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Sample records for gene arg kinase

  1. 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…

  2. 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.

  3. The promoter region of the arg3 gene in Saccharomyces cerevisiae: nucleotide sequence and regulation in an arg3-lacZ gene fusion.

    PubMed

    Crabeel, M; Huygen, R; Cunin, R; Glansdorff, N

    1983-01-01

    We have determined the DNA sequence for the 5' end of the arg3 gene of Saccharomyces cerevisiae, including part of the coding region and the 200 nucleotides immediately upstream. A promoter-deletion mutant was found to have lost all of the sequence lying normally in front of the gene except for the 33 nucleotides preceding the AUG codon. The role of the 5' domain in initiation and regulation of arg3 transcription was assessed by a gene fusion experiment. The Escherichia coli lacZ gene, was truncated of the eight amino-terminal codons substituted in vitro, on a 2mu plasmid, for the carboxy-terminal and 3'-flanking regions of arg3, leaving only the first 19 proximal codons and approximately 1600 nucleotides of the region preceding arg3 on the yeast chromosome. The fused gene was expressed in phase and was still submitted to the two mechanisms regulating the wild-type arg3 gene: the general, probably transcriptional control of amino acid biosynthesis and the specific, apparently post-transcriptional control mediated by the products of the argR genes. These results suggest a determining role for the 5' end portion of the arg3 messenger in the specific arginine-mediated control mechanism. PMID:11894927

  4. Homologous control sites and DNA transcription starts in the related argF and argI genes of Escherichia coli K12.

    PubMed Central

    Piette, J; Cunin, R; Van Vliet, F; Charlier, D; Crabeel, M; Ota, Y; Glansdorff, N

    1982-01-01

    The argF and argI genes code for similar proteins able to assemble into hybrid isoenzymes and are therefore thought to share a common origin. We show here that the nucleotide sequence of the promoter and operator regions of these two genes are highly homologous. DNA regions preceding the control sites also present significant homologies. The results support the notion of divergent evolution of the two genes from a common ancestor. Like argE and argCBH , argF and argI are controlled by a repressor molecule recognizing a family of similar operator sites. Attenuation appears to play no role in this regulation. Images Fig. 5. PMID:6329710

  5. 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

  6. 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.

  7. A conserved Glu-Arg salt bridge connects coevolved motifs that define the eukaryotic protein kinase fold.

    PubMed

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

    2012-01-27

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

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

    PubMed

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

    2013-01-01

    To study the relationship between arsenic resistance of 293T cells and overexpression of ARG1, the ARG1 gene in a recombinant plasmid was transfected into 293T cells via liposomes, and then ARG1 overexpression was examined by real-time PCR and immunocytochemistry. The survival rate, arsenic accumulation and arsenic efflux, GSH level, and GST activity of 293T cells overexpressing ARG1 were assayed by MTT, atomic absorption spectrophotometry, and DTNB, and expression of MRP-2 was detected by Western blot analysis. Compared to that in the control cells, the survival rate of ARG1 gene-overexpressing cells was much higher following exposure to lower sodium arsenite (≤ 8 µM). When cells were exposed to lower sodium arsenite for 24 h, the arsenite content of ARG1 gene-overexpressing cells decreased and arsenic efflux increased. After 48 h, the GSH level, GST activity, and expression of MRP2 increased in a concentration-dependent manner. We conclude that the ARG1 gene increases arsenic resistance of 293T cells. PMID:24391030

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

    PubMed

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

    2013-12-19

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

  10. 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

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

    PubMed

    Meng, Zhigang; Meng, Zhaohong; 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.

  12. Metagenomic profiles of antibiotic resistance genes (ARGs) between human impacted estuary and deep ocean sediments.

    PubMed

    Chen, Baowei; Yang, Ying; Liang, Ximei; Yu, Ke; Zhang, Tong; Li, Xiangdong

    2013-11-19

    Knowledge of the origins and dissemination of antibiotic resistance genes (ARGs) is essential for understanding modern resistomes in the environment. The mechanisms of the dissemination of ARGs can be revealed through comparative studies on the metagenomic profiling of ARGs between relatively pristine and human-impacted environments. The deep ocean bed of the South China Sea (SCS) is considered to be largely devoid of anthropogenic impacts, while the Pearl River Estuary (PRE) in south China has been highly impacted by intensive human activities. Commonly used antibiotics (sulfamethazine, norfloxacin, ofloxacin, tetracycline, and erythromycin) have been detected through chemical analysis in the PRE sediments, but not in the SCS sediments. In the relatively pristine SCS sediments, the most prevalent and abundant ARGs are those related to resistance to macrolides and polypeptides, with efflux pumps as the predominant mechanism. In the contaminated PRE sediments, the typical ARG profiles suggest a prevailing resistance to antibiotics commonly used in human health and animal farming (including sulfonamides, fluoroquinolones, and aminoglycosides), and higher diversity in both genotype and resistance mechanism than those in the SCS. In particular, antibiotic inactivation significantly contributed to the resistance to aminoglycosides, β-lactams, and macrolides observed in the PRE sediments. There was a significant correlation in the levels of abundance of ARGs and those of mobile genetic elements (including integrons and plasmids), which serve as carriers in the dissemination of ARGs in the aquatic environment. The metagenomic results from the current study support the view that ARGs naturally originate in pristine environments, while human activities accelerate the dissemination of ARGs so that microbes would be able to tolerate selective environmental stress in response to anthropogenic impacts.

  13. [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

  14. 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

  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.

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

    PubMed

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

    2013-07-16

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

  18. 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

  19. 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

  20. 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

  1. 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

  2. Leptin receptor gene Gln223Arg polymorphism is not associated with obesity and metabolic syndrome in Turkish children.

    PubMed

    Komşu-Ornek, Zuhal; Demirel, Fatma; Dursun, Ahmet; Ermiş, Bahri; Pişkin, Etem; Bideci, Aysun

    2012-01-01

    The aim of the study was to investigate the relationship between leptin receptor gene (LEPR) Gln223Arg polymorphism and obesity in Turkish children. Ninety-two obese and 99 lean children (between 5-15 years) were included in the study. Twenty-three of the obese children were diagnosed with metabolic syndrome. Blood samples were collected for morning fasting blood glucose, insulin, leptin, and lipid level measurements. LEPR Gln223Arg polymorphism was analyzed by restriction fragment length polymorphism. Significant differences were observed in anthropometric measurements, fasting blood glucose, insulin, leptin, and lipid levels between obese and lean children. Serum leptin levels were markedly higher in obese children. No significant association was noted between Gln223Arg polymorphism and serum leptin, insulin and lipid levels. There were no differences in the genotype frequencies or allele distribution for Gln223Arg polymorphism among obese, obese with metabolic syndrome and lean children. Our findings suggest that there is no association between Gln223Arg polymorphism and obesity in Turkish children.

  3. [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

  4. Beta-2 adrenergic receptor gene polymorphisms Gln27Glu, Arg16Gly in patients with heart failure

    PubMed Central

    Mansur, Alfredo José; Fontes, Rosana Seleri; Canzi, Regina Airoldi; Nishimura, Raphael; Alencar, Airlane Pereira; de Lima, Antonio Carlos Pedroso; Krieger, José Eduardo; Pereira, Alexandre Costa

    2009-01-01

    Background - Beta-2 adrenergic receptor gene polymorphisms Gln27Glu, Arg16Gly and Thr164Ile were suggested to have an effect in heart failure. We evaluated these polymorphisms relative to clinical characteristics and prognosis of alarge cohort of patients with heart failure of different etiologies. Methods - We studied 501 patients with heart failure of different etiologies. Mean age was 58 years (standard deviation 14.4 years), 298 (60%) were men. Polymorphisms were identified by polymerase chain reaction-restriction fragment length polymorphism. Results - During the mean follow-up of 12.6 months (standard deviation 10.3 months), 188 (38%) patients died. Distribution of genotypes of polymorphism Arg16Gly was different relative to body mass index (χ2 = 9.797;p = 0.04). Overall the probability of survival was not significantly predicted by genotypes of Gln27Glu, Arg16Gly, or Thr164Ile. Allele and haplotype analysis also did not disclose any significant difference regarding mortality. Exploratory analysis through classification trees pointed towards a potential association between the Gln27Glu polymorphism and mortality in older individuals. Conclusion - In this study sample, we were not able to demonstrate an overall influence of polymorphisms Gln27Glu and Arg16Gly of beta-2 receptor gene on prognosis. Nevertheless, Gln27Glu polymorphism may have a potential predictive value in older individuals. PMID:19886995

  5. [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.

  6. 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

  7. The β1-adrenoreceptor gene Arg389Gly and Ser49Gly polymorphisms and hypertension: a meta-analysis.

    PubMed

    Kong, Hong; Li, Xiaobo; Zhang, Shanshan; Guo, Shujie; Niu, Wenquan

    2013-06-01

    The gene encoding β1-adrenoreceptor is regarded as a hypertension-susceptibility candidate gene. The association of β1-adrenoreceptor gene Arg389Gly and Ser49Gly polymorphisms with hypertension has been exhaustively investigated; however, the studies have yielded inconsistent results. We sought to shed some light on this inconsistency by performing a systemic meta-analysis. Data were extracted from 17 articles (cases/controls: 7,586/8,441) for Arg389Gly, and eight articles (3,582/2,998) for Ser49Gly. The random-effects model was applied irrespective of between-study heterogeneity. Overall results indicated significance for Ser49Gly under both allelic (odds ratio = 1.13; 95 % confidence interval [95 % CI] 1.03-1.26; P = 0.011) and dominant (1.19; 1.04-1.28; 0.011) models, without evidence of heterogeneity (I (2) = 0.0 %). Grouping studies by ethnicity observed marginally significant association for Arg389Gly (0.82; 0.66-1.0; 0.049) and Ser49Gly (1.3; 1.0-1.68; 0.048) polymorphisms in Caucasians under allelic model. Association was strikingly potentiated for both polymorphisms after restricting analyses to studies published in English journals. When only large studies (≥500 subjects) were considered, 389Gly allele decreased the odds of developing hypertension by 16 % (0.84; 0.74-0.95; 0.007). There was no observable publication bias for both polymorphisms. Taken together, our results provide clarification to the logical candidacy of β1-adrenoreceptor gene in the development of hypertension.

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

    PubMed Central

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

    2016-01-01

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

  9. 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

  10. 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.

  11. Putative modifier genes in mevalonate kinase deficiency.

    PubMed

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

    2016-04-01

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

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

    PubMed

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

    2014-08-01

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

  13. Tyrosine kinase gene rearrangements in epithelial malignancies.

    PubMed

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

    2013-11-01

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

  14. 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

  15. The effect of the Gly16Arg polymorphism of the beta(2)-adrenergic receptor gene on plasma free fatty acid levels is modulated by physical activity.

    PubMed

    Meirhaeghe, A; Luan, J; Selberg-Franks, P; Hennings, S; Mitchell, J; Halsall, D; O'Rahilly, S; Wareham, N J

    2001-12-01

    The lipolytic effects of catecholamines are mediated through members of the beta(2)-adrenergic receptor (BAR-2) family. Previous studies have suggested that genetic variants in the BAR-2 gene may be associated with obesity in some populations. To our knowledge, no studies have directly examined the effects of this polymorphism on circulating nonesterified fatty acid (NEFA) levels. To explore this issue further, a cohort of 604 Caucasian individuals (aged 40-65 yr) was genotyped for a common polymorphism in the BAR-2 gene (Gly16Arg), and the relationships between genotype, body mass index (BMI), NEFA, and lipid levels were examined. Women bearing the Arg16 allele had higher BMI values (P < 0.01) than Gly16Gly women. Women carriers of the Arg16Arg genotype had lower fasting plasma NEFAs (P < 0.01) and greater suppression of NEFAs (P < 0.01) after an oral glucose load than women bearing the Gly16 allele. In multivariate analysis after adjustment for age, sex, and smoking status, the interaction between the BAR-2 genotype and BMI in determining fasting NEFA concentrations was statistically significant (P < 0.05). The availability of objective measures of total energy expenditure in this population permitted the further examination of interactions, particularly that between genotype and physical activity. In the population as a whole, after adjustment for confounding by age, smoking, and BMI, the effect of the Arg16Arg genotype on the suppression of NEFA levels was modified by physical activity level (P for interaction <0.05). These data suggest the existence in this population of a gene-physical activity interaction on NEFA levels.

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

    PubMed

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

    1989-01-01

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

  17. 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.

  18. 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

  19. A heparin binding site Arg79Cys missense mutation in the SERPINC1 gene in a Korean patient with hereditary antithrombin deficiency.

    PubMed

    Yoo, Jong-Ha; Maeng, Ho-Young; Kim, Hee-Jin; Lee, Kyung-A; Choi, Jong-Rak; Song, Jaewoo

    2011-01-01

    We describe a case of heparin binding site Arg79Cys mutation in the gene encoding antithrombin, SERPINC1, in a Korean patient with hereditary antithrombin (AT) deficiency. The patient was a 34-year-old Korean man who presented with deep vein thrombosis (DVT) in his right leg without precipitating factors. On outpatient evaluation, coagulation tests without anticoagulation revealed a decreased AT III activity level at 48%, but normal AT III antigen level at 103%, indicating type II AT deficiency. Family studies revealed that his father (62 years of age) had decreased AT activity (48%) but had normal AT antigen levels (116%), indicating that the proband had a paternally inherited type II AT deficiency. Direct sequencing of the SERPINC1 gene in the patient and his father revealed a heterozygotic missense mutation, a cytosine to thymine substitution at nucleotide position 235 in exon 2 of the SERPINC1 gene (p.Arg79Cys). To our knowledge, this is the first report of Arg79Cys heterozygote mutation in family members with venous thromboembolism.

  20. Effect of β2 -adrenergic receptor gene Arg16Gly polymorphisms on response to long-acting β2-agonist in Chinese Han asthmatic patients

    PubMed Central

    2014-01-01

    Background To evaluate the effect of variation of the Arg16Gly polymorphism of the β2-adrenergic receptor gene on clinical response to salmeterol administered with fluticasone propionate in Chinese Han asthmatic patients. Methods Moderate persistent asthmatic patients (n = 62) currently receiving short-acting β2-agonists were administered twice-daily therapy with salmeterol/fluticasone propionate 50/250 μg in a single inhaler for 12 weeks, followed by a 2-to-4-day run-out period. Using direct DNA sequencing, five single nucleotide polymorphisms (SNPs) in the promoter and coding block regions of β2-adrenergic receptor gene were determined in 62 subjects and haplotypes were combined. Results There was sustained and significant improvement (p < 0.001) over baseline in all measures of asthma control in subjects receiving salmeterol and fluticasone, regardless of Arg16Gly genotype. However, there was no significant difference in the improvement among three genotypes (p > 0.05). Responses to salmeterol did not appear to be modified by haplotype pairs (p > 0.05). During the run-out period, all subjects had similar decreases in measures of asthma control, with no differences between genotypes (p > 0.05). Conclusions Response to salmeterol does not vary with Arg16Gly polymorphisms after chronic dosing with inhaled corticosteroids in Chinese Han asthmatic patients. PMID:24721141

  1. Comparative genomics on nemo-like kinase gene.

    PubMed

    Katoh, Masuko; Katoh, Masaru

    2005-06-01

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

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

    PubMed

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

    2012-08-01

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

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

    PubMed Central

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

    2016-01-01

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

  4. 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

  5. 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.

  6. 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

  7. 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

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

    PubMed

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

    2006-06-01

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

  9. 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

  10. 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

  11. 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

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

    PubMed

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

    2003-02-01

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

  13. 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

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

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

  15. Expression of herpes virus thymidine kinase in Neurospora crassa.

    PubMed Central

    Sachs, M S; Selker, E U; Lin, B; Roberts, C J; Luo, Z; Vaught-Alexander, D; Margolin, B S

    1997-01-01

    The expression of thymidine kinase in fungi, which normally lack this enzyme, will greatly aid the study of DNA metabolism and provide useful drug-sensitive phenotypes. The herpes simplex virus type-1 thymidine kinase gene ( tk ) was expressed in Neurospora crassa. tk was expressed as a fusion to N.crassa arg-2 regulatory sequences and as a hygromycin phosphotransferase-thymidine kinase fusion gene under the control of cytomegalovirus and SV40 sequences. Only strains containing tk showed thymidine kinase enzyme activity. In strains containing the arg-2 - tk gene, both the level of enzyme activity and the level of mRNA were reduced by growth in arginine medium, consistent with control through arg-2 regulatory sequences. Expression of thymidine kinase in N.crassa facilitated radioactive labeling of replicating DNA following addition of [3H]thymidine or [14C]thymidine to the growth medium. Thymidine labeling of DNA enabled demonstration that hydroxyurea can be used to block replication and synchronize the N.crassa mitotic cycle. Strains expressing thymidine kinase were also more sensitive than strains lacking thymidine kinase to anticancer and antiviral nucleoside drugs that are activated by thymidine kinase, including 5-fluoro-2'-deoxyuridine, 1-(2-deoxy-2-fluoro-beta-D-arabinofuranosyl)-5-iodouridine and trifluorothymidine. Finally, expression of thymidine kinase in N. crassa enabled incorporation of bromodeoxyuridine into DNA at levels sufficient to separate newly replicated DNA from old DNA using equilibrium centrifugation. PMID:9171090

  16. 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

  17. ArgR of Streptomyces coelicolor is a versatile regulator.

    PubMed

    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.

  18. Evolutionary expansion, gene structure, and expression of the rice wall-associated kinase gene family.

    PubMed

    Zhang, Shibo; Chen, Calvin; Li, Lei; Meng, Ling; Singh, Jaswinder; Jiang, Ning; Deng, Xing-Wang; He, Zheng-Hui; Lemaux, Peggy G

    2005-11-01

    The wall-associated kinase (WAK) gene family, one of the receptor-like kinase (RLK) gene families in plants, plays important roles in cell expansion, pathogen resistance, and heavy-metal stress tolerance in Arabidopsis (Arabidopsis thaliana). Through a reiterative database search and manual reannotation, we identified 125 OsWAK gene family members from rice (Oryza sativa) japonica cv Nipponbare; 37 (approximately 30%) OsWAKs were corrected/reannotated from earlier automated annotations. Of the 125 OsWAKs, 67 are receptor-like kinases, 28 receptor-like cytoplasmic kinases, 13 receptor-like proteins, 12 short genes, and five pseudogenes. The two-intron gene structure of the Arabidopsis WAK/WAK-Likes is generally conserved in OsWAKs; however, extra/missed introns were observed in some OsWAKs either in extracellular regions or in protein kinase domains. In addition to the 38 OsWAKs with full-length cDNA sequences and the 11 with rice expressed sequence tag sequences, gene expression analyses, using tiling-microarray analysis of the 20 OsWAKs on chromosome 10 and reverse transcription-PCR analysis for five OsWAKs, indicate that the majority of identified OsWAKs are likely expressed in rice. Phylogenetic analyses of OsWAKs, Arabidopsis WAK/WAK-Likes, and barley (Hordeum vulgare) HvWAKs show that the OsWAK gene family expanded in the rice genome due to lineage-specific expansion of the family in monocots. Localized gene duplications appear to be the primary genetic event in OsWAK gene family expansion and the 125 OsWAKs, present on all 12 chromosomes, are mostly clustered. PMID:16286450

  19. Post-transcriptional regulation of the chicken thymidine kinase gene.

    PubMed

    Groudine, M; Casimir, C

    1984-02-10

    In attempting to understand the molecular basis of the control of chicken thymidine kinase (cTK) gene expression, we have examined the steady state cTK RNA content, and the patterns of DNA methylation, chromatin structure and endogenous nuclear runoff transcription of this gene in dividing and non-dividing cells. Our results reveal that the steady state level of cTK poly A+ RNA is correlated with the divisional activity of normal avian cells and tissues. However, no differences in the pattern of Hpa II site methylation or chromatin structure are found among cells containing high or undetectable levels of steady state cTK RNA. In addition, no differences in cTK transcription as assayed by nuclear runoff experiments are detectable in isolated nuclei derived from dividing or non-dividing cells containing high or low levels of steady state cTK RNA. These results suggest that the principal control of chicken thymidine kinase gene expression is post-transcriptional in nature.

  20. 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

  1. The myxoma virus thymidine kinase gene: sequence and transcriptional mapping.

    PubMed

    Jackson, R J; Bults, H G

    1992-02-01

    The myxoma virus thymidine kinase (TK) gene is encoded on a 1.6 kb SacI-SalI restriction fragment located between 57.7 and 59.3 kb on the 163 kb genomic map. The nucleotide sequence of this fragment as well as 228 bp from the adjacent SalI-AA2 fragment was determined and found to encode four major open reading frames (ORFs). Three of these ORFs are similar in nucleotide sequence to ORFs L5R and J1R, and the TK gene of vaccinia virus (VV). The fourth ORF, MF8a, shows similarity to the ORFs found in the same position relative to the TK genes of Shope fibroma virus, Kenya sheep-1 virus and swine-pox virus. A search of the complete VV nucleotide sequence for regions of similarity to MF8a identified the host specificity gene C7L. Northern blot analysis of early viral RNA identified transcripts of approximately 700 nucleotides for both the TK gene and ORF MF8a. The 5' ends of the TK gene and ORF MF8a early mRNAs were mapped by primer extension to initiation sites 13 nucleotides downstream of sequences with similarity to the VV early promoter consensus. The sizes of the TK and MF8a mRNAs are consistent with transcription termination and polyadenylation occurring downstream of the sequence TTTTTNT, which is identical to the consensus sequence for the VV transcription termination signal.

  2. 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

  3. 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.

  4. Association between exonic polymorphism (rs629849, Gly1619Arg) of IGF2R gene and obesity in Korean population

    PubMed Central

    Yang, Seung-Ae

    2015-01-01

    The aim of this study is to investigate the relationship between single nucleotide polymorphisms (SNPs) and susceptibility to obesity. A previous study suggested that insulin-like growth factors (IGFs) may affect obesity and that IGFs regulate cellular signals by receptors that include the insulin-like growth factor 1 receptor (IGF1R) and the insulin-like growth factor 2 receptor (IGF2R). In this research, the rs3743262 and rs2229765 SNPs of IGF1R gene and rs629849 and rs1805075 SNPs of IG-F2R gene were genotyped in 120 overweight and obese patients with a BMI≥23 kg/m2 (Body Mass Index) and 123 healthy controls with a BMI of 18.5–23.0 kg/m2. Genotyping of each SNP was performed by direct sequencing. Among tested SNPs in IGF1R and IGF2R genes, rs629849 SNP of IGF2R gene showed significant association with obesity (OR=1.86, 95% CI=1.02–3.40, P=0.044 in codominant1 model; OR=1.99, 95% CI=1.10–3.57, P=0.020 in dominant model; OR=1.93, 95% CI=1.13–3.31, P=0.013 in log-additive model). And allele distribution between the control group and overweight/obese group also showed significant difference (OR=1.93, 95% CI=1.14–3.28, P=0.015). In conclusion, these results indicate that rs629849 SNP of IGF2R might be contributed to development of obesity in the Korean population. PMID:26535220

  5. A/G Gln20Arg (exon 1) and G/A Val156Met (exon 5) polymorphisms of the human orosomucoid 1 gene in Mexico.

    PubMed

    García-Ortiz, L; Vargas-Alarcón, G; Fragoso, J M; Granados, J; Maldonado Noriega, L; Navas Pérez, A; Huerta Reyes, E; Zenteno-Ruiz, J C; Martínez-Cordero, E

    2008-01-08

    The human orosomucoid 1 gene (ORM1) codes an alpha-1-acid glycoprotein that has been classified as an acute-phase reactive protein, and a major drug-binding serum component, as well as an immunomodulatory protein with genetic polymorphisms. Evaluation of ORM variation through isoelectric focusing and immunobloting has revealed a world-wide distribution of the ORM1 F and ORM1 S alleles. We evaluated and examined the genetic characteristics of two Mexican populations that have different anthropological and cultural antecedents, examining two ORM1 genotypes (exon 1 - A/G (Gln20Arg) and exon 5 G/A (Val156Met)) in 145 individuals, using nested polymerase chain reaction, sequencing, and restricted fragment length polymorphism. Mexican Mestizos had higher frequencies of the exon 1 A allele (P = 0.020) and AA genotype (P = 0.018) and lower frequency of the G allele (P = 0.020) when compared to Teenek Amerindians. When we examined exon 5 G/A (Val156Met) polymorphisms, we found significantly higher frequencies of the G allele (P = 0.0007) and the GG genotype (P = 0.0003) in the Mexican Mestizo population. The Teenek population had a significantly higher frequency of the A allele than has been reported for Chinese and African (P < 0.05) populations, and the G/A genotype was more frequently found in this Mexican population than in Chinese, African and European populations (P < 0.05).

  6. 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.

  7. 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.

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

    PubMed Central

    Gordhandas, Jeenal A.; Pique, Lynn

    2016-01-01

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

  9. [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

  10. 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.

  11. In silico analysis reveals 75 members of mitogen-activated protein kinase kinase kinase gene family in rice.

    PubMed

    Rao, Kudupudi Prabhakara; Richa, Tambi; Kumar, Kundan; Raghuram, Badmi; Sinha, Alok Krishna

    2010-06-01

    Mitogen-Activated Protein Kinase Kinase Kinases (MAPKKKs) are important components of MAPK cascades, which are universal signal transduction modules and play important role in plant growth and development. In the sequenced Arabidopsis genome 80 MAPKKKs were identified and currently being analysed for its role in different stress. In rice, economically important monocot cereal crop only five MAPKKKs were identified so far. In this study using computational analysis of sequenced rice genome we have identified 75 MAPKKKs. EST hits and full-length cDNA sequences (from KOME or Genbank database) of 75 MAPKKKs supported their existence. Phylogenetic analyses of MAPKKKs from rice and Arabidopsis have classified them into three subgroups, which include Raf, ZIK and MEKK. Conserved motifs in the deduced amino acid sequences of rice MAPKKKs strongly supported their identity as members of Raf, ZIK and MEKK subfamilies. Further expression analysis of the MAPKKKs in MPSS database revealed that their transcripts were differentially regulated in various stress and tissue-specific libraries.

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

    SciTech Connect

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

    1996-08-01

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

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

    PubMed

    Kwoh, T J; Engler, J A

    1984-05-11

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

  14. Over-expression of a protein kinase gene enhances the defense of tobacco against Rhizoctonia solani.

    PubMed

    Chacón, Osmany; González, Marleny; López, Yunior; Portieles, Roxana; Pujol, Merardo; González, Ernesto; Schoonbeek, Henk-Jan; Métraux, Jean-Pierre; Borrás-Hidalgo, Orlando

    2010-03-01

    To identify Nicotiana tabacum genes involved in resistance and susceptibility to Rhizoctonia solani, suppression subtractive hybridization was used to generate a cDNA library from transcripts that are differentially expressed during a compatible and incompatible interaction. This allowed the isolation of a protein kinase cDNA that was down-regulated during a compatible and up-regulated during an incompatible interaction. Quantitative RT-PCR analysis of this gene confirmed the differential expression patterns between the compatible and incompatible interactions. Over-expression of this gene in tobacco enhanced the resistance to damping-off produced by an aggressive R. solani strain. Furthermore, silencing of this protein kinase gene reduced the resistance to a non-aggressive R. solani strain. A set of reported tobacco-resistant genes were also evaluated in tobacco plants over-expressing and silencing the protein kinase cDNA. Several genes previously associated with resistance in tobacco, like manganese superoxide dismutase, Hsr203J, chitinases and phenylalanine ammonia-lyase, were up-regulated in tobacco plants over-expressing the protein kinase cDNA. Potentially, the protein kinase gene could be used to engineer resistance to R. solani in tobacco cultivars susceptible to this important pathogen.

  15. Activation of multiple mitogen-activated protein kinases by recombinant calcitonin gene-related peptide receptor.

    PubMed

    Parameswaran, N; Disa, J; Spielman, W S; Brooks, D P; Nambi, P; Aiyar, N

    2000-02-18

    Calcitonin gene-related peptide is a 37-amino-acid neuropeptide and a potent vasodilator. Although calcitonin gene-related peptide has been shown to have a number of effects in a variety of systems, the mechanisms of action and the intracellular signaling pathways, especially the regulation of mitogen-activated protien kinase (MAPK) pathway, is not known. In the present study we investigated the role of calcitonin gene-related peptide in the regulation of MAPKs in human embryonic kidney (HEK) 293 cells stably transfected with a recombinant porcine calcitonin gene-related peptide-1 receptor. Calcitonin gene-related peptide caused a significant dose-dependent increase in cAMP response and the effect was inhibited by calcitonin gene-related peptide(8-37), the calcitonin gene-related peptide-receptor antagonist. Calcitonin gene-related peptide also caused a time- and concentration-dependent increase in extracellular signal-regulated kinase (ERK) and P38 mitogen-activated protein kinase (P38 MAPK) activities, with apparently no significant change in cjun-N-terminal kinase (JNK) activity. Forskolin, a direct activator of adenylyl cyclase also stimulated ERK and P38 activities in these cells suggesting the invovement of cAMP in this process. Calcitonin gene-related peptide-stimulated ERK and P38 MAPK activities were inhibited significantly by calcitonin gene-related peptide receptor antagonist, calcitonin gene-related peptide-(8-37) suggesting the involvement of calcitonin gene-related peptide-1 receptor. Preincubation of the cells with the cAMP-dependent protein kinase inhibitor, H89 [¿N-[2-((p-bromocinnamyl)amino)ethyl]-5-isoquinolinesulfonamide, hydrochloride¿] inhibited calcitonin gene-related peptide-mediated activation of ERK and p38 kinases. On the other hand, preincubation of the cells with wortmannin ¿[1S-(1alpha,6balpha,9abeta,11alpha, 11bbeta)]-11-(acetyloxy)-1,6b,7,8,9a,10,11, 11b-octahydro-1-(methoxymethyl)-9a,11b-dimethyl-3H-furo[4,3, 2-de]indeno[4,5-h]-2

  16. 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

  17. 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.

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

    PubMed Central

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

    1996-01-01

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

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

    PubMed Central

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

    1995-01-01

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

  20. 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.

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

    PubMed

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

    2015-01-01

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

  2. 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

  3. 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

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

    PubMed Central

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

    2013-01-01

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

  5. GRID2 a novel gene possibly associated with mevalonate kinase deficiency.

    PubMed

    Moura, Ronald; Tricarico, Paola Maura; Campos Coelho, Antonio Victor; Crovella, Sergio

    2015-04-01

    Mevalonate kinase deficiency (MKD) is a rare autosomal disease caused by mutations in the mevalonate kinase gene (MVK). The genotype-phenotype correlation is sometimes problematic due to the great genetic and clinical heterogeneity; so we hypothesize that genes other than MVK are able to modulate MKD clinical phenotypes. This hypothesis was tested by analyzing the exome of 22 patients with MKD all carrying MVK gene mutations, and 20 patients with recurrent fevers (RF) not carrying MVK mutations. Our preliminary findings suggest a possible role of GRID2 in the susceptibility to develop MKD. GRID2 gene (4q22.2), encoding for human glutamate receptor delta-2, associated with MKD: The rs1450500 SNP was differently distributed in patients with MKD with respect to those with RF. Being aware of the small number of patients analyzed, we hypothesized a possible role for GRID2 as possible phenotype modifier in MKD patients, especially in those with severe phenotypes.

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

    PubMed

    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

  7. 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

  8. 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

  9. 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

  10. 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

  11. 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.

  12. Arabidopsis MAP kinase 4 regulates gene expression through transcription factor release in the nucleus.

    PubMed

    Qiu, Jin-Long; Fiil, Berthe Katrine; Petersen, Klaus; Nielsen, Henrik Bjørn; Botanga, Christopher J; Thorgrimsen, Stephan; Palma, Kristoffer; Suarez-Rodriguez, Maria Cristina; Sandbech-Clausen, Signe; Lichota, Jacek; Brodersen, Peter; Grasser, Klaus D; Mattsson, Ole; Glazebrook, Jane; Mundy, John; Petersen, Morten

    2008-08-20

    Plant and animal perception of microbes through pathogen surveillance proteins leads to MAP kinase signalling and the expression of defence genes. However, little is known about how plant MAP kinases regulate specific gene expression. We report that, in the absence of pathogens, Arabidopsis MAP kinase 4 (MPK4) exists in nuclear complexes with the WRKY33 transcription factor. This complex depends on the MPK4 substrate MKS1. Challenge with Pseudomonas syringae or flagellin leads to the activation of MPK4 and phosphorylation of MKS1. Subsequently, complexes with MKS1 and WRKY33 are released from MPK4, and WRKY33 targets the promoter of PHYTOALEXIN DEFICIENT3 (PAD3) encoding an enzyme required for the synthesis of antimicrobial camalexin. Hence, wrky33 mutants are impaired in the accumulation of PAD3 mRNA and camalexin production upon infection. That WRKY33 is an effector of MPK4 is further supported by the suppression of PAD3 expression in mpk4-wrky33 double mutant backgrounds. Our data establish direct links between MPK4 and innate immunity and provide an example of how a plant MAP kinase can regulate gene expression by releasing transcription factors in the nucleus upon activation.

  13. 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

  14. 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.

  15. 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

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

    PubMed

    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.

  17. Roots, cycles and leaves. Expression of the phosphoenolpyruvate carboxylase kinase gene family in soybean.

    PubMed

    Sullivan, Stuart; Jenkins, Gareth I; Nimmo, Hugh G

    2004-08-01

    Phosphorylation of phosphoenolpyruvate carboxylase (PEPc; EC 4.1.1.31) plays an important role in the control of central metabolism of higher plants. This phosphorylation is controlled largely at the level of expression of PEPc kinase (PPCK) genes. We have analyzed the expression of both PPCK genes and the PEPC genes that encode PEPc in soybean (Glycine max). Soybean contains at least four PPCK genes. We report the genomic and cDNA sequences of these genes and demonstrate the function of the gene products by in vitro expression and enzyme assays. For two of these genes, GmPPCK2 and GmPPCK3, transcript abundance is highest in nodules and is markedly influenced by supply of photosynthate from the shoots. One gene, GmPPCK4, is under robust circadian control in leaves but not in roots. Its transcript abundance peaks in the latter stages of subjective day, and its promoter contains a sequence very similar to the evening element found in Arabidopsis genes expressed at this time. We report the expression patterns of five PEPC genes, including one encoding a bacterial-type PEPc lacking the phosphorylation site of the plant-type PEPcs. The PEPc expression patterns do not match those of any of the PPCK genes, arguing against the existence of specific PEPc-PPCK expression partners. The PEPC and PPCK gene families in soybean are significantly more complex than previously understood.

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

    PubMed

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

    2015-01-01

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

  19. 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

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

    PubMed

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

    2015-01-01

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

  1. Deoxycytidine kinase and deoxyguanosine kinase of Lactobacillus acidophilus R-26 are colinear products of a single gene.

    PubMed

    Ma, N; Ikeda, S; Guo, S; Fieno, A; Park, I; Grimme, S; Ikeda, T; Ives, D H

    1996-12-10

    Three of the four deoxynucleoside kinases required for growth of Lactobacillus acidophilus R-26 exist as heterodimeric pairs specific for deoxyadenosine (dAK) and deoxycytidine (dCK) or dAK and deoxyguanosine (dGK). However, only two tandem genes, dak/dgk, are found, and are expressed only as dAK/dGK in transformed Escherichia coli. Sequencing peptides spanning 63% of the native dCK subunit revealed a sequence identical to that deduced from dgk (beginning MTVIVL...), except that dCK lacks residues 2 and 3 (dCK is M..IVL; dGK is .TVIVL). Also, mass spectrometry indicates that native dCK and dGK subunits are identical in mass adjusted for the first three residues. Furthermore, the native enzymes have identical isoelectric pH values, indicating an equal number of charged residues. To enable E. coli to express peptide having the native dCK sequence, codons 2 and 3 were deleted from the dgk portion of the tandem genes, resulting in expression of protein having the specificities and regulatory properties of native dAK/dCK, including heterotropic stimulation of dAK activity by deoxycytidine or dCTP (not deoxyguanosine or dGTP) and end-product inhibition of the respective activities by dATP and dCTP. Subcloning normal and mutant dgk yielded homodimeric dGK and dCK, respectively. The dCK homodimer strongly resembles human dCK, with a low K(m) for deoxycytidine, the ability to phosphorylate deoxyadenosine and deoxyguanosine at much higher K(m) values, and end-product inhibition by dCTP. Thus two distinct and specific enzymes evidently are derived from a single Lactobacillus gene. The mechanism by which this occurs in vivo has yet to be elucidated.

  2. 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

  3. Identification of a Wee1–Like Kinase Gene Essential for Procyclic Trypanosoma brucei Survival

    PubMed Central

    Boynak, Natalia Y.; Rojas, Federico; D’Alessio, Cecilia; Vilchez Larrea, Salomé C.; Rodriguez, Vanina; Ghiringhelli, Pablo D.; Téllez-Iñón, María T.

    2013-01-01

    Regulation of eukaryotic cell cycle progression requires sequential activation and inactivation of cyclin-dependent kinases (CDKs). Activation of the cyclin B-cdc2 kinase complex is a pivotal step in mitotic initiation and the tyrosine kinase Wee1 is a key regulator of cell cycle sequence during G2/M transition and inhibits mitotic entry by phosphorylating the inhibitory tyrosine 15 on the cdc2 M-phase-inducing kinase. Wee1 degradation is essential for the exit from the G2 phase. In trypanosomatids, little is known about the genes that regulate cyclin B-cdc2 complexes at the G2/M transition of their cell cycle. Although canonical tyrosine kinases are absent in the genome of trypanosomatids, phosphorylation on protein tyrosine residues has been reported in Trypanosoma brucei. Here, we characterized a Wee1-like protein kinase gene from T. brucei. Expression of TbWee1 in a Schizosaccharomyces pombe strain null for Wee1 inhibited cell division and caused cell elongation. This demonstrates the lengthening of G2, which provided cells with extra time to grow before dividing. The Wee1-like protein kinase was expressed in the procyclic and bloodstream proliferative slender forms of T. brucei and the role of Wee1 in cell cycle progression was analyzed by generating RNA interference cell lines. In the procyclic form of T. brucei, the knock-down of TbWee1 expression by RNAi led to inhibition of parasite growth. Abnormal phenotypes showing an increase in the percentage of cells with 1N0K, 0N1K and 2N1K were observed in these RNAi cell lines. Using parasites with a synchronized cell cycle, we demonstrated that TbWee1 is linked to the G2/M phase. We also showed that TbWee1 is an essential gene necessary for proper cell cycle progression and parasite growth in T. brucei. Our results provide evidence for the existence of a functional Wee1 in T. brucei with a potential role in cell division at G2/M. PMID:24223931

  4. 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.

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

    PubMed

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

    1995-02-14

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

  6. The Inositol 1,4,5-triphosphate kinase1 gene affects olfactory reception in Drosophila melanogaster.

    PubMed

    Gomez-Diaz, Carolina; Martin, Fernando; Alcorta, Esther

    2006-03-01

    The Inositol 1,4,5-triphosphate (IP3) route is one of the two main transduction cascades that mediate olfactory reception in Drosophila melanogaster. The activity of IP3 kinase1 reduces the levels of this substrate by phosphorylation into inositol 1,3,4,5-tetrakiphosphate (IP4). We show here that the gene is expressed in olfactory sensory organs as well as in the rest of the head. To evaluate in vivo the olfactory functional effects of up-regulating IP3K1, individuals with directed genetic changes at the reception level only were generated using the UAS/Gal4 method. In this report, we described the consequences in olfactory perception of overexpressing the IP3Kinase1 gene at eight different olfactory receptor-neuron subsets. Six out of the eight studied Gal-4/UAS-IP3K1 hybrids displayed abnormal behavioral responses to ethyl acetate, acetone, ethanol or propionaldehyde. Specific behavioral defects corresponded to the particular neuronal olfactory profile. These data confirm the role of the IP3kinase1 gene, and consequently the IP3 transduction cascade, in mediating olfactory information at the reception level.

  7. VprBP Has Intrinsic Kinase Activity Targeting Histone H2A and Represses Gene Transcription

    PubMed Central

    Kim, Kyunghwan; Kim, Jin-Man; Kim, Joong-Sun; Choi, Jongkyu; Lee, Yong Suk; Neamati, Nouri; Song, Jin Sook; Heo, Kyu; An, Woojin

    2013-01-01

    SUMMARY Histone modifications play important roles in the regulation of gene expression and chromatin organization. VprBP has been implicated in transcriptionally silent chromatin formation and cell cycle regulation, but the molecular basis underlying such effects remains unclear. Here we report that VprBP possesses an intrinsic protein kinase activity and is capable of phosphorylating histone H2A on threonine 120 (H2AT120p) in a nucleosomal context. VprBP is localized to a large set of tumor suppressor genes and blocks their transcription, in a manner that is dependent on its kinase activity toward H2AT120. The functional significance of VprBP-mediated H2AT120p is further underscored by the fact that RNAi knockdown and small-molecule inhibition of VprBP reactivate growth regulatory genes and impede tumor growth. Our findings establish VprBP as a major kinase responsible for H2AT120p in cancer cells and suggest that VprBP inhibition could be a new strategy for the development of anticancer therapeutics. PMID:24140421

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

    PubMed

    Muchir, Antoine; Worman, Howard J

    2016-01-01

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

  9. Identification and nucleotide sequence of the thymidine kinase gene of Shope fibroma virus

    SciTech Connect

    Upton, C.; McFadden, G.

    1986-12-01

    The thymidine kinase (TK) gene of Shope fibroma virus (SFV), a tumorigenic leporipoxvirus, was localized within the viral genome with degenerate oligonucleotide probes. These probes were constructed to two regions of high sequence conservation between the vaccinia virus TK gene and those of several known eucaryotic cellular TK genes, including human, mouse, hamster, and chicken TK genes. The oligonucleotide probes initially localized the SFV TK gene 50 kilobases (kb) from the right terminus of the 160-kb SFV genome within the 9.5-kb BamHI-HindIII fragment E. Fine-mapping analysis indicated that the TK Gene was within a 1.2-kb AvaI-HaeIII fragment, and DNA sequencing of this region revealed an open reading frame capable of encoding a polypeptide of 187 amino acids possessing considerable homology to the TK genes of the vaccinia, variola, and monkeypox orthopoxviruses and also to a variety of cellular TK genes. Homology matrix analysis and homology scores suggest that the SFV TK gene has diverged significantly from its counterpart members in the orthopoxvirus genus. Nevertheless, the presence of conserved upstream open reading frames on the 5' side of all of the poxvirus TK genes indicates a similarity of functional organization between the orthopoxviruses and leporipoxviruses. These data suggest a common ancestral origin for at least some of the unique internal regions of the leporipoxviruses and orthopoxviruses as exemplified by SFV and vaccinia virus, respectively.

  10. Serum- and glucocorticoid-regulated kinase (SGK1) gene and blood pressure.

    PubMed

    Busjahn, Andreas; Aydin, Atakan; Uhlmann, Regina; Krasko, Christine; Bähring, Sylvia; Szelestei, Tamas; Feng, Yuxi; Dahm, Stephan; Sharma, Arya M; Luft, Friedrich C; Lang, Florian

    2002-09-01

    The serum- and glucose-regulated kinase (SGK1) gene has recently been identified as an important aldosterone-induced protein kinase that mediates trafficking of the renal epithelial Na(+) channel (ENaC) to the cell membrane. Thus, SGK1 is an appealing candidate for blood pressure regulation and possibly essential hypertension. To test this hypothesis, we recruited monozygotic (126 pairs) and dizygotic (70 pairs) normotensive twin subjects and parents of dizygotic twins. Blood pressure was measured in a controlled fashion: recumbent, sitting, and upright. We documented genetic variance on blood pressure in all positions. We then relied on microsatellite markers at the SGK1 gene locus (D6S472, D6S1038, and D6S270) and 2 single nucleotide polymorphisms within the SGK1 gene. We found significant linkage of the SGK1 gene locus to diastolic blood pressure (P<0.0002) and suggestive evidence for linkage for systolic blood pressure (P<0.04), documenting the locus as a quantitative trait locus for blood pressure. We next performed association, using all dizygotic twins and a monozygotic member from each pair. We found significant associations between both single nucleotide polymorphism variants and blood pressure, as well as a significant interaction between the single nucleotide polymorphisms enhancing the effect. This combined effect of the polymorphisms was confirmed in an independent sample of 260 young normotensive men. We conclude that the SGK1 gene is relevant to blood pressure regulation and probably to hypertension in man. PMID:12215463

  11. Endotheliotropic elephant herpesvirus, the first betaherpesvirus with a thymidine kinase gene.

    PubMed

    Ehlers, Bernhard; Dural, Güzin; Marschall, Manfred; Schregel, Vera; Goltz, Michael; Hentschke, Jochen

    2006-10-01

    Endotheliotropic elephant herpesvirus (elephantid herpesvirus 1; ElHV-1) is apathogenic for African elephants (Loxodonta africana), but causes fatal haemorrhagic disease in Asian elephants (Elephas maximus). This is thought to occur through transmission from African elephants in places where both species are housed, such as zoological gardens. The virus has caused considerable losses in North American and European zoological gardens and thus severely impedes breeding of the endangered Asian elephant. Previously, the ultrastructural and genetic characterization of ElHV-1 from a male Asian elephant that died from the disease at the Berlin zoological gardens in 1998 have been reported. Here, a partial characterization of the ElHV-1 genome is presented. A 60 kbp locus, spanning 34 open reading frames, was analysed. Most of the detected genes were found to be conserved among the herpesviruses and showed an overall arrangement most similar to that of betaherpesviruses, in particular Human herpesvirus 6 and Human herpesvirus 7. Most importantly, in addition to a protein kinase gene that is homologous to the human cytomegalovirus UL97 gene, a thymidine kinase (TK) gene was found, which is generally missing in betaherpesvirus genomes. Thus, ElHV-1 is the only known betaherpesvirus to encode a TK gene. This peculiarity might contribute to the fulminant pathogenicity of ElHV-1, but also provide a crucial enzymic activity for developing an efficient antiviral therapy with currently available nucleoside analogues.

  12. 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

  13. 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.

  14. A novel mammalian protein kinase gene (mak) is highly expressed in testicular germ cells at and after meiosis.

    PubMed Central

    Matsushime, H; Jinno, A; Takagi, N; Shibuya, M

    1990-01-01

    We isolated a novel gene designated mak (male germ cell-associated kinase) by using weak cross-hybridization with a tyrosine kinase gene (v-ros). Sequence analysis of the cDNA corresponding to the 2.6-kilobase transcript revealed that the predicted product of rat mak consisted of 622 amino acids and contained protein kinase consensus motifs in its amino-terminal region. Comparison of the deduced amino acid sequence of mak in the kinase domain with those of other protein kinase genes demonstrated that mak was approximately 40% identical to the cdc2-CDC28 gene family in Schizosaccharomyces pombe, Saccharomyces cerevisiae, and humans but less identical to most other protein kinase gene products. Expression of mak was highly tissue specific, and its transcripts were detected almost exclusively in testicular cells entering and after meiosis but hardly detectable in ovarian cells including oocytes, after the dictyotene stage. These results suggest that the mak gene plays an important role in spermatogenesis. Images PMID:2183027

  15. 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

  16. 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

  17. 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

  18. Reversion of argE3 to Arg(+) in Escherichia coli AB1157 -an informative bacterial system for mutation detection.

    PubMed

    Sikora, Anna; Grzesiuk, Elżbieta

    2010-01-01

    This review concerns reversion of the argE3 (ochre) nonsense mutation to prototrophy in E. coli AB1157 strain as an informative system for mutation detection. Strain AB1157 bears the argE3 (ochre), hisG4 (ochre) and thr-1 (amber) mutations, and the supE44 amber suppressor on its chromosome. The Arg(+) phenotype can be restored by (i) any base substitution at the argE3 site that changes the nonsense UAA codon to any sense nucleotide triplet or to UAG recognized by the supE44 amber suppressor, or (ii) suppressor mutations enabling the reading of the UAA nonsense codon. The argE3 → Arg(+) reversion-based system enables (i) determination of the spontaneous or induced mutation level; (ii) determination of base substitutions (suppressor analysis); (iii) examination of transcription-coupled repair (TCR) since targets for DNA damage are situated on the transcribed or coding strand of DNA; (iv) detection of mutations resulting from single stranded DNA damage. This review focuses on studies carried out since the early 1990s till now with the application of the AB1157-based mutation detection system. Recently, the system has been used to obtain new data on the processes of methyl methanesulfonate-induced mutagenesis and DNA repair in E. coli alkB⁻ mutants. PMID:20978633

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

    DOEpatents

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

    2002-10-15

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

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

    DOEpatents

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

    2003-03-04

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

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

    PubMed Central

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

    2016-01-01

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

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

    PubMed

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

    2016-01-01

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

  4. 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

  5. Chromosomal localization of three mouse diacylglycerol kinase (DAGK) genes: Genes sharing sequence homology to the Drosophila retinal degeneration A (rdgA) gene

    SciTech Connect

    Pilz, A.; Hunt, D.; Fitzgibbon, J.

    1995-04-10

    There is growing evidence to support some form of light-activated phosphoinositide signal transduction pathway in the mammalian retina. Although this pathway plays no obvious role in mammalian phototransduction, mutations in this pathway cause retinal degenerations in Drosophila. These include the retinal-degeneration A mutant, which is caused by an alteration in an eye-specific diacylglycerol kinase (DAGK) gene. In our efforts to consider genes mutated in Drosophila as candidates for mammalian eye disease, we have initially determined the map position of three DAGK genes in the mouse. 21 refs., 2 figs.

  6. 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

  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. 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.

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

    SciTech Connect

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

    1996-04-01

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

  10. Comprehensive genomic analysis and expression profiling of diacylglycerol kinase gene family in Malus prunifolia (Willd.) Borkh.

    PubMed

    Li, Yali; Tan, Yanxiao; Shao, Yun; Li, Mingjun; Ma, Fengwang

    2015-05-01

    Diacylglycerol kinase (DGK) is a pivotal enzyme that phosphorylates diacylglycerol (DAG) to form phosphatidic acid (PA). The production of PA from phospholipase D (PLD) and the coupled phospholipase C (PLC)/DGK route is a critical signaling process in animal and plant cells. Next to PLD, DGK is the second most important generator of PA in biotic and abiotic stress responses. We identified 8 DGK members within the apple genome and all of their putative proteins contain one DGK catalytic domain and one DGK accessory domain. Four coding sequences were confirmed by cloning from Malus prunifolia. Phylogenetic and gene structure analyses showed that the apple DGK genes could be assigned to Clusters I, II, or III. Expression analysis of 6 of them revealed that their transcript levels were highest in stems. Some apple DGK genes were also significantly up-regulated in response to salt and drought stresses. This suggested their possible roles in plant defenses against environmental challenges. As a first step toward genome-wide analyses of the DGK genes in woody plants, our results imply that apple DGK genes are involved in the signaling of stress responses. These findings will contribute to further functional dissection of this gene family.

  11. 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

  12. Two PAK kinase genes, CHM1 and MST20, have distinct functions in Magnaporthe grisea.

    PubMed

    Li, Lei; Xue, Chaoyang; Bruno, Kenneth; Nishimura, Marie; Xu, Jin-Rong

    2004-05-01

    In the rice blast fungus Magnaporthe grisea, the Pmk1 mitogen-activated protein (MAP) kinase is essential for appressorium formation and infectious growth. PMK1 is homologous to yeast Fus3 and Kss1 MAP kinases that are known to be regulated by the Ste20 PAK kinase for activating the pheromone response and filamentation pathways. In this study, we isolated and characterized two PAK genes, CHM1 and MST20, in M. grisea. Mutants disrupted in MST20 were reduced in aerial hyphae growth and conidiation, but normal in growth rate, appressorium formation, penetration, and plant infection. In chm1 deletion mutants, growth, conidiation, and appressorium formation were reduced significantly. Even though appressoria formed by chm1 mutants were defective in penetration, chm1 mutants were able to grow invasively on rice leaves and colonize through wounds. The chm1 mutants were altered in conidiogenesis and produced conidia with abnormal morphology. Hyphae of chm1 mutants had normal septation, but the length of hyphal compartments was reduced. On nutritionally poor oatmeal agar, chm1 mutants were unstable and produced sectors that differed from original chm1 mutants in growth rate, conidiation, or colony morphology. However, none of the monoconidial cultures derived from these spontaneous sectors were normal in appressorial penetration and fungal pathogenesis. These data suggest that MST20 is dispensable for plant infection in M. grisea, but CHM1 plays a critical role in appressorium formation and penetration. Both mst20 and chm1 deletion mutants were phenotypically different from the pmk1 mutant that is defective in appressorium formation and infectious hyphae growth. It is likely that MST20 and CHM1 individually play no critical role in activating the PMK1 MAP kinase pathway during appressorium formation and infectious hyphae growth. However, CHM1 appears to be essential for appressorial penetration and CHM1 and MST20 may have redundant functions in M. grisea. PMID:15141959

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

    PubMed Central

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

    2013-01-01

    Mitogen-Activated Protein Kinase (MAPK) genes encode proteins that mediate various signaling pathways associated with biotic and abiotic stress responses in eukaryotes. The MAPK genes form a 3-tier signal transduction cascade between cellular stimuli and physiological responses. Recent identification of soybean MAPKs and availability of genome sequences from other legume species allowed us to identify their MAPK genes. The main objectives of this study were to identify MAPKs in 3 legume species, Lotus japonicus, Medicago truncatula, and Phaseolus vulgaris, and to assess their phylogenetic relationships. We used approaches in comparative genomics for MAPK gene identification and named the newly identified genes following Arabidopsis MAPK nomenclature model. We identified 19, 18, and 15 MAPKs and 7, 4, and 9 MAPKKs in the genome of Lotus japonicus, Medicago truncatula, and Phaseolus vulgaris, respectively. Within clade placement of MAPKs and MAPKKs in the 3 legume species were consistent with those in soybean and Arabidopsis. Among 5 clades of MAPKs, 4 founder clades were consistent to MAPKs of other plant species and orthologs of MAPK genes in the fifth clade-"Clade E" were consistent with those in soybean. Our results also indicated that some gene duplication events might have occurred prior to eudicot-monocot divergence. Highly diversified MAPKs in soybean relative to those in 3 other legume species are attributable to the polyploidization events in soybean. The identification of the MAPK genes in the legume species is important for the legume crop improvement; and evolutionary relationships and functional divergence of these gene members provide insights into plant genome evolution. PMID:24317362

  15. 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.

  16. Expression pattern of the CsPK3 auxin-responsive protein kinase gene.

    PubMed

    Chono, M; Suzuki, Y; Nemoto, K; Yamane, H; Murofushi, N; Yamaguchi, I

    2001-03-01

    We have previously cloned a cDNA of a putative serine/threonine protein kinase gene named CsPK3 from cucumber, the mRNA level of which was up-regulated by auxin and down-regulated by light irradiation. To examine the CsPK3 gene expression in detail, we cloned a genomic DNA of CsPK3 gene and made transgenic tobacco (Nicotiana tabacum L. cv. Petit Havana SR1) plants containing the fused CsPK3 promoter-beta-glucuronidase gene. The beta-glucuronidase expression was detected in the shoot apex, vascular tissues, and the outermost layer of cortex. The histological distribution of CsPK3 mRNA in cucumber seedlings was supported by in situ hybridization, where the positive signals were observed in similar tissues as those observed by beta-glucuronidase staining. The responsiveness of the CsPK3 gene to auxin and light was also confirmed for beta-glucuronidase activity. The pattern of beta-glucuronidase staining changed during the development of the tobacco seedlings. The results of our experiment showed that CsPK3 was expressed in a wide variety of tissues and cells in which the developmental and growth controls by auxin are suggested.

  17. 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

  18. Developmental effect of the XmnI site on Ggamma-globin gene expression among newborn Hb F-Malta-I [Ggamma117(G19)His-->Arg, CAT-->CGT] heterozygotes and adult beta+ -Thalassemia homozygotes.

    PubMed

    Pulis, Svetlana; Scerri, Christian A; Wismayer, Pierre Schembri; Galdies, Ruth; Wettinger, Stephanie Bezzina; Felice, Alex E

    2007-01-01

    Hb F-Malta-I [Ggamma117(19)His-->Arg, CAT-->CGT] is a stable and benign variant of Hb F found in 1.8% of Maltese newborn. We studied 120 Hb F-Malta-I heterozygotes and four Hb F-Malta-I homozygotes. The mean proportion of Ggamma-F-Malta-I in Hb F was 0.26 +/- 0.03 for the Hb F-Malta-I heterozygotes and 0.58 +/- 0.06 for the Hb F-Malta-I homozygotes. The Hb F-Malta-I allele was shown to occur on a background of the common Mediterranean haplotype Va [+ + - - - - - + + -]. Furthermore, the common Mediterranean haplotypes Va, IIIb [- + + + - + + + + -], I [+ + - - - - - + + +] and II [- + - + + - + + + +] accounted for most (66.2%) of the wild-type alleles among the tested Hb F-Malta-I heterozygotes. Different genotypes at the 5' epsilon HincII, Ggamma and Agamma HindIII, and 3'psibeta HincII sites (but not at the 5' Ggamma XmnI site) were found to be linked to significant variations in the proportion of Ggamma-F-Malta-I and Ggamma-globins in the Hb F of newborn Hb F-Malta-I heterozygotes. Moreover, the 5' Ggamma XmnI site was found to be associated with variations in Hb F and Ggamma-globin levels in a population of adult Maltese beta-thalassemia (thal) homozygotes. This implies that a determinant linked to the XmnI site which effects Ggamma-globin gene expression is active in anemic adults but not in normal infants.

  19. MKK3- and MKK6-regulated gene expression is mediated by the p38 mitogen-activated protein kinase signal transduction pathway.

    PubMed Central

    Raingeaud, J; Whitmarsh, A J; Barrett, T; Dérijard, B; Davis, R J

    1996-01-01

    The p38 mitogen-activated protein (MAP) kinase signal transduction pathway is activated by proinflammatory cytokines and environmental stress. The detection of p38 MAP kinase in the nucleus of activated cells suggests that p38 MAP kinase can mediate signaling to the nucleus. To test this hypothesis, we constructed expression vectors for activated MKK3 and MKK6, two MAP kinase kinases that phosphorylate and activate p38 MAP kinase. Expression of activated MKK3 and MKK6 in cultured cells caused a selective increase in p38 MAP kinase activity. Cotransfection experiments demonstrated that p38 MAP kinase activation causes increased reporter gene expression mediated by the transcription factors ATF2 and Elk-1. These data demonstrate that the nucleus is one target of the p38 MAP kinase signal transduction pathway. PMID:8622669

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

    PubMed

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

    2014-12-18

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

  1. 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

  2. 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.

  3. [Receptor tyrosine kinase KIT may regulate expression of genes involved in spontaneous regression of neuroblastoma].

    PubMed

    Lebedev, T D; Spirin, P V; Suntsova, M V; Ivanova, A V; Buzdin, A A; Prokofjeva, M M; Rubtsov, P M; Prassolov, V S

    2015-01-01

    Hallmark of neuroblastoma is an ability of this malignant tumor to undergo spontaneous regression or differentiation into benign tumor during any stage of the disease, but it is little known about mechanisms of these phenomena. We studied effect of receptor tyrosine kinase receptor KIT on expression of genes, which may be involved in tumor spontaneous regression. Downregulation of KIT expression by RNA interference in SH-SY5Y cells causes suppression of neurotrophin receptor NGFR expression that may promote the loss of sensibility of cells to nerve growth factors, also it causes upregulation of TrkA receptor expression which can stimulate cell differentiation or apoptosis in NGF dependent manner. Furthermore there is an upregulation of genes which stimulate malignant cell detection by immune system, such as genes of major histocompatibility complex HLA class I HLA-B and HLA-C, and interferon-γ receptors IFNGR1 and IFNGR2 genes. Thus KIT can mediate neuroblastoma cell sensibility to neurotrophins and immune system components--two factors directly contributing to spontaneous regression of neuroblastoma.

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

    PubMed

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

    2015-01-01

    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.

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

    PubMed Central

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

    2015-01-01

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

  6. 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.

  7. 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

  8. 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

  9. 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

  10. Estrogen-related receptors stimulate pyruvate dehydrogenase kinase isoform 4 gene expression.

    PubMed

    Zhang, Yi; Ma, Ke; Sadana, Prabodh; Chowdhury, Farhana; Gaillard, Stephanie; Wang, Fang; McDonnell, Donald P; Unterman, Terry G; Elam, Marshall B; Park, Edwards A

    2006-12-29

    The pyruvate dehydrogenase complex (PDC) catalyzes the conversion of pyruvate to acetyl-CoA in mitochondria and is a key regulatory enzyme in the oxidation of glucose to acetyl-CoA. Phosphorylation of PDC by the pyruvate dehydrogenase kinases (PDK2 and PDK4) inhibits PDC activity. Expression of the PDK genes is elevated in diabetes, leading to the decreased oxidation of pyruvate to acetyl-CoA. In these studies we have investigated the transcriptional regulation of the PDK4 gene by the estrogen-related receptors (ERRalpha and ERRgamma). The ERRs are orphan nuclear receptors whose physiological roles include the induction of fatty acid oxidation in heart and muscle. Previously, we found that the peroxisome proliferator-activated receptor gamma coactivator (PGC-1alpha) stimulates the expression of PDK4. Here we report that ERRalpha and ERRgamma stimulate the PDK4 gene in hepatoma cells, suggesting a novel role for ERRs in controlling pyruvate metabolism. In addition, both ERR isoforms recruit PGC-1alpha to the PDK4 promoter. Insulin, which decreases the expression of the PDK4 gene, inhibits the induction of PDK4 by ERRalpha and ERRgamma. The forkhead transcription factor (FoxO1) binds the PDK4 gene and contributes to the induction of PDK4 by ERRs and PGC-1alpha. Insulin suppresses PDK4 expression in part through the dissociation of FoxO1 and PGC-1alpha from the PDK4 promoter. Our data demonstrate a key role for the ERRs in the induction of hepatic PDK4 gene expression. PMID:17079227

  11. Pectin activation of MAP kinase and gene expression is WAK2 dependent.

    PubMed

    Kohorn, Bruce D; Johansen, Susan; Shishido, Akira; Todorova, Tanya; Martinez, Rhysly; Defeo, Elita; Obregon, Pablo

    2009-12-01

    The angiosperm extracellular matrix, or cell wall, is composed of a complex array of cellulose, hemicellulose, pectins and proteins, the modification and regulated synthesis of which are essential for cell growth and division. The wall associated kinases (WAKs) are receptor-like proteins that have an extracellular domain that bind pectins, the more flexible portion of the extracellular matrix, and are required for cell expansion as they have a role in regulating cellular solute concentrations. We show here that both recombinant WAK1 and WAK2 bind pectin in vitro. In protoplasts pectins activate, in a WAK2-dependent fashion, the transcription of vacuolar invertase, and a wak2 mutant alters the normal pectin regulation of mitogen-activated protein kinases. Microarray analysis shows that WAK2 is required for the pectin activation of numerous genes in protoplasts, many of which are involved in cell wall biogenesis. Thus, WAK2 plays a major role in signaling a diverse array of cellular events in response to pectin in the extracellular matrix.

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

    PubMed

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

    1997-10-31

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

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

    PubMed

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

    2005-06-01

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

  14. Characterization of a protein kinase gene responsive to auxin and gibberellin in cucumber hypocotyls.

    PubMed

    Chono, M; Nemoto, K; Yamane, H; Yamaguchi, I; Murofushi, N

    1998-09-01

    By means of the PCR, cDNA clones encoding putative protein kinases have been obtained from cucumber hypocotyls. The abundance of the transcript of one of these genes, which was named CsPK3, increased on treatment with gibberellin (GA4) and/or auxin (IAA). We screened a cucumber cDNA library to clone CsPK3 cDNA. The cDNA clone (cCsPK3) encodes an open reading frame of 1,413 bp (471 amino acids), and its predicted amino acid sequence showed homology with those of serine/threonine protein kinases. Northern blot analysis indicated that IAA was more active than GA4 in increasing the level of CsPK3 mRNA in cucumber hypocotyls and that the increase in the level of CsPK3 mRNA on treatment with IAA was not inhibited by pretreatment with a protein synthesis inhibitor. The level of CsPK3 mRNA was high in hypocotyls of dark-grown cucumber seedlings and decreased to less than 50% of the original level within 15 min of the start of irradiation with white light.

  15. Gene expression related to synaptogenesis, neuritogenesis, and MAP kinase in behavioral sensitization to psychostimulants.

    PubMed

    Ujike, Hiroshi; Takaki, Manabu; Kodama, Masafumi; Kuroda, Shigetoshi

    2002-06-01

    The most important characteristic of behavioral sensitization to psychostimulants, such as amphetamine and cocaine, is the very long-lasting hypersensitivity to the drug after cessation of exposure. Rearrangement and structural modification of neural networks in CNS must be involved in behavioral sensitization. Previous microscopic studies have shown that the length of dendrites and density of dendritic spines increased in the nucleus accumbens and frontal cortex after repeated exposure to amphetamine and cocaine, but the molecular mechanisms responsible are not well understood. We investigated a set of genes related to synaptogenesis, neuritogenesis, and mitogen-activated protein (MAP) kinase after exposure to methamphetamine. Synaptophysin mRNA, but not VAMP2 (synaptobrevin 2) mRNA, which are considered as synaptogenesis markers, increased in the accumbens, striatum, hippocampus, and several cortices, including the medial frontal cortex, after a single dose of 4 mg/kg methamphetamine. Stathmin mRNA, but not neuritin or narp mRNA, which are markers for neuritic sprouting, increased in the striatum, hippocampus, and cortices after a single dose of methamphetamine. The mRNA of arc, an activity-regulated protein associated with cytoskeleton, but not of alpha-tubulin, as markers for neuritic elongation, showed robust increases in the striatum, hippocampus, and cortices after a single dose of methamphetamine. The mRNAs of MAP kinase phosphatase-1 (MKP-1), MKP-3, and rheb, a ras homologue abundant in brain, were investigated to assess the MAP kinase cascades. MKP-1 and MKP-3 mRNAs, but not rheb mRNA, increased in the striatum, thalamus, and cortices, and in the striatum, hippocampus, and cortices, respectively, after a single methamphetamine. Synaptophysin and stathmin mRNAs did not increase again after chronic methamphetamine administration, whereas the increases in arc, MKP-1, and MKP-3 mRNAs persisted in the brain regions after chronic methamphetamine administration

  16. 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. PMID:27588186

  17. 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.

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

    PubMed

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

    2009-10-27

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

  19. A single ataxia telangiectasia gene with a product similar to PI-3 kinase

    SciTech Connect

    Savitsky, K.; Bar-Shira, A.; Gilad, S.; Rotman, G.; Ziv, Y.; Vanagaite, L.; Smith, S.; Uziel, T.; Sfez, S.; Ashkenazi, M.

    1995-06-23

    A gene, ATM, that is mutated in the autosomal recessive disorder ataxia telangiectasia (AT) was identified by positional cloning on chromosome 11q22-23. AT is characterized by cerebellar degeneration, immunodeficiency, chromosomal instability, cancer predisposition, radiation sensitivity, and cell cycle abnormalities. The disease is genetically heterogeneous, with four complementation groups that have been suspected to represent different genes. ATM, which has a transcript of 12 kilobases, was found to be mutated in AT patients from all complementation groups, indicating that it is probably the sole gene responsible for this disorder. A partial ATM complementary DNA clone of 5.9 kilobases encoded a putative protein that is similar to several yeast and mammalian phosphatidylinositol-3{prime} kinases that are involved in mitogenic signal transduction, meiotic recombination, and cell cycle control. The discovery of ATM should enhance understanding of AT and related syndromes and may allow the identification of AT heterozygotes, who are at increased risk of cancer. 54 refs., 5 figs., 1 tab.

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

    PubMed Central

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

    2016-01-01

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

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

    PubMed

    Weir, J P; Moss, B

    1987-05-01

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

  2. 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

  3. 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.

  4. 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...

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

    PubMed Central

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

    1995-01-01

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

  6. Diverse Transcriptional Programs Associated with Environmental Stress and Hormones in the Arabidopsis Receptor-Like Kinase Gene Family

    PubMed Central

    Chae, Lee; Sudat, Sylvia; Dudoit, Sandrine; Zhu, Tong; Luan, Sheng

    2009-01-01

    The genome of Arabidopsis thaliana encodes more than 600 receptor-like kinase (RLK) genes, by far the dominant class of receptors found in land plants. Although similar to the mammalian receptor tyrosine kinases, plant RLKs are serine/threonine kinases that represent a novel signaling innovation unique to plants and, consequently, an excellent opportunity to understand how extracellular signaling evolved and functions in plants as opposed to animals. RLKs are predicted to be major components of the signaling pathways that allow plants to respond to environmental and developmental conditions. However, breakthroughs in identifying these processes have been limited to only a handful of individual RLKs. Here, we used a Syngenta custom Arabidopsis GeneChip array to compile a detailed profile of the transcriptional activity of 604 receptor-like kinase genes after exposure to a cross-section of known signaling factors in plants, including abiotic stresses, biotic stresses, and hormones. In the 68 experiments comprising the study, we found that 582 of the 604 RLK genes displayed a two-fold or greater change in expression to at least one of 12 types of treatments, thereby providing a large body of experimental evidence for targeted functional screens of individual RLK genes. We investigated whether particular subfamilies of RLK genes are responsive to specific types of signals and found that each subfamily displayed broad ranges of expression, as opposed to being targeted towards particular signal classes. Finally, by analyzing the divergence of sequence and gene expression among the RLK subfamilies, we present evidence as to the functional basis for the expansion of the RLKs and how this expansion may have affected conservation and divergences in their function. Taken as a whole, our study represents a preliminary, working model of processes and interactions in which the members of the RLK gene family may be involved, where such information has remained elusive for so many

  7. ADRB2 Arg16Gly Polymorphism, Lung Function, and Mortality: Results from the Atherosclerosis Risk in Communities Study

    PubMed Central

    Ferdinands, Jill M.; Mannino, David M.; Gwinn, Marta L.; Bray, Molly S.

    2007-01-01

    Background 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 the Atherosclerosis Risk in Communities study. Methodology and Principal Findings We genotyped study participants and analyzed the association of the Arg16Gly polymorphism and beta-agonist use with lung function at baseline and clinical examination three years later and with all-cause mortality during 10 years of follow-up. Lung function was characterized by percent-predicted forced expiratory volume in 1 second. Associations were examined separately for blacks and whites. Black beta-agonist users with the Arg/Arg genotype had better lung function at baseline and at the second clinical visit than those with Arg/Gly and Gly/Gly genotypes. Adjusted mean percent-predicted FEV1 was 21% higher in Arg/Arg subjects compared to Gly/Gly at baseline (p = 0.01) and 20% higher than Gly/Gly at visit 2 (p = 0.01). Arg/Gly subjects had adjusted percent-predicted FEV1 17% lower than Arg/Arg at baseline but were similar to Arg/Arg subjects at visit 2. Although black beta-agonist users with the Arg/Arg genotype appeared to have better crude survival rates, the association between genotype and all-cause mortality was inconclusive. We found no difference in lung function or mortality by genotype among blacks who did not use beta-agonists or among whites, regardless of beta-agonist use. Conclusions Black beta-agonist users with the ADRB2 Arg16Arg genotype had better lung function, and, possibly, better overall survival compared to black beta-agonist users with the Gly16Gly genotype. Our findings highlight the need for additional studies of sufficient size and statistical power to allow examination of outcomes among beta-agonist users of different races and genotypes. PMID

  8. Down-regulation of rat mitochondrial branched-chain 2-oxoacid dehydrogenase kinase gene expression by glucocorticoids.

    PubMed Central

    Huang, Y S; Chuang, D T

    1999-01-01

    The mammalian mitochondrial branched-chain 2-oxoacid dehydrogenase (BCOD) complex is regulated by a reversible phosphorylation (inactivation)/dephosphorylation (activation) cycle. In the present study, the effects of glucocorticoids on the level of BCOD kinase mRNA were investigated in rat hepatoma cell lines (H4IIE and FTO-2B), as well as in the rat. In H4IIE cells, dexamethasone was found to significantly reduce steady-state concentrations of BCOD kinase mRNA after a 48 h culture, and this was correlated with a 2-fold increase in the dephosphorylated form of the BCOD complex. The half-life of the kinase mRNA in H4IIE cells was not affected by dexamethasone treatment. Therefore, the decrease in the steady-state kinase mRNA level resulting from dexamethasone treatment was not caused by changes in mRNA stability, which raised the possibility of regulation at the level of gene transcription. To identify the negative glucocorticoid-responsive element in the kinase promoter, nested deletion constucts in the 3.0 kb promoter region were examined in H4IIE cells cultured in the presence or absence of dexamethasone. No significant differences in promoter activity were observed on either transient or stable transfection. The data showed that the glucocorticoid-responsive element was located outside the 3. 0 kb promoter region. At the physiological level, hepatic BCOD kinase mRNA levels were reduced in rats injected intraperitoneally with dexamethasone. This effect was liver-specific, and was not detected in other tissues. These results suggest that the down-regulation of kinase gene expression by glucocorticoids is mediated through a liver-specific or -enriched transcription factor(s). PMID:10215586

  9. Plc1p, Arg82p, and Kcs1p, enzymes involved in inositol pyrophosphate synthesis, are essential for phosphate regulation and polyphosphate accumulation in Saccharomyces cerevisiae.

    PubMed

    Auesukaree, Choowong; Tochio, Hidehito; Shirakawa, Masahiro; Kaneko, Yoshinobu; Harashima, Satoshi

    2005-07-01

    In Saccharomyces cerevisiae, the phosphate signal transduction PHO pathway is involved in regulating several phosphate-responsive genes such as PHO5, which encodes repressible acid phosphatase. In this pathway, a cyclin-dependent kinase inhibitor (Pho81p) regulates the kinase activity of the cyclin-cyclin-dependent kinase complex Pho80p-Pho85p, which phosphorylates the transcription factor Pho4p in response to intracellular phosphate levels. However, how cells sense phosphate availability and transduce the phosphate signal to Pho81p remains unknown. To identify additional components of the PHO pathway, we have screened a collection of yeast deletion strains. We found that disruptants of PLC1, ARG82, and KCS1, which are involved in the synthesis of inositol polyphosphate, and ADK1, which encodes adenylate kinase, constitutively express PHO5. Each of these factors functions upstream of Pho81p and negatively regulates the PHO pathway independently of intracellular orthophosphate levels. Overexpression of KCS1, but not of the other genes, suppressed PHO5 expression in the wild-type strain under low phosphate conditions. These results raise the possibility that diphosphoinositol tetrakisphosphate and/or bisdiphosphoinositol triphosphate may be essential for regulation of the PHO pathway. Furthermore, the Deltaplc1, Deltaarg82, and Deltakcs1 deletion strains, but not the Deltaipk1 deletion strain, had significantly reduced intracellular polyphosphate levels, suggesting that enzymes involved in inositol pyrophosphate synthesis are also required for polyphosphate accumulation.

  10. Antitumor activity of combined endostatin and thymidine kinase gene therapy in C6 glioma models.

    PubMed

    Chen, Yan; Huang, Honglan; Yao, Chunshan; Su, Fengbo; Guan, Wenming; Yan, Shijun; Ni, Zhaohui

    2016-09-01

    The combination of Endostatin (ES) and Herpes Simplex Virus thymidine kinase (HSV-TK) gene therapy is known to have antitumor activity in bladder cancer. The potential effect of ES and TK therapy in glioma has not yet been investigated. In this study, pTK-internal ribosome entry site (IRES), pIRES-ES, and pTK-IRES-ES plasmids were constructed; pIRES empty vector served as the negative control. The recombinant constructs were transfected into human umbilical vein endothelial cells (HUVECs) ECV304 and C6 rat glioma cell line. Ganciclovir (GCV) was used to induce cell death in transfected C6 cells. We found that ECV304 cells expressing either ES or TK-ES showed reduced proliferation, decreased migration capacity, and increased apoptosis, as compared to untransfected cells or controls. pTK-IRES-ES/GCV or pTK-IRES/GCV significantly suppressed cell proliferation and induced cell apoptosis in C6 cells, as compared to the control. In addition, the administration of pIRES-ES, pTK-IRES/GCV, or pTK-IRES-ES/GCV therapy improved animal activity and behavior; was associated with prolonged animal survival, and a lower microvessel density (MVD) value in tumor tissues of C6 glioma rats. In comparison to others, dual gene therapy in form of pTK-IRES-ES/GCV had a significant antitumor activity against C6 glioma. These findings indicate combined TK and ES gene therapy was associated with a superior antitumor efficacy as compared to single gene therapy in C6 glioma. PMID:27366865

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

    PubMed Central

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

    2012-01-01

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

  12. Gene regulation of ENaC subunits by serum- and glucocorticoid-inducible kinase-1.

    PubMed

    Boyd, Cary; Náray-Fejes-Tóth, Anikó

    2005-03-01

    Aldosterone is a key regulator of epithelial Na+ channels (ENaC) in renal cortical collecting ducts (CCD). The goal of this study was to examine whether serum- and glucocorticoid-inducible kinase-1 (SGK1), an aldosterone-induced gene, is vital to the delayed effect of aldosterone by increasing the gene expression of ENaC subunits. To test this hypothesis, we compared the levels of ENaC mRNA in mouse CCD cells that stably express either full-length (FL)-SGK1 or a kinase-dead dominant negative (K127M)-SGK1. Our results revealed that SGK1 regulates gene expression of ENaC, whether cells are maintained in steroid-free media or in the presence of corticosteroids (CS) and/or other growth factors. Under all conditions, the loss of function of SGK1 caused a significant decrease in the expression of alpha- and beta-ENaC, but not gamma-ENaC. Compared with cells expressing FL-SGK1, K127M-SGK1 decreased the expression of alpha- and beta-subunit mRNA by approximately 45 and approximately 90%, respectively. Next, to determine whether SGK1 is one of the proteins mediating the induction of alpha-ENaC mRNA by CS, we compared steroid induction of alpha-ENaC in cells expressing K127M-SGK1 vs. FL-SGK1. The maximum level of alpha-ENaC mRNA levels following CS was significantly (approximately 45%) higher in FL-SGK1- vs. K127M-SGK1-expressing cells, although the fold-induction by CS was similar in both FL-SGK1- and K127M-SGK1-expressing cells. In summary, we report for the first time that SGK1 regulates transcription of ENaC subunits. We propose that the effect of SGK1 on ENaC transcription is mediated by the activation of unidentified transcription factors. PMID:15536167

  13. 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.

  14. Differential gene expression of phosphoglyceric kinase (PGK) and hypoxic adaptation in chicken.

    PubMed

    Wang, CunFang; Yuan, CunZhong; Zhang, Lao; Wu, ChangXin; Li, Ning

    2007-06-01

    Four single-nucleotide polymorphisms (SNP) of the Phosphoglyceric Kinase (PGK) gene were discovered based on comparison of the sequences from an altiplano chicken breed (Tibetan chicken) and two lowland breeds (White Leghorn and Shouguang chicken). Gel-shift results indicate that one of these SNPs, an A-->G mutation at position 59 in exon10, is able to bind hypoxia-induced factor-l (HIF-1), functioning as a hypoxia response element (HRE). The mutant gene results in M-->T mutation at position 379 amino acid. The combined activity of this HRE and HIF-1 could increase correspondingly under a hypoxic stimulus. Hypoxia leads to increased death rates of chicken embryos; while the M-->T mutation described herein is prevalent in healthy embryos grown under hypoxic conditions, thus it may represent an adaptation to hypoxia. Fluorescence quantitative reverse transcription PCR results revealed that HIF-1 upregulates the transcript level of the glycolytic enzyme PGK in the brain and skeletal muscle of animals subjected to hypoxia. Thus, a large amount of ATP is produced by increased glycolysis, allowing the organism to meet energy metabolism demands. As such, we believe this SNP to be an adaptation to the external anoxic environment.

  15. 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.

  16. 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

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

    PubMed

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

    2014-12-01

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

  18. 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

  19. Clinical Relevance of Liver Kinase B1(LKB1) Protein and Gene Expression in Breast Cancer

    PubMed Central

    Chen, I-Chun; Chang, Yuan-Ching; Lu, Yen-Shen; Chung, Kuei-Pin; Huang, Chiun-Sheng; Lu, Tzu-Pin; Kuo, Wen-Hung; Wang, Ming-Yang; Kuo, Kuan-Ting; Wu, Pei-Fang; Hsueh, Tsu-Hsin; Shen, Chen-Yang; Lin, Ching-Hung; Cheng, Ann-Lii

    2016-01-01

    Liver kinase B1 (LKB1) is a tumor suppressor, and its loss might lead to activation of the mammalian target of rapamycin (mTOR) and tumorigenesis. This study aimed to determine the clinical relevance of LKB1 gene and protein expression in breast cancer patients. LKB1 protein expression was evaluated using immunohistochemistry in tumors from early breast cancer patients in two Taiwanese medical centers. Data on LKB1 gene expression were obtained from the Molecular Taxonomy of Breast Cancer International Consortium (METABRIC) data set. The correlations between LKB1 expression, clinicopathologic factors, and patient outcome were analyzed. LKB1 expression was significantly associated with estrogen receptor (ER) expression in 2 of the 4 cohorts, but not with other clinicopathologic factors. LKB1 expression was not a predictor for relapse-free survival, overall survival (OS), or breast cancer-specific survival. In a subgroup analysis of the two Taiwanese cohorts, high LKB1 protein expression was predictive of high OS in human epidermal growth factor receptor 2 (HER2)-positive breast cancer patients (P = 0.013). Our study results indicate that LKB1 expression is not prognostic in the whole population of breast cancer patients, but it is a potential predictor of OS in the subset of HER2-positive patients PMID:26877155

  20. 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

  1. 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

  2. Cloning of the rat pyruvate dehydrogenase kinase 4 gene promoter: activation of pyruvate dehydrogenase kinase 4 by the peroxisome proliferator-activated receptor gamma coactivator.

    PubMed

    Ma, Ke; Zhang, Yi; Elam, Marshall B; Cook, George A; Park, Edwards A

    2005-08-19

    The pyruvate dehydrogenase complex catalyzes the conversion of pyruvate to acetyl-CoA in mitochondria and is a key regulatory enzyme in the metabolism of glucose to acetyl-CoA. Phosphorylation of pyruvate dehydrogenase by the pyruvate dehydrogenase kinases (PDK) inhibits pyruvate dehydrogenase complex activity. There are four PDK isoforms, and expression of PDK4 and PDK2 genes is elevated in starvation and diabetes, allowing glucose to be conserved while fatty acid oxidation is increased. In these studies we have investigated the transcriptional mechanisms by which the expression of the PDK4 gene is increased. The peroxisome proliferator-activated receptor gamma coactivator (PGC-1alpha) stimulates the expression of genes involved in hepatic gluconeogenesis and mitochondrial fatty acid oxidation. We have found that PGC-1alpha will induce the expression of both the PDK2 and PDK4 genes in primary rat hepatocytes and ventricular myocytes. We cloned the promoter for the rat PDK4 gene. Hepatic nuclear factor 4 (HNF4), which activates many genes in the liver, will induce PDK4 expression. Although HNF4 and PGC-1alpha interact to stimulate several genes encoding gluconeogenic enzymes, the induction of PDK4 does not involve interactions of PGC-1alpha with HNF4. Using the chromatin immunoprecipitation assay, we have demonstrated that HNF4 and PGC-1alpha are associated with the PDK4 gene in vivo. Our data suggest that by inducing PDK genes PGC-1alpha will direct pyruvate away from metabolism into acetyl-CoA and toward the formation of oxaloacetate and into the gluconeogenic pathway. PMID:15967803

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

    PubMed

    Lee, Sang-Eun; Chang, Sunghoe

    2016-09-01

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

  4. 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

  5. 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.

  6. Decreased Activity in Neuropathic Pain Form and Gene Expression of Cyclin-Dependent Kinase5 and Glycogen Synthase Kinase-3 Beta in Soleus Muscle of Wistar Male Rats

    PubMed Central

    Rahmati, Masoud; Taherabadi, Seyed Jalal; Mehrabi, Mahmoud

    2015-01-01

    Background: The relationship between decreased activity/neuropathic pain and gene expression alterations in soleus muscle has remained elusive. Objectives: In this experimental study, we investigated the effects of decreased activity in neuropathic pain form on Cyclin-Dependent Kinase 5 (CDK5) and Glycogen Synthase Kinase-3 β (GSK-3β) gene expression in soleus muscle of rats. Materials and Methods: Twelve male Wistar rats were randomly divided into three groups: (1) tight ligation of the L5 spinal nerve (SNL: n = 4); (2) sham surgery (Sham: n = 4), and (3) control (C: n = 4). The threshold to produce a withdrawal response to a mechanical and thermal stimulus was measured using von Frey filaments and radiation heat apparatus, respectively. Following 4 weeks after surgery, the left soleus muscle was removed and mRNA levels were determined by real-time Polymerase Chain Reaction (PCR). Results: Compared to control animals, L5 ligated animals developed mechanical and heat hypersensitivity during total period of study. Soleus muscle weight as well as CDK5 mRNA levels (less than ~ 0.4 fold) was decreased and GSK-3β mRNA levels (up to ~ 7 folds) increased in L5 ligated animals. Conclusions: These results showed enhanced muscle atrophy processes following peripheral nerve damage and might provide a useful approach to study underlying muscle mechanisms associated with clinical neuropathic pain syndromes. PMID:26290750

  7. 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

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

    PubMed Central

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

    2016-01-01

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

  9. 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

  10. The genome-wide identification of mitogen-activated protein kinase kinase (MKK) genes in Yesso scallop Patinopecten yessoensis and their expression responses to bacteria challenges.

    PubMed

    Zou, Jiajun; Wang, Ruijia; Li, Ruojiao; Kong, Yifan; Wang, Jing; Ning, Xianhui; Zhang, Lingling; Wang, Shi; Hu, Xiaoli; Bao, Zhenmin

    2015-08-01

    Mitogen-activated protein kinase kinases (MKK) are the essential components of the evolutionarily conserved MAPK signaling cascade, which regulates a variety of cellular activities and innate immune responses. Although MKK genes have been extensively studied in various vertebrate and invertebrate species, they have not been systematically characterized in bivalves. In this study, we identified and characterized five MKK genes (PyMKK1/2, PyMKK4, PyMKK5, PyMKK3/6 and PyMKK7) in the Yesso scallop (Patinopecten yessoensis). Phylogenetic and protein structural analyses were conducted to determine their identities and evolutionary relationships. To gain insights into the possible roles of MKK genes during scallop innate immune responses, quantitative real-time PCR (qRT-PCR) was used to investigate their expression profiles during different developmental stages in samples taken from healthy adult tissues and hemocytes after Micrococcus luteus and Vibrio anguillarum bacterial infections. The Yesso scallop MKKs (PyMKKs) were found to have highly conserved structural features compared to the MKK genes from other invertebrate species. Using qRT-PCR analysis, three distinct expression patterns were detected among the PyMKKs over the course of ten different developmental stages. In adult scallops, the majority of the PyMKKs were highly expressed in mantle, gill, muscle and hemocytes. The differential expression patterns of the five PyMKKs after M. luteus (Gram-positive) and V. anguillarum (Gram-negative) bacterial infections suggested their possible involvement in the innate immune response and provide the foundation and resource for the further study on innate immune response of MAPK signal pathway in mollusk. PMID:26067168

  11. Definition of regulatory sequence elements in the promoter region and the first intron of the myotonic dystrophy protein kinase gene.

    PubMed

    Storbeck, C J; Sabourin, L A; Waring, J D; Korneluk, R G

    1998-04-10

    Myotonic dystrophy is the most common inherited adult neuromuscular disorder with a global frequency of 1/8000. The genetic defect is an expanding CTG trinucleotide repeat in the 3'-untranslated region of the myotonic dystrophy protein kinase gene. We present the in vitro characterization of cis regulatory elements controlling transcription of the myotonic dystrophy protein kinase gene in myoblasts and fibroblasts. The region 5' to the initiating ATG contains no consensus TATA or CCAAT box. We have mapped two transcriptional start sites by primer extension. Deletion constructs from this region fused to the bacterial chloramphenicol acetyltransferase reporter gene revealed only subtle muscle specific cis elements. The strongest promoter activity mapped to a 189-base pair fragment. This sequence contains a conserved GC box to which the transcription factor Sp1 binds. Reporter gene constructs containing a 2-kilobase pair first intron fragment of the myotonic dystrophy protein kinase gene enhances reporter activity up to 6-fold in the human rhabdomyosarcoma myoblast cell line TE32 but not in NIH 3T3 fibroblasts. Co-transfection of a MyoD expression vector with reporter constructs containing the first intron into 10 T1/2 fibroblasts resulted in a 10-20-fold enhancement of expression. Deletion analysis of four E-box elements within the first intron reveal that these elements contribute to enhancer activity similarly in TE32 myoblasts and 10 T1/2 fibroblasts. These data suggest that E-boxes within the myotonic dystrophy protein kinase first intron mediate interactions with upstream promoter elements to up-regulate transcription of this gene in myoblasts.

  12. 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.

  13. 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.

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

    PubMed Central

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

    2016-01-01

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

  15. 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

  16. 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.

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

    PubMed

    Tang, Dingzhong; Innes, Roger W

    2002-12-01

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

  18. 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.

  19. 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

  20. 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

  1. 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

  2. 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

  3. 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.

  4. Phenotypic switching in cells transformed with the herpes simplex virus thymidine kinase gene

    SciTech Connect

    Ostrander, M.; Vogel, S.; Silverstein, S.

    1982-06-01

    Biochemical transformation of Ltk/sup -/ cells with the herpes simplex virus thymidine kinase (tk) gene resulted in numerous TK/sup +/ colonies that survived selection in hypoxanthine-aminopterin-thymidine medium. Many of these TK/sup +/ cell lines switched phenotypes and reverted to the TK/sup -/ state. In this report, the authors describe the biological and biochemical characteristics of three TK/sup -/ revertant lines. One (K/sub 1/B/sub 5/) transiently expressed TK in the presence of bromodeoxyuridine, which selects for the TK/sup -/ phenotype. Another TK/sup -/ sibling (K/sub 1/B/sub 6//sup n/) expressed TK only after removal from bromodeoxyuridine-containing medium. The last variant (K/sub 1/B/sub 6//sup me/) lost the ability to switch to the TK/sup +/ phenotype, although it maintained the herpes simplex virus sequences coding for TK. Loss of the ability of K/sub 1/B/sub 6//sup me/ cells to express TK was correlated with extensive methylation of the sequence recognized by the restriction endonuclease HpaII (pCpCpGpG). After these cells were treated with 5-azacytidine, they regained the ability to clone in hypoxanthine-aminopterin-thymidine medium and reexpressed virus tk mRNA and enzyme. In addition, the HpaII sites that were previously shown to be refractile to enzyme digestion were converted to a sensitive state, demonstrating that they were no longer methylated.

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

    PubMed

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

    2014-08-15

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

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

    PubMed Central

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

    1999-01-01

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

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

    PubMed Central

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

    2014-01-01

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

  8. Transcriptional and post-transcriptional regulation of tyrosine hydroxylase gene by protein kinase C.

    PubMed Central

    Vyas, S; Faucon Biguet, N; Mallet, J

    1990-01-01

    The role played by protein kinase C (PKC) in TH gene regulation was investigated at transcriptional and post-transcriptional levels using PC12 cells. The cells were treated with the phorbol ester TPA, which not only activates PKC but also causes down-regulation. PKC levels were monitored by [3H]PDBU binding assay and by using an anti-PKC antibody that detected intact PKC (79 kd) as well as its catalytic and regulatory domains. The [3H]PDBU binding to the membrane-associated PKC increased within 15-30 min of TPA treatment; thereafter total cellular [3H]PDBU binding decreased to a minimum of 20% of the control at 8 h. The rate of decrease in binding was greater than the decrease in the intensity of the staining of PKC holo enzyme visualized by anti-PKC antibody. TH mRNA levels, measured over the same time period, rose within 15 min of TPA treatment to peak at 4 h and subsequently declined below control level, paralleling the depletion of PKC. If cells depleted of PKC were reincubated in the normal medium, a recovery in PKC level was seen and, in parallel, TH mRNA levels increased to above control level. Furthermore, if down-regulation of PKC was prevented by incubating the cells with the protease inhibitor leupeptin, a decrease beyond control level in TH mRNA was not observed. TPA rapidly induced TH gene transcription; a maximal increase of two-fold was observed at 15 min, but the transcriptional rate then declined although it did not decrease beyond control values after 8 and 24 h of TPA treatment.(ABSTRACT TRUNCATED AT 250 WORDS) Images Fig.2 Fig.3 Fig.6 PMID:1976513

  9. 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.

  10. 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.

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

    SciTech Connect

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

    1991-10-01

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

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

    PubMed

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

    1997-02-01

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

  13. 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

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

    PubMed Central

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

    2008-01-01

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

  15. Insulin inhibits glucocorticoid-stimulated L-type 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase gene expression by activation of the c-Jun N-terminal kinase pathway.

    PubMed Central

    De Los Pinos E; Fernández De Mattos S; Joaquin, M; Tauler, A

    2001-01-01

    The hepatic isoform of 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase (PF2K/Fru-2,6-BPase) is transcriptionally stimulated by glucocorticoids, whereas insulin blocks this stimulatory effect. Although this inhibitory effect has been extensively reported, nothing is known about the signalling pathway responsible. We have used well-characterized inhibitors for proteins involved in different signalling cascades to assess the involvement of these pathways on the transcriptional regulation of glucocorticoid-stimulated PF2K/Fru-2,6-BPase by insulin. Our results demonstrate that the phosphoinositide 3-kinase, p70/p85 ribosomal S6 kinase, extracellular signal-regulated protein kinase (ERK)1/2 and p38 mitogen-activated protein (MAP) kinase pathways are not involved in the inhibitory effect of insulin on glucocorticoid-stimulated PF2K/Fru-2,6-BPase. To evaluate the implication of the MAP kinase/ERK kinase (MEK)-4-stress-activated protein kinase-c-Jun-N-terminal protein kinase ('JNK-SAPK') pathway we overexpressed the N-terminal JNK-binding domain of the JNK-interacting protein 1 ('JIP-1'), demonstrating that activation of JNK is necessary for the insulin inhibitory effect. Moreover, overexpression of MEK kinase 1 and JNK-haemagglutinin resulted in the inhibition of the glucocorticoid-stimulated PF2K/Fru-2,6-BPase. These results provide clear and specific evidence for the role of JNK in the insulin inhibition of glucocorticoid-stimulated PF2K/Fru-2,6-BPase gene expression. In addition, we performed experiments with a mutant of the glucocorticoid receptor in which the JNK phosphorylation target Ser-246 had been mutated to Ala. Our results demonstrate that the phosphorylation of the glucocorticoid receptor on Ser-246 is not responsible for the JNK repression of glucocorticoid-stimulated PF2K/Fru-2,6-BPase gene expression. PMID:11139390

  16. 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.

  17. 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.

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

    PubMed Central

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

    2014-01-01

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

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

    SciTech Connect

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

    1993-10-01

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

  20. 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

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

    PubMed

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

    2015-11-25

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

  2. 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

  3. 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.

  4. 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

  5. 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

  6. ARG portable neutron radiography. Final report

    SciTech Connect

    Barton, J.P.

    1995-04-01

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

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

    PubMed Central

    Horlick, R A; Benfield, P A

    1989-01-01

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

  8. 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.

  9. The Association Between TP53 Arg72pro Polymorphism and Non-Melanoma Skin Cancer Risk: A Meta-Analysis Including 7,107 Subjects

    PubMed Central

    Yang, Xueling; Yang, Baohong; Liu, Ya; Xu, Shanshan; Li, Bo

    2013-01-01

    Background: The p53 gene is a critical molecular in the protection of cells from DNA damage due to Ultraviolet (UV) exposure, and TP53 mutation is very common in non-melanoma skin cancer. Objectives: To assess the association between the TP53 Arg72Pro polymorphism and non-melanoma skin cancer (NMSC) risk. Methods: We performed this meta–analysis with 13 case-control studies involving 3,520 cases and 3,587 controls. Results: Our meta-analysis showed that TP53 Arg72Pro polymorphism was not associated with non-melanoma skin cancer susceptibility in overall population.(for Arg/Arg vs. Pro/Pro: OR 0.98, 95% CI 0.80-1.19; for Arg/Pro vs. Pro/Pro: OR 0.99, 95% CI 0.84-1.17; for the recessive model Arg/Arg vs. Arg/Pro + Pro/Pro: OR 1.10, 95% CI 0.89-1.35; for the dominant model Arg/Arg + Arg/Pro vs. Pro/Pro: OR 1.00, 95% CI 0.85-1.18). We also detected no effect of this polymorphism on any subtype of non-melanoma skin cancer, such as squamous cell carcinoma (SCC), and basal cell carcinoma (BCC). Furthermore, no significant association in any subgroup was detected in stratified analyses according to ethnicity. However, in the stratified analysis by sample collection resources, Arg/Arg carriers from tumor tissue subgroup had 3.42 times risk of cancer (95% CI, 1.19 to 9.84) as compared with the variant type Pro/Pro in NMSC. Conclusions: TP53 Arg72Pro polymorphism may have little involvement in the pathogenesis of NMSC, regardless of type, including SCC, and BCC. PMID:23723465

  10. 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

  11. Protein signaling and regulation of gene transcription in leukemia: role of the Casein Kinase II-Ikaros axis.

    PubMed

    Gowda, Chandrika S; Song, Chunhua; Ding, Yali; Kapadia, Malika; Dovat, Sinisa

    2016-03-01

    Protein signaling and regulation of gene expression are the two major mechanisms that regulate cellular proliferation in leukemia. Discerning the function of these processes is essential for understanding the pathogenesis of leukemia and for developing the targeted therapies. Here, we provide an overview of one of the mechanisms that regulates gene transcription in leukemia. This mechanism involves the direct interaction between Casein Kinase II (CK2) and the Ikaros transcription factor. Ikaros (IKZF1) functions as a master regulator of hematopoiesis and a tumor suppressor in acute lymphoblastic leukemia (ALL). Impaired Ikaros function results in the development of high-risk leukemia. Ikaros binds to the upstream regulatory elements of its target genes and regulates their transcription via chromatin remodeling. In vivo, Ikaros is a target for CK2, a pro-oncogenic kinase. CK2 directly phosphorylates Ikaros at multiple amino acids. Functional experiments showed that CK2-mediated phosphorylation of Ikaros, regulates Ikaros' DNA binding affinity, subcellular localization and protein stability. Recent studies revealed that phosphorylation of Ikaros by CK2 regulates Ikaros binding and repression of the terminal deoxytransferase (TdT) gene in normal thymocytes and in T-cell ALL. Available data suggest that the oncogenic activity of CK2 in leukemia involves functional inactivation of Ikaros and provide a rationale for CK2 inhibitors as a potential treatment for ALL. PMID:26912004

  12. The mechanisms regulating cyclin-dependent kinase 5 in hippocampus during systemic inflammatory response: The effect on inflammatory gene expression.

    PubMed

    Czapski, Grzegorz A; Gąssowska, Magdalena; Wilkaniec, Anna; Chalimoniuk, Małgorzata; Strosznajder, Joanna B; Adamczyk, Agata

    2016-02-01

    Cyclin-dependent kinase 5 (Cdk5) is critical for nervous system's development and function, and its aberrant activation contributes to pathomechanism of Alzheimer's disease and other neurodegenerative disorders. It was recently suggested that Cdk5 may participate in regulation of inflammatory signalling. The aim of this study was to analyse the mechanisms involved in regulating Cdk5 activity in the brain during systemic inflammatory response (SIR) as well as the involvement of Cdk5 in controlling the expression of inflammatory genes. Genetic and biochemical alterations in hippocampus were analysed 3 and 12 h after intraperitoneal injection of lipopolysaccharide. We observed an increase in both Cdk5 gene expression and protein level. Moreover, phosphorylation of Cdk5 on Ser159 was significantly enhanced. Also transcription of Cdk5-regulatory protein (p35/Cdk5r1) was augmented, and the level of p25, calpain-dependent cleavage product of p35, was increased. All these results demonstrated rapid activation of Cdk5 in the brain during SIR. Hyperactivity of Cdk5 contributed to enhanced phosphorylation of tau and glycogen synthase kinase 3β. Inhibition of Cdk5 with Roscovitine reduced activation of NF-κB and expression of inflammation-related genes, demonstrating the critical role of Cdk5 in regulation of gene transcription during SIR.

  13. 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

  14. 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

  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. 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

  17. 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

  18. 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.

  19. 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.

  20. Cancer progression and tumor cell motility are associated with the FGFR4 Arg(388) allele.

    PubMed

    Bange, Johannes; Prechtl, Dieter; Cheburkin, Yuri; Specht, Katja; Harbeck, Nadia; Schmitt, Manfred; Knyazeva, Tatjana; Müller, Susanne; Gärtner, Silvia; Sures, Irmi; Wang, Hongyang; Imyanitov, Evgeny; Häring, Hans-Ulrich; Knayzev, Pjotr; Iacobelli, Stefano; Höfler, Heinz; Ullrich, Axel

    2002-02-01

    Expression analysis of genes encoding components of the phosphotyrosine signaling system by cDNA array hybridization revealed elevated levels of FGFR4 transcripts in several mammary carcinoma cell lines. In the FGFR4 gene transcript from MDA-MB-453 mammary carcinoma cells, a G to A conversion was discovered that results in the substitution of glycine by arginine at position 388 in the transmembrane domain of the receptor. The Arg(388) allele was also found in cell lines derived from a variety of other tumor types as well as in the germ-line of cancer patients and healthy individuals. Analysis of three geographically separated groups indicated that it occurs in approximately 50% of the human population. Investigation of the clinical data of 84 breast cancer patients revealed that homo- or heterozygous carriers of the Arg(388) allele had a significantly reduced disease-free survival time (P = 0.01) within a median follow-up of 62 months. Moreover, the FGFR4 Arg(388) allele was associated with early lymph node metastasis and advanced tumor-node-metastasis (TNM) stage in 82 colon cancer patients. Consistent with this finding, MDA-MB-231 mammary tumor cells expressing FGFR4 Arg(388) exhibited increased motility relative to cells expressing the FGFR4 Gly(388) isotype. Our results support the conclusion that the FGFR4 Arg(388) allele represents a determinant that is innocuous in healthy individuals but predisposes cancer patients for significantly accelerated disease progression.

  1. Implication of Arg213 and Arg337 on the kinetic and structural stability of firefly luciferase.

    PubMed

    Riahi-Madvar, Ali; Hosseinkhani, Saman; Rezaee, Fatemeh

    2013-01-01

    Possible roles of two different Arginine (Arg; R) 213 and 337 on kinetic and structural stability of Photinus pyralis luciferase have been investigated using thermal and chemical denaturation studies. This enzyme is highly sensitive to protease digestion and temperature, which limits its fieldability, particularly for in vivo imaging. In order to generate more stable luciferases against trypsin digestion, site-directed mutagenesis was conducted to block two representative tryptic sites on the surface of N-terminal domain, via substitution of Arg213 and Arg337 by methionine (Met; M) and glutamine (Gln; Q), respectively [A. Riahi-Madvar, S. Hosseinkhani, Protein engineering, design and selection 22 (2009) 655-663]. The improvement of mutant enzymes stability against protease hydrolysis may be attributed to the more rigidity of the enzyme structure upon mutations, as can be deducted from elevated levels of m(U-N) values and decrease of activation energy. Furthermore, mutation at position 337 which is accompanied with more alteration on the basic kinetic properties relative to mutation at position 213, revealed the high values of the ΔG(H(2)O), half-time of inactivation at 30°C and T(m) for R337Q where Arg213 is maintained in structure. Based on the results, it can be concluded that whilst Arg213 affects structural stability, Arg337 is critical for kinetic stability.

  2. 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.

  3. 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.

  4. Induction of a cytosolic pyruvate kinase 1 gene during the resistance response to Tobacco mosaic virus in Capsicum annuum.

    PubMed

    Kim, Ki-Jeong; Park, Chang-Jin; Ham, Byung-Kook; Choi, Soo Bok; Lee, Boo-Ja; Paek, Kyung-Hee

    2006-04-01

    Hot pepper (Capsicum annuum L. cv. Bugang) plants exhibit a hypersensitive response (HR) upon infection by Tobacco mosaic virus (TMV) pathotype P(0). Previously, to elucidate molecular mechanism that underlies this resistance, hot pepper cv. Bugang leaves were inoculated with TMV-P(0) and genes specifically up-regulated during the HR were isolated by microarray analysis. One of the clones, Capsicum annuum cytosolic pyruvate kinase 1 (CaPK(c)1) gene was increased specifically in the incompatible interaction with TMV-P(0). The expression of CaPK(c)1 gene was also triggered not only by various hormones such as salicylic acid (SA), ethylene, and methyl jasmonate (MeJA), but also NaCl and wounding. These results suggest that CaPK(c)1 responds to several defense-related abiotic stresses in addition to TMV infection.

  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. Involvement of a Magnaporthe grisea Serine/Threonine Kinase Gene, MgATG1, in Appressorium Turgor and Pathogenesis▿

    PubMed Central

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

    2007-01-01

    We isolated an MgATG1 gene encoding a serine/threonine protein kinase from the rice blast fungus Magnaporthe grisea. In the ΔMgatg1 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 ΔMgatg1 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

  7. Protein kinasegene expression is oppositely regulated by GCN5 and EBF1 in immature B cells.

    PubMed

    Kikuchi, Hidehiko; Nakayama, Masami; Kuribayashi, Futoshi; Imajoh-Ohmi, Shinobu; Nishitoh, Hideki; Takami, Yasunari; Nakayama, Tatsuo

    2014-05-01

    In this study, we revealed that GCN5 and early B cell factor 1 (EBF1) participate in regulation of protein kinase Cθ (PKCθ) gene expression in an opposite manner in immature B cells. GCN5-deficiency in DT40 caused drastic down-regulation of transcription of PKCθ. In contrast, EBF1-deficiency brought about remarkable up-regulation of that of PKCθ, and re-expression of EBF1 dramatically suppressed transcription of PKCθ. Chromatin immunoprecipitation assay revealed that GCN5 binds to the 5'-flanking region of the chicken PKCθ gene and acetylates histone H3, and EBF1 binds to the 5'-flanking region of the gene surrounding putative EBF1 binding motifs.

  8. Lipopolysaccharide and Raf-1 kinase regulate secretory interleukin-1 receptor antagonist gene expression by mutually antagonistic mechanisms.

    PubMed Central

    Guthridge, C J; Eidlen, D; Arend, W P; Gutierrez-Hartmann, A; Smith, M F

    1997-01-01

    Lipopolysaccharide (LPS) treatment of monocytic cells has been shown to activate the Raf-1/mitogen-activated protein kinase (MAPK) signaling pathway and to increase secretory interleukin-1 receptor antagonist (sIL-1Ra) gene expression. The significance of the activation of the Raf-1/MAPK signaling pathway to LPS regulation of sIL-1Ra gene expression, however, has not been determined. This study addresses the role of the Raf-1/MAPK signaling pathway in regulation of sIL-1Ra gene expression by LPS. Cotransfection of the murine macrophage cell line RAW 264.7 with a 294-bp sIL-1Ra promoter/luciferase construct (pRA-294-luc) and a constitutively active Raf-1 kinase expression vector (pRSV-Raf-BXB) resulted in induction of sIL-1Ra promoter activity, indicating that Raf-1, like LPS, can regulate sIL-1Ra promoter activity. An in vitro MAPK analysis indicated that both LPS treatment and pRSV-Raf-BXB transfection of RAW 264.7 cells increases p42 MAPK activity. An in vitro Raf-1 kinase assay, however, failed to detect LPS-induced Raf-1 kinase activity in RAW 264.7 cells, suggesting that in RAW 264.7 cells, Raf-1 kinase is not an activating component of the LPS signaling pathway regulating MAPK activity or sIL-1Ra promoter activity. This observation was supported by results from transfection studies which demonstrated that expression of a dominant-inhibitory Raf-1 mutant in RAW 264.7 cells does not inhibit LPS-induced MAPK activity or sIL-1Ra promoter activity, indicating that LPS-induced sIL-1Ra promoter activation occurs independent of the Raf-1/MAPK signaling pathway. In additional studies, cotransfection of RAW 264.7 cells with pRA-294-luc and increasing amounts of pRSV-Raf-BXB caused a dose-dependent inhibition of LPS-induced sIL-1Ra promoter activity, indicating that the role of the Raf-1 pathway in the regulation of sIL-1Ra promoter activity by LPS is as an antagonizer. Interestingly, LPS treatment of RAW 264.7 cells, cotransfected with pRA-294-luc and p

  9. Lipopolysaccharide and Raf-1 kinase regulate secretory interleukin-1 receptor antagonist gene expression by mutually antagonistic mechanisms.

    PubMed

    Guthridge, C J; Eidlen, D; Arend, W P; Gutierrez-Hartmann, A; Smith, M F

    1997-03-01

    Lipopolysaccharide (LPS) treatment of monocytic cells has been shown to activate the Raf-1/mitogen-activated protein kinase (MAPK) signaling pathway and to increase secretory interleukin-1 receptor antagonist (sIL-1Ra) gene expression. The significance of the activation of the Raf-1/MAPK signaling pathway to LPS regulation of sIL-1Ra gene expression, however, has not been determined. This study addresses the role of the Raf-1/MAPK signaling pathway in regulation of sIL-1Ra gene expression by LPS. Cotransfection of the murine macrophage cell line RAW 264.7 with a 294-bp sIL-1Ra promoter/luciferase construct (pRA-294-luc) and a constitutively active Raf-1 kinase expression vector (pRSV-Raf-BXB) resulted in induction of sIL-1Ra promoter activity, indicating that Raf-1, like LPS, can regulate sIL-1Ra promoter activity. An in vitro MAPK analysis indicated that both LPS treatment and pRSV-Raf-BXB transfection of RAW 264.7 cells increases p42 MAPK activity. An in vitro Raf-1 kinase assay, however, failed to detect LPS-induced Raf-1 kinase activity in RAW 264.7 cells, suggesting that in RAW 264.7 cells, Raf-1 kinase is not an activating component of the LPS signaling pathway regulating MAPK activity or sIL-1Ra promoter activity. This observation was supported by results from transfection studies which demonstrated that expression of a dominant-inhibitory Raf-1 mutant in RAW 264.7 cells does not inhibit LPS-induced MAPK activity or sIL-1Ra promoter activity, indicating that LPS-induced sIL-1Ra promoter activation occurs independent of the Raf-1/MAPK signaling pathway. In additional studies, cotransfection of RAW 264.7 cells with pRA-294-luc and increasing amounts of pRSV-Raf-BXB caused a dose-dependent inhibition of LPS-induced sIL-1Ra promoter activity, indicating that the role of the Raf-1 pathway in the regulation of sIL-1Ra promoter activity by LPS is as an antagonizer. Interestingly, LPS treatment of RAW 264.7 cells, cotransfected with pRA-294-luc and p

  10. In vivo identification of solar radiation-responsive gene network: role of the p38 stress-dependent kinase.

    PubMed

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

    2010-05-21

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

  11. 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.

  12. Estrogen-induced genes, WISP-2 and pS2, respond divergently to protein kinase pathway.

    PubMed

    Inadera, Hidekuni

    2003-09-19

    Recently, we identified WISP-2 (Wnt-1 inducible signaling pathway protein 2) as a novel estrogen-inducible gene in the MCF-7 human breast cancer cell line. In this study, we examined whether WISP-2 expression is modulated by PK activators. Treatment with protein kinase A (PKA) activators [cholera toxin plus 3-isobutyl-1-methylxanthine (CT/IBMX)] induced WISP-2 expression. CT/IBMX induced expression of the other estrogen-responsive gene, pS2, more dramatically than maximum stimulation by 17beta-estradiol (E2). Treatment with 12-O-tetradecanoylphorbol-13-acetate (TPA), which directly stimulates protein kinase C (PKC) activity, completely prevented WISP-2 mRNA induction by E2, whereas it increased pS2 mRNA expression more dramatically than maximum stimulation by E2. Results of treatments with the protein synthesis inhibitor cycloheximide and the pure antiestrogen ICI182,780 suggest that these PK pathways modulate WISP-2 gene expression via different molecular mechanisms than those for pS2. Because TPA inhibits cell proliferation, we investigated whether WISP-2 induction was dependent on cell growth. Cells were treated with insulin-like growth factor-1 (IGF-1) or interleukin-1alpha (IL-1alpha) to stimulate or inhibit cell growth, respectively. These treatments had no effect on WISP-2 mRNA expression either alone or in combination with E2, suggesting that WISP-2 induction is independent of cell growth.

  13. Cloning of a phosphatidylinositol 4-kinase gene based on fiber strength transcriptome QTL mapping in the cotton species Gossypium barbadense.

    PubMed

    Liu, H W; Shi, R F; Wang, X F; Pan, Y X; Zang, G Y; Ma, Z Y

    2012-09-25

    Sea Island cotton (Gossypium barbadense) is highly valued for its superior fiber qualities, especially fiber strength. Based on a transcript-derived fragment originated from transcriptome QTL mapping, a fiber strength related candidate gene of phosphatidylinositol 4-kinase cDNA, designated as GbPI4K, was first cloned, and its expression was characterized in the secondary cell wall thickening stage of G. barbadense fibers. The ORF of GbPI4K was found to be 1926 bp in length and encoded a predicted protein of 641 amino acid residues. The putative protein contained a clear PI3/4K kinase catalytic domain and fell into the plant type II PI4K cluster in phylogenetic analysis. In this study, the expression of cotton PI4K protein was also induced in Escherichia coli BL21 (DE3) as a fused protein. Semi-quantitative RT-PCR analysis showed that the gene expressed in the root, hypocotyl and leaf of the cotton plants. Real-time RT-PCR indicated that this gene in Sea Island cotton fibers expressed 10 days longer than that in Upland cotton fibers, and the main expression difference of PI4K between Sea Island cotton and Upland cotton in fibers was located in the secondary cell wall thickening stage of the fiber. Further analysis indicated that PI4K is a crucial factor in the ability of Rac proteins to regulate phospholipid signaling pathways.

  14. 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

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

    PubMed

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

    2002-02-01

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

  16. Analysis of promoter hypermethylation of death-associated protein kinase and p16 tumor suppressor genes in actinic keratoses and squamous cell carcinomas of the skin.

    PubMed

    Tyler, Lisa N; Ai, Lingbao; Zuo, Chunlai; Fan, Chun-Yang; Smoller, Bruce R

    2003-07-01

    Death-associated protein kinase is a serine/threonine protein kinase implicated in promoting apoptosis and tumor suppression, whereas p16 is a tumor suppressor gene that inhibits cyclin-dependent kinase 4 and 6 activity and arrests the cell cycle in the G1 phase. Hypermethylation of death-associated protein kinase or p16 gene with resultant gene inactivation has been described in a wide variety of human cancers. Promoter methylation of the death-associated protein kinase and p16 gene has been found in about 55% and 30% cases of head and neck squamous cell carcinoma respectively but has not yet been analyzed in cutaneous premalignant and malignant lesions. A total of 33 cases were examined for evidence of death-associated protein kinase and p16 hypermethylation and these consist of 9 cases of spongiotic dermatitis as nonneoplastic skin control, 9 cases of actinic keratosis, 8 cases of squamous cell carcinoma in situ, and 7 cases of invasive squamous cell carcinoma. Death-associated protein kinase promoter methylation was detected in 1 case of squamous cell carcinoma in situ and 1 case of nonneoplastic skin control but none of the cases of invasive squamous cell carcinoma or actinic keratosis. P16 promoter methylation was detected in 1 case of invasive squamous cell carcinoma and 1 case of nonneoplastic skin control but none of the cases of squamous cell carcinoma in situ or actinic keratosis. Promoter hypermethylation of the death-associated protein kinase and p16 genes does not appear to play an important role in the development of cutaneous squamous cell carcinoma. The data thus suggest that the mechanisms of ultraviolet-induced cutaneous carcinomas differ from those involved in the development of head and neck squamous cell carcinoma, a malignant disease induced by tobacco and alcohol exposure.

  17. A previously unknown mutation in the pyruvate kinase gene (PKLR) identified from a neonate with severe jaundice.

    PubMed

    Yaish, Hassan M; Nussenzveig, Roberto H; Agarwal, Archana M; Siddiqui, Abdul H; Christensen, Robert D

    2014-01-01

    We report a neonate with early and severe hemolytic jaundice and low erythrocyte pyruvate kinase enzymatic activity (<2 U/g hemoglobin, reference interval 9-22). We found her asymptomatic mother to be heterozygous for a novel PKLR mutation (c.1573delT) with an erythrocyte PK activity of 6.2 U/g hemoglobin. Her asymptomatic father was heterozygous for the common Northern European PKLR mutation (c.1529A) with an erythrocyte PK activity of 3.6 U/g. The neonate was a compound heterozygote with both mutations, but with no other mutations identified by sequencing a panel of 27 genes involved in severe neonatal jaundice.

  18. ATPase-promoting dead end inhibitors of the cAMP-dependent protein kinase.

    PubMed

    Mendelow, M; Prorok, M; Salerno, A; Lawrence, D S

    1993-06-15

    The cAMP-dependent protein kinase is a bifunctional enzyme, catalyzing the phosphorylation of the serine and threonine residues in peptides and proteins (kinase activity) as well as the phosphorylation of water (ATPase activity). We have found that several peptides, which serve as inhibitors of the kinase reaction, will either maintain or enhance the ATPase reaction catalyzed by the enzyme. Positively charged dipeptides (e.g. Arg-Arg), as well as small guanidino-containing compounds (e.g. guanethidine) block protein kinase activity yet enhance ATPase activity up to 3.5-fold over that exhibited by the enzyme in the absence of these compounds. In contrast, several nonphosphorylatable peptides, whose primary sequences are based on that of a known substrate (i.e. Leu-Arg-Arg-Ala-Ser-Leu-Gly), such as Leu-Arg-Arg-Ala-Ala-Leu-Gly, Leu-Arg-Arg-Ala-Phe-Leu-Gly, and Leu-Arg-Arg-Ala-Tyr-Leu-Gly, have little or no effect on the rate of the kinase-catalyzed hydrolysis of ATP. An exception to the latter observation is Leu-Arg-Arg-Ala-Cys-Leu-Gly, a cysteine-containing peptide that promotes the protein kinase-catalyzed ATPase reaction by 2.2-fold. We have also found that peptides that possess relatively large amino acid side chain moieties immediately following the arginine dyad (i.e. such as Phe, Tyr, Cys, or Asn at Xaa in Leu-Arg-Arg-Xaa-Ala-Leu-Gly) sharply reduce the rate of enzyme-catalyzed ATP hydrolysis. This suggests that in the presence of peptides containing an -Arg-Arg-Ala- sequence, the enzyme-bound gamma-phosphate of ATP is relatively accessible to water. In contrast, when the latter alanine moiety is replaced by a larger residue, access by water to ATP appears to be hindered. These results indicate that certain structural features associated with the substrate or substrate analog have a profound influence on the manner by which these species interact with the protein kinase. Furthermore, the work described herein demonstrates that it is possible to block the

  19. 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

  20. 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.

  1. Gene Expression Patterns Define Key Transcriptional Events InCell-Cycle Regulation By cAMP And Protein Kinase A

    SciTech Connect

    Zambon, Alexander C.; Zhang, Lingzhi; Minovitsky, Simon; Kanter, Joan R.; Prabhakar, Shyam; Salomonis, Nathan; Vranizan, Karen; Dubchak Inna,; Conklin, Bruce R.; Insel, Paul A.

    2005-06-01

    Although a substantial number of hormones and drugs increase cellular cAMP levels, the global impact of cAMP and its major effector mechanism, protein kinase A (PKA), on gene expression is not known. Here we show that treatment of murine wild-type S49 lymphoma cells for 24 h with 8-(4-chlorophenylthio)-cAMP (8-CPTcAMP), a PKA-selective cAMP analog, alters the expression of approx equal to 4,500 of approx. equal to 13,600 unique genes. By contrast, gene expression was unaltered in Kin- S49 cells (that lack PKA) incubated with 8-CPTcAMP. Changes in mRNA and protein expression of several cell cycle regulators accompanied cAMP-induced G1-phase cell-cycle arrest of wild-type S49 cells. Within 2h, 8-CPT-cAMP altered expression of 152 genes that contain evolutionarily conserved cAMP-response elements within 5 kb of transcriptional start sites, including the circadian clock gene Per1. Thus, cAMP through its activation of PKA produces extensive transcriptional regulation in eukaryotic cells. These transcriptional networks include a primary group of cAMP-response element-containing genes and secondary networks that include the circadian clock.

  2. 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

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

    PubMed Central

    Long, Shaojun; Wang, Qiuling

    2016-01-01

    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

  4. Abscisic Acid and Gibberellin Differentially Regulate Expression of Genes of the SNF1-Related Kinase Complex in Tomato Seeds1

    PubMed Central

    Bradford, Kent J.; Downie, A. Bruce; Gee, Oliver H.; Alvarado, Veria; Yang, Hong; Dahal, Peetambar

    2003-01-01

    The SNF1/AMP-activated protein kinase subfamily plays central roles in metabolic and transcriptional responses to nutritional or environmental stresses. In yeast (Saccharomyces cerevisiae) and mammals, activating and anchoring subunits associate with and regulate the activity, substrate specificity, and cellular localization of the kinase subunit in response to changing nutrient sources or energy demands, and homologous SNF1-related kinase (SnRK1) proteins are present in plants. We isolated cDNAs corresponding to the kinase (LeSNF1), regulatory (LeSNF4), and localization (LeSIP1 and LeGAL83) subunits of the SnRK1 complex from tomato (Lycopersicon esculentum Mill.). LeSNF1 and LeSNF4 complemented yeast snf1 and snf4 mutants and physically interacted with each other and with LeSIP1 in a glucose-dependent manner in yeast two-hybrid assays. LeSNF4 mRNA became abundant at maximum dry weight accumulation during seed development and remained high when radicle protrusion was blocked by abscisic acid (ABA), water stress, far-red light, or dormancy, but was low or undetected in seeds that had completed germination or in gibberellin (GA)-deficient seeds stimulated to germinate by GA. In leaves, LeSNF4 was induced in response to ABA or dehydration. In contrast, LeSNF1 and LeGAL83 genes were essentially constitutively expressed in both seeds and leaves regardless of the developmental, hormonal, or environmental conditions. Regulation of LeSNF4 expression by ABA and GA provides a potential link between hormonal and sugar-sensing pathways controlling seed development, dormancy, and germination. PMID:12857836

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

    PubMed Central

    Thach, Trung Thanh; Luong, Truc Thanh; Lee, Sangho; Rhee, Dong-Kwon

    2014-01-01

    Streptococcus pneumoniae (pneumococcus) infection causes more than 1.6 million deaths worldwide. Pneumococcal growth is a prerequisite for its virulence and requires an appropriate supply of cellular energy. Adenylate kinases constitute a major family of enzymes that regulate cellular ATP levels. Some bacterial adenylate kinases (AdKs) are known to be critical for growth, but the physiological effects of AdKs in pneumococci have been poorly understood at the molecular level. Here, by crystallographic and functional studies, we report that the catalytic activity of adenylate kinase from S.pneumoniae (SpAdK) serotype 2 D39 is essential for growth. We determined the crystal structure of SpAdK in two conformations: ligand-free open form and closed in complex with a two-substrate mimic inhibitor adenosine pentaphosphate (Ap5A). Crystallographic analysis of SpAdK reveals Arg-89 as a key active site residue. We generated a conditional expression mutant of pneumococcus in which the expression of the adk gene is tightly regulated by fucose. The expression level of adk correlates with growth rate. Expression of the wild-type adk gene in fucose-inducible strains rescued a growth defect, but expression of the Arg-89 mutation did not. SpAdK increased total cellular ATP levels. Furthermore, lack of functional SpAdK caused a growth defect in vivo. Taken together, our results demonstrate that SpAdK is essential for pneumococcal growth in vitro and in vivo. PMID:25180151

  6. Identification of regulated genes during permanent focal cerebral ischaemia: characterization of the protein kinase 9b5/MARKL1/MARK4.

    PubMed

    Schneider, Armin; Laage, Rico; von Ahsen, Oliver; Fischer, Achim; Rossner, Moritz; Scheek, Sigrid; Grünewald, Sylvia; Kuner, Rohini; Weber, Daniela; Krüger, Carola; Klaussner, Bettina; Götz, Bernhard; Hiemisch, Holger; Newrzella, Dieter; Martin-Villalba, Ana; Bach, Alfred; Schwaninger, Markus

    2004-03-01

    Cerebral ischaemia induces transcriptional changes in a number of pathophysiologically important genes. Here we have systematically studied gene expression changes after 90 min and 24 h of permanent focal ischaemia in the mouse by an advanced fragment display technique (restriction-mediated differential display). We identified 56 transcriptionally altered genes, many of which provide novel hints to ischaemic pathophysiology. Particularly interesting were two pro-apoptotic genes (Grim19 and Tdag51), whose role in cerebral ischaemia and neuronal cell death has not been recognized so far. Among the unknown sequences, we identified a gene that was rapidly and transiently up-regulated. The encoded protein displayed high homology to the MARK family of serine-threonine protein kinases and has recently been described as MARKL1/MARK4. Here we demonstrate that this protein is a functional protein kinase with the ability to specifically phosphorylate a cognate peptide substrate for the AMP-kinase family. Upon overexpression in heterologous cells, the functional wild-type protein, but not its kinase-dead mutant, led to decreased cell viability. We conclude that the up-regulation of this kinase during focal ischaemia may represent an interesting new target for pharmacological intervention. PMID:15009667

  7. 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

  8. 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.

  9. 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

  10. 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

  11. 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

  12. Focal adhesion kinase protein regulates Wnt3a gene expression to control cell fate specification in the developing neural plate

    PubMed Central

    Fonar, Yuri; Gutkovich, Yoni E.; Root, Heather; Malyarova, Anastasia; Aamar, Emil; Golubovskaya, Vita M.; Elias, Sarah; Elkouby, Yaniv M.; Frank, Dale

    2011-01-01

    Focal adhesion kinase (FAK) is a cytoplasmic tyrosine kinase protein localized to regions called focal adhesions, which are contact points between cells and the extracellular matrix. FAK protein acts as a scaffold to transfer adhesion-dependent and growth factor signals into the cell. Increased FAK expression is linked to aggressive metastatic and invasive tumors. However, little is known about its normal embryonic function. FAK protein knockdown during early Xenopus laevis development anteriorizes the embryo. Morphant embryos express increased levels of anterior neural markers, with reciprocally reduced posterior neural marker expression. Posterior neural plate folding and convergence-extension is also inhibited. This anteriorized phenotype resembles that of embryos knocked down zygotically for canonical Wnt signaling. FAK and Wnt3a genes are both expressed in the neural plate, and Wnt3a expression is FAK dependent. Ectopic Wnt expression rescues this FAK morphant anteriorized phenotype. Wnt3a thus acts downstream of FAK to balance anterior–posterior cell fate specification in the developing neural plate. Wnt3a gene expression is also FAK dependent in human breast cancer cells, suggesting that this FAK–Wnt linkage is highly conserved. This unique observation connects the FAK- and Wnt-signaling pathways, both of which act to promote cancer when aberrantly activated in mammalian cells. PMID:21551070

  13. Characterization of a mitogen-activated protein kinase gene from cucumber required for trichoderma-conferred plant resistance.

    PubMed

    Shoresh, Michal; Gal-On, Amit; Leibman, Diana; Chet, Ilan

    2006-11-01

    The fungal biocontrol agent Trichoderma asperellum has been recently shown to induce systemic resistance in plants through a mechanism that employs jasmonic acid and ethylene signal transduction pathways. Mitogen-activated protein kinase (MAPK) proteins have been implicated in the signal transduction of a wide variety of plant stress responses. Here we report the identification and characterization of a Trichoderma-induced MAPK (TIPK) gene function in cucumber (Cucumis sativus). Similar to its homologs, wound-induced protein kinase, MPK3, and MPK3a, TIPK is also induced by wounding. Normally, preinoculation of roots with Trichoderma activates plant defense mechanisms, which result in resistance to the leaf pathogen Pseudomonas syringae pv lachrymans. We used a unique attenuated virus vector, Zucchini yellow mosaic virus (ZYMV-AGII), to overexpress TIPK protein and antisense (AS) RNA. Plants overexpressing TIPK were more resistant to pathogenic bacterial attack than control plants, even in the absence of Trichoderma preinoculation. On the other hand, plants expressing TIPK-AS revealed increased sensitivity to pathogen attack. Moreover, Trichoderma preinoculation could not protect these AS plants against subsequent pathogen attack. We therefore demonstrate that Trichoderma exerts its protective effect on plants through activation of the TIPK gene, a MAPK that is involved in signal transduction pathways of defense responses.

  14. Construction of a differentiated human hepatocyte cell line expressing the herpes simplex virus-thymidine kinase gene.

    PubMed

    Kobayashi, N; Miyazaki, M; Westerman, K A; Noguchi, H; Sakaguchi, M; Totsugawa, T; Watanabe, T; Matsumura, T; Fujiwara, T; Leboulch, P; Tanaka, N; Namba, M

    2001-01-01

    Transient support using a hybrid artificial liver (HAL) device is a promising treatment for the patients with acute liver failure. Primary human hepatocytes are an ideal source for HAL therapy; however, the number of human livers available for hepatocyte isolation is limited by competition for use in whole organ transplantation. To overcome this problem, we previously established a highly differentiated human fetal hepatocyte cell line OUMS-29. Considering the potential risk when using these genetically engineered cells in humans, additional safeguards should be added to make the cells more clinically useful. In this work, the herpes simplex virus thymidine kinase (HSVtk) gene was retrovirally introduced into OUMS-29 cells. One of the HSVtk-expressed clones, OUMS-29/thymidine kinase (TK), grew in chemically defined serum free medium and expressed the genes of albumin, asialoglycoprotein receptor, glutamine synthetase, glutathione-S-transferase pi, and blood coagulation factor X. In vitro sensitivity of the cells to ganciclovir was evaluated. Intrasplenic transplantation of 50 x 10(6) OUMS-29/TK cells prolonged the survival of 90% hepatectomized rats compared with medium injection alone (control). In the present study, we have established highly differentiated immortalized human hepatocytes with tight regulation. The cells may be clinically useful for HAL treatment. PMID:11575821

  15. c-Jun N-Terminal Kinase 1 Is Required for Toll-Like Receptor 1 Gene Expression in Macrophages▿

    PubMed Central

    Izadi, Hooman; Motameni, Amirreza T.; Bates, Tonya C.; Olivera, Elias R.; Villar-Suarez, Vega; Joshi, Ila; Garg, Renu; Osborne, Barbara A.; Davis, Roger J.; Rincón, Mercedes; Anguita, Juan

    2007-01-01

    The regulation of innate immune responses to pathogens occurs through the interaction of Toll-like receptors (TLRs) with pathogen-associated molecular patterns and the activation of several signaling pathways whose contribution to the overall innate immune response to pathogens is poorly understood. We demonstrate a mechanism of control of murine macrophage responses mediated by TLR1/2 heterodimers through c-Jun N-terminal kinase 1 (JNK1) activity. JNK controls tumor necrosis factor alpha production and TLR-mediated macrophage responses to Borrelia burgdorferi, the causative agent of Lyme disease, and the TLR1/TLR2-specific agonist PAM3CSK4. JNK1, but not JNK2, activity regulates the expression of the tlr1 gene in the macrophage cell line RAW264.7, as well as in primary CD11b+ cells. We also show that the proximal promoter region of the human tlr1 gene contains an AP-1 binding site that is subjected to regulation by the kinase and binds two complexes that involve the JNK substrates c-Jun, JunD, and ATF-2. These results demonstrate that JNK1 regulates the response to TLR1/2 ligands and suggest a positive feedback loop that may serve to increase the innate immune response to the spirochete. PMID:17664270

  16. 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

  17. 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

  18. 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

  19. 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

  20. Identification of cis-acting regulatory elements in the promoter region of the rat brain creatine kinase gene.

    PubMed Central

    Hobson, G M; Molloy, G R; Benfield, P A

    1990-01-01

    The functional organization of the rat brain creatine kinase (ckb) promoter was analyzed by deletion, linker scanning, and substitution mutagenesis. Mutations were introduced into the ckb promoter of hybrid ckb/neo (neomycin resistance gene) genes, and the mutant genes were expressed transiently in HeLa cells. Expression was assayed by primer extension analysis of neo RNA, which allowed the transcription start sites and the amount of transcription to be determined. Transfections and primer extension reactions were internally controlled by simultaneous analysis of transcription from the adenovirus VA gene located on the same plasmid as the hybrid ckb/neo gene. We demonstrate that 195 bp of the ckb promoter is sufficient for efficient in vivo expression in HeLa cells. A nonconsensus TTAA element at -28 bp appears to provide the TATA box function for the ckb promoter in vivo. Two CCAAT elements, one at -84 bp and the other at -54 bp, and a TATAAA TA element (a consensus TATA box sequence) at -66 bp are required for efficient transcription from the TTAA element. In addition, we present evidence that the consensus beta-globin TATA box responds to the TATAAATA element in the same way as the ckb nonconsensus TTAA element. Images PMID:2247071

  1. The receiver domain of hybrid histidine kinase VirA: an enhancing factor for vir gene expression in Agrobacterium tumefaciens.

    PubMed

    Wise, Arlene A; Fang, Fang; Lin, Yi-Han; He, Fanglian; Lynn, David G; Binns, Andrew N

    2010-03-01

    The plant pathogen Agrobacterium tumefaciens expresses virulence (vir) genes in response to chemical signals found at the site of a plant wound. VirA, a hybrid histidine kinase, and its cognate response regulator, VirG, regulate vir gene expression. The receiver domain at the carboxyl end of VirA has been described as an inhibitory element because its removal increased vir gene expression relative to that of full-length VirA. However, experiments that characterized the receiver region as an inhibitory element were performed in the presence of constitutively expressed virG. We show here that VirA's receiver domain is an activating factor if virG is expressed from its native promoter on the Ti plasmid. When virADeltaR was expressed from a multicopy plasmid, both sugar and the phenolic inducer were essential for vir gene expression. Replacement of wild-type virA on pTi with virADeltaR precluded vir gene induction, and the cells did not accumulate VirG or induce transcription of a virG-lacZ fusion in response to acetosyringone. These phenotypes were corrected if the virG copy number was increased. In addition, we show that the VirA receiver domain can interact with the VirG DNA-binding domain. PMID:20081031

  2. 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.

  3. 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

  4. 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

  5. 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

  6. 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

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

    SciTech Connect

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

    2012-01-20

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

  8. Diverging regulation of pyruvate dehydrogenase kinase isoform gene expression in cultured human muscle cells.

    PubMed

    Abbot, Emily L; McCormack, James G; Reynet, Christine; Hassall, David G; Buchan, Kevin W; Yeaman, Stephen J

    2005-06-01

    The pyruvate dehydrogenase complex occupies a central and strategic position in muscle intermediary metabolism and is primarily regulated by phosphorylation/dephosphorylation. The identification of multiple isoforms of pyruvate dehydrogenase kinase (PDK1-4) and pyruvate dehydrogenase phosphatase (PDP1-2) has raised intriguing new possibilities for chronic pyruvate dehydrogenase complex control. Experiments to date suggest that PDK4 is the major isoenzyme responsible for changes in pyruvate dehydrogenase complex activity in response to various different metabolic conditions. Using a cultured human skeletal muscle cell model system, we found that expression of both PDK2 and PDK4 mRNA is upregulated in response to glucose deprivation and fatty acid supplementation, the effects of which are reversed by insulin treatment. In addition, insulin directly downregulates PDK2 and PDK4 mRNA transcript abundance via a phosphatidylinositol 3-kinase-dependent pathway, which may involve glycogen synthase kinase-3 but does not utilize the mammalian target of rapamycin or mitogen-activated protein kinase signalling pathways. In order to further elucidate the regulation of PDK, the role of the peroxisome proliferators-activated receptors (PPAR) was investigated using highly potent subtype selective agonists. PPARalpha and PPARdelta agonists were found to specifically upregulate PDK4 mRNA expression, whereas PPARgamma activation selectively decreased PDK2 mRNA transcript abundance. PDP1 mRNA expression was unaffected by all conditions analysed. These results suggest that in human muscle, hormonal and nutritional conditions may control PDK2 and PDK4 mRNA expression via a common signalling mechanism. In addition, PPARs appear to independently regulate specific PDK isoform transcipt levels, which are likely to impart important metabolic mediation of fuel utilization by the muscle. PMID:15955060

  9. Cloning of the two pyruvate kinase isoenzyme structural genes from Escherichia coli: the relative roles of these enzymes in pyruvate biosynthesis.

    PubMed Central

    Ponce, E; Flores, N; Martinez, A; Valle, F; Bolívar, F

    1995-01-01

    We report the cloning of the pykA and pykF genes from Escherichia coli, which code for the two pyruvate kinase isoenzymes (ATP:pyruvate 2-O-phosphotransferases; EC 2.7.1.40) in this microorganism. These genes were insertionally inactivated with antibiotic resistance markers and utilized to interrupt one or both pyk genes in the E. coli chromosome. With these constructions, we were able to study the role of these isoenzymes in pyruvate biosynthesis. PMID:7559366

  10. 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

  11. Resistance to gemcitabine in a human follicular lymphoma cell line is due to partial deletion of the deoxycytidine kinase gene

    PubMed Central

    Galmarini, Carlos María; Clarke, Marilyn L; Jordheim, Lars; Santos, Cheryl L; Cros, Emeline; Mackey, John R; Dumontet, Charles

    2004-01-01

    Background Gemcitabine is an analogue of deoxycytidine with activity against several solid tumors. In order to elucidate the mechanisms by which tumor cells become resistant to gemcitabine, we developed the resistant subline RL-G from the human follicular lymphoma cell line RL-7 by prolonged exposure of parental cells to increasing concentrations of gemcitabine. Results In vitro, the IC50 increased from 0.015 μM in parental RL-7 cells to 25 μM in the resistant variant, RL-G. Xenografts of both cell lines developed in nude mice were treated with repeated injections of gemcitabine. Under conditions of gemcitabine treatment which totally inhibited the development of RL-7 tumors, RL-G derived tumors grew similarly to those of untreated animals, demonstrating the in vivo resistance of RL-G cells to gemcitabine. HPLC experiments showed that RL-G cells accumulated and incorporated less gemcitabine metabolites into DNA and RNA than RL-7 cells. Gemcitabine induced an S-phase arrest in RL-7 cells but not in RL-G cells. Exposure to gemcitabine induced a higher degree of apoptosis in RL-7 than in RL-G cells, with poly-(ADP-ribose) polymerase cleavage in RL-7 cells. No modifications of Bcl-2 nor of Bax expression were observed in RL-7 or RL-G cells exposed to gemcitabine. These alterations were associated with the absence of the deoxycytidine kinase mRNA expression observed by quantitative RT-PCR in RL-G cells. PCR amplification of désoxycytidine kinase gene exons showed a partial deletion of the dCK gene in RL-G cells. Conclusions These results suggest that partial deletion of the dCK gene observed after selection in the presence of gemcitabine is involved with resistance to this agent both in vitro and in vivo. PMID:15157282

  12. Chimeric plant calcium/calmodulin-dependent protein kinase gene with a neural visinin-like calcium-binding domain.

    PubMed Central

    Patil, S; Takezawa, D; Poovaiah, B W

    1995-01-01

    Calcium, a universal second messenger, regulates diverse cellular processes in eukaryotes. Ca2+ and Ca2+/calmodulin-regulated protein phosphorylation play a pivotal role in amplifying and diversifying the action of 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 Ca2+/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 (approximately 56 kDa) binds calmodulin in a Ca(2+)-dependent manner. Furthermore, 45Ca-binding assays revealed that CCaMK directly binds Ca2+. 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 Ca2+ signaling in plants. Images Fig. 3 Fig. 4 Fig. 5 Fig. 6 PMID:7761420

  13. Pyrroloquinoline quinone and a quinoprotein kinase support γ-radiation resistance in Deinococcus radiodurans and regulate gene expression.

    PubMed

    Rajpurohit, Yogendra Singh; Desai, Shruti Sumeet; Misra, Hari Sharan

    2013-06-01

    Deinococcus radiodurans is known for its extraordinary resistance to various DNA damaging agents including γ-radiation and desiccation. The pqqE:cat and Δdr2518 mutants making these cells devoid of pyrroloquinoline quinone (PQQ) and a PQQ inducible Ser/Thr protein kinase, respectively, became sensitive to γ-radiation. Transcriptome analysis of these mutants showed differential expression of the genes including those play roles in oxidative stress tolerance and (DSB) repair in D. radiodurans and in genome maintenance and stress response in other bacteria. Escherichia coli cells expressing DR2518 and PQQ showed improved resistance to γ-radiation, which increased further when both DR2518 and PQQ were present together. Although, profiles of genes getting affected in these mutants were different, there were still a few common genes showing similar expression trends in both the mutants and some others as reported earlier in oxyR and pprI mutant of this bacterium. These results suggested that PQQ and DR2518 have independent roles in γ-radiation resistance of D. radiodurans but their co-existence improves radioresistance further, possibly by regulating differential expression of the genes important for bacterial response to oxidative stress and DNA damage.

  14. 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

  15. 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.

  16. 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

  17. Prodigiosin induces the proapoptotic gene NAG-1 via glycogen synthase kinase-3beta activity in human breast cancer cells.

    PubMed

    Soto-Cerrato, Vanessa; Viñals, Francesc; Lambert, James R; Kelly, Julie A; Pérez-Tomás, Ricardo

    2007-01-01

    Prodigiosin (2-methyl-3-pentyl-6-methoxyprodigiosene) is a bacterial metabolite that has anticancer and antimetastatic properties. However, the molecular mechanisms responsible for these abilities are not fully understood. Gene expression profiling of the human breast cancer cell line MCF-7 treated with prodigiosin was analyzed by cDNA array technology. The majority of the significantly modified genes were related to apoptosis, cell cycle, cellular adhesion, or transcription regulation. The dramatic increase of the nonsteroidal anti-inflammatory drug-activated gene 1 (NAG-1) made this gene an interesting candidate regarding the possible mechanism by which prodigiosin induces cytotoxicity in MCF-7 cells. Our results show that prodigiosin triggers accumulation of the DNA-damage response tumor-suppressor protein p53 but that NAG-1 induction was independent of p53 accumulation. Moreover, prodigiosin caused AKT dephosphorylation and glycogen synthase kinase-3beta (GSK-3beta) activation, which correlated with NAG-1 expression. Prodigiosin-induced apoptosis was recovered by inhibiting GSK-3beta, which might be due, at least in part, to the blockade of the GSK-3beta-dependent up-regulation of death receptors 4 and 5 expression. These findings suggest that prodigiosin-mediated GSK-3beta activation is a key event in regulating the molecular pathways that trigger the apoptosis induced by this anticancer agent.

  18. Extracellular simian virus 40 induces an ERK/MAP kinase-independent signalling pathway that activates primary response genes and promotes virus entry.

    PubMed

    Dangoria, N S; Breau, W C; Anderson, H A; Cishek, D M; Norkin, L C

    1996-09-01

    Simian virus 40 (SV40) binding to growth-arrested cells activated an intracellular signalling pathway that induced the up-regulation of the primary response genes c-myc, c-jun and c-sis within 30 min and of JE within 90 min. The up-regulation of the primary response genes occurred in the presence of cycloheximide and when UV-inactivated SV40 was adsorbed to cells. SV40 binding did not activate Raf or mitogen-activated protein kinase (MAP/ERK1), or mobilize intracellular Ca2+. The SV40-induced up-regulation of c-myc and c-jun was blocked by the tyrosine kinase inhibitor, genistein, and by the protein kinase C (PKC) inhibitor, calphostin C, but not by expression of the MAP kinase-specific phosphatase, MKP-1. These results suggest that the SV40-induced signalling pathway includes the activities of a tyrosine kinase and a Ca(2+)-independent isoform of PKC, but not of Raf or MAP kinase. Finally, SV40 infectious entry into cells was specifically and reversibly blocked by genistein.

  19. 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.

  20. 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

  1. beta(2)-adrenergic receptor Arg16/Arg16 genotype is associated with reduced lung function, but not with asthma, in the Hutterites.

    PubMed

    Summerhill, E; Leavitt, S A; Gidley, H; Parry, R; Solway, J; Ober, C

    2000-08-01

    In a genome-wide screen for asthma loci in the Hutterites, a marker locus on chromosome 5q23-31 showed evidence of linkage to asthma (C. Ober and colleagues, Hum. Molec. Genet. 1998;7:1393). To determine whether the beta(2)-adrenergic receptor (beta(2)AR) gene is the 5q-linked asthma locus in the Hutterites, we genotyped this sample for polymorphisms in the beta(2)AR gene. Neither the Arg16Gly nor Gln27Glu polymorphisms showed evidence of linkage to qualitative measures of asthma and bronchial hyperresponsiveness (BHR) (p > 0.10) or to quantitative measures of serum IgE and airway reactivity (p > 0.10). In contrast, FEV(1) percentage of predicted and FVC percentage of predicted were significantly lower among individuals homozygous for the Arg16 allele (FEV(1) %: 98.3 +/- 13.2% versus 103. 8 +/- 14.9%, p = 0.003; FVC %: 104.2 +/- 12.3% versus 108.3 +/- 13. 2%, p = 0.02 by t test). These findings held true for adolescents and adults, but not for children gene. However, it is the first study to suggest that the beta(2)AR Arg16Gly polymorphism influences either lung growth or the rate of decline of lung function with age. PMID:10934093

  2. 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

  3. GENETIC VARIATION IN THE BETA-3-ADRENORECEPTOR GENE (TRP64ARG POLYMORPHISM) AND THEIR INFLUENCE ON ANTHROPOMETRIC PARAMETERS AND INSULIN RESISTANCE AFTER A HIGH PROTEIN/LOW CARBOHYDRATE VERSUS A STANDARD HYPOCALORIC DIET.

    PubMed

    de Luis, Daniel Antonio; Aller, Rocío; Izaola, Olatz; de la Fuente, Beatriz; Romero, Enrique

    2015-08-01

    Introducción: la variante Trp64Arg del receptor Beta ha sido relacionada con un aumento del peso corporal y resistencia a la insulina. Objetivo: el objetivo de nuestro estudio fue investigar la influencia del polimorfismo (rs 4994) del gen del receptor adrenérgico-Beta-3 en la respuesta metabólica y la pérdida de peso en un estudio de intervención a medio plazo con una dieta con alto contenido en proteínas/baja en carbohidratos vs una dieta hipocalórica estándar (1.000 kcal / día). Material y métodos: se evaluó una muestra de 284 sujetos obesos con un diseño de ensayo aleatorio. Se realizó una evaluación nutricional al inicio y al final de un período de 9 meses en el que los sujetos recibieron una de las dos dietas (dieta HP: alta en proteínas/baja en carbohidratos vs dieta S: dieta estándar). Resultados: no hubo diferencias significativas entre los efectos positivos (sobre el peso, el índice de masa corporal, la circunferencia de la cintura, la masa grasa, la presión arterial sistólica y los niveles de leptina) en los dos genotipos con ambas dietas. Con ambas dietas y solo en el genotipo salvaje (dieta HP vs dieta S), colesterol total (-10,1 ± 3,9 mg / dl vs -10,1 ± 2,2 mg / dl; p> 0,05), colesterol LDL (-9,5 ± 2,1 mg / dl vs -8,5 ± 2,3 mg / dl; p> 0,05) y los triglicéridos (-19,1 ± 2,1 mg / dl vs -14,3 ± 2,1 mg / dl; p> 0,05) disminuyeron. La mejoría de estos parámetros fue similar en sujetos con dieta HP vs dieta HS. Con la dieta HP y solo en el genotipo salvaje, los niveles de insulina (-3,7 ± 1,9 UI / L; p.

  4. 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

  5. A genomewide overexpression screen identifies genes involved in the phosphatidylinositol 3-kinase pathway in the human protozoan parasite Entamoeba histolytica.

    PubMed

    Koushik, Amrita B; Welter, Brenda H; Rock, Michelle L; Temesvari, Lesly A

    2014-03-01

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

  6. Regulation of sterol regulatory-element binding protein 1 gene expression in liver: role of insulin and protein kinase B/cAkt.

    PubMed Central

    Fleischmann, M; Iynedjian, P B

    2000-01-01

    Insulin stimulates the transcription of the sterol regulatory- element binding protein (SREBP) 1/ADD1 gene in liver. Hepatocytes in primary culture were used to delineate the insulin signalling pathway for induction of SREBP1 gene expression. The inhibitors of phosphoinositide 3-kinase (PI 3-kinase), wortmannin and LY 294002, abolished the insulin-dependent increase in SREBP1 mRNA, whereas the inhibitor of the mitogen- activated protein kinase cascade, PD 98059, was without effect. To investigate the role of protein kinase B (PKB)/cAkt downstream of PI 3-kinase, hepatocytes were transduced with an adenovirus encoding a PKB--oestrogen receptor fusion protein. The PKB activity of this recombinant protein was rapidly activated in hepatocytes challenged with 4-hydroxytamoxifen (OHT), as was endogenous PKB in hepatocytes challenged with insulin. The addition of OHT to transduced hepatocytes resulted in accumulation of SREBP1 mRNA, with a time-course and magnitude similar to the effect of insulin in non-transduced cells. The level of SREBP1 mRNA was not increased by OHT in hepatocytes expressing a mutant form of the recombinant protein whose PKB activity was not activated by OHT. Thus acute activation of PKB is sufficient to induce SREBP1 mRNA accumulation in primary hepatocytes, and might be the major signalling event by which insulin induces SREBP1 gene expression in the liver. PMID:10861205

  7. Prevention of allograft rejection in heart transplantation through concurrent gene silencing of TLR and Kinase signaling pathways

    PubMed Central

    Wang, Hongmei; Zhang, Xusheng; Zheng, Xiufen; Lan, Zhu; Shi, Jun; Jiang, Jifu; Zwiep, Terry; Li, Qing; Quan, Douglas; Zhang, Zhu-Xu; Min, Weiping

    2016-01-01

    Toll-like receptors (TLRs) act as initiators and conductors responsible for both innate and adaptive immune responses in organ transplantation. The mammalian target of rapamycin (mTOR) is one of the most critical signaling kinases that affects broad aspects of cellular functions including metabolism, growth, and survival. Recipients (BALB/c) were treated with MyD88, TRIF and mTOR siRNA vectors, 3 and 7 days prior to heart transplantation and 7, 14 and 21 days after transplantation. After siRNA treatment, recipients received a fully MHC-mismatched C57BL/6 heart. Treatment with mTOR siRNA significantly prolonged allograft survival in heart transplantation. Moreover, the combination of mTOR siRNA with MyD88 and TRIF siRNA further extended the allograft survival; Flow cytometric analysis showed an upregulation of FoxP3 expression in spleen lymphocytes and a concurrent downregulation of CD40, CD86 expression, upregulation of PD-L1 expression in splenic dendritic cells in MyD88, TRIF and mTOR treated mice. There is significantly upregulated T cell exhaustion in T cells isolated from tolerant recipients. This study is the first demonstration of preventing immune rejection of allogeneic heart grafts through concurrent gene silencing of TLR and kinase signaling pathways, highlighting the therapeutic potential of siRNA in clinical transplantation. PMID:27659428

  8. Modulation of protein kinase activity and gene expression by reactive oxygen species and their role in vascular physiology and pathophysiology.

    PubMed

    Griendling, K K; Sorescu, D; Lassègue, B; Ushio-Fukai, M

    2000-10-01

    Emerging evidence indicates that reactive oxygen species, especially superoxide and hydrogen peroxide, are important signaling molecules in cardiovascular cells. Their production is regulated by hormone-sensitive enzymes such as the vascular NAD(P)H oxidases, and their metabolism is coordinated by antioxidant enzymes such as superoxide dismutase, catalase, and glutathione peroxidase. Both of these reactive oxygen species serve as second messengers to activate multiple intracellular proteins and enzymes, including the epidermal growth factor receptor, c-Src, p38 mitogen-activated protein kinase, Ras, and Akt/protein kinase B. Activation of these signaling cascades and redox-sensitive transcription factors leads to induction of many genes with important functional roles in the physiology and pathophysiology of vascular cells. Thus, reactive oxygen species participate in vascular smooth muscle cell growth and migration; modulation of endothelial function, including endothelium-dependent relaxation and expression of a proinflammatory phenotype; and modification of the extracellular matrix. All of these events play important roles in vascular diseases such as hypertension and atherosclerosis, suggesting that the sources of reactive oxygen species and the signaling pathways that they modify may represent important therapeutic targets.

  9. The phylogenetic distribution and evolution of enzymes within the thymidine kinase 2-like gene family in metazoa.

    PubMed

    Konrad, Anke; Lai, Jason; Mutahir, Zeeshan; Piškur, Jure; Liberles, David A

    2014-04-01

    Deoxyribonucleoside kinases (dNKs) carry out the rate-determining step in the nucleoside salvage pathway within all domains of life where the pathway is present, and, hence, are an indication on whether or not a species/genus retains the ability to salvage deoxyribonucleosides. Here, a phylogenetic tree is constructed for the thymidine kinase 2-like dNK gene family in metazoa. Each enzyme class (deoxycytidine, deoxyguanosine, and deoxythymidine kinases, as well as the multisubstrate dNKs) falls into a monophyletic clade. However, in vertebrates, dCK contains an apparent duplication with one paralog lost in mammals, and a number of crustacean genomes (like Caligus rogercresseyi and Lepeophtheirus salmonis) unexpectedly contain not only the multisubstrate dNKs, related to Drosophila multisubstrate dNK, but also a TK2-like kinase. Additionally, crustaceans (Daphnia, Caligus, and Lepeophtheirus) and some insects (Tribolium, Danaus, Pediculus, and Acyrthosiphon) contain several multisubstrate dNK-like enzymes which group paraphyletically within the arthropod clade. This might suggest that the multisubstrate dNKs underwent multiple rounds of duplications with differential retention of duplicate copies between insect families and more complete retention within some crustaceans and insects. Genomes of several basal animalia contain more than one dNK-like sequence, some of which group outside the remaining eukaryotes (both plants and animals) and/or with bacterial dNKs. Within the vertebrates, the mammalian genomes do not contain the second dCK, while birds, fish, and amphibians do retain it. Phasianidae (chicken and turkey) have lost dGK, while it has been retained in other bird lineages, like zebra finch. Reconstruction of the ancestral sequence between the multisubstrate arthropod dNKs and the TK2 clade of vertebrates followed by homology modeling and discrete molecular dynamics calculations on this sequence were performed to examine the evolutionary path which led to

  10. 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.

  11. 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

  12. A novel calcium-dependent protein kinase gene from Populus euphratica, confers both drought and cold stress tolerance.

    PubMed

    Chen, Jinhuan; Xue, Bin; Xia, Xinli; Yin, Weilun

    2013-11-22

    Populus species are the most important timber trees over the Northern hemisphere. Most of them are cold- and drought-sensitive except the Populus euphratica Oliv. Here, a calcium-dependent protein kinase (CDPK) gene cloned from P. euphratica, designated as PeCPK10, was rapidly induced by salt, cold, and drought stresses. The protein encoded by PeCPK10 was localized within the nucleus and cytosol, which may be important for its specific regulation in cellular functions. To elucidate the physiological functions of PeCPK10, we generated transgenic Arabidopsis plants overexpressing PeCPK10. The results showed that PeCPK10-transgenic lines experienced better growth than vector control plants when treated with drought. Stronger abscisic acid-induced promotion of stomatal closing has been showed in transgenic lines. Particularly, overexpression of PeCPK10 showed enhanced freezing tolerance. Constitutive expression of PeCPK10 enhanced the expression of several abscisic acid-responsive genes and multiple abiotic stress-responsive genes such as RD29B and COR15A. Accordingly, a positive regulator responsive to cold and drought stresses in P. euphratica is proposed. PMID:24177011

  13. The muscle creatine kinase gene is regulated by multiple upstream elements, including a muscle-specific enhancer

    SciTech Connect

    Jaynes, J.B.; Johnson, J.E.; Buskin, J.N.; Gartside, C.L.; Hauschka, S.D.

    1988-01-01

    Muscle creatine kinase (MCK) is induced to high levels during skeletal muscle differentiation. The authors examined the upstream regulatory elements of the mouse MCK gene which specify its activation during myogenesis in culture. Fusion genes containing up to 3,300 nucleotides (nt) of MCK 5' flanking DNA in various positions and orientations relative to the bacterial chloramphenicol acetyltransferase (CAT) structural gene were transfected into cultured cells. Transient expression of CAT was compared between proliferating and differentiated MM14 mouse myoblasts and with nonmyogenic mouse L cells. The major effector of high-level expression was found to have the properties of a transcriptional enhancer. This element, located between 1,050 and 1,256 nt upstream of the transcription start site, was also found to have a major influence on the tissue and differentiation specificity of MCK expression; it activated either the MCK promoter or heterologous promoters only in differentiated muscle cells. Comparisons of viral and cellular enhancer sequences with the MCK enhancer revealed some similarities to essential regions of the simian virus 40 enhancer as well as to a region of the immunoglobulin heavy-chain enhancer, which has been implicated in tissue-specific protein binding. Even in the absence of the enhancer, low-level expression from a 776-nt MCK promoter retained differentiation specificity. In addition to positive regulatory elements, our data provide some evidence for negative regulatory elements with activity in myoblasts. These may contribute to the cell type and differentiation specificity of MCK expression.

  14. 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

  15. Genetic Characterization of Plasmodium Putative Pantothenate Kinase Genes Reveals Their Essential Role in Malaria Parasite Transmission to the Mosquito.

    PubMed

    Hart, Robert J; Cornillot, Emmanuel; Abraham, Amanah; Molina, Emily; Nation, Catherine S; Ben Mamoun, Choukri; Aly, Ahmed S I

    2016-01-01

    The metabolic machinery for the biosynthesis of Coenzyme A (CoA) from exogenous pantothenic acid (Vitamin B5) has long been considered as an excellent target for the development of selective antimicrobials. Earlier studies in the human malaria parasite Plasmodium falciparum have shown that pantothenate analogs interfere with pantothenate phosphorylation and block asexual blood stage development. Although two eukaryotic-type putative pantothenate kinase genes (PanK1 and PanK2) have been identified in all malaria parasite species, their role in the development of Plasmodium life cycle stages remains unknown. Here we report on the genetic characterization of PanK1 and PanK2 in P. yoelii. We show that P. yoelii parasites lacking either PanK1 or PanK2 undergo normal asexual stages development and sexual stages differentiation, however they are severely deficient in ookinete, oocyst and sporozoite formation inside the mosquito vector. Quantitative transcriptional analyses in wild-type and knockout parasites demonstrate an important role for these genes in the regulation of expression of other CoA biosynthesis genes. Together, our data provide the first genetic evidence for the importance of the early steps of pantothenate utilization in the regulation of CoA biosynthesis and malaria parasite transmission to Anopheles mosquitoes. PMID:27644319

  16. 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

  17. 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

  18. Genetic Characterization of Plasmodium Putative Pantothenate Kinase Genes Reveals Their Essential Role in Malaria Parasite Transmission to the Mosquito

    PubMed Central

    Hart, Robert J.; Cornillot, Emmanuel; Abraham, Amanah; Molina, Emily; Nation, Catherine S.; Ben Mamoun, Choukri; Aly, Ahmed S. I.

    2016-01-01

    The metabolic machinery for the biosynthesis of Coenzyme A (CoA) from exogenous pantothenic acid (Vitamin B5) has long been considered as an excellent target for the development of selective antimicrobials. Earlier studies in the human malaria parasite Plasmodium falciparum have shown that pantothenate analogs interfere with pantothenate phosphorylation and block asexual blood stage development. Although two eukaryotic-type putative pantothenate kinase genes (PanK1 and PanK2) have been identified in all malaria parasite species, their role in the development of Plasmodium life cycle stages remains unknown. Here we report on the genetic characterization of PanK1 and PanK2 in P. yoelii. We show that P. yoelii parasites lacking either PanK1 or PanK2 undergo normal asexual stages development and sexual stages differentiation, however they are severely deficient in ookinete, oocyst and sporozoite formation inside the mosquito vector. Quantitative transcriptional analyses in wild-type and knockout parasites demonstrate an important role for these genes in the regulation of expression of other CoA biosynthesis genes. Together, our data provide the first genetic evidence for the importance of the early steps of pantothenate utilization in the regulation of CoA biosynthesis and malaria parasite transmission to Anopheles mosquitoes. PMID:27644319

  19. Genetic Characterization of Plasmodium Putative Pantothenate Kinase Genes Reveals Their Essential Role in Malaria Parasite Transmission to the Mosquito.

    PubMed

    Hart, Robert J; Cornillot, Emmanuel; Abraham, Amanah; Molina, Emily; Nation, Catherine S; Ben Mamoun, Choukri; Aly, Ahmed S I

    2016-01-01

    The metabolic machinery for the biosynthesis of Coenzyme A (CoA) from exogenous pantothenic acid (Vitamin B5) has long been considered as an excellent target for the development of selective antimicrobials. Earlier studies in the human malaria parasite Plasmodium falciparum have shown that pantothenate analogs interfere with pantothenate phosphorylation and block asexual blood stage development. Although two eukaryotic-type putative pantothenate kinase genes (PanK1 and PanK2) have been identified in all malaria parasite species, their role in the development of Plasmodium life cycle stages remains unknown. Here we report on the genetic characterization of PanK1 and PanK2 in P. yoelii. We show that P. yoelii parasites lacking either PanK1 or PanK2 undergo normal asexual stages development and sexual stages differentiation, however they are severely deficient in ookinete, oocyst and sporozoite formation inside the mosquito vector. Quantitative transcriptional analyses in wild-type and knockout parasites demonstrate an important role for these genes in the regulation of expression of other CoA biosynthesis genes. Together, our data provide the first genetic evidence for the importance of the early steps of pantothenate utilization in the regulation of CoA biosynthesis and malaria parasite transmission to Anopheles mosquitoes.

  20. 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.

  1. 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

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

    PubMed Central

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

    1995-01-01

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

  3. Changes in the expression of transthyretin and protein kinasegenes in the prefrontal cortex in response to naltrexone.

    PubMed

    Yu, Jaehak; Halder, Debasish; Baek, Mi Na; Das, Nando Dulal; Choi, Mi Ran; Oh, Dong Yul; Choi, Ihn Geun; Jung, Kyoung Hwa; Chai, Young Gyu

    2011-01-25

    Naltrexone, an opioid receptor antagonist, has been approved for clinical use in the treatment of alcohol dependence. In the present study, we examined the underlying mechanisms of naltrexone by investigating the pharmacogenomic variations in the brain regions associated with alcohol consumption. A complementary DNA microarray analysis was used to profile gene expression changes in the hippocampus and prefrontal cortex (PFC) of C57BL/6 mice injected with naltrexone following ethanol treatment. Intraperitoneal administration of 200μl (16mg/kg) of naltrexone for 4 weeks caused alterations in the expression of a wide range of hippocampal (394) and PFC (566) genes in ethanol-treated mice. Ingenuity Pathway Analysis (IPA) software was used to search for biological pathways and interrelationships between gene networks in the subsets of candidate genes that were altered in the naltrexone-treated PFC and hippocampus. We found gene networks associated with cell morphology, cell death, nervous system development and function, and neurological disease. Confirmation studies using quantitative reverse transcription-polymerase chain reaction (qRT-PCR) revealed that the expression of transthyretin (TTR) and protein kinase C (PKC)γ were increased in the PFC but not in the hippocampus of naltrexone-treated mice. In conclusion, the present study demonstrates a pharmacogenomic response to naltrexone in the brains of ethanol-consuming mice. These findings provide a basis for conducting pharmacogenetic research on the effect of naltrexone in specific brain areas, which would enhance our understanding of the underlying causes and possible treatments of alcohol use disorders. PMID:21111029

  4. 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

  5. Adenylate kinase 1 gene deletion disrupts muscle energetic economy despite metabolic rearrangement

    PubMed Central

    Janssen, Edwin; Dzeja, Petras P.; Oerlemans, Frank; Simonetti, Arjan W.; Heerschap, Arend; Haan, Arnold de; Rush, Paula S.; Terjung, Ronald R.; Wieringa, Bé; Terzic, Andre

    2000-01-01

    Efficient cellular energy homeostasis is a critical determinant of muscle performance, providing evolutionary advantages responsible for species survival. Phosphotransfer reactions, which couple ATP production and utilization, are thought to play a central role in this process. Here, we provide evidence that genetic disruption of AK1-catalyzed β-phosphoryl transfer in mice decreases the potential of myofibers to sustain nucleotide ratios despite up-regulation of high-energy phosphoryl flux through glycolytic, guanylate and creatine kinase phosphotransfer pathways. A maintained contractile performance of AK1-deficient muscles was associated with higher ATP turnover rate and larger amounts of ATP consumed per contraction. Metabolic stress further aggravated the energetic cost in AK1–/– muscles. Thus, AK1-catalyzed phosphotransfer is essential in the maintenance of cellular energetic economy, enabling skeletal muscle to perform at the lowest metabolic cost. PMID:11101510

  6. Minimum requirements for inhibition of smooth-muscle myosin light-chain kinase by synthetic peptides.

    PubMed Central

    Hunt, J T; Floyd, D M; Lee, V G; Little, D K; Moreland, S

    1989-01-01

    Although the amino acid residues that are important for peptide substrates of myosin light-chain kinase have been reported, those that are important for peptide inhibitors of this enzyme have not previously been investigated. Synthetic peptides based on the sequence Lys11-Lys12-Arg13-Ala-Ala-Arg16-Ala-Thr-Ser19 -Asn-Val21-Phe22-Ala of the chicken gizzard myosin light chain were tested as inhibitors of pig carotid-artery myosin light-chain kinase. The basic amino acid residues of the known myosin light-chain kinase inhibitor Lys-Lys-Arg-Ala-Ala-Arg-Ala-Thr-Ser-NH2 (IC50 = 14 microM) [Pearson, Misconi & Kemp (1986) J. Biol. Chem. 261, 25-27] were shown to be the important residues that contribute to inhibitor potency, as evidence by the finding that the hexapeptide Lys-Lys-Arg-Ala-Ala-Arg-NH2 had an IC50 value of 22 microM. This indicates that binding of the phosphorylatable serine residue to myosin light-chain kinase, which is of obvious importance for a substrate, does not enhance the potency of an inhibitor. With the aim of preparing more potent inhibitors, peptides Lys-Lys-Arg-Ala-Ala-Arg-Ala-Ala-Xaa-NH2 were prepared with a variety of amino acids substituted for the phosphorylatable serine residue. None of these peptides was a more potent inhibitor than the serine peptide. PMID:2920029

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

    PubMed

    Greuber, Emileigh K; Pendergast, Ann Marie

    2012-12-01

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

  8. Association between p53 Arg72Pro polymorphism and recurrent pregnancy loss: an updated systematic review and meta-analysis.

    PubMed

    Chen, Hui; Yang, Xiaorong; Wang, Zhiping

    2015-08-01

    The p53 tumour suppressor gene plays an important role in angiogenesis and apoptosis. The association between Arg72Pro polymorphism and recurrent pregnancy loss (RPL) has been studied but with inconsistent results. A meta-analysis was conducted to examine whether p53 Arg72Pro polymorphism is a risk factor for RPL. Electronic searches of PubMed, Embase and Web of Knowledge were conducted to identify relevant studies. The final meta-analysis included six published articles with 730 RPL cases and 613 controls. The pooled results suggested that the variant genotype was associated with the RPL risk in additive model (Pro versus Arg; odds ratio [OR] = 1.28; 95% confidence interval [CI]: 1.06 to 1.54) and recessive model (Pro/Pro versus Arg/Pro + Arg/Arg; OR = 1.82; 95% CI: 1.21 to 2.73). In the stratified analysis by ethnicity, for white people the results were consistent. The Egger's regression asymmetry test suggested a lack of publication bias. Results of this meta-analysis suggest that p53 codon 72 polymorphism is associated with RPL, especially in white people. Identification of p53 codon 72 mutation would have some implication for primary prevention of RPL and screening of high-risk individuals. Large well-designed studies are needed to fully describe the association between this polymorphism and RPL. PMID:26099444

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

    SciTech Connect

    Eipers, P.G.

    1992-01-01

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

  10. 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.

  11. 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

  12. The Global Arginine Regulator ArgR Controls Expression of argF in Pseudomonas syringae pv. phaseolicola but Is Not Required for the Synthesis of Phaseolotoxin or for the Regulated Expression of argK†

    PubMed Central

    Hernández-Flores, José Luis; López-López, Karina; Garcidueñas-Piña, Rogelio; Jofre-Garfias, Alba E.; Alvarez-Morales, Ariel

    2004-01-01

    In Pseudomonas syringae pv. phaseolicola the enzyme ornithine carbamoyltransferase (OCTase), encoded by argF, is negatively regulated by argR, similar to what has been reported for Pseudomonas aeruginosa. However, production of the phaseolotoxin-resistant OCTase encoded by argK, synthesis of phaseolotoxin, and infectivity for bean pods occur independently of the ArgR protein. PMID:15150254

  13. Gene for the catalytic subunit of the human DNA-activated protein kinase maps to the site of the XRCC7 gene on chromosome 8

    SciTech Connect

    Sipley, J.D.; Anderson, C.W.; Menninger, J.C.

    1995-08-01

    The DNA-activated serine/threonine protein kinase (DNA-PK) is composed for a large ({approximately}460 kDa) catalytic polypeptide (DNA-PK{sub cs}) and Ku, a heterodimeric DNA-binding component (p70/p80) that targets DNA-PK{sub cs} to DNA. A 41-kbp segment of the DNA-PK{sub cs} gene was isolated, and a 7902-bp segment was sequenced. The sequence contains a polymorphic Pvu II restriction enzyme site, and comparing the sequence with that of the cDNA revealed the positions of nine exons. The DNA-PK{sub cs} gene was mapped to band q11 of chromosome 8 by in situ hybridization. This location is coincident with that of XRCC7, the gene that complements the DNA double-strand break repair and V(D)J recombination defects (where V is variable, D is diversity, and J is joining) of hamster V3 and murine severe combined immunodeficient (scid) cells. 50 refs., 4 figs., 2 tabs.

  14. ArgR is an essential local transcriptional regulator of the arcABC operon in Streptococcus suis and is crucial for biological fitness in an acidic environment.

    PubMed

    Fulde, Marcus; Willenborg, Joerg; de Greeff, Astrid; Benga, Laurentiu; Smith, Hilde E; Valentin-Weigand, Peter; Goethe, Ralph

    2011-02-01

    Streptococcus suis is one of the most important pathogens in pigs and can also cause severe infections in humans. Despite its clinical relevance, very little is known about the factors that contribute to its virulence. Recently, we identified a new putative virulence factor in S. suis, the arginine deiminase system (ADS), an arginine catabolic enzyme system encoded by the arcABC operon, which enables S. suis to survive in an acidic environment. In this study, we focused on ArgR, an ADS-associated regulator belonging to the ArgR/AhrC arginine repressor family. Using an argR knockout strain we were able to show that ArgR is essential for arcABC operon expression and necessary for the biological fitness of S. suis. By cDNA expression microarray analyses and quantitative real-time RT-PCR we found that the arcABC operon is the only gene cluster regulated by ArgR, which is in contrast to the situation in many other bacteria. Reporter gene analysis with gfp under the control of the arcABC promoter demonstrated that ArgR is able to activate the arcABC promoter. Electrophoretic mobility shift assays with fragments of the arcABC promoter and recombinant ArgR, and chromatin immunoprecipitation with antibodies directed against ArgR, revealed that ArgR interacts with the arcABC promoter in vitro and in vivo by binding to a region from -147 to -72 bp upstream of the transcriptional start point. Overall, our results show that in S. suis, ArgR is an essential, system-specific transcriptional regulator of the ADS that interacts directly with the arcABC promoter in vivo.

  15. 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

  16. 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

  17. Conserved interactions of a compact highly active enhancer/promoter upstream of the rhodopsin kinase (GRK1) gene.

    PubMed

    Young, Joyce E; Kasperek, Eileen M; Vogt, Todd M; Lis, Agnieszka; Khani, Shahrokh C

    2007-08-01

    Rhodopsin kinase (RK) is a conserved component of the light adaptation and recovery pathways shared among rod and cone photoreceptors of a variety of species. To gain insight into transcriptional mechanisms driving RK and potentially other genes of similar spatial profile, the components and the interactions of the highly compact enhancer/promoter region (E/P) upstream of the human RK gene were examined. Cross-species comparison outlined an active 49-bp widely shared E/P core as the major site of conservation in the entire 5' flanking sequence. The area consisted of a bicoid-type homeodomain recognition cassette and a unique T-rich module interacting with TATA-binding proteins. Homeodomain interactions involved primarily Crx and secondarily Otx2. Both strongly stimulated the E/P. In the absence of Crx, persistent E/P activity shifted from the outer retina to the inner to follow the Otx2 pattern. The spatial patterns were largely unaffected by the absence of rod transcription factors, Nrl and Nr2e3, and the RK transcriptional activity preceded the surge in rod-specific transcription. Conserved bicoid homeodomain factors thus appear to be the key factors governing localization of RK E/P activity in retina and photoreceptors.

  18. Activation protein 1-dependent transcriptional activation of interleukin 2 gene by Ca2+/calmodulin kinase type IV/Gr

    PubMed Central

    1996-01-01

    The Ca2+/calmodulin-dependent protein kinase (CaMK) type IV/Gr is selectively expressed in T lymphocytes and is activated after signaling via the T cell antigen receptor (TCR), indicating that it mediates some of the Ca(2+)-dependent transcriptional events that follow TCR engagement. Here we show that CaMKIV/Gr induces the transcription factor activation protein 1 (AP-1) alone or in synergy with T cell mitogens and with the p21ras oncoprotein. CaMKIV/ Gr signaling is associated with transcriptional activation of c-fos but is independent of p21ras or calcineurin. AP-1 is an integral component of the nuclear factor of activated T cells (NFAT) transcriptional complex, which is required for interleukin 2 gene expression in T cells. We demonstrate that CaMKIV/Gr reconstitutes the capacity of the cytosolic component of NFAT to direct transcription from NFAT sites in non-T cells. These results reveal a central role for CaMKIV/Gr as a Ca(2+)-regulated activator of gene transcription in T lymphocytes. PMID:8691123

  19. 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

  20. TaCIPK29, a CBL-interacting protein kinase gene from wheat, confers salt stress tolerance in transgenic tobacco.

    PubMed

    Deng, Xiaomin; Hu, Wei; Wei, Shuya; 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 Ca(2+) 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.

  1. 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.

  2. Structure-function analysis of a lupus anti-DNA autoantibody: central role of the heavy chain complementarity-determining region 3 Arg in binding of double- and single-stranded DNA.

    PubMed

    Li, Z; Schettino, E W; Padlan, E A; Ikematsu, H; Casali, P

    2000-07-01

    To determine the contribution of the somatic point mutations and that of the complementarity-determining region (CDR)3 Arg to DNA binding, we engineered the germline V(H) and V(kappa) gene revertant and site-mutagenized the CDR3 Arg residues of the mutated and "antigen-selected" mAb 412.67. This anti-DNA autoantibody was derived from B-1 cells of a lupus patient and bore two H-CDR3 Arg, Arg105 and Arg107, encoded by N segment additions, and one kappa-CDR3 Arg, Arg97, resulting from a point mutation (Kasaian et al. 1994. J. Immunol. 152: 3137-3151; Kasaian et al. 1995. Ann. N.Y Acad. Sci. 764: 410-423). The germ-line revertant bound double-stranded (ds) DNA and single-stranded (ss) DNA as effectively as its wild-type counterpart (relative avidity: 6.4x10(-7) and 9.9x10(-9) vs. 6.7x10(-7) and 9.1 x10(-9) g/microl), raising the possibility that an antigen other than DNA was responsible for the selection of the mAb 412.67 V(H) and V(kappa) point mutations. H-CDR3 Arg105 and Arg107 were both required for dsDNA binding, but either Arg105 or Arg107 was sufficient for ssDNA binding. The central role of Arg105 and Arg107 in DNA binding reflected their solvent-exposed orientation at the apex of the H-CDR3 main loop. Consistent with its inward orientation afar from the antigen-binding surface, the kappa-CDR3 Arg97 played no role in either dsDNA or ssDNA binding.

  3. The SNF1-type serine-threonine protein kinase SAPK4 regulates stress-responsive gene expression in rice

    PubMed Central

    Diédhiou, Calliste J; Popova, Olga V; Dietz, Karl-Josef; Golldack, Dortje

    2008-01-01

    Background Plants respond to extracellularly perceived abiotic stresses such as low temperature, drought, and salinity by activation of complex intracellular signaling cascades that regulate acclimatory biochemical and physiological changes. Protein kinases are major signal transduction factors that have a central role in mediating acclimation to environmental changes in eukaryotic organisms. In this study, we characterized the function of the sucrose nonfermenting 1-related protein kinase2 (SnRK2) SAPK4 in the salt stress response of rice. Results Translational fusion of SAPK4 with the green fluorescent protein (GFP) showed subcellular localization in cytoplasm and nucleus. To examine the role of SAPK4 in salt tolerance we generated transgenic rice plants with over-expression of rice SAPK4 under control of the CaMV-35S promoter. Induced expression of SAPK4 resulted in improved germination, growth and development under salt stress both in seedlings and mature plants. In response to salt stress, the SAPK4-overexpressing rice accumulated less Na+ and Cl- and showed improved photosynthesis. SAPK4-regulated genes with functions in ion homeostasis and oxidative stress response were identified: the vacuolar H+-ATPase, the Na+/H+ antiporter NHX1, the Cl- channel OsCLC1 and a catalase. Conclusion Our results show that SAPK4 regulates ion homeostasis and growth and development under salinity and suggest function of SAPK4 as a regulatory factor in plant salt stress acclimation. Identification of signaling elements involved in stress adaptation in plants presents a powerful approach to identify transcriptional activators of adaptive mechanisms to environmental changes that have the potential to improve tolerance in crop plants. PMID:18442365

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

    PubMed

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

    1996-01-01

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

  5. Global Effects of Inactivation of the Pyruvate Kinase Gene in the Mycobacterium tuberculosis Complex▿ †

    PubMed Central

    Chavadi, Sivagamisundaram; Wooff, Esen; Coldham, Nicholas G.; Sritharan, Manjula; Hewinson, R. Glyn; Gordon, Stephen V.; Wheeler, Paul R.

    2009-01-01

    To better understand the global effects of “natural” lesions in genes involved in the pyruvate metabolism in Mycobacterium bovis, null mutations were made in the Mycobacterium tuberculosis H37Rv ald and pykA genes to mimic the M. bovis situation. Like M. bovis, the M. tuberculosis ΔpykA mutant yielded dysgonic colonies on solid medium lacking pyruvate, whereas colony morphology was eugonic on pyruvate-containing medium. Global effects of the loss of the pykA gene, possibly underlying colony morphology, were investigated by using proteomics on cultures grown in the same conditions. The levels of Icd2 increased and those of Icl and PckA decreased in the ΔpykA knockout. Proteomics suggested that the synthesis of enzymes involved in fatty acid and lipid biosynthesis were decreased, whereas those involved in β-oxidation were increased in the M. tuberculosis ΔpykA mutant, as confirmed by direct assays for these activities. Thus, the loss of pykA from M. tuberculosis results in fatty acids being used principally for energy production, in contrast to the situation in the host when carbon from fatty acids is conserved through the glyoxylate cycle and gluconeogenesis; when an active pykA gene was introduced into M. bovis, the opposite effects occurred. Proteins involved in oxidative stress—AhpC, KatG, and SodA—showed increased synthesis in the ΔpykA mutant, and iron-regulated proteins were also affected. Ald levels were decreased in the ΔpykA knockout, explaining why an M. tuberculosis ΔpykA Δald double mutant showed little additional phenotypic effect. Overall, these data show that the loss of the pykA gene has powerful, global effects on proteins associated with central metabolism. PMID:19820096

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

    PubMed

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

    2007-06-01

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

  7. 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

  8. Identification of upstream and intragenic regulatory elements that confer cell-type-restricted and differentiation-specific expression on the muscle creatine kinase gene

    SciTech Connect

    Sternberg, E.A.; Spizz, G.; Perry, W.M.; Vizard, D.; Weil, T.; Olson, E.N.

    1988-07-01

    Terminal differentiation of skeletal myobalsts is accompanied by induction of a series of tissue-specific gene products, which includes the muscle isoenzymte of creatine kinase (MCK). To begin to define the sequences and signals involved in MCK regulation in developing muscle cells, the mouse MCK gene has been isolated. Sequence analysis of 4,147 bases of DNA surrounding the transcription initiation site revealed several interesting structural features, some of which are common to other muscle-specific genes and to cellular and viral enhancers.

  9. 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.

  10. Regulation of gene expression by glucose in pancreatic beta -cells (MIN6) via insulin secretion and activation of phosphatidylinositol 3'-kinase.

    PubMed

    da Silva Xavier, G; Varadi, A; Ainscow, E K; Rutter, G A

    2000-11-17

    Increases in glucose concentration control the transcription of the preproinsulin (PPI) gene and several other genes in the pancreatic islet beta-cell. Although recent data have demonstrated that secreted insulin may regulate the PPI gene (Leibiger, I. B., Leibiger, B., Moede, T., and Berggren, P. O. (1998) Mol. Cell 1, 933-938), the role of insulin in the control of other beta-cell genes is unexplored. To study the importance of insulin secretion in the regulation of the PPI and liver-type pyruvate kinase (L-PK) genes by glucose, we have used intranuclear microinjection of promoter-luciferase constructs into MIN6 beta-cells and photon-counting imaging. The activity of each promoter was increased either by 30 (versus 3) mm glucose or by 1-20 nm insulin. These effects of insulin were not due to enhanced glucose metabolism since culture with the hormone had no impact on the stimulation of increases in intracellular ATP concentration caused by 30 mm glucose. Furthermore, the islet-specific glucokinase promoter and cellular glucokinase immunoreactivity were unaffected by 30 mm glucose or 20 nm insulin. Inhibition of insulin secretion with the Ca(2+) channel blocker verapamil, the ATP-sensitive K(+) channel opener diazoxide, or the alpha(2)-adrenergic agonist clonidine blocked the effects of glucose on L-PK gene transcription. Similarly, 30 mm glucose failed to induce the promoter after inhibition of phosphatidylinositol 3'-kinase activity with LY294002 and the expression of dominant negative-acting phosphatidylinositol 3'-kinase (Deltap85) or the phosphoinositide 3'-phosphatase PTEN (phosphatase and tensin homologue). LY294002 also diminished the activation of the L-PK gene caused by inhibition of 5'-AMP-activated protein kinase with anti-5'-AMP-activated protein kinase alpha2 antibodies. Conversely, stimulation of insulin secretion with 13 mm KCl or 10 microm tolbutamide strongly activated the PPI and L-PK promoters. These data indicate that, in MIN6 beta

  11. 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

  12. 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.

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

    SciTech Connect

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

    1994-09-01

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

  14. 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

  15. A screen for upstream components of the yeast protein kinase C signal transduction pathway identifies the product of the SLG1 gene.

    PubMed

    Jacoby, J J; Nilius, S M; Heinisch, J J

    1998-04-01

    We employed the constitutive BCK1-20 allele of the gene for the MAP kinase kinase kinase (MAP-KKK) in the yeast Pkc signal transduction pathway to develop a genetic screen for mutants in genes encoding upstream components. Transposon mutagenesis yielded a mutant that was completely dependent on the active allele in the absence of osmotic stabilization. The transposon had integrated at the yeast SLG1 (HCS77) locus. This gene encodes a putative membrane protein. Haploid slg1 deletion strains are sensitive to caffeine, as expected for mutants in the Pkc pathway, as well as a variety of other drugs. The response to elevated temperatures and the dependence on osmotic stabilization depends on the genetic background. Thus, in the strain used for mutagenesis, disruption of SLG1 causes the cells to become non-viable in the absence of osmotic stabilization at both 30 degrees C and 37 degrees C. In a different genetic background this phenotype was not observed. Sensitivity of the haploid deletion mutants to caffeine can be partially suppressed by overexpression of genes for other components of the Pkc pathway, such as PKC1, SLT2, ROM2, and STE20. In addition, a SLG1-lacZ reporter construct shows higher expression in the presence of caffeine or magnesium chloride in a wild-type diploid background.

  16. High humidity suppresses ssi4-mediated cell death and disease resistance upstream of MAP kinase activation, H2O2 production and defense gene expression.

    PubMed

    Zhou, Fasong; Menke, Frank L H; Yoshioka, Keiko; Moder, Wolfgang; Shirano, Yumiko; Klessig, Daniel F

    2004-09-01

    The Arabidopsis ssi4 mutant, which exhibits spontaneous lesion formation, constitutive expression of pathogenesis-related (PR) genes and enhanced resistance to virulent bacterial and oomycete pathogens, contains a gain-of-function mutation in a TIR-NBS-LRR type R gene. Epistatic analyses revealed that both PR gene expression and disease resistance are activated via a salicylic acid (SA)- and EDS1-dependent, but NPR1- and NDR1-independent signaling pathway. In this study, we demonstrate that in moderate relative humidity (RH; 60%), the ssi4 mutant accumulates H(2)O(2) and SA prior to lesion formation and displays constitutive activation of the MAP kinases AtMPK6 and AtMPK3. It also constitutively expresses a variety of defense-associated genes, including those encoding the WRKY transcription factors AtWRKY29 and AtWRKY6, the MAP kinases AtMPK6 and AtMPK3, the powdery mildew R proteins RPW8.1 and RPW8.2, EDS1 and PR proteins. All of these ssi4-induced responses, as well as the chlorotic, stunted morphology and enhanced disease resistance phenotype, are suppressed by high RH (95%) growth conditions. Thus, a humidity sensitive factor (HSF) appears to function at an early point in the ssi4 signaling pathway. All ssi4 phenotypes, except for MAP kinase activation, also were suppressed by the eds1-1 mutation. Thus, ssi4-induced MAP kinase activation occurs downstream of the HSF but either upstream of EDS1 or on a separate branch of the ssi4 signaling pathway. SA is a critical signaling component in ssi4-mediated defense responses. However, exogenously supplied SA failed to restore lesion formation in high RH-grown ssi4 plants, although it induced defense gene expression. Thus, additional signals also are involved.

  17. Recurrent BCAM-AKT2 fusion gene leads to a constitutively activated AKT2 fusion kinase in high-grade serous ovarian carcinoma

    PubMed Central

    Kannan, Kalpana; Coarfa, Cristian; Chao, Pei-Wen; Luo, Liming; Wang, Yan; Brinegar, Amy E.; Hawkins, Shannon M.; Milosavljevic, Aleksandar; Matzuk, Martin M.; Yen, Laising

    2015-01-01

    High-grade serous ovarian cancer (HGSC) is among the most lethal forms of cancer in women. Excessive genomic rearrangements, which are expected to create fusion oncogenes, are the hallmark of this cancer. Here we report a cancer-specific gene fusion between BCAM, a membrane adhesion molecule, and AKT2, a key kinase in the PI3K signaling pathway. This fusion is present in 7% of the 60 patient cancers tested, a significant frequency considering the highly heterogeneous nature of this malignancy. Further, we provide direct evidence that BCAM-AKT2 is translated into an in-frame fusion protein in the patient’s tumor. The resulting AKT2 fusion kinase is membrane-associated, constitutively phosphorylated, and activated as a functional kinase in cells. Unlike endogenous AKT2, whose activity is tightly regulated by external stimuli, BCAM-AKT2 escapes the regulation from external stimuli. Moreover, a BCAM-AKT2 fusion gene generated via chromosomal translocation using the CRISPR/Cas9 system leads to focus formation in both OVCAR8 and HEK-293T cell lines, suggesting that BCAM-AKT2 is oncogenic. Together, the results indicate that BCAM-AKT2 expression is a new mechanism of AKT2 kinase activation in HGSC. BCAM-AKT2 is the only fusion gene in HGSC that is proven to translate an aberrant yet functional kinase fusion protein with oncogenic properties. This recurrent genomic alteration is a potential therapeutic target and marker of a clinically relevant subtype for tailored therapy of HGSC. PMID:25733895

  18. NcoI RFLP at the creatine kinase-muscle type gene locus (CKMM, chromosome 19)

    SciTech Connect

    Coerwinkel-Driessen, M.; Schepens, J.; van Zandvoort, P.; van Oost, B.; Mariman, E.; Wieringa, B. )

    1988-09-12

    A 3.2 kbp human genomic DNA fragment (BamHI-Sau3A) of the 3{prime} untranslated and 3{prime} flanking region of the CKMM gene was isolated and subcloned into the BamHI site of vector pSP64. The CKMM 3{prime}-probe identifies a 2-allele polymorphism with bands at 2.3 and 1.0 kbp (allele A) and 3.3 kbp (allele B). In addition a weak constant 4.2 kbp band is observed. This probe also detects a 2-allele TaqI RFLP reported previously, as either a 4.3 kbp (A) or a 4.2 kbp (B) band. The CKMM locus previously has been assigned to 19q13.2-q13.3. By Southern blot analysis of human-rodent somatic cell hybrids containing unique subregional fragments of chromosome 19 of man the authors have assigned the gene to 19q13.2. Co-dominant segregation was observed in 8 families with 3 generations.

  19. 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

  20. 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.

  1. [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

  2. Regenerating gene family member 4 promotes growth and migration of gastric cancer through protein kinase B pathway.

    PubMed

    Huang, Jiamiao; Yang, Ya; Yang, Jian; Li, Xian

    2014-01-01

    Regenerating gene family member 4 (REG4), a secreted protein, is overexpressed in several cancers, including gastric cancer. The present study was undertaken to determine the roles of REG4 in the growth of gastric cancer in the nude mice and in the proliferation and migration in human gastric cancer cell line and its downstream signaling pathway. Gastric cancer models were elicited by intraperitoneally injecting MKN45 human gastric cancer cells and the tumor size was measured every other day. The expressions of REG4 mRNA and protein were increased in the gastric cancer tissues from gastric cancer patients. REG4 increased the gastric tumor weight and size in the nude mice, and promoted the proliferation and migration of gastric cancer cells MKN45. Adeno-associated viral (AAV)-mediated knockdown of REG4 decreased the gastric tumor weight and size in the nude mice, and suppressed the proliferation and migration of MKN45 cells. REG4 increased the expression of phosphorylated protein kinase B (Akt). Triciribine hydrate (TCN), the inhibitor of Akt, decreased the gastric tumor weight and size in the nude mice and abolished REG4-induced weight and size increase of the tumor. TCN also inhibited proliferation and migration and abolished REG4-induced proliferation and migration increase of human gastric cell line MKN45. These results indicate that REG4 promotes the growth, proliferation and migration of gastric cancer through Akt pathway. PMID:25356179

  3. 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.

  4. Dynamic gene and protein expression patterns of the autism-associated Met receptor tyrosine kinase in the developing mouse forebrain

    PubMed Central

    Judson, Matthew C.; Bergman, Mica Y.; Campbell, Daniel B.; Eagleson, Kathie L.; Levitt, Pat

    2009-01-01

    The establishment of appropriate neural circuitry depends upon the coordination of multiple developmental events across space and time. These events include proliferation, migration, differentiation, and survival - all of which can be mediated by hepatocyte growth factor (HGF) signaling through the Met receptor tyrosine kinase. We previously found a functional promoter variant of the MET gene to be associated with autism spectrum disorder, suggesting that forebrain circuits governing social and emotional function may be especially vulnerable to developmental disruptions in HGF/Met signaling. However, little is known about the spatiotemporal distribution of Met expression in the forebrain during the development of such circuits. To advance our understanding of the neurodevelopmental influences of Met activation, we employed complementary Western blotting, in situ hybridization and immunohistochemistry to comprehensively map Met transcript and protein expression throughout perinatal and postnatal development of the mouse forebrain. Our studies reveal complex and dynamic spatiotemporal patterns of expression during this period. Spatially, Met transcript is localized primarily to specific populations of projection neurons within the neocortex and in structures of the limbic system, including the amygdala, hippocampus and septum. Met protein appears to be principally located in axon tracts. Temporally, peak expression of transcript and protein occurs during the second postnatal week. This period is characterized by extensive neurite outgrowth and synaptogenesis, supporting a role for the receptor in these processes. Collectively, these data suggest that Met signaling may be necessary for the appropriate wiring of forebrain circuits with particular relevance to social and emotional dimensions of behavior. PMID:19226509

  5. Fibrinopeptide A binds Gly-Pro-Arg-Pro.

    PubMed Central

    Root-Bernstein, R S; Westall, F C

    1984-01-01

    The tetrapeptide Gly-Pro-Arg-Pro inhibits fibrinogen aggregation, probably by binding to the same sites used during initiation of fibrin formation. The Gly-Pro-Arg-Pro binding sites have not yet been identified. However, their possible sequence and locations have been predicted on the basis of the amino acid pairing hypothesis. One of these predicted sites is on fibrinopeptide A. We report here that nuclear magnetic resonance studies indicate that Gly-Pro-Arg-Pro binds to fibrinopeptide A with a binding constant, K, of ca. 10(4) per mol. We also report results of 19 related peptide combinations used as controls. PMID:6589598

  6. Role for AMP-activated protein kinase in glucose-stimulated insulin secretion and preproinsulin gene expression.

    PubMed Central

    da Silva Xavier, Gabriela; Leclerc, Isabelle; Varadi, Aniko; Tsuboi, Takashi; Moule, S Kelly; Rutter, Guy A

    2003-01-01

    AMP-activated protein kinase (AMPK) has recently been implicated in the control of preproinsulin gene expression in pancreatic islet beta-cells [da Silva Xavier, Leclerc, Salt, Doiron, Hardie, Kahn and Rutter (2000) Proc. Natl. Acad. Sci. U.S.A. 97, 4023-4028]. Using pharmacological and molecular strategies to regulate AMPK activity in rat islets and clonal MIN6 beta-cells, we show here that the effects of AMPK are exerted largely upstream of insulin release. Thus forced increases in AMPK activity achieved pharmacologically with 5-amino-4-imidazolecarboxamide riboside (AICAR), or by adenoviral overexpression of a truncated, constitutively active form of the enzyme (AMPK alpha 1.T(172)D), blocked glucose-stimulated insulin secretion. In MIN6 cells, activation of AMPK suppressed glucose metabolism, as assessed by changes in total, cytosolic or mitochondrial [ATP] and NAD(P)H, and reduced increases in intracellular [Ca(2+)] caused by either glucose or tolbutamide. By contrast, inactivation of AMPK by expression of a dominant-negative form of the enzyme mutated in the catalytic site (AMPK alpha 1.D(157)A) did not affect glucose-stimulated increases in [ATP], NAD(P)H or intracellular [Ca(2+)], but led to the unregulated release of insulin. These results indicate that inhibition of AMPK by glucose is essential for the activation of insulin secretion by the sugar, and may contribute to the transcriptional stimulation of the preproinsulin gene. Modulation of AMPK activity in the beta-cell may thus represent a novel therapeutic strategy for the treatment of type 2 diabetes mellitus. PMID:12589707

  7. Transcription of the phosphoglycerate kinase gene of Saccharomyces cerevisiae increases when fermentative cultures are stressed by heat-shock.

    PubMed

    Piper, P W; Curran, B; Davies, M W; Lockheart, A; Reid, G

    1986-12-15

    The single gene for phosphoglycerate kinase (PGK) in the haploid genome of Saccharomyces cerevisiae is expressed to a very high level in cultures fermenting glucose. Despite this it responds to heat-shock. When S. cerevisiae growing exponentially on glucose media was shifted from 25 degrees C to 38 degrees C transient increases of 6-7-fold in cellular PGK mRNA were observed. This elevation in PGK mRNA still occurred in the presence of the protein-synthesis inhibitor cycloheximide, but was not observed in cells bearing the rna1.1 mutation. From the kinetics of continuous labelling of PGK mRNA, relative to the labelling of other RNAs in the same cultures whose levels do not alter with heat-shock, it was shown that the elevation in PGK mRNA in response to temperature upshift reflects primarily an increased synthesis of this mRNA and not an alteration of its half-life. PGK mRNA synthesis is therefore one target of a response mechanism to thermal stress. Synthesis of PGK enzyme in glucose-grown cultures is efficient after mild (25 degrees C to 38 degrees C) or severe (25 degrees C to 42 degrees C) heat-shocks. Following the severe shock, the synthesis of most proteins is abruptly terminated, but synthesis of PGK and a few other glycolytic enzymes continues at levels comparable to the levels of synthesis of most of those proteins dramatically induced by heat (heat-shock proteins). Cells that overproduce PGK due to the presence of multiple copies of the PGK gene on a high-copy-number plasmid continue their overproduction of this enzyme during severe thermal stress. Therefore PGK mRNA is both elevated in level in response to heat-shock and translated efficiently at supra-optimal temperatures.

  8. Association between polymorphisms of the α-kinase 1 gene and type 2 diabetes mellitus in community-dwelling individuals

    PubMed Central

    SHIMOKATA, SHIGETAKA; OGURI, MITSUTOSHI; FUJIMAKI, TETSUO; HORIBE, HIDEKI; KATO, KIMIHIKO; YAMADA, YOSHIJI

    2013-01-01

    We previously demonstrated that the α-kinase 1 gene (ALPK1) is a susceptibility locus for chronic kidney disease in individuals with diabetes mellitus (DM) by a genome-wide association study. Although genetic variants of ALPK1 have been associated with chronic kidney disease in individuals with DM, whether ALPK1 is a susceptibility locus for DM has not been elucidated. The purpose of the present study was to investigate a possible association of the rs2074388 (A→G, Asp565Gly) or rs2074379 (A→G, Ile732Met) variants of ALPK1 with type 2 DM in community-dwelling individuals. The study subjects comprised 5,959 community-dwelling individuals (495 subjects with type 2 DM and 5,464 controls) who were recruited to a population-based cohort study in Inabe, Mie, Japan. The comparisons of allele frequencies or genotype distributions using the Chi-square test revealed that the rs2074388 and rs2074379 variants of ALPK1 were significantly associated with type 2 DM (P<0.05). A multivariable logistic regression analysis with adjustment for age, gender, body mass index and smoking status revealed that the rs2074388 (P=0.0051; odds ratio, 1.32) and rs2074379 (P=0.0058; odds ratio, 1.32) variants were significantly associated with type 2 DM. The haplotype analysis of these polymorphisms revealed that the frequency of the major haplotype, A (rs2074388)-A (rs2074379), was significantly lower, whereas that of the minor haplotype G-G was significantly higher in subjects with type 2 DM compared to controls. Thus, ALPK1 may be a susceptible gene for type 2 DM in community-dwelling Japanese individuals. PMID:24649057

  9. Modulation of the chromatin phosphoproteome by the Haspin protein kinase.

    PubMed

    Maiolica, Alessio; de Medina-Redondo, Maria; Schoof, Erwin M; Chaikuad, Apirat; Villa, Fabrizio; Gatti, Marco; Jeganathan, Siva; Lou, Hua Jane; Novy, Karel; Hauri, Simon; Toprak, Umut H; Herzog, Franz; Meraldi, Patrick; Penengo, Lorenza; Turk, Benjamin E; Knapp, Stefan; Linding, Rune; Aebersold, Ruedi

    2014-07-01

    Recent discoveries have highlighted the importance of Haspin kinase activity for the correct positioning of the kinase Aurora B at the centromere. Haspin phosphorylates Thr(3) of the histone H3 (H3), which provides a signal for Aurora B to localize to the centromere of mitotic chromosomes. To date, histone H3 is the only confirmed Haspin substrate. We used a combination of biochemical, pharmacological, and mass spectrometric approaches to study the consequences of Haspin inhibition in mitotic cells. We quantified 3964 phosphorylation sites on chromatin-associated proteins and identified a Haspin protein-protein interaction network. We determined the Haspin consensus motif and the co-crystal structure of the kinase with the histone H3 tail. The structure revealed a unique bent substrate binding mode positioning the histone H3 residues Arg(2) and Lys(4) adjacent to the Haspin phosphorylated threonine into acidic binding pockets. This unique conformation of the kinase-substrate complex explains the reported modulation of Haspin activity by methylation of Lys(4) of the histone H3. In addition, the identification of the structural basis of substrate recognition and the amino acid sequence preferences of Haspin aided the identification of novel candidate Haspin substrates. In particular, we validated the phosphorylation of Ser(137) of the histone variant macroH2A as a target of Haspin kinase activity. MacroH2A Ser(137) resides in a basic stretch of about 40 amino acids that is required to stabilize extranucleosomal DNA, suggesting that phosphorylation of Ser(137) might regulate the interactions of macroH2A and DNA. Overall, our data suggest that Haspin activity affects the phosphorylation state of proteins involved in gene expression regulation and splicing.

  10. AMF/PGI transactivates the MMP-3 gene through the activation of Src-RhoA-phosphatidylinositol 3-kinase signaling to induce hepatoma cell migration.

    PubMed

    Shih, Wen-Ling; Liao, Ming-Huei; Yu, Feng-Ling; Lin, Ping-Yuan; Hsu, Hsue-Yin; Chiu, Shu-Jun

    2008-11-01

    We have previously shown that AMF/PGI induces hepatoma cell migration through the induction of MMP-3. This work investigates how AMF/PGI activates the MMP-3 gene. We demonstrated that AMF/PGI transactivates the MMP-3 gene promoter through AP-1. The transactivation and induction of cell migration effect of AMF/PGI directly correlates with its enzymatic activity. Various analyses showed that AMF/PGI stimulated the Src-RhoA-PI3-kinase signaling pathway, and these three signaling molecules could form a complex. Our results demonstrate a new mechanism of AMF/PGI-induced cell migration and a link between Src-RhoA-PI3-kinase, AP-1, MMP-3 and hepatoma cell migration.

  11. 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

  12. 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.

  13. 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

  14. Role of Arg-Gingipain A in Virulence of Porphyromonas gingivalis

    PubMed Central

    Tokuda, Masayuki; Karunakaran, Thonthi; Duncan, Margaret; Hamada, Nobushiro; Kuramitsu, Howard

    1998-01-01

    In order to access the role of the Porphyromonas gingivalis Arg-gingipain proteases in the virulence of this organism, a mutant defective in the rgpA gene was constructed in strain 381. This mutant, MT10, displayed only 40% of the Arg-specific cysteine protease activity of the wild-type strain. In addition, MT10, as well as the recently characterized protease mutant G-102, which is defective in the rgpB gene, displayed reduced self-aggregation, hemagglutination, and the ability to bind to immobilized type I collagen compared to levels of the wild-type parent. However, unlike mutant G-102, the rgpA mutant displayed increased binding to epithelial cells relative to that of the parental organism. Mutant MT10 also did not express detectable levels of the FimA protein as assessed by both Western and Northern blotting or fimbriae visible by electron microscopy of the cells. Furthermore, the ability of MT10 to degrade rat tail collagen fibers when it was cultured at 37°C was markedly attenuated compared to that of strain 381. These results suggest that Arg-gingipain A may play a significant role in the pathogenicity of P. gingivalis by altering the colonization and toxic properties of the organism. PMID:9488409

  15. 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

  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. Chronic SO2 inhalation above environmental standard impairs neuronal behavior and represses glutamate receptor gene expression and memory-related kinase activation via neuroinflammation in rats.

    PubMed

    Yao, Gaoyi; Yue, Huifeng; Yun, Yang; Sang, Nan

    2015-02-01

    Sulfur dioxide (SO2), as a ubiquitous air pollutant implicated in the genesis of pulmonary disease, is now being considered to be involved in neurotoxicity and increased risk for hospitalization of brain disorders. However, comparatively little is known about the impact of chronically SO2 inhalation on neuronal function. In the present study, by exposing male Wistar rats to SO2 at 3.50 and 7.00 mg/m(3) (approximately 1225 and 2450 ppb, 4.08-8.16 (24h average concentration) times higher than the EPA standard for environmental air concentrations) or filtered air for 90 days, we investigated the impact of chronic SO2 inhalation on performance in Morris water maze, and probed the accompanying neurobiological effects, including activity-regulated cytoskeletal associated gene (Arc) and glutamate receptor gene expression, memory-related kinase level and inflammatory cytokine release in the hippocampus. Here, we found that SO2 exposure reduced the number of target zone crossings and time spent in the target quadrant during the test session in the spatial memory retention of the Morris water maze. Following the neuro-functional abnormality, we detected that SO2 inhalation reduced the expression of Arc and glutamate receptor subunits (GluR1, GluR2, NR1, NR2A, and NR2B) with a concentration-dependent property in comparison to controls. Additionally, the expression of memory kinases was attenuated statistically in the animals receiving the higher concentration, including protein kinase A (PKA), protein kinase C (PKC) and calcium/calmodulin-dependent protein kinaseIIα (CaMKIIα). And the inflammatory cytokine release was increased in rats exposed to SO2. Taken together, our results suggest that long-term exposure to SO2 air pollution at concentrations above the environmental standard in rats impaired spatial learning and memory, and indicate a close link between the neurobiological changes highlighted in the brain and the behavioral disturbances.

  18. Co-Expression of Wild-Type P2X7R with Gln460Arg Variant Alters Receptor Function

    PubMed Central

    Aprile-Garcia, Fernando; Metzger, Michael W.; Paez-Pereda, Marcelo; Stadler, Herbert; Acuña, Matías; Liberman, Ana C.; Senin, Sergio A.; Gerez, Juan; Hoijman, Esteban; Refojo, Damian; Mitkovski, Mišo; Panhuysen, Markus; Stühmer, Walter; Holsboer, Florian; Deussing, Jan M.; Arzt, Eduardo

    2016-01-01

    The P2X7 receptor is a member of the P2X family of ligand-gated ion channels. A single-nucleotide polymorphism leading to a glutamine (Gln) by arginine (Arg) substitution at codon 460 of the purinergic P2X7 receptor (P2X7R) has been associated with mood disorders. No change in function (loss or gain) has been described for this SNP so far. Here we show that although the P2X7R-Gln460Arg variant per se is not compromised in its function, co-expression of wild-type P2X7R with P2X7R-Gln460Arg impairs receptor function with respect to calcium influx, channel currents and intracellular signaling in vitro. Moreover, co-immunoprecipitation and FRET studies show that the P2X7R-Gln460Arg variant physically interacts with P2X7R-WT. Specific silencing of either the normal or polymorphic variant rescues the heterozygous loss of function phenotype and restores normal function. The described loss of function due to co-expression, unique for mutations in the P2RX7 gene so far, explains the mechanism by which the P2X7R-Gln460Arg variant affects the normal function of the channel and may represent a mechanism of action for other mutations. PMID:26986975

  19. The ubiquitous mitochondrial creatine kinase gene maps to a conserved region on human chromosome 15q15 and mouse chromosome 2 bands F1-F3

    SciTech Connect

    Steeghs, K.; Wieringa, B.; Merkx, G.

    1994-11-01

    Members of the creatine kinase isoenzyme family (CKs; EC 2.7.3.2) are found in mitochondria and specialized subregions of the cytoplasm and catalyze the reversible exchange of high-energy phosphoryl between ATP and phosphocreatine. At least four functionally active genes, which encode the distinct CK subunits CKB, CKM, CKMT1 (ubiquitous), and CKMT2 (sarcomeric), and a variable number of CKB pseudogenes have been identified. Here, we report the use of a CKMT1 containing phage to map the CKMT1 gene by in situ hybridization on both human and mouse chromosomes.

  20. Regulation of expression of ABCB1 and LRP genes by mitogen-activated protein kinase/extracellular signal-regulated kinase pathway and its role in generation of side population cells in canine lymphoma cell lines.

    PubMed

    Tomiyasu, Hirotaka; Watanabe, Manabu; Goto-Koshino, Yuko; Fujino, Yasuhito; Ohno, Koichi; Sugano, Sumio; Tsujimoto, Hajime

    2013-06-01

    The concept of the cancer stem cell (CSC) has been recognized as key for elucidation of the mechanisms that confer the multidrug resistance (MDR) phenotype to tumor cells, and the side population (SP) fraction has been shown to be enriched by cells with the CSC phenotype. The purpose of the present study was to identify the mechanism that induces a difference of phenotype between the SP and the remaining major population (MP) using two canine lymphoma cell lines. Expression levels of ABCB1 and LRP genes, which encode efflux pumps, were significantly higher in the SP than in the MP. Microarray analysis revealed up-regulation of the expression of transforming growth factor-β (TGF-β) type II receptor in SP compared with MP, and the mitogen-activated protein kinase/extracellular signal-regulated kinase (MAPK/ERK) pathway was more up-regulated in the SP than in the MP. Stimulation of the MAPK/ERK pathway significantly increased the mRNA expression of both ABCB1 and LRP genes. These results indicate increased expression of the efflux pumps through the MAPK/ERK pathway in SP cells.

  1. The zinc finger domain of Tzfp binds to the tbs motif located at the upstream flanking region of the Aie1 (aurora-C) kinase gene.

    PubMed

    Tang, C J; Chuang, C K; Hu, H M; Tang, T K

    2001-06-01

    Our previous studies showed that Aie1 (aurora-C), is a novel testis kinase belonging to the aurora kinase family (). In this report, we describe a testis zinc finger protein (Tzfp) that binds to the upstream flanking sequence of the Aie1 gene. The mouse Tzfp gene, mapped to chromosome 7 B2-B3, encodes a 465-amino acid transcription factor containing a conserved N-terminal BTB/POZ domain and three C-terminal PLZF-like C(2)H(2) zinc fingers. The zinc finger domain of Tzfp binds to the TGTACAGTGT motif (Tzfp binding site, termed tbs) located at the upstream flanking sequence of the Aie1 gene by gel mobility shift, DNase I footprinting, and competition analyses. When the C-terminal zinc fingers of Tzfp were fused to the transactivation domain of VP16, the chimera activated transcription of a reporter construct containing multiple copies of the tbs. In contrast, the same chimera did not activate the reporter gene when an essential nucleotide fifth C was mutated to A at the tbs. Furthermore, we showed that the N-terminal BTB/POZ domain of TZFP has a repressor activity. Taken together, our results indicate that Tzfp recognizes a sequence-specific motif (tbs) and may play a role in the regulation of the genes carrying the tbs.

  2. Effects of doxepin on brain-derived neurotrophic factor, tumor necrosis factor alpha, mitogen-activated protein kinase 14, and AKT1 genes expression in rat hippocampus

    PubMed Central

    Eidelkhani, Nastaran; Radahmadi, Maryam; Kazemi, Mohammad; Rafiee, Laleh; Alaei, Hojjatallah; Reisi, Parham

    2015-01-01

    Background: It has been suggested that doxepin in addition to enhancement of noradrenaline and serotonin levels may have neuroprotective effects. Therefore, this study investigated the effect of doxepin on gene expression of brain-derived neurotrophic factor (BDNF), tumor necrosis factor alpha (TNF-α), mitogen-activated protein kinase 14 (MAPK14), and serine-threonine protein kinase AKT1 in rat hippocampus. Materials and Methods: Male rats were divided randomly into three groups: Control, doxepin 1 mg/kg, and doxepin 5 mg/kg. Rats received an i.p injection of doxepin for 21 days. Then the hippocampi were dissected for the measurement of the expression of BDNF, TNF-α, MAPK14, and AKT1 genes. Results: Our results showed no significant effects of doxepin on gene expression of BDNF, TNF-α, MAPK14, and AKT1 genes in the hippocampus. Conclusions: These results did not show significant effects of doxepin on the genes that affect the neuronal survival in intact animals. However, more studies need to be done, especially in models associated with neuronal damage. PMID:26601091

  3. 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

  4. The Abi-domain Protein Abx1 Interacts with the CovS Histidine Kinase to Control Virulence Gene Expression in Group B Streptococcus

    PubMed Central

    Firon, Arnaud; Tazi, Asmaa; Da Cunha, Violette; Brinster, Sophie; Sauvage, Elisabeth; Dramsi, Shaynoor; Golenbock, Douglas T.; Glaser, Philippe; Poyart, Claire; Trieu-Cuot, Patrick

    2013-01-01

    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. Cloning, sequencing, and expression of the Zymomonas mobilis fructokinase gene and structural comparison of the enzyme with other hexose kinases.

    PubMed Central

    Zembrzuski, B; Chilco, P; Liu, X L; Liu, J; Conway, T; Scopes, R

    1992-01-01

    The frk gene encoding the enzyme fructokinase (fructose 6-phosphotransferase [EC 2.7.1.4]) from Zymomonas mobilis has been isolated on a partial TaqI digest fragment of the genome and sequenced. An open reading frame of 906 bp corresponding to 302 amino acids was identified on a 3-kbp TaqI fragment. The deduced amino acid sequence corresponds to the first 20 amino acids (including an N-terminal methionine) determined by amino acid sequencing of the purified protein. The 118 bp preceding the methionine codon on this fragment does not appear to contain a promoter sequence. There was weak expression of the active enzyme in the recombinant Escherichia coli clone under control of the lac promoter on the pUC plasmid. Comparison of the amino acid sequence with that of the glucokinase enzyme (EC 2.7.1.2) from Z. mobilis reveals relatively little homology, despite the fact that fructokinase also binds glucose and has kinetic and structural properties similar to those of glucokinase. Also, there is little homology with hexose kinases that have been sequenced from other organisms. Northern (RNA) blot analysis showed that the frk transcript is 1.2 kb long. Fructokinase activity is elevated up to twofold when Z. mobilis was grown on fructose instead of glucose, and there was a parallel increase in frk mRNA levels. Differential mRNA stability was not a factor, since the half-lives of the frk transcript were 6.2 min for glucose-grown cells and 6.6 min for fructose-grown cells. Images PMID:1317376

  6. 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

  7. Insulin-Mediated Down-Regulation of Apolipoprotein A5 Gene Expression through the Phosphatidylinositol 3-Kinase Pathway: Role of Upstream Stimulatory Factor

    PubMed Central

    Nowak, Maxime; Helleboid-Chapman, Audrey; Jakel, Heidelinde; Martin, Geneviève; Duran-Sandoval, Daniel; Staels, Bart; Rubin, Edward M.; Pennacchio, Len A.; Taskinen, Marja-Riitta; Fruchart-Najib, Jamila; Fruchart, Jean-Charles

    2005-01-01

    The apolipoprotein A5 gene (APOA5) has been repeatedly implicated in lowering plasma triglyceride levels. Since several studies have demonstrated that hyperinsulinemia is associated with hypertriglyceridemia, we sought to determine whether APOA5 is regulated by insulin. Here, we show that cell lines and mice treated with insulin down-regulate APOA5 expression in a dose-dependent manner. Furthermore, we found that insulin decreases human APOA5 promoter activity, and subsequent deletion and mutation analyses uncovered a functional E box in the promoter. Electrophoretic mobility shift and chromatin immunoprecipitation assays demonstrated that this APOA5 E box binds upstream stimulatory factors (USFs). Moreover, in transfection studies, USF1 stimulates APOA5 promoter activity, and the treatment with insulin reduced the binding of USF1/USF2 to the APOA5 promoter. The inhibition of the phosphatidylinositol 3-kinase (PI3K) pathway abolished insulin's effect on APOA5 gene expression, while the inhibition of the P70 S6 kinase pathway with rapamycin reversed its effect and increased APOA5 gene expression. Using an oligonucleotide precipitation assay for USF from nuclear extracts, we demonstrate that phosphorylated USF1 fails to bind to the APOA5 promoter. Taken together, these data indicate that insulin-mediated APOA5 gene transrepression could involve a phosphorylation of USFs through the PI3K and P70 S6 kinase pathways that modulate their binding to the APOA5 E box and results in APOA5 down-regulation. The effect of exogenous hyperinsulinemia in men showed a decrease in the plasma ApoAV level. These results suggest a potential contribution of the APOA5 gene in hypertriglyceridemia associated with hyperinsulinemia. PMID:15684402

  8. 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.

  9. A YAC contig spanning a cluster of human type III receptor protein tyrosine kinase genes (PDGFRA-KIT-KDR) in chromosome segment 4q12

    SciTech Connect

    Spritz, R.A.; Strunk, K.M.; Lee, S.T.

    1994-07-15

    The authors have mapped five genes encoding protein tyrosine kinases (PTKs) to the pericentromeric region of human chromosome 4. PTK4 and TYRO4, which encode nonreceptor intracellular PTKs, are located at 4p12 and 4q13, respectively. The other three genes, PDGFRA, KIT, and KDR, encode type III transmembrane receptor PTKs for known ligands. The authors have developed a contig of 29 yeast artificial chromosomes (YACs) spanning approximately 2 Mb of DNA at 4q12 that includes PDGFRA, KIT, and KDR, and have used this YAC contig to map 12 different sequence-tagged sites in this region. PDGFRA, KIT, and KDR thus constitute a cluster of genes at 4q12 encoding closely related type III receptor PTKs. Mutations of the human KIT gene result in piebaldism, an autosomal dominant disorder of melanocyte development. 42 refs., 3 figs., 2 tabs.

  10. 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

  11. 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

  12. 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

  13. Expression profiling of 519 kinase genes in matched malignant peripheral nerve sheath tumor/plexiform neurofibroma samples is discriminatory and identifies mitotic regulators BUB1B, PBK and NEK2 as overexpressed with transformation.

    PubMed

    Stricker, Thomas P; Henriksen, Kammi J; Tonsgard, James H; Montag, Anthony G; Krausz, Thomas N; Pytel, Peter

    2013-07-01

    About 50% of all malignant peripheral nerve sheath tumors (MPNSTs) arise as neurofibromatosis type 1 associated lesions. In those patients malignant peripheral nerve sheath tumors are thought to arise through malignant transformation of a preexisting plexiform neurofibroma. The molecular changes associated with this transformation are still poorly understood. We sought to test the hypothesis that dysregulation of expression of kinases contributes to this malignant transformation. We analyzed expression of all 519 kinase genes in the human genome using the nanostring nCounter system. Twelve cases of malignant peripheral nerve sheath tumor arising in a background of preexisting plexiform neurofibroma were included. Both components were separately sampled. Statistical analysis compared global changes in expression levels as well as changes observed in the pairwise comparison of samples taken from the same surgical specimen. Immunohistochemical studies were performed on tissue array slides to confirm expression of selected proteins. The expression pattern of kinase genes can separate malignant peripheral nerve sheath tumors and preexisting plexiform neurofibromas. The majority of kinase genes is downregulated rather than overexpressed with malignant transformation. The patterns of expression changes are complex without simple recurring alteration. Pathway analysis demonstrates that differentially expressed kinases are enriched for kinases involved in the direct regulation of mitosis, and several of these show increased expression in malignant peripheral nerve sheath tumors. Immunohistochemical studies for the mitotic regulators BUB1B, PBK and NEK2 confirm higher expression levels at the protein level. These results suggest that the malignant transformation of plexiform neurofibroma is associated with distinct changes in the expression of kinase genes. The patterns of these changes are complex and heterogeneous. There is no single unifying alteration. Kinases involved

  14. 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

  15. VaCPK20, a calcium-dependent protein kinase gene of wild grapevine Vitis amurensis Rupr., mediates cold and drought stress tolerance.

    PubMed

    Dubrovina, Alexandra S; Kiselev, Konstantin V; Khristenko, Valeriya S; Aleynova, Olga A

    2015-08-01

    Abiotic stresses, such as drought, salinity, cold and heat, are major environmental factors that limit crop productivity. Vitis amurensis Rupr. is a wild grapevine species displaying a high level of abiotic and biotic stress resistance. Protein kinases, including Ca(2+)-dependent protein kinases (CDPKs), are known to mediate plant acclimation to various environmental changes. However, the functions of most grape CDPKs have not been clarified. A recent CDPK gene expression analysis revealed that 10 CDPK genes of V. amurensis were up-regulated under different abiotic stress treatments. The expression of the VaCPK20 gene was significantly up-regulated under low and high temperature stress in V. amurensis. In the current study, the effects of overexpressing the VaCPK20 gene in callus cell lines of V. amurensis and transgenic plants of A. thaliana on their responses to abiotic stresses were investigated. Transgenic Arabidopsis overexpressing the VaCPK20 gene showed higher tolerance to freezing and drought stresses, and transgenic grape cell cultures overexpressing the VaCPK20 gene showed higher resistance to cold stress in comparison with the controls transformed by the "empty" vector. Heat and salt stress resistance of the transgenic V. amurensis calli and A. thaliana was comparable to that of the wild type and vector controls. Overexpression of the VaCPK20 gene increased the expression of stress-responsive genes, such as COR47, NHX1, KIN1, or ABF3, in the transgenic Arabidopsis plants under non-stress conditions, after freezing, and under drought stress. The results imply that VaCPK20 may act as a regulatory factor involved in cold and drought stress response pathways. PMID:26264965

  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. Involvement of adenosine monophosphate-activated protein kinase in the influence of timed high-fat evening diet on the hepatic clock and lipogenic gene expression in mice.

    PubMed

    Huang, Yan; Zhu, Zengyan; Xie, Meilin; Xue, Jie

    2015-09-01

    A high-fat diet may result in changes in hepatic clock gene expression, but potential mechanisms are not yet elucidated. Adenosine monophosphate-activated protein kinase (AMPK) is a serine/threonine protein kinase that is recognized as a key regulator of energy metabolism and certain clock genes. Therefore, we hypothesized that AMPK may be involved in the alteration of hepatic clock gene expression under a high-fat environment. This study aimed to examine the effects of timed high-fat evening diet on the activity of hepatic AMPK, clock genes, and lipogenic genes. Mice with hyperlipidemic fatty livers were induced by orally administering high-fat milk via gavage every evening (19:00-20:00) for 6 weeks. Results showed that timed high-fat diet in the evening not only decreased the hepatic AMPK protein expression and activity but also disturbed its circadian rhythm. Accordingly, the hepatic clock genes, including clock, brain-muscle-Arnt-like 1, cryptochrome 2, and period 2, exhibited prominent changes in their expression rhythms and/or amplitudes. The diurnal rhythms of the messenger RNA expression of peroxisome proliferator-activated receptorα, acetyl-CoA carboxylase 1α, and carnitine palmitoyltransferase 1 were also disrupted; the amplitude of peroxisome proliferator-activated receptorγcoactivator 1α was significantly decreased at 3 time points, and fatty liver was observed. These findings demonstrate that timed high-fat diet at night can change hepatic AMPK protein levels, activity, and circadian rhythm, which may subsequently alter the circadian expression of several hepatic clock genes and finally result in the disorder of hepatic lipogenic gene expression and the formation of fatty liver.

  18. Chromatin-wide profiling of DYRK1A reveals a role as a gene-specific RNA polymerase II CTD kinase.

    PubMed

    Di Vona, Chiara; Bezdan, Daniela; Islam, Abul B M M K; Salichs, Eulàlia; López-Bigas, Nuria; Ossowski, Stephan; de la Luna, Susana

    2015-02-01

    DYRK1A is a dosage-sensitive protein kinase that fulfills key roles during development and in tissue homeostasis, and its dysregulation results in human pathologies. DYRK1A is present in both the nucleus and cytoplasm of mammalian cells, although its nuclear function remains unclear. Genome-wide analysis of DYRK1A-associated loci reveals that the kinase is recruited preferentially to promoters of genes actively transcribed by RNA polymerase II (RNAPII), which are functionally associated with translation, RNA processing, and cell cycle. DYRK1A-bound promoter sequences are highly enriched in a conserved palindromic motif, which is necessary to drive DYRK1A-dependent transcriptional activation. DYRK1A phosphorylates the C-terminal domain (CTD) of RNAPII at Ser2 and Ser5. Depletion of DYRK1A results in reduced association of RNAPII at the target promoters as well as hypophosphorylation of the RNAPII CTD along the target gene bodies. These results are consistent with DYRK1A being a transcriptional regulator by acting as a CTD kinase. PMID:25620562

  19. Loss of kinase activity in a patient with Wolcott-Rallison syndrome caused by a novel mutation in the EIF2AK3 gene.

    PubMed

    Biason-Lauber, Anna; Lang-Muritano, Mariarosaria; Vaccaro, Tindara; Schoenle, Eugen J

    2002-07-01

    Wolcott-Rallison syndrome (WRS) is an autosomal recessive disorder characterized by neonatal or early infancy type 1 diabetes, epiphyseal dysplasia, and growth retardation. Mutations in the EIF2AK3 gene, encoding the eukaryotic initiation factor 2alpha-kinase 3 (EIF2AK3), have been found in WRS patients. Here we describe a girl who came to our attention at 2 months of age with severe hypertonic dehydration and diabetic ketoacidosis. A diagnosis of type 1 diabetes was made and insulin treatment initiated. Growth retardation and microcephaly were also present. Anti-islet cell autoantibodies were negative, and mitochondrial diabetes was excluded. Imaging revealed a hypoplastic pancreas and typical signs of spondylo-epiphyseal dysplasia. The diagnosis of WRS was therefore made at age 5 years. Sequencing analysis of her EIF2AK3 gene revealed the presence of a homozygous T to C exchange in exon 13 leading to the missense serine 877 proline mutation. The mutated kinase, although it partly retains the ability of autophosphorylation, is unable to phosphorylate its natural substrate, eukaryotic initiation factor 2alpha (eIF2alpha). This is the first case in which the pathophysiological role of EIF2AK3 deficiency in WRS is confirmed at the molecular level. Our data demonstrate that EIF2AK3 kinase activity is essential for pancreas islet function and bone development in humans, and we suggest EIF2AK3 as a possible target for therapeutic intervention in diabetes. PMID:12086964

  20. Leucine-rich Repeat Kinase 2 (LRRK2) Pharmacological Inhibition Abates α-Synuclein Gene-induced Neurodegeneration*

    PubMed Central

    Daher, João P. L.; Abdelmotilib, Hisham A.; Hu, Xianzhen; Volpicelli-Daley, Laura A.; Moehle, Mark S.; Fraser, Kyle B.; Needle, Elie; Chen, Yi; Steyn, Stefanus J.; Galatsis, Paul; Hirst, Warren D.; West, Andrew B.

    2015-01-01

    Therapeutic approaches to slow or block the progression of Parkinson disease (PD) do not exist. Genetic and biochemical studies implicate α-synuclein and leucine-rich repeat kinase 2 (LRRK2) in late-onset PD. LRRK2 kinase activity has been linked to neurodegenerative pathways. However, the therapeutic potential of LRRK2 kinase inhibitors is not clear because significant toxicities have been associated with one class of LRRK2 kinase inhibitors. Furthermore, LRRK2 kinase inhibitors have not been tested previously for efficacy in models of α-synuclein-induced neurodegeneration. To better understand the therapeutic potential of LRRK2 kinase inhibition in PD, we evaluated the tolerability and efficacy of a LRRK2 kinase inhibitor, PF-06447475, in preventing α-synuclein-induced neurodegeneration in rats. Both wild-type rats as well as transgenic G2019S-LRRK2 rats were injected intracranially with adeno-associated viral vectors expressing human α-synuclein in the substantia nigra. Rats were treated with PF-06447475 or a control compound for 4 weeks post-viral transduction. We found that rats expressing G2019S-LRRK2 have exacerbated dopaminergic neurodegeneration and inflammation in response to the overexpression of α-synuclein. Both neurodegeneration and neuroinflammation associated with G2019S-LRRK2 expression were mitigated by LRRK2 kinase inhibition. Furthermore, PF-06447475 provided neuroprotection in wild-type rats. We could not detect adverse pathological indications in the lung, kidney, or liver of rats treated with PF-06447475. These results demonstrate that pharmacological inhibition of LRRK2 is well tolerated for a 4-week period of time in rats and can counteract dopaminergic neurodegeneration caused by acute α-synuclein overexpression. PMID:26078453

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

    PubMed

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

    2001-10-01

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

  2. 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

  3. 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

  4. 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

  5. ZmCPK1, a calcium-independent kinase member of the Zea mays CDPK gene family, functions as a negative regulator in cold stress signalling.

    PubMed

    Weckwerth, Philipp; Ehlert, Britta; Romeis, Tina

    2015-03-01

    Calcium-dependent protein kinases (CDPKs) have been shown to play important roles in plant environmental stress signal transduction. We report on the identification of ZmCPK1 as a member of the maize (Zea mays) CDPK gene family involved in the regulation of the maize cold stress response. Based upon in silico analysis of the Z. mays cv. B73 genome, we identified that the maize CDPK gene family consists of 39 members. Two CDPK members were selected whose gene expression was either increased (Zmcpk1) or decreased (Zmcpk25) in response to cold exposure. Biochemical analysis demonstrated that ZmCPK1 displays calcium-independent protein kinase activity. The C-terminal calcium-binding domain of ZmCPK1 was sufficient to mediate calcium independency of a previously calcium-dependent enzyme in chimeric ZmCPK25-CPK1 proteins. Furthermore, co-transfection of maize mesophyll protoplasts with active full-length ZmCPK1 suppressed the expression of a cold-induced marker gene, Zmerf3 (ZmCOI6.21). In accordance, heterologous overexpression of ZmCPK1 in Arabidopsis thaliana yielded plants with altered acclimation-induced frost tolerance. Our results identify ZmCPK1 as a negative regulator of cold stress signalling in maize.

  6. PTK1, a mitogen-activated-protein kinase gene, is required for conidiation, appressorium formation, and pathogenicity of Pyrenophora teres on barley.

    PubMed

    Ruiz-Roldán, M C; Maier, F J; Schäfer, W

    2001-02-01

    Mitogen-activated protein kinases (MAPKs) are a group of protein kinases that execute a wide variety of roles in cellular signal transduction pathways such as osmoregulation, cell wall biosynthesis, growth, and differentiation. A polymerase chain reaction (PCR) with degenerate primers based on conserved regions of known MAPKs was used to clone the MAPK gene PTK1 from the leaf pathogen Pyrenophora teres (anamorph Drechslera teres), the causal agent of net blotch of barley (Hordeum vulgare L.). The predicted amino acid sequence shows high homology with MAPKs from other phytopathogenic fungi. The gene is present in the genome as a single copy. PTK1 is expressed during in vitro growth on complete medium, under conidiation-inducing conditions and during infection of barley leaves, as shown by reverse transcription-PCR studies. In order to assess the role of PTK1 in the life cycle of P. teres, targeted gene disruption was conducted. Mutants carrying an interrupted copy of the gene were deficient in conidiation, did not form appressoria on glass surfaces or on barley leaves, lost their ability to infect barley leaves, and could not colonize host tissues following artificial wounding.

  7. 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

  8. Repressing the expression of the SUCROSE NONFERMENTING-1-RELATED PROTEIN KINASE gene in pea embryo causes pleiotropic defects of maturation similar to an abscisic acid-insensitive phenotype.

    PubMed

    Radchuk, Ruslana; Radchuk, Volodymyr; Weschke, Winfriede; Borisjuk, Ljudmilla; Weber, Hans

    2006-01-01

    The classic role of SUCROSE NONFERMENTING-1 (Snf1)-like kinases in eukaryotes is to adapt metabolism to environmental conditions such as nutrition, energy, and stress. During pea (Pisum sativum) seed maturation, developmental programs of growing embryos are adjusted to changing physiological and metabolic conditions. To understand regulation of the switch from cell proliferation to differentiation, SUCROSE NONFERMENTING-1-RELATED PROTEIN KINASE (SnRK1) was antisense repressed in pea seeds. Transgenic seeds show maturation defects, reduced conversion of sucrose into storage products, lower globulin content, frequently altered cotyledon surface, shape, and symmetry, as well as occasional precocious germination. Gene expression analysis of embryos using macroarrays of 5,548 seed-specific genes revealed 183 differentially expressed genes in two clusters, either delayed down-regulated or delayed up-regulated during transition. Delayed down-regulated genes are related to mitotic activity, gibberellic acid/brassinosteroid synthesis, stress response, and Ca2+ signal transduction. This specifies a developmentally younger status and conditional stress. Higher gene expression related to respiration/gluconeogenesis/fermentation is consistent with a role of SnRK1 in repressing energy-consuming processes in maturing cotyledons under low oxygen/energy availability. Delayed up-regulated genes are mainly related to storage protein synthesis and stress tolerance. Most of the phenotype resembles abscisic acid (ABA) insensitivity and may be explained by reduced Abi-3 expression. This may cause a reduction in ABA functions and/or a disconnection between metabolic and ABA signals, suggesting that SnRK1 is a mediator of ABA functions during pea seed maturation. SnRK1 repression also impairs gene expression associated with differentiation, independent from ABA functions, like regulation and signaling of developmental events, chromatin reorganization, cell wall synthesis, biosynthetic

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

    PubMed Central

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

    1995-01-01

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

  10. Effect of ZIP2 Gln/Arg/Leu (rs2234632) polymorphism on zinc homeostasis and inflammatory response following zinc supplementation.

    PubMed

    Giacconi, Robertina; Costarelli, Laura; Malavolta, Marco; Cardelli, Maurizio; Galeazzi, Roberta; Piacenza, Francesco; Gasparini, Nazzarena; Basso, Andrea; Mariani, Erminia; Fulop, Tamas; Rink, Lothar; Dedoussis, George; Herbein, Georges; Jajte, Jolanta; Provinciali, Mauro; Busco, Franco; Mocchegiani, Eugenio

    2015-01-01

    Zinc dyshomeostasis may lead to an augmented production of proinflammatory cytokines promoting chronic inflammation and increasing the susceptibility to age-related diseases. Several studies suggest that the zinc transporter protein ZIP2 may play a relevant role in the immune system especially during zinc deficiency, while a polymorphism on the coding region of ZIP2 gene (Gln/Arg/Leu) has been associated with severe carotid artery disease. The aim of this study is to investigate the role of ZIP2 SNP on zinc and inflammatory status in 1090 elderly healthy free-living subjects enrolled in the ZincAge project and to assess the effect of zinc supplementation on zinc status, inflammatory mediators, and zinc transporter expression depending on ZIP2 genotype. ZIP2 Leu- (Arg43Arg) carriers showed enhanced IL-6, TNF-α, and RANTES plasma levels associated with decreased free cytosolic zinc in PBMCs and an upregulation of zinc transporters ZIP2, ZIP8, and Znt1. Moreover, Leu- subjects displayed significant decrement of inflammatory mediators such as MCP-1, TNF-α, and RANTES following zinc supplementation. In summary, this investigation provides new evidence on the effect of ZIP2 Gln/Arg/Leu polymorphism on proinflammatory mediators and zinc homeostasis in elderly population with a more pronounced anti-inflammatory effect of zinc supplementation in subjects carrying ZIP2 Leu- (Arg43Arg) genotype. These novel findings could be useful in identifying elderly subjects who may benefit of zinc intervention to decrease the inflammatory status and to prevent or delay the development of age-related diseases.

  11. Expression analysis of sugarcane shaggy-like kinase (SuSK) gene identified through cDNA subtractive hybridization in sugarcane (Saccharum officinarum L.).

    PubMed

    Patade, Vikas Yadav; Rai, Archana Neeraj; Suprasanna, Penna

    2011-07-01

    Identification of genes whose expression enables plants to adapt to any kind of stresses is integral to developing stress tolerance in crop plants. In this study, PCR-based cDNA suppression subtractive hybridization technique was used to construct sugarcane salt (NaCl) stress specific forward and reverse subtracted cDNA library. For this, mRNAs were pooled from the shoot and root tissues stressed with NaCl (200 mM) for various time intervals (0.5 to 18 h). Sequencing the clones from the forward subtracted cDNA library, we identified shaggy-like protein kinase (hereafter referred as sugarcane shaggy-like protein kinase, SuSK; NCBI GenBank EST database Acc: FG804674). The sequence analysis of the SuSK revealed homology to Arabidopsis thaliana shaggy-related protein kinase delta (E value, 1e(-108)), dzeta and iota. Alignment of the catalytic domain sequence of GSK-3/shaggy-like kinase with partial sequence of SuSK performed using ClustalW tool indicated kinase active-site signature sequence. Spatial and temporal transcript expression profiling of the SuSK gene based on Real-Time PCR revealed significant induction of transcript expression in response to short-term salt (NaCl 200 mM) or polyethylene glycol-8,000 (PEG; 20% w/v) induced osmotic stress in leaves and shoots of sugarcane plants. The transcript expression increased progressively under salt stress and reached to 1.5-fold of the control up to 8 h treatment. In response to PEG stress, the transcript expression increased by 1.5-fold over the control in 2-h treatment in leaf, whereas in shoots, the expression remained unchanged in response to the various treatments. Differences in growth parameters, relative water content, and membrane damage rate were statistically insignificant in the short-term salt or PEG-stressed plants as compared to the control, non-stressed plants. Expression analysis revealed the differential and temporal regulation of this gene under salt and PEG stress and that its early induction may

  12. The control in cis of the position and the amount of the ARG4 meiotic double-strand break of Saccharomyces cerevisiae.

    PubMed Central

    de Massy, B; Nicolas, A

    1993-01-01

    During meiosis, a transient DNA double-strand break (DSB) occurs in the promoter region (positions -200/-185) of the Saccharomyces cerevisiae ARG4 gene and is a likely intermediate in the initiation of meiotic gene conversion events in this region. We report here a functional analysis of the ARG4 DSB based on the study of various deletions in this chromosomal region. We have identified several cis-acting elements located within the -465/+3 region of the ARG4 promoter that control the formation of this DSB. The -465/-317 region includes a transcription terminator and is necessary for a normal amount of ARG4 DSB, but not for its positioning. The -316/-140 region can be replaced by an unrelated DNA sequence where a meiotic DSB then occurs, suggesting that the site of DSB is not sequence-specific, but is positioned at a fixed distance from the adjacent -139/+3 region. Also, in all strains constructed, the amount of meiotic DSB is correlated with the frequency of gene conversion in ARG4, which provides a strong argument for the initiation of gene conversion by a DSB in this region of the yeast genome. Images PMID:8467798

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

    PubMed

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

    1992-08-01

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

  14. 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.

  15. 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 adult, and this co-expression was also especially evident in the svz of the hypothalamic region. xUlk4 was also expressed in the radial glia along the whole brain. We have also found minor expression of xUlk4 in some DCX- or MAP2-positive cells but not in TH-positive neurons. These findings suggest that Ulk4 may play roles in neural stem/progenitor cells during neurogenesis both in development and in the adulthood, in migrating cells as well as in cells committed to neuronal fate in Xenopus. Moreover, the results obtained in this study argue for an involvement of Ulk4 in the control of the neuroendocrine homeostatic functions in the brain. PMID:25637795

  16. Eradication of Myc-overexpressing small cell lung cancer cells transfected with herpes simplex virus thymidine kinase gene containing Myc-Max response elements.

    PubMed

    Kumagai, T; Tanio, Y; Osaki, T; Hosoe, S; Tachibana, I; Ueno, K; Kijima, T; Horai, T; Kishimoto, T

    1996-01-15

    Herpes simplex virus thymidine kinase (HSV-TK) gene was ligated with four repeats of the Myc-Max response elements (a core nucleotide sequence CACGTG), and its utility for gene therapy was examined by the treatment of either c-, L- or N-myc-overexpressing the small cell lung cancer (SCLC) cell line with ganciclovir (GCV). The chloramphenicol acetyltransferase assay demonstrated that the overexpression of any myc genes activated transcription from the CAT gene depending on the Myc-Max binding sites. The transduction of the HSV-TK gene ligated with the CACGTG core rendered all three SCLC lines to be more sensitive to GCV than parental ones in vitro. In addition, the growth of c- or L-myc-overexpressing SCLC cells containing the hybrid HSV-TK gene were significantly suppressed by GCV in vivo. When parental SCLC cells were mixed with HSV-TK-expressing tumor cells at a ratio of 1:3, GCV treatment inhibited tumor growth by 90% compared with parental cells only, indicating the existence of the "bystander effect." These data suggest that the CACGTG-driven HSV-TK gene may be useful for the treatment of SCLC overexpressing any type of myc family oncogenes.

  17. Three members of the human pyruvate dehydrogenase kinase gene family are direct targets of the peroxisome proliferator-activated receptor beta/delta.

    PubMed

    Degenhardt, Tatjana; Saramäki, Anna; Malinen, Marjo; Rieck, Markus; Väisänen, Sami; Huotari, Anne; Herzig, Karl-Heinz; Müller, Rolf; Carlberg, Carsten

    2007-09-14

    The nuclear receptors peroxisome proliferator-activated receptors (PPARs) are known for their critical role in the metabolic syndrome. Here, we show that they are direct regulators of the family of pyruvate dehydrogenase kinase (PDK) genes, whose products act as metabolic homeostats in sensing hunger and satiety levels in key metabolic tissues by modulating the activity of the pyruvate dehydrogenase complex. Mis-regulation of this tightly controlled network may lead to hyperglycemia. In human embryonal kidney cells we found the mRNA expression of PDK2, PDK3 and PDK4 to be under direct primary control of PPAR ligands, and in normal mouse kidney tissue Pdk2 and Pdk4 are PPAR targets. Both, treatment of HEK cells with PPARbeta/delta-specific siRNA and the genetic disruption of the Pparbeta/delta gene in mouse fibroblasts resulted in reduced expression of Pdk genes and abolition of induction by PPARbeta/delta ligands. These findings suggest that PPARbeta/delta is a key regulator of PDK genes, in particular the PDK4/Pdk4 gene. In silico analysis of the human PDK genes revealed two candidate PPAR response elements in the PDK2 gene, five in the PDK3 gene and two in the PDK4 gene, but none in the PDK1 gene. For seven of these sites we could demonstrate both PPARbeta/delta ligand responsiveness in context of their chromatin region and simultaneous association of PPARbeta/delta with its functional partner proteins, such as retinoidXreceptor, co-activator and mediator proteins and phosphorylated RNA polymerase II. In conclusion, PDK2, PDK3 and PDK4 are primary PPARbeta/delta target genes in humans underlining the importance of the receptor in the control of metabolism. PMID:17669420

  18. A Multi-Scale Settlement Matching Algorithm Based on ARG

    NASA Astrophysics Data System (ADS)

    Yue, Han; Zhu, Xinyan; Chen, Di; Liu, Lingjia

    2016-06-01

    Homonymous entity matching is an important part of multi-source spatial data integration, automatic updating and change detection. Considering the low accuracy of existing matching methods in dealing with matching multi-scale settlement data, an algorithm based on Attributed Relational Graph (ARG) is proposed. The algorithm firstly divides two settlement scenes at different scales into blocks by small-scale road network and constructs local ARGs in each block. Then, ascertains candidate sets by merging procedures and obtains the optimal matching pairs by comparing the similarity of ARGs iteratively. Finally, the corresponding relations between settlements at large and small scales are identified. At the end of this article, a demonstration is presented and the results indicate that the proposed algorithm is capable of handling sophisticated cases.

  19. Evolution of BCR/ABL Gene Mutation in CML Is Time Dependent and Dependent on the Pressure Exerted by Tyrosine Kinase Inhibitor

    PubMed Central

    Vaidya, Shantashri; Vundinti, Babu Rao; Shanmukhaiah, Chandrakala; Chakrabarti, Prantar; Ghosh, Kanjaksha

    2015-01-01

    Background Mutations in the ABL kinase domain and SH3-SH2 domain of the BCR/ABL gene and amplification of the Philadelphia chromosome are the two important BCR/ABL dependent mechanisms of imatinib resistance. Here, we intended to study the role played by TKI, imatinib, in selection of gene mutations and development of chromosomal abnormalities in Indian CML patients. Methods Direct sequencing methodology was employed to detect mutations and conventional cytogenetics was done to identify Philadelphia duplication. Results Among the different mechanisms of imatinib resistance, kinase domain mutations (39%) of the BCR/ABL gene were seen to be more prevalent, followed by mutations in the SH3-SH2 domain (4%) and then BCR/ABL amplification with the least frequency (1%). The median duration of occurrence of mutation was significantly shorter for patients with front line imatinib than those pre-treated with hydroxyurea. Patients with high Sokal score (p = 0.003) showed significantly higher incidence of mutations, as compared to patients with low/intermediate score. Impact of mutations on the clinical outcome in AP and BC was observed to be insignificant. Of the 94 imatinib resistant patients, only 1 patient exhibited duplication of Philadelphia chromosome, suggesting a less frequent occurrence of this abnormality in Indian CML patients. Conclusion Close monitoring at regular intervals and proper analysis of the disease resistance would facilitate early detection of resistance and thus aid in the selection of the most appropriate therapy. PMID:25629972

  20. The Receiver of the Agrobacterium tumefaciens VirA Histidine Kinase Forms a Stable Interaction with VirG to Activate Virulence Gene Expression.

    PubMed

    Wise, Arlene A; Binns, Andrew N

    2015-01-01

    The plant pathogen Agrobacterium tumefaciens carries a virulence gene system that is required for the initiation of crown gall tumors on susceptible plants. Expression of the vir genes is activated by the VirA/VirG two component regulatory system. VirA is a histidine kinase which signals the presence of certain chemicals found at the site of a plant wound. The receiver domain located at its carboxyl terminus defines VirA as a hybrid histidine kinase. Here, we show that the VirA receiver interacts with the DNA-binding domain of VirG. This finding supports the hypothesis that the receiver acts as a recruiting factor for VirG. In addition, we show that removal of the VirA receiver allowed vir gene expression in response to glucose in a dose dependent manner, indicating that the receiver controls VirG activation and suggesting that the supplementary ChvE-sugar signal increases this activity. PMID:26779177

  1. The Receiver of the Agrobacterium tumefaciens VirA Histidine Kinase Forms a Stable Interaction with VirG to Activate Virulence Gene Expression

    PubMed Central

    Wise, Arlene A.; Binns, Andrew N.

    2016-01-01

    The plant pathogen Agrobacterium tumefaciens carries a virulence gene system that is required for the initiation of crown gall tumors on susceptible plants. Expression of the vir genes is activated by the VirA/VirG two component regulatory system. VirA is a histidine kinase which signals the presence of certain chemicals found at the site of a plant wound. The receiver domain located at its carboxyl terminus defines VirA as a hybrid histidine kinase. Here, we show that the VirA receiver interacts with the DNA-binding domain of VirG. This finding supports the hypothesis that the receiver acts as a recruiting factor for VirG. In addition, we show that removal of the VirA receiver allowed vir gene expression in response to glucose in a dose dependent manner, indicating that the receiver controls VirG activation and suggesting that the supplementary ChvE-sugar signal increases this activity. PMID:26779177

  2. Systematic Mutational Analysis of Histidine Kinase Genes in the Nosocomial Pathogen Stenotrophomonas maltophilia Identifies BfmAK System Control of Biofilm Development

    PubMed Central

    Zheng, Liu; Wang, Fang-Fang; Ren, Bao-Zhen; Liu, Wei

    2016-01-01

    The Gram-negative bacterium Stenotrophomonas maltophilia lives in diverse ecological niches. As a result of its formidable capabilities of forming biofilm and its resistance to multiple antibiotic agents, the bacterium is also a nosocomial pathogen of serious threat to the health of patients whose immune systems are suppressed or compromised. Besides the histidine kinase RpfC, the two-component signal transduction system (TCS), which is the canonical regulatory machinery used by most bacterial pathogens, has never been experimentally investigated in S. maltophilia. Here, we annotated 62 putative histidine kinase genes in the S. maltophilia genome and successfully obtained 51 mutants by systematical insertional inactivation. Phenotypic characterization identified a series of mutants with deficiencies in bacterial growth, swimming motility, and biofilm development. A TCS, named here BfmA-BfmK (Smlt4209-Smlt4208), was genetically confirmed to regulate biofilm formation in S. maltophilia. Together with interacting partner prediction and chromatin immunoprecipitation screens, six candidate promoter regions bound by BfmA in vivo were identified. We demonstrated that, among them, BfmA acts as a transcription factor that binds directly to the promoter regions of bfmA-bfmK and Smlt0800 (acoT), a gene encoding an acyl coenzyme A thioesterase that is associated with biofilm development, and positively controls their transcription. Genome-scale mutational analyses of histidine kinase genes and functional dissection of BfmK-BfmA regulation in biofilm provide genetic information to support more in-depth studies on cellular signaling in S. maltophilia, in the context of developing novel approaches to fight this important bacterial pathogen. PMID:26873318

  3. No association of a casein kinase 1ε (CK1ε) gene polymorphism with personality traits in healthy Chinese-Han subjects.

    PubMed

    Li, Jingying; Ma, Huan; Huang, Yinglin; Wu, Lijuan; Li, Jun; Zhao, Xiaofeng; Jin, Qiu; Zhu, Gang

    2012-07-01

    The human casein kinase 1 (CK1) family is comprised of seven monomeric serine/threonine kinases (α, β, γ1-γ3, δ, and ε) encoded by seven highly conserved genes. Casein kinases modulate numerous biological and pathological processes by regulating the phosphorylation of the 32 kDa dopamine- and cAMP-regulated phosphoprotein DARPP-32, a major downstream regulator of dopamine signaling. Individual variation in the dopamine signaling system is thought to determine certain dimensions of personality, but there have been no published studies investigating the involvement of CK1 in the biological determination of temperament. We examined the association between the rs135745C/G polymorphism of the CK1ε gene CSNK1E and personality traits as measured by the tridimensional personality questionnaire in healthy Chinese-Han subjects. There were no differences in the total scores for novelty seeking (NS, χ (2) = 4.151, P = 0.125), harm avoidance (χ (2) = 3.299, P = 0.192), or reward dependence (χ (2) = 0.816, P = 0.665) between the rs135745C/G genotypes. In the sub-item analyses, the NS1 scores were significantly different (χ (2) = 7.024, P = 0.030) between rs135745C/G genotypes. However, this difference did not remain statistically significant after Bonferroni correction. Thus, our results did not provide evidence for the association between CK1ε gene and personality traits in healthy Chinese-Han subjects.

  4. OsAGSW1, an ABC1-like kinase gene, is involved in the regulation of grain size and weight in rice.

    PubMed

    Li, Tao; Jiang, Jieming; Zhang, Shengchun; Shu, Haoran; Wang, Yaqin; Lai, Jianbin; Du, Jinju; Yang, Chengwei

    2015-09-01

    Grain shape and weight are two determining agronomic traits of rice yield. ABC1 (Activity of bc1 complex) is a newly found atypical kinase in plants. Here, we report on an ABC1 protein kinase gene, OsAGSW1 (ABC1-like kinase related to Grain size and Weight). Expression of OsAGSW1-GFP in rice revealed that OsAGSW1 is localized to the chloroplasts in rice. Analysis of OsAGSW1 promoter::β-glucuronidase transgenic rice indicated that this gene was highly expressed in vascular bundles in shoot, hull and caryopsis. Furthermore, OsAGSW1-RNAi and overexpressed transgenic rice lines were generated. Stable transgenic lines overexpressing OsAGSW1 exhibited a phenotype with a significant increase in grain size, grain weight, grain filling rate and 1000-grain weight compared with the wild-type and RNAi transgenic plants. Microscopy analysis showed that spikelet hulls just before heading were different in the OsAGSW1-overexpressed plants compared with wild-type and OsAGSW1 RNAi rice. Further cytological analysis showed that the number of external parenchyma cells in rice hulls of OsAGSW1-overexpressed plants increased, leading to wider and longer spikelet hulls than those of the wild-type and OsAGSW1-RNAi plants. The vascular cross-sectional area in lemma, carpopodium and ovules also strikingly increased and area of both xylem and phloem were enlarged in the OsAGSW1-overexpressed plants. Thus, our results demonstrated that OsAGSW1 plays an important role in seed shape and size of rice by regulating the number of external parenchyma cells and the development of vascular bundles, providing a new insight into the functions of ABC1 genes in plants.

  5. Multiple genes in the left half of the cag pathogenicity island of Helicobacter pylori are required for tyrosine kinase-dependent transcription of interleukin-8 in gastric epithelial cells.

    PubMed

    Li, S D; Kersulyte, D; Lindley, I J; Neelam, B; Berg, D E; Crabtree, J E

    1999-08-01

    Helicobacter pylori strains that contain the cag pathogenicity island (PAI) elicit increased synthesis of gastric C-X-C chemokines, promote neutrophilic infiltration into the gastric epithelium, and stimulate the synthesis of interleukin-8 (IL-8) in cultured gastric epithelial cells. To investigate the effects of cag PAI genes on the transcription of the IL-8 gene, the Kato-3 gastric epithelial cell line was stably transfected with plasmid DNA containing the IL-8 gene promoter fused to a luciferase reporter gene. The resulting reporter cell line, L5F11, was used to monitor the effects of infection in cell culture by H. pylori 26695 and isogenic derivatives with null mutations in genes in the cag PAI on transcription of the IL-8 gene. We found that null mutations in eight open reading frames, including homologs of the Agrobacterium virB9, virB10, and virB11 genes, in the left half of the cag PAI abrogated the induction of IL-8 gene transcription. Further studies with the L5F11 cell line showed that IL-8 gene transcription induced by H. pylori was blocked by the protein tyrosine kinase inhibitor herbimycin A but not by the protein kinase C inhibitor calphostin C or by the protein kinase G inhibitor KT5823. IL-8 gene transcription in L5F11 cells could also be induced by the cytokine tumor necrosis factor alpha (TNF-alpha) without exposure to H. pylori. This TNF-alpha-induced IL-8 transcription was inhibited by the protein kinase A inhibitor H7, which had no significant effect on H. pylori-induced IL-8 transcription. These studies show that multiple genes in the left half of the cag PAI are essential for the transcription of the IL-8 gene in gastric epithelial cells and that this depends on protein tyrosine kinase activation. PMID:10417153

  6. Toward understanding the cationicity of defensins. Arg and Lys versus their noncoded analogs.

    PubMed

    Zou, Guozhang; de Leeuw, Erik; Li, Chong; Pazgier, Marzena; Li, Changqing; Zeng, Pengyun; Lu, Wei-Yue; Lubkowski, Jacek; Lu, Wuyuan

    2007-07-01

    Human defensins are a family of small antimicrobial proteins found predominantly in leukocytes and epithelial cells that play important roles in the innate and adaptive immune defense against microbial infection. The most distinct molecular feature of defensins is cationicity, manifested by abundant Arg and/or Lys residues in their sequences. Sequence analysis indicates that Arg is strongly selected over Lys in alpha-defensins but not in beta-defensins. To understand this Arg/Lys disparity in defensins, we chemically synthesized human alpha-defensin 1 (HNP1) and several HNP1 analogs where three Arg residues were replaced by each of the following six alpha-amino acids: Lys, ornithine (Orn), diaminobutyric acid (Dab), diaminopropionic acid (Dap), N,N-dimethyl-Lys ((diMe)Lys), and homo-Arg ((homo)Arg). In addition, we prepared human beta-defensin 1 (hBD1) and (Lys-->Arg)hBD1 in which all four Lys residues were substituted for Arg. Bactericidal activity assays revealed the following. 1) Arg-containing HNP1 and (Lys-->Arg)hBD1 are functionally better than Lys-HNP1 and hBD1, respectively; the difference between Arg and Lys is more evident in the alpha-defensin than in the beta-defensin and is more evident at low salt concentrations than at high salt concentrations. 2) For HNP1, the Arg/Lys disparity is much more pronounced with Staphylococcus aureus than with Escherichia coli, and the Arg-rich HNP1 kills bacteria faster than its Lys-rich analog. 3) Arg and Lys appear to have optimal chain lengths for bacterial killing as shortening Lys or lengthening Arg in HNP1 invariably becomes functionally deleterious. Our findings provide insights into the Arg/Lys disparity in defensins, and shed light on the cationicity of defensins with respect to their antimicrobial activity and specificity.

  7. Homeodomain-interacting protein kinases (Hipks) promote Wnt/Wg signaling through stabilization of beta-catenin/Arm and stimulation of target gene expression.

    PubMed

    Lee, Wendy; Swarup, Sharan; Chen, Joanna; Ishitani, Tohru; Verheyen, Esther M

    2009-01-01

    The Wnt/Wingless (Wg) pathway represents a conserved signaling cascade involved in diverse biological processes. Misregulation of Wnt/Wg signal transduction has profound effects on development. Homeodomain-interacting protein kinases (Hipks) represent a novel family of serine/threonine kinases. Members of this group (in particular Hipk2) are implicated as important factors in transcriptional regulation to control cell growth, apoptosis and development. Here, we provide genetic and phenotypic evidence that the sole Drosophila member of this family, Hipk, functions as a positive regulator in the Wg pathway. Expression of hipk in the wing rescues loss of the Wg signal, whereas loss of hipk can enhance decreased wg signaling phenotypes. Furthermore, loss of hipk leads to diminished Arm protein levels, whereas overexpression of hipk promotes the Wg signal by stabilizing Arm, resulting in activation of Wg responsive targets. In Wg transcriptional assays, Hipk enhanced Tcf/Arm-mediated gene expression in a kinase-dependent manner. In addition, Hipk can bind to Arm and Drosophila Tcf, and phosphorylate Arm. Using both in vitro and in vivo assays, Hipk was found to promote the stabilization of Arm. We observe similar molecular interactions between Lef1/beta-catenin and vertebrate Hipk2, suggesting a direct and conserved role for Hipk proteins in promoting Wnt signaling. PMID:19088090

  8. Transcriptome analysis of cyclic AMP-dependent protein kinase A-regulated genes reveals the production of the novel natural compound fumipyrrole by Aspergillus fumigatus.

    PubMed

    Macheleidt, Juliane; Scherlach, Kirstin; Neuwirth, Toni; Schmidt-Heck, Wolfgang; Straßburger, Maria; Spraker, Joseph; Baccile, Joshua A; Schroeder, Frank C; Keller, Nancy P; Hertweck, Christian; Heinekamp, Thorsten; Brakhage, Axel A

    2015-04-01

    Aspergillus fumigatus is an opportunistic human pathogenic fungus causing life-threatening infections in immunocompromised patients. Adaptation to different habitats and also virulence of the fungus depends on signal perception and transduction by modules such as the cyclic AMP-dependent protein kinase A (PKA) pathway. Here, by transcriptome analysis, 632 differentially regulated genes of this important signaling cascade were identified, including 23 putative transcriptional regulators. The highest upregulated transcription factor gene was located in a previously unknown secondary metabolite gene cluster, which we named fmp, encoding an incomplete non-ribosomal peptide synthetase, FmpE. Overexpression of the regulatory gene fmpR using the Tet(On) system led to the specific expression of the other six genes of the fmp cluster. Metabolic profiling of wild type and fmpR overexpressing strain by HPLC-DAD and HPLC-HRESI-MS and structure elucidation by NMR led to identification of 5-benzyl-1H-pyrrole-2-carboxylic acid, which we named fumipyrrole. Fumipyrrole was not described as natural product yet. Chemical synthesis of fumipyrrole confirmed its structure. Interestingly, deletion of fmpR or fmpE led to reduced growth and sporulation of the mutant strains. Although fmp cluster genes were transcribed in infected mouse lungs, deletion of fmpR resulted in wild-type virulence in a murine infection model.

  9. Enhanced green fluorescent protein fusion proteins of herpes simplex virus type 1 thymidine kinase and cytochrome P450 4B1: applications for prodrug-activating gene therapy.

    PubMed

    Steffens, S; Frank, S; Fischer, U; Heuser, C; Meyer, K L; Dobberstein, K U; Rainov, N G; Kramm, C M

    2000-05-01

    To monitor therapeutic transgene expression, we developed fusion genes of enhanced green fluorescent protein (EGFP) with two different prodrug-activating enzyme genes: herpes simplex virus type 1 thymidine kinase (HSV-tk) and rabbit cytochrome P450 4B1 (cyp4b1). Expression of the resulting fusion proteins, TK-EGFP and 4B1-EGFP, rendered transduced human and rodent glioma cells sensitive to cytotoxic treatment with the corresponding prodrugs ganciclovir and 4-ipomeanol. Ganciclovir and 4-ipomeanol sensitivity was comparable with that achieved with the native HSV-TK and CYP4B1 proteins. As shown by fluorescence microscopy, TK-EGFP was expressed predominantly intranuclearly, whereas 4B1-EGFP was detectable in the cytoplasm, thereby displaying the orthotopic subcellular distribution of the corresponding native enzymes. The fluorescence intensity correlated well with the corresponding prodrug sensitivity, as shown by fluorescence-activated cell sorter analysis. EGFP expression was also used for the selection of stably HSV-tk-transduced cells by flow cytometric cell sorting. Resulting cell populations showed a homogeneity of fluorescence intensity similar to single-cell clones after antibiotic selection. In conclusion, tk-egfp and 4b1-egfp fusion genes are valuable tools for monitoring prodrug-activating gene therapy in living cells. EGFP fusion genes/proteins provide a simple and reproducible means for the detection, selection, and characterization of cells expressing enzyme genes for prodrug activation.

  10. Haem-regulated eIF2α kinase is necessary for adaptive gene expression in erythroid precursors under the stress of iron deficiency

    PubMed Central

    Liu, Sijin; Bhattacharya, Sanchita; Han, Anping; Suragani, Rajasekhar N. V. S.; Zhao, Wanting; Fry, Rebecca C.; Chen, Jane-Jane

    2016-01-01

    Summary Haem-regulated eIF2α kinase (HRI) is essential for the regulation of globin gene translation and the survival of erythroid precursors in iron/haem deficiency. This study found that that in iron deficiency, fetal definitive erythropoiesis is inhibited at the basophilic erythroblast stage with increased proliferation and elevated apoptosis. This hallmark of ineffective erythropoiesis is more severe in HRI deficiency. Microarray gene profiling analysis showed that HRI was required for adaptive gene expression in erythroid precursors during chronic iron deficiency. The number of genes with expression affected more than twofold increased, from 213 in iron deficiency and 73 in HRI deficiency, to 3135 in combined iron and HRI deficiencies. Many of these genes are regulated by Gata1 and Fog1. We demonstrate for the first time that Gata1 expression in developing erythroid precursors is decreased in iron deficiency, and is decreased further in combined iron and HRI deficiencies. Additionally, Fog1 expression is decreased in combined deficiencies, but not in iron or HRI deficiency alone. Our results indicate that HRI confers adaptive gene expression in developing erythroblasts during iron deficiency through maintaining Gata1/Fog1 expression. PMID:18665838

  11. Arg16Gly and Gln27Glu β2 adrenergic polymorphisms influence cardiac autonomic modulation and baroreflex sensitivity in healthy young Brazilians

    PubMed Central

    Atala, Magda M; Goulart, Alessandra; Guerra, Grazia M; Mostarda, Cristiano; Rodrigues, Bruno; Mello, Priscila R; Casarine, Dulce E; Irigoyen, Maria-Claudia; Pereira, Alexandre C; Consolim-Colombo, Fernanda M

    2015-01-01

    The association between functional β2 adrenergic receptor (β2-AR) polymorphisms and cardiac autonomic modulation is still unclear. Thus, two common polymorphisms in the β2-AR gene (Gln27Glu β2 and Arg16Gly β2) were studied to determine whether they might affect tonic and reflex cardiac sympathetic activity in healthy young subjects. A total of 213 healthy young white subjects of both genders (53% female), aged 18-30 years (23.5±3.4 y), had their continuous blood pressure curves noninvasively recorded by Finometer at baseline, and other hemodynamic parameters, as cardiac autonomic modulation, baroreflex sensitivity, and allele, genotype, and diplotype frequencies calculated. Associations were made between Arg16Gly β2 and Gln27Glu β2 polymorphisms and between β2-AR diplotypes and all variables. The heart rate was significantly lower (P<0.001) in the presence of homozygous Arg/Arg alleles (60.9±1.5 bpm) than in that of Arg/Gly heterozygotes (65.9±1.0 bpm) or Gly/Gly homozygotes (66.3±1.2 bpm). Homozygous carriers of Arg16 allele had an alpha index (19.2±1.3) significantly higher (P<0.001) than that of the subjects with the Gly allele Gly/Gly (14.5±0.7) or Arg/Gly (14.6±0.7). Furthermore, the recessive Glu27Glu and the heterozygous Gln27Glu genotypes had a higher percentage of low-frequency components (LF%) than the homozygous Gln27Gln (15.1% vs. 16.0% vs. 8.2%, P=0.03, respectively). In healthy young subjects, the presence of β2-AR Arg16 allele in a recessive model was associated with higher baroreflex sensitivity, and increased parasympathetic modulation in studied individuals. PMID:25755837

  12. Characterization of 5' end of human thromboxane receptor gene. Organizational analysis and mapping of protein kinase C--responsive elements regulating expression in platelets.

    PubMed

    D'Angelo, D D; Davis, M G; Houser, W A; Eubank, J J; Ritchie, M E; Dorn, G W

    1995-09-01

    Platelet thromboxane receptors are acutely and reversibly upregulated after acute myocardial infarction. To determine if platelet thromboxane receptors are under transcriptional control, we isolated and characterized human genomic DNA clones containing the 5' flanking region of the thromboxane receptor gene. The exon-intron structure of the 5' portion of the thromboxane receptor gene was determined initially by comparing the nucleotide sequence of the 5' flanking genomic clone with that of a novel human uterine thromboxane receptor cDNA that extended the mRNA 141 bp further upstream than the previously identified human placental cDNA. A major transcription initiation site was located in three human tissues approximately 560 bp upstream from the translation initiation codon and 380 bp upstream from any previously identified transcription initiation site. The thromboxane receptor gene has neither a TATA nor a CAAT consensus site. Promoter function of the 5' flanking region of the thromboxane receptor gene was evaluated by transfection of thromboxane receptor gene promoter/chloramphenicol acetyltransferase (CAT) chimera plasmids into platelet-like K562 cells. Thromboxane receptor promoter activity, as assessed by CAT expression, was relatively weak but was significantly enhanced by phorbol ester treatment. Functional analysis of 5' deletion constructs in transfected K562 cells and gel mobility shift localized the major phorbol ester-responsive motifs in the thromboxane receptor gene promoter to a cluster of activator protein-2 (AP-2) binding consensus sites located approximately 1.8 kb 5' from the transcription initiation site. These studies are the first to determine the structure and organization of the 5' end of the thromboxane receptor gene and demonstrate that thromboxane receptor gene expression can be regulated by activation of protein kinase C via induction of an AP-2-like nuclear factor binding to upstream promoter elements. These findings strongly suggest

  13. Pegylated recombinant human arginase (rhArg-peg5,000mw) inhibits the in vitro and in vivo proliferation of human hepatocellular carcinoma through arginine depletion.

    PubMed

    Cheng, Paul Ning-Man; Lam, Tin-Lun; Lam, Wai-Man; Tsui, Sam-Mui; Cheng, Anthony Wai-Ming; Lo, Wai-Hung; Leung, Yun-Chung

    2007-01-01

    Hepatocellular carcinoma (HCC) is believed to be auxotrophic for arginine through the lack of expression of argininosuccinate synthetase (ASS). The successful use of the arginine-depleting enzyme arginine deiminase (ADI) to treat ASS-deficient tumors has opened up new possibilities for effective cancer therapy. Nevertheless, many ASS-positive HCC cell lines are found to be resistant to ADI treatment, although most require arginine for proliferation. Thus far, an arginine-depleting enzyme for killing ASS-positive tumors has not been reported. Here, we provide direct evidence that recombinant human arginase (rhArg) inhibits ASS-positive HCCs. All the five human HCC cell lines we used were sensitive to rhArg but ADI had virtually no effect on these cells. They all expressed ASS, but not ornithine transcarbamylase (OTC), the enzyme that converts ornithine, the product of degradation of arginine with rhArg, to citrulline, which is converted back to arginine via ASS. Transfection of HCC cells with OTC resulted in resistance to rhArg. Thus, OTC expression alone may be sufficient to induce rhArg resistance in ASS-positive HCC cells. This surprising correlation between the lack of OTC expression and sensitivity of ASS-positive HCC cells shows that OTC-deficient HCCs are sensitive to rhArg-mediated arginine depletion. Therefore, pretreatment tumor gene expression profiling of ASS and OTC could aid in predicting tumor response to arginine depletion with arginine-depleting enzymes. We have also shown that the rhArg native enzyme and the pegylated rhArg (rhArg-peg(5,000mw)) gave similar anticancer efficacy in vitro. Furthermore, the growth of the OTC-deficient Hep3B tumor cells (ASS-positive and ADI-resistant) in mice was inhibited by treatment with rhArg-peg(5,000mw), which is active alone and is synergistic in combination with 5-fluorouracil. Thus, our data suggest that rhArg-peg(5,000mw) is a novel agent for effective cancer therapy.

  14. N-cadherin mediated distribution of beta-catenin alters MAP kinase and BMP-2 signaling on chondrogenesis-related gene expression.

    PubMed

    Modarresi, Rozbeh; Lafond, Toulouse; Roman-Blas, Jorge A; Danielson, Keith G; Tuan, Rocky S; Seghatoleslami, M Reza

    2005-05-01

    We have examined the effect of calcium-dependent adhesion, mediated by N-cadherin, on cell signaling during chondrogenesis of multipotential embryonic mouse C3H10T1/2 cells. The activity of chondrogenic genes, type II collagen, aggrecan, and Sox9 were examined in monolayer (non-chondrogenic), and micromass (chondrogenic) cultures of parental C3H10T1/2 cells and altered C3H10T1/2 cell lines that express a dominant negative form of N-cadherin (delta390-T1/2) or overexpress normal N-cadherin (MNCD2-T1/2). Our findings show that missexpression or inhibition of N-cadherin in C3H10T1/2 cells results in temporal and spatial changes in expression of the chondrogenic genes Sox9, aggrecan, and collagen type II. We have also analyzed activity of the serum response factor (SRF), a nuclear target of MAP kinase signaling implicated in chondrogenesis. In semi-confluent monolayer cultures (minimum cell-cell contact) of C3H10T1/2, MNCD2-T1/2, or delta390-T1/2 cells, there was no significant change in the pattern of MAP kinase or bone morphogenetic protein-2 (BMP-2) regulation of SRF. However, in micromass cultures, the effect of MAP kinase and BMP-2 on SRF activity was proportional to the nuclear localization of beta-catenin, a Wnt stabilized cytoplasmic factor that can associate with lymphoid enhancer-binding factor (LEF) to serve as a transcription factor. Our findings suggest that the extent of adherens junction formation mediated by N-cadherin can modulate the potential Wnt-induced nuclear activity of beta-catenin. PMID:15723280

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

    PubMed

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

    2000-12-01

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

  16. [Role of the activating mutation Val617Phe of Janus kinase 2 gene in myeloproliferative diseases and significance of its detection].

    PubMed

    Andrikovics, Hajnalka; Szilvási, Anikó; Meggyesi, Nóra; Király, Viktória; Halm, Gabriella; Lueff, Sándor; Nahajevszky, Sarolta; Mikala, Gábor; Sipos, Andrea; Lovas, Nóra; Csukly, Zoltán; Mátrai, Zoltán; Tamáska, Júlia; Tordai, Attila; Masszi, Tamás

    2007-02-01

    The Val617Phe point mutation of Janus kinase 2 gene is believed to participate in the pathogenesis of myeloproliferative syndrome characterised by the clonal alteration of hematopoietic stem cells. According to current results, the frequency of Val617Phe activating mutation is around 80% in polycythaemia vera, 35% in essential thrombocythemia, and 50% in chronic idiopathic myelofibrosis. The diagnoses of polycythemia vera, essential thrombocythemia and idiopathic myelofibrosis were so far based on the exclusion of secondary factors as well as bone marrow biopsy histology. The goal of the present work was to establish simple molecular genetic techniques for the routine testing of Janus kinase 2 gene Val617Phe mutation, and to compare the clinical phenotypes of Val617Phe mutation positive and negative myeloproliferative syndromes. We employed the allele specific polymerase chain technique for detection of Val617Phe mutation in 252 patients with myeloproliferative syndrome. We measured Val617Phe frequency as 85,4% (117/137) in polycythemia vera, 56,6% (56/99) in essential thrombocythemia, and 87,5% (14/16) in idiopathic myelofibrosis. We found significantly elevated hemoglobin levels and white blood cell counts (measured at the time of diagnosis) in Val617Phe-positive polycythemia vera and essential thrombocythemia patient groups compared to Val617Phe-negative patients. However, the frequencies of splenomegaly and other complications (thrombosis, bleeding, transformation to acute leukemia) were not significantly different between the mutation-positive and negative groups. In conclusion, the non-invasive mutation analysis of the Janus kinase 2 Val617Phe is suitable for routine laboratory application and helps the differential diagnosis of myeloproliferative syndrome. Although the exact role of Val617Phe mutation testing has not yet been identified on the basis of a broad professional consensus, the testing is suggested in cases of erythrocytoses and thrombocytoses of

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

    PubMed Central

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

    2000-01-01

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

  18. Hepatic fibrinogen storage disease due to the fibrinogen γ375 Arg → Trp mutation "fibrinogen Aguadilla" is present in Arabs.

    PubMed

    Al-Hussaini, Abdulrahman; Altalhi, Abdulhadi; El Hag, Imad; AlHussaini, Hussa; Francalanci, Paola; Giovannoni, Isabella; Callea, Francesco

    2014-01-01

    The mutation γ375Arg → Trp (fibrinogen Aguadilla) is one of four mutations (Brescia, Aguadilla, Angers, and AI duPont) capable of causing hepatic storage of fibrinogen. It has been observed in four children from the Caribbean, Europe, and Japan, suffering from cryptogenic liver disease. We report the first case of hepatic fibrinogen storage disease in Arabs due to a mutation in the fibrinogen γ-chain gene in a 3-year-old Syrian girl presenting with elevated liver enzymes. The finding of an impressive accumulation of fibrinogen in liver cells raised the suspicion of endoplasmic reticulum storage disease. Sequencing of the fibrinogen genes revealed a γ375Arg → Trp mutation (fibrinogen Aguadilla) in the child and in her father. In conclusion, when confronted with chronic hepatitis of unknown origin, one should check the plasma fibrinogen level and look carefully for the presence of hepatocellular intracytoplasmic globular inclusions to exclude hepatic fibrinogen storage disease.

  19. Cyclin-dependent kinase 5 represses Foxp3 gene expression and Treg development through specific phosphorylation of Stat3 at Serine 727.

    PubMed

    Lam, Eric; Choi, Sung Hee; Pareek, Tej K; Kim, Byung-Gyu; Letterio, John J

    2015-10-01

    Cyclin-dependent kinase 5 (Cdk5) is known as a unique member of the cyclin-dependent family of serine/threonine kinases. Previously, we demonstrated Cdk5 to be an important regulator of T cell function and that disruption of Cdk5 expression ameliorates T cell mediated neuroinflammation. Here, we show a novel role of Cdk5 in the regulation of Foxp3 expression in murine CD4(+) T cells. Our data indicate that disruption of Cdk5 activity in T cells abrogates the IL-6 suppression of Foxp3 expression. This effect is achieved through Cdk5 phosphorylation of the signal transducer and activator of transcription 3 (Stat3) specifically at Serine 727 in T cells, and we show this post-translational modification is required for proper Stat3 DNA binding to the Foxp3 gene on the enhancer II region. Taken together, our data point to an essential role for Cdk5 in the differentiation of T cells as it regulates Foxp3 gene expression through phosphorylation of Stat3. PMID:26198700

  20. Down-regulation of the tumor suppressor gene retinoic acid receptor beta2 through the phosphoinositide 3-kinase/Akt signaling pathway.

    PubMed

    Lefebvre, Bruno; Brand, Céline; Flajollet, Sébastien; Lefebvre, Philippe

    2006-09-01

    The retinoic acid receptor beta2 (RARbeta2) is a potent, retinoid-inducible tumor suppressor gene, which is a critical molecular relay for retinoid actions in cells. Its down-regulation, or loss of expression, leads to resistance of cancer cells to retinoid treatment. Up to now, no primary mechanism underlying the repression of the RARbeta2 gene expression, hence affecting cellular retinoid sensitivity, has been identified. Here, we demonstrate that the phosphoinositide 3-kinase/Akt signaling pathway affects cellular retinoid sensitivity, by regulating corepressor recruitment to the RARbeta2 promoter. Through direct phosphorylation of the corepressor silencing mediator for retinoic and thyroid hormone receptors (SMRT), Akt stabilized RAR/SMRT interaction, leading to an increased tethering of SMRT to the RARbeta2 promoter, decreased histone acetylation, down-regulation of the RARbeta2 expression, and impaired cellular differentiation in response to retinoid. The phosphoinositide 3-kinase/Akt signaling pathway, an important modulator of cellular survival, has thus a direct impact on cellular retinoid sensitivity, and its deregulation may be the triggering event in retinoid resistance of cancer cells.

  1. The product of the Saccharomyces cerevisiae cell cycle gene DBF2 has homology with protein kinases and is periodically expressed in the cell cycle.

    PubMed Central

    Johnston, L H; Eberly, S L; Chapman, J W; Araki, H; Sugino, A

    1990-01-01

    Several Saccharomyces cerevisiae dbf mutants defective in DNA synthesis have been described previously. In this paper, one of them, dbf2, is characterized in detail. The DBF2 gene has been cloned and mapped, and its nucleotide sequence has been determined. This process has identified an open reading frame capable of encoding a protein of molecular weight 64,883 (561 amino acids). The deduced amino acid sequence contains all 11 conserved domains found in various protein kinases. DBF2 was periodically expressed in the cell cycle at a time that clearly differed from the time of expression of either the histone H2A or DNA polymerase I gene. Its first function was compl