to an iron-bound diatomic molecule we have interpreted as dioxygen. Notably, the Arg position is not compatible with the mode of Cys binding seen in both rat CDO and BsCDO. As sequence alignments show that this newly discovered active site Arg is well conserved among “Gln-type” CDO enzymes, we conclude that the “Gln-type” CDO homologs are not authentic CDOs but will have substrate specificity more similar to 3-mercaptopropionate dioxygenases.

  3. Co-Expression of Wild-Type P2X7R with Gln460Arg Variant Alters Receptor Function

    PubMed Central

    Aprile-Garcia, Fernando; Metzger, Michael W.; Paez-Pereda, Marcelo; Stadler, Herbert; Acuña, Matías; Liberman, Ana C.; Senin, Sergio A.; Gerez, Juan; Hoijman, Esteban; Refojo, Damian; Mitkovski, Mišo; Panhuysen, Markus; Stühmer, Walter; Holsboer, Florian; Deussing, Jan M.; Arzt, Eduardo

    2016-01-01

    The P2X7 receptor is a member of the P2X family of ligand-gated ion channels. A single-nucleotide polymorphism leading to a glutamine (Gln) by arginine (Arg) substitution at codon 460 of the purinergic P2X7 receptor (P2X7R) has been associated with mood disorders. No change in function (loss or gain) has been described for this SNP so far. Here we show that although the P2X7R-Gln460Arg variant per se is not compromised in its function, co-expression of wild-type P2X7R with P2X7R-Gln460Arg impairs receptor function with respect to calcium influx, channel currents and intracellular signaling in vitro. Moreover, co-immunoprecipitation and FRET studies show that the P2X7R-Gln460Arg variant physically interacts with P2X7R-WT. Specific silencing of either the normal or polymorphic variant rescues the heterozygous loss of function phenotype and restores normal function. The described loss of function due to co-expression, unique for mutations in the P2RX7 gene so far, explains the mechanism by which the P2X7R-Gln460Arg variant affects the normal function of the channel and may represent a mechanism of action for other mutations. PMID:26986975

  4. The Abi-domain protein Abx1 interacts with the CovS histidine kinase to control virulence gene expression in group B Streptococcus.

    PubMed

    Firon, Arnaud; Tazi, Asmaa; Da Cunha, Violette; Brinster, Sophie; Sauvage, Elisabeth; Dramsi, Shaynoor; Golenbock, Douglas T; Glaser, Philippe; Poyart, Claire; Trieu-Cuot, Patrick

    2013-02-01

    Group B Streptococcus (GBS), a common commensal of the female genital tract, is the leading cause of invasive infections in neonates. Expression of major GBS virulence factors, such as the hemolysin operon cyl, is regulated directly at the transcriptional level by the CovSR two-component system. Using a random genetic approach, we identified a multi-spanning transmembrane protein, Abx1, essential for the production of the GBS hemolysin. Despite its similarity to eukaryotic CaaX proteases, the Abx1 function is not involved in a post-translational modification of the GBS hemolysin. Instead, we demonstrate that Abx1 regulates transcription of several virulence genes, including those comprising the hemolysin operon, by a CovSR-dependent mechanism. By combining genetic analyses, transcriptome profiling, and site-directed mutagenesis, we showed that Abx1 is a regulator of the histidine kinase CovS. Overexpression of Abx1 is sufficient to activate virulence gene expression through CovS, overcoming the need for an additional signal. Conversely, the absence of Abx1 has the opposite effect on virulence gene expression consistent with CovS locked in a kinase-competent state. Using a bacterial two-hybrid system, direct interaction between Abx1 and CovS was mapped specifically to CovS domains involved in signal processing. We demonstrate that the CovSR two-component system is the core of a signaling pathway integrating the regulation of CovS by Abx1 in addition to the regulation of CovR by the serine/threonine kinase Stk1. In conclusion, our study reports a regulatory function for Abx1, a member of a large protein family with a characteristic Abi-domain, which forms a signaling complex with the histidine kinase CovS in GBS. PMID:23436996

  5. Identification of four novel phosphorylation sites in estrogen receptor α: impact on receptor-dependent gene expression and phosphorylation by protein kinase CK2

    PubMed Central

    2009-01-01

    Background Estrogen receptor α (ERα) phosphorylation is important for estrogen-dependent transcription of ER-dependent genes, ligand-independent receptor activation and endocrine therapy response in breast cancer. However ERα phosphorylation at the previously identified sites does not fully account for these receptor functions. To determine if additional ERα phosphorylation sites exist, COS-1 cells expressing human ERα were labeled with [32P]H3PO4 in vivo and ERα tryptic phosphopeptides were isolated to identify phosphorylation sites. Results Previously uncharacterized phosphorylation sites at serines 46/47, 282, 294, and 559 were identified by manual Edman degradation and phosphoamino acid analysis and confirmed by mutagenesis and phospho-specific antibodies. Antibodies detected phosphorylation of endogenous ERα in MCF-7, MCF-7-LCC2, and Ishikawa cancer cell lines by immunoblot. Mutation of Ser-282 and Ser-559 to alanine (S282A, S559A) resulted in ligand independent activation of ERα as determined by both ERE-driven reporter gene assays and endogenous pS2 gene expression in transiently transfected HeLa cells. Mutation of Ser-46/47 or Ser-294 to alanine markedly reduced estradiol dependent reporter activation. Additionally protein kinase CK2 was identified as a kinase that phosphorylated ERα at S282 and S559 using motif analysis, in vitro kinase assays, and incubation of cells with CK2 kinase inhibitor. Conclusion These novel ERα phosphorylation sites represent new means for modulation of ERα activity. S559 represents the first phosphorylation site identified in the extreme C-terminus (F domain) of a steroid receptor. PMID:20043841

  6. Identification of the tuberous sclerosis complex-2 tumor suppressor gene product tuberin as a target of the phosphoinositide 3-kinase/akt pathway.

    PubMed

    Manning, Brendan D; Tee, Andrew R; Logsdon, M Nicole; Blenis, John; Cantley, Lewis C

    2002-07-01

    The S/T-protein kinases activated by phosphoinositide 3-kinase (PI3K) regulate a myriad of cellular processes. Here, we show that an approach using a combination of biochemistry and bioinformatics can identify substrates of these kinases. This approach identifies the tuberous sclerosis complex-2 gene product, tuberin, as a potential target of Akt/PKB. We demonstrate that, upon activation of PI3K, tuberin is phosphorylated on consensus recognition sites for PI3K-dependent S/T kinases. Moreover, Akt/PKB can phosphorylate tuberin in vitro and in vivo. We also show that S939 and T1462 of tuberin are PI3K-regulated phosphorylation sites and that T1462 is constitutively phosphorylated in PTEN(-/-) tumor-derived cell lines. Finally, we find that a tuberin mutant lacking the major PI3K-dependent phosphorylation sites can block the activation of S6K1, suggesting a means by which the PI3K-Akt pathway regulates S6K1 activity. PMID:12150915

  7. Inactivation of the tuberous sclerosis complex-1 and -2 gene products occurs by phosphoinositide 3-kinase/Akt-dependent and -independent phosphorylation of tuberin.

    PubMed

    Tee, Andrew R; Anjum, Rana; Blenis, John

    2003-09-26

    The tuberous sclerosis complex (TSC) is a genetic disorder that is caused through mutations in either one of the two tumor suppressor genes, TSC1 and TSC2, that encode hamartin and tuberin, respectively. Interaction of hamartin with tuberin forms a heterodimer that inhibits signaling by the mammalian target of rapamycin to its downstream targets: eukaryotic initiation factor 4E-binding protein 1 (4E-BP1) and ribosomal protein S6 kinase 1 (S6K1). During mitogenic sufficiency, the phosphoinositide 3-kinase (PI3K)/Akt pathway phosphorylates tuberin on Ser-939 and Thr-1462 that inhibits the tumor suppressor function of the TSC complex. Here we show that tuberin-hamartin heterodimers block protein kinase C (PKC)/MAPK- and phosphatidic acid-mediated signaling toward mammalian target of rapamycin-dependent targets. We also show that two TSC2 mutants derived from TSC patients are defective in repressing phorbol 12-myristate 13-acetate-induced 4E-BP1 phosphorylation. PKC/MAPK signaling leads to phosphorylation of tuberin at sites that overlap with and are distinct from Akt phosphorylation sites. Phosphorylation of tuberin by phorbol 12-myristate 13-acetate was reduced by treatment of cells with either bisindolylmaleimide I or UO126, inhibitors of PKC and MAPK/MEK (MAPK/ERK kinase), respectively, but not by wortmannin (an inhibitor of PI3K). This work reveals that both PI3K-independent and -dependent mechanisms modulate tuberin phosphorylation in vivo. PMID:12867426

  8. In Silico Prediction of the Effects of Mutations in the Human Mevalonate Kinase Gene: Towards a Predictive Framework for Mevalonate Kinase Deficiency.

    PubMed

    Browne, Claire; Timson, David J

    2015-11-01

    Mevalonate kinase (MVK) catalyses the phosphorylation of mevalonate. Deficiency of MVK is associated with two rare periodic fever syndromes, mevalonic aciduria (MA), a severe form and hyper-immunoglobulin-D syndrome (HIDS), a milder form. An in silico approach was used to analyse the physicochemical and structural effects of 47 disease-associated variants of MVK. A further 20 variants, which are present in human genome databases, were also analysed. Variants associated with MA are clustered into a "hotspot" consisting of residues 8-35 and 234-338 and tended to result in a prediction of severely reduced protein stability. Four of the uncharacterised variants, p.H24P, p.G198R, p. R253W, and p.G335S, were likely to be associated with MA. This method could be used as the basis for initial predictions of severity when new MVK variants are discovered. PMID:26420133

  9. Mapping of the NEP receptor tyrosine kinase gene to human chromosome 6p21.3 and mouse chromosome 17C

    SciTech Connect

    Edelhoff, S.; Disteche, C.M.; Sweetser, D.A.

    1995-01-01

    The mouse receptor tyrosine kinase (RTK) NEP, also called Ptk-3, is widely expressed, with high levels in proliferating neuroepithelia of mouse embryos. The recently described human discoidin domain receptor (DDR) has a predicted amino acid sequence 93% identical to that of murine NEP and may be its human homologue. We have mapped the gene encoding NEP in human and mouse by fluorescence in situ hybridization using a mouse cDNA probe. The NEP/Nep gene maps to human chromosome 6p21.3 and mouse chromosome 17C, respectively. This places the NEP/Nep gene at, or near, the major histocompatibility (MHC) locus-HLA in human and H2 in mouse, respectively. Based on its pattern of expression during development, NEP and Nep represent candidate genes for several MHC-linked developmental abnormalities in human and mouse. 19 refs., 1 fig.

  10. Micro-PET/CT Monitoring of Herpes Thymidine Kinase Suicide Gene Therapy in a Prostate Cancer Xenograft: The Advantage of a Cell-specific Transcriptional Targeting Approach

    PubMed Central

    Johnson, Mai; Sato, Makoto; Burton, Jeremy; Gambhir, Sanjiv S.; Carey, Michael; Wu, Lily

    2010-01-01

    Cancer gene therapy based on tissue-restricted expression of cytotoxic gene should achieve superior therapeutic index over an unrestricted method. This study compared the therapeutic effects of a highly augmented, prostate-specific gene expression method to a strong constitutive promoter-driven approach. Molecular imaging was coupled to gene therapy to ascertain real-time therapeutic activity. The imaging reporter gene (luciferase) and the cytotoxic gene (herpes simplex thymidine kinase) were delivered by adenoviral vectors injected directly into human prostate tumors grafted in SCID mice. Serial bioluminescence imaging, positron emission tomography, and computed tomography revealed restriction of gene expression to the tumors when prostate-specific vector was employed. In contrast, administration of constitutive active vector resulted in strong signals in the liver. Liver serology, tissue histology, and frail condition of animals confirmed liver toxicity suffered by the constitutive active cohorts, whereas the prostate-targeted group was unaffected. The extent of tumor killing was analyzed by apoptotic staining and human prostate marker (prostate-specific antigen). Overall, the augmented prostate-specific expression system was superior to the constitutive approach in safeguarding against systemic toxicity, while achieving effective tumor killing. Integrating noninvasive imaging into cytotoxic gene therapy will provide a useful strategy to monitor gene expression and therapeutic efficacy in future clinical protocols. PMID:16285908

  11. Phosphate Concentration and the Putative Sensor Kinase Protein CckA Modulate Cell Lysis and Release of the Rhodobacter capsulatus Gene Transfer Agent

    PubMed Central

    Westbye, A. B.; Leung, M. M.; Florizone, S. M.; Taylor, T. A.; Johnson, J. A.; Fogg, P. C.

    2013-01-01

    The gene transfer agent of Rhodobacter capsulatus (RcGTA) is a bacteriophage-like genetic element with the sole known function of horizontal gene transfer. Homologues of RcGTA genes are present in many members of the alphaproteobacteria and may serve an important role in microbial evolution. Transcription of RcGTA genes is induced as cultures enter the stationary phase; however, little is known about cis-active sequences. In this work, we identify the promoter of the first gene in the RcGTA structural gene cluster. Additionally, gene transduction frequency depends on the growth medium, and the reason for this is not known. We report that millimolar concentrations of phosphate posttranslationally inhibit the lysis-dependent release of RcGTA from cells in both a complex medium and a defined medium. Furthermore, we found that cell lysis requires the genes rcc00555 and rcc00556, which were expressed and studied in Escherichia coli to determine their predicted functions as an endolysin and holin, respectively. Production of RcGTA is regulated by host systems, including a putative histidine kinase, CckA, and we found that CckA is required for maximal expression of rcc00555 and for maturation of RcGTA to yield gene transduction-functional particles. PMID:23995641

  12. Organization and nucleotide sequences of the Spiroplasma citri genes for ribosomal protein S2, elongation factor Ts, spiralin, phosphofructokinase, pyruvate kinase, and an unidentified protein.

    PubMed Central

    Chevalier, C; Saillard, C; Bové, J M

    1990-01-01

    The gene for spiralin, the major membrane protein of the helical mollicute Spiroplasma citri, was cloned in Escherichia coli as a 5-kilobase-pair (kbp) DNA fragment. The complete nucleotide sequence of the 5.0-kbp spiroplasmal DNA fragment was determined (GenBank accession no. M31161). The spiralin gene was identified by the size and amino acid composition of its translational product. Besides the spiralin gene, the spiroplasmal DNA fragment was found to contain five additional open reading frames (ORFs). The translational products of four of these ORFs were identified by their amino acid sequence homologies with known proteins: ribosomal protein S2, elongation factor Ts, phosphofructokinase, and pyruvate kinase, respectively encoded by the genes rpsB, tsf, pfk, and pyk. The product of the fifth ORF remains to be identified and was named protein X (X gene). The order of the above genes was tsf--X--spiralin gene--pfk--pyk. These genes were transcribed in one direction, while the gene for ribosomal protein S2 (rpsB) was transcribed in the opposite direction. Images PMID:2139649

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

    PubMed Central

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

    1996-01-01

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

  14. Identification of the glycerol kinase gene and its role in diapause embryo restart and early embryo development of Artemia sinica.

    PubMed

    Cheng, Cheng; Yao, Feng; Chu, Bing; Li, Xuejie; Liu, Yan; Wu, Yang; Mei, Yanli; Wang, Peisheng; Hou, Lin; Zou, Xiangyang

    2014-03-01

    Glycerol kinase (GK) catalyzes the rate-limiting step in glycerol utilization by transferring a phosphate from ATP to glycerol, yielding glycerol 3-phosphate, which is an important intermediate for both energy metabolism and glycerolipid production. Artemia sinica has an unusual diapause process under stress conditions of high salinity, low temperature and lack of food. In the process, diapause embryos of A. sinica (brine shrimp) accumulate high concentrations of glycerol as a cryoprotectant to prevent low temperature damage to embryos. Upon embryo restart, glycerol is converted into glucose and other carbohydrates. Therefore, GK plays an important role in the diapause embryo restart process. However, the role of GK in diapause termination of embryo development in A. sinica remains unknown. In the present study, a 2096 bp full-length cDNA of gk from A. sinica (As-gk) was obtained, encoding putative 551 amino acids, 60.6 kDa protein. As a crucial enzyme in glycerol uptake and metabolism, GK has been conserved structurally and functionally during evolution. The expression pattern of As-gk was investigated by quantitative real-time PCR and Western blotting. Expression locations of As-gk were analyzed using in situ hybridization. As-gk was widely distributed in the early embryo and several main parts of Artemia after differentiation. The expression of As-GK was also induced by stresses such as cold exposure and high salinity. This initial research into the expression pattern and stress response of GK in Artemia provides a sound basis for further understanding of the function and regulation of genes in early embryonic development in A. sinica and the stress response. PMID:24365596

  15. Clinical characteristics of megaconial congenital muscular dystrophy due to choline kinase beta gene defects in a series of 15 patients.

    PubMed

    Haliloglu, Goknur; Talim, Beril; Sel, Cigdem Genc; Topaloglu, Haluk

    2015-11-01

    A new form of congenital muscular dystrophy (CMD) with multisystem involvement and characteristic mitochondrial structural changes, due to choline kinase beta (CHKB) gene defects has been characterized by intellectual disability, autistic features, ichthyosis-like skin changes, and dilated cardiomyopathy. We define the clinical characteristics in 15 patients, from 14 unrelated families with so-called 'megaconial CMD', all having mutations in CHKB. Core clinical phenotype included global developmental delay prominent in gross-motor and language domains, severe intellectual disability (ID), and/or muscle weakness in all cases. Muscle biopsies were equivocally 'megaconial' in all. Other peculiarities were: ichthyosis-like skin changes (n = 11), increased serum CK levels (n = 12), microcephaly (n = 6), dysmorphic facial features (n = 7), neonatal hypotonia (n = 3), seizures (n = 3), epileptiform activity without clinically overt seizures (n = 2), dilated cardiomyopathy (n = 2), decreased left ventricular systolic function (n = 2), congenital heart defects (n = 3), sensorineural (n = 1), and conductive hearing loss (n = 1). Ten patients had cranial neuroimaging (MRI-MRS) study, which was notably normal in all, other than one patient having a decreased choline: creatine peak. Intra-familial variability in clinical expression of the disease is noted in four families. Two siblings from the same family, one presenting with global developmental delay and dilated cardiomyopathy, and the other with ichthyosis, ID and proximal weakness without cardiomyopathy died at the ages of 2 years 1 month, and 7 years 4 months respectively. Evolution was progressive (n = 13) and static (n = 2). PMID:26067811

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

    PubMed Central

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

    2012-01-01

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

  17. Sphingosine kinase-1 mediates TNF-alpha-induced MCP-1 gene expression in endothelial cells: upregulation by oscillatory flow.

    PubMed

    Chen, Xi-Lin; Grey, Janice Y; Thomas, Suzanne; Qiu, Fei-Hua; Medford, Russell M; Wasserman, Martin A; Kunsch, Charles

    2004-10-01

    Atherosclerosis is a focal inflammatory disease and preferentially occurs in areas of low fluid shear stress and oscillatory flow, whereas the risk of atherosclerosis is decreased in regions of high fluid shear stress and steady laminar flow. Sphingosine kinase-1 (SphK1) catalyzes the conversion of sphingosine to sphingosine-1 phosphate (S1P), a sphingolipid metabolite that plays important roles in angiogenesis, inflammation, and cell growth. In the present study, we demonstrated that exposure of human aortic endothelial cells to oscillatory flow (shear stress, +/-5 dyn/cm(2) for 48 h) resulted in a marked increase in SphK1 mRNA levels compared with endothelial cells kept in static culture. In contrast, laminar flow (shear stress, 20 dyn/cm(2) for 48 h) decreased SphK1 mRNA levels. We further investigated the role of SphK1 in TNF-alpha-induced expression of inflammatory genes, such as monocyte chemoattractant protein-1 (MCP-1) and VCAM-1 by using small interfering RNA (siRNA) specifically for SphK1. Treatment of endothelial cells with SphK1 siRNA suppressed TNF-alpha-induced increase in MCP-1 mRNA levels, MCP-1 protein secretion, and activation of p38 MAPK. SphK1 siRNA also inhibited TNF-alpha-induced cell surface expression of VCAM-1, but not ICAM-1, protein. Exposure of endothelial cells to S1P led to an increase in MCP-1 protein secretion and MCP-1 mRNA levels and activation of NF-kappaB-mediated transcriptional activity. Treatment of endothelial cells with the p38 MAPK inhibitor SB-203580 suppressed S1P-induced MCP-1 protein secretion. These data suggest that SphK1 mediates TNF-alpha-induced MCP-1 gene expression through a p38 MAPK-dependent pathway and may participate in oscillatory flow-mediated proinflammatory signaling pathway in the vasculature. PMID:15191888

  18. Hypothesis: Do miRNAs Targeting the Leucine-Rich Repeat Kinase 2 Gene (LRRK2) Influence Parkinson's Disease Susceptibility?

    PubMed

    Yılmaz, Şenay Görücü; Geyik, Sırma; Neyal, Ayşe Münife; Soko, Nyarai D; Bozkurt, Hakan; Dandara, Collet

    2016-04-01

    Parkinson's disease (PD) is a frequently occurring neurodegenerative motor disorder adversely impacting global health. There is a paucity of biomarkers and diagnostics that can forecast susceptibility to PD. A new research frontier for PD pathophysiology is the study of variations in microRNA (miRNA) expression whereby miRNAs serve as "upstream regulators" of gene expression in relation to functioning of the dopamine neuronal pathways. Leucine-Rich Repeat Kinase 2 (LRRK2) is a frequently studied gene in PD. Little is known about the ways in which expression of miRNAs targeting LRKK2 impact PD susceptibility. In a sample of 204 unrelated subjects (102 persons with PD and 102 healthy controls), we report here candidate miRNA expression in whole blood samples as measured by real-time PCR (hsa-miR-4671-3p, hsa-miR-335-3p, hsa-miR-561-3p, hsa-miR-579-3p, and hsa-miR-3143) that target LRRK2. Using step-wise logistic regression, and controlling for covariates such as age, gender, PD disease severity, concomitant medications, and co-morbidity, we found that the combination of has-miR-335-3p, has-miR-561-3p, and has-miR-579-3p account for 50% of the variation in regards to PD susceptibility (p < 0.0001). Notably, the hsa-miR-561-3p expression was the most robust predictor of PD in both univariate and multivariate analyses (p < 0.001). Moreover, the biological direction (polarity) of the association was plausible in that the candidate miRNAs displayed a diminished expression in patients. This is consistent with the hypothesis that decreased levels of miRNAs targeting LRRK2 might result in a gain of function for LRRK2, and by extension, loss of neuronal viability. To the best of our knowledge, this is the first clinical association study of the above candidate miRNAs' expression in PD using peripheral samples. These observations may guide future clinical diagnostics research on PD. PMID:27093107

  19. Leptin modulates the expression of catabolic genes in rat nucleus pulposus cells through the mitogen-activated protein kinase and Janus kinase 2/signal transducer and activator of transcription 3 pathways

    PubMed Central

    MIAO, DAOYI; ZHANG, LINGZHOU

    2015-01-01

    Obesity has been demonstrated to be involved in the progress of intervertebral disc degeneration (IDD). However, the associated mechanisms remain to be elucidated. The purpose the present study was to examine the effect of leptin on the expression of degeneration-associated genes in rat nucleus pulposus (NP) cells, and determine the possible mechanism. Normal NP cells, obtained from Sprague Dawley rats, were identified using immunocytochemistry for the expression of collagen II and CA125, and treated with leptin and/or interleukin (IL)-β. Subsequently, the mRNA expression levels of matrix metalloproteinase (MMP)-1, MMP-3, MMP-9, MMP-13, a disintegrin and metalloproteinase with thrombospondin motifs (ADAMTS)-4, ADAMTS-5, aggrecan and COL2A1 were detected by reverse transcription-quantitative polymerase chain reaction (RT-q-PCR). Alcian staining and immunocytochemistry were used to examine the expression levels of proteoglycan and collagen II. The pathway activation was investigated using western blotting, and inhibitors of the pathways were used to reveal the effect of these pathways on the NP cells. The results of the RT-qPCR demonstrated that leptin alone upregulated the mRNA expression levels of MMP-1, MMP-13, ADAMTS-4, ADAMTS-5 and COL2A1. Synergy of leptin and IL-β was found in the increased expression levels of MMP-1, MMP-3 and ADAMTS-5. The leptin-treated NP cells exhibited decreased expression of collagen II. The mitrogen-activated protein kinase (MAPK) pathway (c-Jun-N-terminal kinase, phosphorylated extracellular signal-regulated kinase and p38), phosphatidylinositol 3-kinase (PI3K)/Akt pathway and Janus kinase (JAK)2/signal transducer and activator of transcription 3 pathway were all activated by leptin, however, inhibitors of all the pathways, with the exception of the PI3K/Akt pathway, reversed the expression levels of MMP-1 and MMP-13. These results suggested that leptin promoted catabolic metabolism in the rat NP cells via the MAPK and JAK2/STAT3

  20. Leptin modulates the expression of catabolic genes in rat nucleus pulposus cells through the mitogen-activated protein kinase and Janus kinase 2/signal transducer and activator of transcription 3 pathways.

    PubMed

    Miao, Daoyi; Zhang, Lingzhou

    2015-08-01

    Obesity has been demonstrated to be involved in the progress of intervertebral disc degeneration (IDD). However, the associated mechanisms remain to be elucidated. The purpose the present study was to examine the effect of leptin on the expression of degeneration-associated genes in rat nucleus pulposus (NP) cells, and determine the possible mechanism. Normal NP cells, obtained from Sprague Dawley rats, were identified using immunocytochemistry for the expression of collagen II and CA125, and treated with leptin and/or interleukin (IL)-β. Subsequently, the mRNA expression levels of matrix metalloproteinase (MMP)-1, MMP-3, MMP-9, MMP-13, a disintegrin and metalloproteinase with thrombospondin motifs (ADAMTS)-4, ADAMTS-5, aggrecan and COL2A1 were detected by reverse transcription-quantitative polymerase chain reaction (RT-q-PCR). Alcian staining and immunocytochemistry were used to examine the expression levels of proteoglycan and collagen II. The pathway activation was investigated using western blotting, and inhibitors of the pathways were used to reveal the effect of these pathways on the NP cells. The results of the RT-qPCR demonstrated that leptin alone upregulated the mRNA expression levels of MMP-1, MMP-13, ADAMTS-4, ADAMTS-5 and COL2A1. Synergy of leptin and IL-β was found in the increased expression levels of MMP-1, MMP-3 and ADAMTS-5. The leptin-treated NP cells exhibited decreased expression of collagen II. The mitrogen-activated protein kinase (MAPK) pathway (c-Jun-N-terminal kinase, phosphorylated extracellular signal-regulated kinase and p38), phosphatidylinositol 3-kinase (PI3K)/Akt pathway and Janus kinase (JAK)2/signal transducer and activator of transcription 3 pathway were all activated by leptin, however, inhibitors of all the pathways, with the exception of the PI3K/Akt pathway, reversed the expression levels of MMP-1 and MMP-13. These results suggested that leptin promoted catabolic metabolism in the rat NP cells via the MAPK and JAK2/STAT3

  1. A novel de novo mutation in the serine-threonine kinase STK11 gene in a Korean patient with Peutz-Jeghers syndrome

    PubMed Central

    Yoo, Jong-Ha; Yoo, Jee-Hyoung; Choi, Yoon-Jung; Kang, Jung-Gu; Sun, Young-Kyu; Ki, Chang-Seok; Lee, Kyung-A; Choi, Jong-Rak

    2008-01-01

    Background Peutz-Jeghers syndrome (PJS) is an unusual autosomal dominant disorder characterized by mucocutaneous pigmentation and multiple gastrointestinal hamartomatous polyps. Patients with PJS are at an increased risk of developing multi-organ cancer, most frequently those involving the gastrointestinal tract. Germline mutation of the STK11 gene, which encodes a serine-threonine kinase, is responsible for PJS. Methods Using DNA samples obtained from the patient and his family members, we sequenced nine exons and flanking intron regions of the STK11 gene using polymerase chain reaction (PCR) and direct sequencing. Results Sequencing of the STK11 gene in the proband of the family revealed a novel 1-base pair deletion of guanine (G) in exon 6 (c.826delG; Gly276AlafsX11). This mutation resulted in a premature termination at codon 286, predicting a partial loss of the kinase domain and complete loss of the C-terminal domain. We did not observe this mutation in both parents of the PJS patient. Therefore, it is considered a novel de novo mutation. Conclusion The results presented herein enlarge the spectrum of mutations of the STK11 gene by identifying a novel de novo mutation in a PJS patient and further support the hypothesis that STK11 mutations are disease-causing mutations for PJS with or without a positive family history. PMID:18495044

  2. An SNP in Exon 11 of Chicken 5'-AMP-Activated Protein Kinase Gamma 3 Subunit Gene was Associated with Meat Water Holding Capacity.

    PubMed

    Yang, Yunzhou; Xiong, Dan; Yao, Ling; Zhao, Chunjiang

    2016-01-01

    The 5'-Adenosine-monophosphate -activated protein kinase plays a key role in regulating cellular energy homeostasis, and it was reported that nucleotide variants in the genes coding the protein were associated with meat quality. In the present study, genetic variations in the exons of gamma non-catalytic subunit genes of the protein kinase were screened among 284 White Plymouth Rock chickens from 7 families with denaturing gradient gel electrophoresis, and their meat quality traits including drip loss and cooked meat rate which reflect water holding capacity and serum biochemical indices were also measured, and the association between the genotypes and the traits was analyzed with a SAS GLM procedure. Our results showed that there were three G/A nucleotide variants including one in exon 6 of the gamma subunit 2 gene and two in exon 11 of the gamma subunit 3 gene, which resulted in amino acid substitutions V150I, V315 M, and A337 T, respectively. And locus V315 M was associated with water holding capacity significantly (P < 0.05). The studied polymorphic locus has the potential to be used as a genetic marker for poultry breeding work. PMID:26597656

  3. The Neuronal-Specific SGK1.1 (SGK1_v2) Kinase as a Transcriptional Modulator of BAG4, Brox, and PPP1CB Genes Expression

    PubMed Central

    González-Fernández, Rebeca; Ávila, Julio; Arteaga, María F.; Canessa, Cecilia M.; Martín-Vasallo, Pablo

    2015-01-01

    The Serum- and Glucocorticoid-induced Kinase 1, SGK1, exhibits a broad range of cellular functions that include regulation of the number of ion channels in plasma membrane and modulation of signaling pathways of cell survival. This diversity of functions is made possible by various regulatory processes acting upon the SGK1 gene, giving rise to various isoforms: SGK1_v1–5, each with distinct properties and distinct aminotermini that serve to target proteins to different subcellular compartments. Among cellular effects of SGK1 expression is to indirectly modulate gene transcription by phosphorylating transcriptional factors of the FOXO family. Here we examined if SGK1.1 (SGK1_v2; NM_001143676), which associates primarily to the plasma membrane, is also able to regulate gene expression. Using a differential gene expression approach we identified six genes upregulated by SGK1.1 in HeLa cells. Further analysis of transcript and protein levels validated two genes: BCL2-associated athanogene 4 (BAG-4) and Brox. The results indicate that SGK1.1 regulates gene transcription upon a different set of genes some of which participate in cell survival pathways (BAG-4) and others in intracellular vesicular traffic (Brox). PMID:25849655

  4. Dominant and recessive mutations in the Raf-like kinase HT1 gene completely disrupt stomatal responses to CO2 in Arabidopsis

    PubMed Central

    Hashimoto-Sugimoto, Mimi; Negi, Juntaro; Monda, Keina; Higaki, Takumi; Isogai, Yasuhiro; Nakano, Toshiaki; Hasezawa, Seiichiro; Iba, Koh

    2016-01-01

    HT1 (HIGH LEAF TEMPERATURE 1) is the first component associated with changes in stomatal aperture in response to CO2 to be isolated by forward genetic screening. The HT1 gene encodes a protein kinase expressed mainly in guard cells. The loss-of-function ht1-1 and ht1-2 mutants in Arabidopsis thaliana have CO2-hypersensitive stomatal closure with concomitant reductions in their kinase activities in vitro. In addition to these mutants, in this study we isolate or obtaine five new ht1 alleles (ht1-3, ht1-4, ht1-5, ht1-6, and ht1-7). Among the mutants, only ht1-3 has a dominant mutant phenotype and has widely opened stomata due to CO2 insensitivity. The ht1-3 mutant has a missense mutation affecting a non-conserved residue (R102K), whereas the other six recessive mutants have mutations in highly conserved residues in the catalytic domains required for kinase activity. We found that the dominant mutation does not affect the expression of HT1 or the ability to phosphorylate casein, a universal kinase substrate, but it does affect autophosphorylation activity in vitro. A 3D structural model of HT1 also shows that the R102 residue protrudes from the surface of the kinase, implying a role for the formation of oligomers and/or interaction with its targets. We demonstrate that both the loss-of-function and gain-of-function ht1 mutants have completely disrupted CO2 responses, although they have normal responses to ABA. Furthermore, light-induced stomatal opening is smaller in ht1-3 and much smaller in ht1-2. Taken together, these results indicate that HT1 is a critical regulator for CO2 signaling and is partially involved in the light-induced stomatal opening pathway. PMID:27034327

  5. Dominant and recessive mutations in the Raf-like kinase HT1 gene completely disrupt stomatal responses to CO2 in Arabidopsis.

    PubMed

    Hashimoto-Sugimoto, Mimi; Negi, Juntaro; Monda, Keina; Higaki, Takumi; Isogai, Yasuhiro; Nakano, Toshiaki; Hasezawa, Seiichiro; Iba, Koh

    2016-05-01

    HT1 (HIGH LEAF TEMPERATURE 1) is the first component associated with changes in stomatal aperture in response to CO2 to be isolated by forward genetic screening. The HT1 gene encodes a protein kinase expressed mainly in guard cells. The loss-of-function ht1-1 and ht1-2 mutants in Arabidopsis thaliana have CO2-hypersensitive stomatal closure with concomitant reductions in their kinase activities in vitro In addition to these mutants, in this study we isolate or obtaine five new ht1 alleles (ht1-3, ht1-4, ht1-5, ht1-6, and ht1-7). Among the mutants, only ht1-3 has a dominant mutant phenotype and has widely opened stomata due to CO2 insensitivity. The ht1-3 mutant has a missense mutation affecting a non-conserved residue (R102K), whereas the other six recessive mutants have mutations in highly conserved residues in the catalytic domains required for kinase activity. We found that the dominant mutation does not affect the expression of HT1 or the ability to phosphorylate casein, a universal kinase substrate, but it does affect autophosphorylation activity in vitro A 3D structural model of HT1 also shows that the R102 residue protrudes from the surface of the kinase, implying a role for the formation of oligomers and/or interaction with its targets. We demonstrate that both the loss-of-function and gain-of-function ht1 mutants have completely disrupted CO2 responses, although they have normal responses to ABA. Furthermore, light-induced stomatal opening is smaller in ht1-3 and much smaller in ht1-2 Taken together, these results indicate that HT1 is a critical regulator for CO2 signaling and is partially involved in the light-induced stomatal opening pathway. PMID:27034327

  6. Transcriptional responses to loss or gain of function of the leucine-rich repeat kinase 2 (LRRK2) gene uncover biological processes modulated by LRRK2 activity

    PubMed Central

    Nikonova, Elena V.; Xiong, Yulan; Tanis, Keith Q.; Dawson, Valina L.; Vogel, Robert L.; Finney, Eva M.; Stone, David J.; Reynolds, Ian J.; Kern, Jonathan T.; Dawson, Ted M.

    2012-01-01

    Mutations in the leucine-rich repeat kinase 2 gene (LRRK2) are the most common genetic cause of Parkinson's disease (PD) and cause both autosomal dominant familial and sporadic PD. Currently, the physiological and pathogenic activities of LRRK2 are poorly understood. To decipher the biological functions of LRRK2, including the genes and pathways modulated by LRRK2 kinase activity in vivo, we assayed genome-wide mRNA expression in the brain and peripheral tissues from LRRK2 knockout (KO) and kinase hyperactive G2019S (G2019S) transgenic mice. Subtle but significant differences in mRNA expression were observed relative to wild-type (WT) controls in the cortex, striatum and kidney of KO animals, but only in the striatum in the G2019S model. In contrast, robust, consistent and highly significant differences were identified by the direct comparison of KO and G2019S profiles in the cortex, striatum, kidney and muscle, indicating opposite effects on mRNA expression by the two models relative to WT. Ribosomal and glycolytic biological functions were consistently and significantly up-regulated in LRRK2 G2019S compared with LRRK2 KO tissues. Genes involved in membrane-bound organelles, oxidative phosphorylation, mRNA processing and the endoplasmic reticulum were down-regulated in LRRK2 G2019S mice compared with KO. We confirmed the expression patterns of 35 LRRK2-regulated genes using quantitative reverse transcription polymerase chain reaction. These findings provide the first description of the transcriptional responses to genetically modified LRRK2 activity and provide preclinical target engagement and/or pharmacodynamic biomarker strategies for LRRK2 and may inform future therapeutic strategies for LRRK2-associated PD. PMID:21972245

  7. Tomato thymidine kinase-based suicide gene therapy for malignant glioma--an alternative for Herpes Simplex virus-1 thymidine kinase.

    PubMed

    Stedt, H; Samaranayake, H; Kurkipuro, J; Wirth, G; Christiansen, L S; Vuorio, T; Määttä, A-M; Piškur, J; Ylä-Herttuala, S

    2015-04-01

    Malignant gliomas (MGs) are the most common malignant primary brain tumors with a short life estimate accompanied by a marked reduction in the quality of life. Herpes Simplex virus-1 thymidine kinase ganciclovir (HSV-TK/GCV) system is the best characterized enzyme prodrug therapy in use. However, lipophobicity of GCV and low enzymatic activity of HSV-TK reduce the treatment efficacy. Tomato TK (ToTK) has shown high activity in combination with its specific substrate azidothymidine (AZT). The aim of this study was to evaluate whether ToTK/AZT could be used as an alternative to HSV-TK/GCV therapy. Both treatments demonstrated cytotoxicity in human MG cells in vitro. In vivo, both treatments decreased tumor growth and tumors were smaller in comparison with controls in mouse orthotopic MG model. Survival of ToTK/AZT-treated mice was significantly increased compared with control mice (*P<0.05) but not as compared with HSV-TK/GCV-treated mice. No significant differences were observed in clinical chemistry safety analyses. We conclude that both treatments showed a beneficial treatment response in comparison to controls on tumor growth and ToTK/AZT also on survival. There were no significant differences between these treatments. Therefore ToTK/AZT could be considered as an alternative treatment option for MG because of its favorable therapeutic characteristics. PMID:25613481

  8. Association of a functional polymorphism (Gln261Arg) in 12-lipoxygenase with breast cancer

    PubMed Central

    PRASAD, VIDUDALA V.T.S.; KOLLI, PADMA; MOGANTI, DIVYA

    2011-01-01

    The overexpression of arachidonyl lipoxygenase-12 (ALOX12) in breast cancer has been reported. Hence, we examined whether a non-synonymous polymorphism of ALOX12 (mRNA, A835G; Gln261Arg) is associated with breast cancer in females. The polymorphism was detected in genomic DNA by PCR-RFLP. The association between the A835G polymorphism and breast cancer risk was measured by odds ratio (OR) with 95% confidence intervals (CIs) using Fisher's exact test, and differences were considered significant at p<0.05. The frequencies of AA (wild-type), GG (homozygous variant) and AG (heterozygous variant) were 59.5, 0.9 and 39.6% in the controls, and 39.3, 2.5 and 58.2% in the breast cancer cases, respectively. The frequency of the AG genotype was higher in the patients compared to the controls (p<0.0014). The frequency of the GG variant was 2.5 and 0.9% in the cancer subjects and controls, respectively. The relative risk of breast cancer was 2 times greater (OR=2.227) at 95% CI when compared to the relative risk of the heterozygous variant. For the GG genotype, the risk was 4 times greater (OR=4.125) at 95% CI than that of the controls, suggesting a positive association of the AG genotype with the occurrence of breast cancer. The frequencies of the polymorphism were different in different populations. The Arg/Gln and Arg/Arg variants were associated with an increased risk of breast cancer, and the frequencies of the variants differed considerably among various populations. The identification of a gene with links to breast cancer may impact screening, diagnosis and drug development. PMID:22977504

  9. The human gene (CSNK2A1) coding for the casein kinase II subunit [alpha] is located on chromosome 20 and contains tandemly arranged Alu repeats

    SciTech Connect

    Wirkner, U.; Lichter, P.; Pyerin, W. ); Voss, H.; Ansorge, W. )

    1994-01-15

    The authors have isolated and characterized an 18.9-kb genomic clone representing a central portion of the human casein kinase II (CKII) subunit [alpha] gene (CSNK2A1). Using the whole clone as a probe, the gene was localized on chromosome 20p13. The clone contains eight exons whose sequences comprise bases 102 to 824 of the coding region of the human CKII[alpha]. The exon/intron splice junctions conform to the gt/ag rule. Three of the nine introns are located at positions corresponding to those in the CKII[alpha] gene of the nematode Caenorhabditis elegans. The introns contain eight complete and eight incomplete Alu repeats. Some of the Alu sequences are arranged in tandems of two or three, which seem to originate from insertions of younger Alu sequences into the poly(A) region of previously integrated Alu sequences, as indicated by flanking direct repeats. 50 refs., 5 figs., 1 tab.

  10. Database on natural polymorphisms and resistance-related non-synonymous mutations in thymidine kinase and DNA polymerase genes of herpes simplex virus types 1 and 2.

    PubMed

    Sauerbrei, Andreas; Bohn-Wippert, Kathrin; Kaspar, Marisa; Krumbholz, Andi; Karrasch, Matthias; Zell, Roland

    2016-01-01

    The use of genotypic resistance testing of herpes simplex virus types 1 and 2 (HSV-1 and HSV-2) is increasing because the rapid availability of results significantly improves the treatment of severe infections, especially in immunocompromised patients. However, an essential precondition is a broad knowledge of natural polymorphisms and resistance-associated mutations in the thymidine kinase (TK) and DNA polymerase (pol) genes, of which the DNA polymerase (Pol) enzyme is targeted by the highly effective antiviral drugs in clinical use. Thus, this review presents a database of all non-synonymous mutations of TK and DNA pol genes of HSV-1 and HSV-2 whose association with resistance or natural gene polymorphism has been clarified by phenotypic and/or functional assays. In addition, the laboratory methods for verifying natural polymorphisms or resistance mutations are summarized. This database can help considerably to facilitate the interpretation of genotypic resistance findings in clinical HSV-1 and HSV-2 strains. PMID:26433780

  11. Leptin receptor polymorphism Gln223Arg (rs1137101) in oral squamous cell carcinoma and potentially malignant oral lesions.

    PubMed

    Domingos, Patrícia Luciana Batista; Farias, Lucyana Conceição; Pereira, Camila Santos; das Graças Pena, Geórgia; Reis, Tatiana Carvalho; Silva, Rosângela Ramos Veloso; Fraga, Carlos Alberto de Carvalho; de Souza, Marcela Gonçalves; Soares, Mariana Batista; Jones, Kimberly Marie; Menezes, Elytania Veiga; Nobre, Sérgio Avelino Mota; Rodrigues Neto, João Felício; de Paula, Alfredo Maurício Batista; Velásquez-Meléndez, Jorge Gustavo; Sena Guimarães, André Luiz

    2014-01-01

    The purpose of this study was to assess the LEPR gene Gln223Arg polymorphism (rs1137101) in oral squamous cell carcinoma (OSCC) and in potentially malignant oral lesions (PMOL) in comparison to normal oral mucosa in a Brazilian population. Smokers (n = 89) were selected from a representative sample of 471 individuals from the general population of Montes Claros, Brazil. Participants were age and gender matched to patients with OSCC (n = 25) and oral epithelial dysplasia (n = 25). We investigated the LEPR Gln223Arg polymorphism (A>G; rs1137101) in these groups. Genotype variants were assessed by RFLP-PCR, using MspI (HPAII) restriction endonuclease. The institutional review board of the Universidade Estadual de Montes Claros approved the study (process number 2667/2011). Written informed consent for this study was obtained from all participants. The GG genotype (Arg223Arg) appears to be the more relevant polymorphic variant in OSCC. It occurred, approximately, twice as frequently in OSCC patients than in the general population. In contrast, the A allele in its homozygosis form (Gln223Gln) is significantly associated with the development of PMOL; 80% of the samples from the PMOL group exhibit AA genotype. Our findings suggest new insights regarding LEPR gene variations in the development of OSCC and PMOL. PMID:26034683

  12. Genome-wide analysis of differential mRNA expression of Amsacta moorei entomopoxvirus, mediated by the gene encoding a viral protein kinase (AMV197).

    PubMed

    Muratoglu, Hacer; Nalcacioglu, Remziye; Arif, Basil M; Demirbag, Zihni

    2016-04-01

    Insect-born entomopoxviruses (Fam: Poxviridae) are potentially important bio-pesticide against insect pests and expression vectors as well as vectors for transient human gene therapies including recombinant viral vaccines. For these reasons, it is necessary to understand the regulatory genes functions to improve its biotechnological potential. Here, we focused on the characterization of serine/threonine (Ser/Thr; ORF AMV197) protein kinase gene from the Amsacta moorei entomopoxvirus (AMEV), the type species of the genus Betaentomopoxvirus. Transcription of the parental and an amv197-null recombinant AMEV was compared by whole-genome gene expression microarray analysis. Blast2GO analysis reflected a broad diversity of upregulated and downregulated genes. Results showed that expression levels of 102 genes (45%) out of 226 tested genes changed significantly in the recombinant AMEV infected cells. Of these transcripts, 72 (70.58%) were upregulated and 30 (29.41%) were downregulated throughout the infection period. Genes involved in DNA repair, replication and nucleotide metabolism, transcription and RNA modification, and protein modification were mostly upregulated at different times in cells infected with the recombinant virus. Furthermore, transcription of all studied cellular genes including metabolism of apoptosis (Nedd2-like caspase, hemolin and elongation factor-1 alpha (ef1a) gene) was downregulated in the absence of amv197. Quantitative real time reverse transcription-PCR confirmed viral transcriptional changes obtained by microarray. The results of this study indicated that the product of amv197 appears to affect the transcriptional regulation of most viral and many cellular genes. Further investigations are, however, needed to narrow down the role of AMV197 throughout the infection process. PMID:26820433

  13. Expression of the herpes thymidine kinase gene in Xenopus laevis oocytes: an assay for the study of deletion mutants constructed in vitro.

    PubMed Central

    McKnight, S L; Gavis, E R

    1980-01-01

    When Xenopus laevis oocyte nuclei are injected with a recombinant plasmid containing the Herpes Simplex Virus (HSV) thymidine kinase (tk) gene, a 100-fold increase in tk enzymatic activity is observed. Three lines of evidence show that this increase in tk activity is a result of the expression of the HSV tk gene. First, the enzymatic activity is selectively inactivated by the IgG fraction of antiserum raised against HSV tk protein. Second, a polypeptide that comigrates with authentic HSV tk on polyacrylamide gels is synthesized uniquely by oocytes injected with the HSV tk gene. Third, the induced tk activity found in injected oocytes is capable of phosphorylating deoxycytidine, a substrate that is utilized by HSV tk but not by cellular tk. We have used these observations to establish an assay for examining the activity of mutated variants of the HSV tk gene. Two sets of deletion mutants of the tk gene were constructed in vitro. In one set varying amounts of 5' flanking and intragenic sequences are deleted. The other set is deleted at the 3' end of the gene. By testing the activity of each mutant in the oocyte injection assay we have delimited functional boundaries corresponding to the 5' and 3' termini of the HSV tk gene. Images PMID:6258155

  14. Disruption of SRM1, a mitogen-activated protein kinase gene, affects sensitivity to osmotic and ultraviolet stressors in the phytopathogenic fungus Bipolaris oryzae.

    PubMed

    Moriwaki, Akihiro; Kubo, Emiko; Arase, Sakae; Kihara, Junichi

    2006-04-01

    Mitogen-activated protein kinases (MAPKs) play key roles in biological processes including differentiation, growth, proliferation, survival, and stress responses. We isolated and characterized the SRM1 gene, which encodes an MAPK related to yeast High-osmolarity glycerol 1 (Hog1), from the rice leaf pathogen Bipolaris oryzae. The deduced amino sequence of the SRM1 gene showed significant homology with Hog1-type MAPK homologues from other phytopathogenic fungi and contained a TGY motif for phosphorylation. The B. oryzae mutants with disruption of the SRM1 gene (Deltasrm1) showed growth inhibition under hyperosmotic, hydrogen peroxide, and UV exposure conditions. The Deltasrm1 mutants showed moderate resistance to dicarboximide and phenylpyrrole fungicides. The Deltasrm1 mutations caused a defect in the expression of the gene that encodes antioxidant enzyme catalase (CAT2) under UV and hyperosmotic conditions. Furthermore, the transcriptional patterns of the three melanin biosynthesis genes (PKS1, THR1, and SCD1) and of a gene of unknown function, uvi-1, which are specifically induced by near-ultraviolet (NUV) radiation, gradually decreased in comparison with the wild-type expression patterns. These results suggest that Srm1 contributes to responses to not only osmostress but also to hydrogen peroxide and UV stress, whereas Srm1 does not appear to regulate directly the expression of genes related to NUV-induced photomorphogenesis. PMID:16553861

  15. Lack of association between XPG Asp1104His and XPF Arg415Gln polymorphism and breast cancer risk: a meta-analysis of case-control studies.

    PubMed

    Ding, Da-Peng; He, Xiao-Feng; Zhang, Ying

    2011-08-01

    The xeroderma pigmentosum group G (XPG or ERCC5) and group F (XPF or ERCC4) play an important role in DNA repair, and produce dual incision 3' and 5' to the damaged nucleotide fragment. Several polymorphisms in the XPF and XPG gene have been described, including the commonly occurring Asp1104His in XPG and Arg415Gln in XPF. The published data on the association between these polymorphisms and breast cancer remained controversial. This meta-analysis of literatures was performed to derive a more precise estimation of the relationship. A total of 17 studies were identified to the meta-analysis, including 5,235 cases and 5,685 controls for XPG Asp1104His (from ten studies) and 3,910 cases and 3,985 controls for XPF Arg415Gln (from seven studies). Overall, no significantly elevated breast cancer risk was found in all genetic models when all studies were pooled into the meta-analysis (for XPG Asp1104His Asp/His vs. Asp/Asp: OR 1.02, 95% CI 0.94-1.11; His/His vs. Asp/Asp: OR 0.96, 95% CI 0.83-1.11; dominant model: OR 1.01, 95% CI 0.94-1.09; and for XPF Arg415Gln Arg/Gln vs. Arg/Arg: OR 1.00, 95% CI 0.89-1.12; Gln/Gln vs. Arg/Arg: OR 2.40, 95% CI 0.62-9.22; dominant model: OR 1.03, 95% CI 0.90-1.18). In stratified analyses, we observed an overall OR of 5.20 (95% CI 2.08-12.95) for breast cancer developing risk in the Caucasian ethnicity, comparing Gln/Gln type to wild-type Arg/Arg for Arg415Gln polymorphism. In conclusion, this meta-analysis suggests that XPG Asp1104His polymorphism is not associated with increased breast cancer risk, and XPF Arg415Gln may be a low-penetrant risk factor in the Caucasian ethnicity for developing breast cancer. PMID:21424776

  16. Deciphering the Arginine-Binding Preferences at the Substrate-Binding Groove of Ser/Thr Kinases by Computational Surface Mapping

    PubMed Central

    Ben-Shimon, Avraham; Niv, Masha Y.

    2011-01-01

    Protein kinases are key signaling enzymes that catalyze the transfer of γ-phosphate from an ATP molecule to a phospho-accepting residue in the substrate. Unraveling the molecular features that govern the preference of kinases for particular residues flanking the phosphoacceptor is important for understanding kinase specificities toward their substrates and for designing substrate-like peptidic inhibitors. We applied ANCHORSmap, a new fragment-based computational approach for mapping amino acid side chains on protein surfaces, to predict and characterize the preference of kinases toward Arginine binding. We focus on positions P−2 and P−5, commonly occupied by Arginine (Arg) in substrates of basophilic Ser/Thr kinases. The method accurately identified all the P−2/P−5 Arg binding sites previously determined by X-ray crystallography and produced Arg preferences that corresponded to those experimentally found by peptide arrays. The predicted Arg-binding positions and their associated pockets were analyzed in terms of shape, physicochemical properties, amino acid composition, and in-silico mutagenesis, providing structural rationalization for previously unexplained trends in kinase preferences toward Arg moieties. This methodology sheds light on several kinases that were described in the literature as having non-trivial preferences for Arg, and provides some surprising departures from the prevailing views regarding residues that determine kinase specificity toward Arg. In particular, we found that the preference for a P−5 Arg is not necessarily governed by the 170/230 acidic pair, as was previously assumed, but by several different pairs of acidic residues, selected from positions 133, 169, and 230 (PKA numbering). The acidic residue at position 230 serves as a pivotal element in recognizing Arg from both the P−2 and P−5 positions. PMID:22125489

  17. Screening ADAMTS10 in Dog Populations Supports Gly661Arg as the Glaucoma-Causing Variant in Beagles

    PubMed Central

    Kuchtey, John; Kunkel, Jessica; Esson, Douglas; Sapienza, John S.; Ward, Daniel A.; Plummer, Caryn E.; Gelatt, Kirk N.; Kuchtey, Rachel W.

    2013-01-01

    Purpose. Previously, we mapped the disease locus in the beagle model of autosomal recessive primary open angle glaucoma (POAG) to a 4-Mb interval on chromosome 20, and identified a Gly661Arg variant in ADAMTS10 as the candidate disease-causing variant. The purpose of this study was to test the hypothesis that the Gly661Arg variant of ADAMTS10 causes glaucoma by genotyping dogs of various breeds affected and unaffected by primary glaucoma. Methods. Dogs of various breeds, affected or unaffected with primary glaucoma, were genotyped for the Gly661Arg variant of ADAMTS10, as well as 7 other nonsynonymous single nucleotide polymorphisms (SNPs) in other genes in the beagle POAG locus that segregate with disease. Alternate allele frequencies were calculated with 95% confidence intervals and comparisons made to expected allele frequency relative to disease prevalence or between cases and controls. Results. For the nonsynonymous SNPs other than the ADAMTS10 variant, control dogs were identified that were homozygous for the alternative alleles, ruling out those variants as causative. None of the nonsynonymous SNPs were found associated with primary glaucoma in American cocker spaniels. The Gly661Arg variant of ADAMTS10 was the only variant with minor allele frequency consistent with the prevalence of primary glaucoma in the general beagle population. The only dog found homozygous for the Gly661Arg variant of ADAMTS10 was an affected beagle, unrelated to the POAG colony. Conclusions. These findings support the Gly661Arg mutation of ADAMTS10 as the likely cause of POAG in beagles. PMID:23422823

  18. Homeodomain-interacting protein kinase 2, a novel autoimmune regulator interaction partner, modulates promiscuous gene expression in medullary thymic epithelial cells.

    PubMed

    Rattay, Kristin; Claude, Janine; Rezavandy, Esmail; Matt, Sonja; Hofmann, Thomas G; Kyewski, Bruno; Derbinski, Jens

    2015-02-01

    Promiscuous expression of a plethora of tissue-restricted Ags (TRAs) by medullary thymic epithelial cells (mTECs) plays an essential role in T cell tolerance. Although the cellular mechanisms by which promiscuous gene expression (pGE) imposes T cell tolerance have been well characterized, the underlying molecular mechanisms remain poorly understood. The autoimmune regulator (AIRE) is to date the only validated molecule known to regulate pGE. AIRE is part of higher-order multiprotein complexes, which promote transcription, elongation, and splicing of a wide range of target genes. How AIRE and its partners mediate these various effects at the molecular level is still largely unclear. Using a yeast two-hybrid screen, we searched for novel AIRE-interacting proteins and identified the homeodomain-interacting protein kinase 2 (HIPK2) as a novel partner. HIPK2 partially colocalized with AIRE in nuclear bodies upon cotransfection and in human mTECs in situ. Moreover, HIPK2 phosphorylated AIRE in vitro and suppressed the coactivator activity of AIRE in a kinase-dependent manner. To evaluate the role of Hipk2 in modulating the function of AIRE in vivo, we compared whole-genome gene signatures of purified mTEC subsets from TEC-specific Hipk2 knockout mice with control mice and identified a small set of differentially expressed genes. Unexpectedly, most differentially expressed genes were confined to the CD80(lo) mTEC subset and preferentially included AIRE-independent TRAs. Thus, although it modulates gene expression in mTECs and in addition affects the size of the medullary compartment, TEC-specific HIPK2 deletion only mildly affects AIRE-directed pGE in vivo. PMID:25552543

  19. A root-specific wall-associated kinase gene, HvWAK1, regulates root growth and is highly divergent in barley and other cereals.

    PubMed

    Kaur, Ravneet; Singh, Kashmir; Singh, Jaswinder

    2013-06-01

    Wall-associated receptor-like kinases (WAKs) are important candidates for directly linking the extracellular matrix with intracellular compartments and are involved in developmental processes and stress response. WAK gene family has been identified in plants such as Arabidopsis and rice. Here, we present a detailed analysis of the WAK1 gene from barley cv. Golden Promise, mapped to chromosome 5H. Three BAC clones corresponding to the WAK fragment were sequenced and the full-length WAK1 gene was characterized. The gene has three exons and two short introns with a coding region of 2,178 bp encoding a protein of 725 amino acids. A regulatory region was analyzed in -1,000 bp sequence upstream to start codon. Using conserved domains database and SMART, various conserved domains such as GUB WAK Bind, epidermal growth factor CA, and protein kinase C as well as other regions like signal peptides, active sites, and transmembrane domains were identified. The gene organization of HvWAK1 was compared with wheat (TaWAK1) and Arabidopsis (AtWAK1), suggesting that the WAK1 gene organization has remained highly conserved. Nonetheless, WAK1 was found to be highly divergent when compared with sequences available from barley cv. Haruna Nijo (50 %), rice (46 %), wheat (21 %), Arabidopsis (25 %), and maize (19 %). This divergence may have facilitated a better adaptation to surrounding environments due to its role in communication between the extracellular matrix, cell, and outer environment. Semiquantitative RT-PCR-based expression analysis indicates HvWAK1 expression is specific to roots. Significant differences in root growth between GP wild type and GP-Ds mutant seedlings were observed under control and salt stress conditions. PMID:23443578

  20. Expression of a novel serine/threonine kinase gene, Ulk4, in neural progenitors during Xenopus laevis forebrain development.

    PubMed

    Domínguez, L; Schlosser, G; Shen, S

    2015-04-01

    We have analyzed the expression pattern of a novel serine/threonine kinase gene Ulk4 during forebrain development in Xenopus laevis. To this aim, we firstly cloned a Ulk4 cDNA fragment from X.laevis and generated a RNA probe that was used for its detection by in situ hybridization. Throughout development xUlk4 expression was detected along the ventricular (vz) and subventricular zones (svz) of all forebrain regions, with the exception of the vz of the striatum. In the adult, xUlk4 was also mainly located in the vz, with some xUlk4 expressing cells reaching the svz/mantle zone (mz). This xUlk4 expression was especially remarkable in forebrain regions involving the homeostatic control of the brain such as the preoptic region, the hypothalamic territory and some neurosecretory circumventricular organs (CVOs). We further combined in situ hybridization for xUlk4 with immunohistochemistry for the neural progenitor cell marker SOX3, the radial glial marker brain lipid-binding protein (BLBP), neuronal markers MAP2 and doublecortin (DCX) and the specific neuronal marker tyrosine hydroxylase (TH). xUlk4 was co-expressed with the neural stem/progenitor cell marker SOX3 in the vz of all the forebrain regions throughout development and in the