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Sample records for pten-induced putative kinase

  1. Roles of PTEN-induced putative kinase 1 and dynamin-related protein 1 in transient global ischemia-induced hippocampal neuronal injury

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    Chen, Shang-Der, E-mail: chensd@adm.cgmh.org.tw [Department of Neurology, Kaohsiung Chang Gung Memorial Hospital, Chang Gung University College of Medicine, Taiwan (China); Center for Translational Research in Biomedical Sciences, Kaohsiung Chang Gung Memorial Hospital, Chang Gung University College of Medicine, Taiwan (China); Lin, Tsu-Kung [Department of Neurology, Kaohsiung Chang Gung Memorial Hospital, Chang Gung University College of Medicine, Taiwan (China); Yang, Ding-I. [Institute of Brain Science and Brain Research Center, National Yang-Ming University, Taipei, Taiwan (China); Lee, Su-Ying [Department of Neurology, Kaohsiung Chang Gung Memorial Hospital, Chang Gung University College of Medicine, Taiwan (China); Shaw, Fu-Zen [Department of Psychology, National Cheng Kung University, Tainan, Taiwan (China); Liou, Chia-Wei [Department of Neurology, Kaohsiung Chang Gung Memorial Hospital, Chang Gung University College of Medicine, Taiwan (China); Chuang, Yao-Chung, E-mail: ycchuang@adm.cgmh.org.tw [Department of Neurology, Kaohsiung Chang Gung Memorial Hospital, Chang Gung University College of Medicine, Taiwan (China); Center for Translational Research in Biomedical Sciences, Kaohsiung Chang Gung Memorial Hospital, Chang Gung University College of Medicine, Taiwan (China)

    2015-05-01

    Recent studies showed that increased mitochondrial fission is an early event of cell death during cerebral ischemia and dynamin-related protein 1 (Drp1) plays an important role in mitochondrial fission, which may be regulated by PTEN-induced putative kinase 1 (PINK1), a mitochondrial serine/threonine-protein kinase thought to protect cells from stress-induced mitochondrial dysfunction and regulate mitochondrial fission. However, the roles of PINK1 and Drp1 in hippocampal injury caused by transient global ischemia (TGI) remain unknown. We therefore tested the hypothesis that TGI may induce PINK1 causing downregulation of Drp1 phosphorylation to enhance hippocampal neuronal survival, thus functioning as an endogenous neuroprotective mechanism. We found progressively increased PINK1 expression in the hippocampal CA1 subfield1-48 h following TGI, reaching the maximal level at 4 h. Despite lack of changes in the expression level of total Drp1 and phosphor-Drp1 at Ser637, TGI induced a time-dependent increase of Drp1 phosphorlation at Ser616 that peaked after 24 h. Notably, PINK1-siRNA increased p-Drp1(Ser616) protein level in hippocampal CA1 subfield 24 h after TGI. The PINK1 siRNA also aggravated the TGI-induced oxidative DNA damage with an increased 8-hydroxy-deoxyguanosine (8-OHdG) content in hippocampal CA1 subfield. Furthermore, PINK1 siRNA also augmented TGI-induced apoptosis as evidenced by the increased numbers of TUNEL-positive staining and enhanced DNA fragmentation. These findings indicated that PINK1 is an endogenous protective mediator vital for neuronal survival under ischemic insult through regulating Drp1 phosphorylation at Ser616. - Highlights: • Transient global ischemia increases expression of PINK1 and p-Drp1 at Ser616 in hippocampal CA1 subfield. • PINK1-siRNA decreases PINK1 expression but increases p-Drp1 at Ser616 in hippocampal CA1 subfield. • PINK1-siRNA augments oxidative stress and neuronal damage in hippocampal CA1 subfield.

  2. Putative modifier genes in mevalonate kinase deficiency.

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    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.

  3. A novel putative tyrosine kinase receptor with oncogenic potential.

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    Janssen, J W; Schulz, A S; Steenvoorden, A C; Schmidberger, M; Strehl, S; Ambros, P F; Bartram, C R

    1991-11-01

    We have detected transforming activity by a tumorigenicity assay using NIH3T3 cells transfected with DNA from a chronic myeloproliferative disorder patient. Here, we report the cDNA cloning of the corresponding oncogene, designated UFO, in allusion to the as yet unidentified function of its protein. Nucleotide sequence analysis of a 3116bp cDNA clone revealed a 2682-bp-long open reading frame capable of directing the synthesis of a 894 amino acid polypeptide. The predicted UFO protein exhibits characteristic features of a transmembrane receptor with associated tyrosine kinase activity. The UFO proto-oncogene maps to human chromosome 19q13.1 and is transcribed into two 5.0 kb and 3.2 kb mRNAs in human bone marrow and human tumor cell lines. The UFO locus is evolutionarily conserved between vertebrate species. A 4.0 kb mRNA of the murine UFO homolog is expressed in a variety of different mouse tissues. We thus have identified a novel element of the complex signaling network involved in the control of cell proliferation and differentiation.

  4. PUTATIVE CREATINE KINASE M-ISOFORM IN HUMAN SPERM IS IDENTIFIED AS THE 70-KILODALTON HEAT SHOCK PROTEIN HSPA2

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    THE PUTATIVE CREATINE KINASE M-ISOFORM IN HUMAN SPERM IS IDENTIFIED AS THE 70 kDa HEAT SHOCK PROTEIN HSPA2* Gabor Huszar1, Kathryn Stone2, David Dix3 and Lynne Vigue11The Sperm Physiology Laboratory, Department of Obstetrics and Gynecology, 2 W.M. Keck Foundatio...

  5. Mutations within the putative active site of heterodimeric deoxyguanosine kinase block the allosteric activation of the deoxyadenosine kinase subunit.

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    Park, Inshik; Ives, David H

    2002-03-31

    Replacement of the Asp-84 residue of the deoxyguanosine kinase subunit of the tandem deoxyadenosine kinase/ deoxyguanosine kinase (dAK/dGK) from Lactobacillus acidophilus R-26 by Ala, Asn, or Glu produced increased Km values for deoxyguanosine on dGK. However, it did not seem to affect the binding of Mg-ATP. The Asp-84 dGK replacements had no apparent effect on the binding of deoxyadenosine by dAK. However, the mutant dGKs were no longer inhibited by dGTP, normally a potent distal endproduct inhibitor of dGK. Moreover, the allosteric activation of dAK activity by dGTP or dGuo was lost in the modified heterodimeric dAK/dGK enzyme. Therefore, it seems very likely that Asp-84 participates in dGuo binding at the active site of the dGK subunit of dAK/dGK from Lactobacillus acidophilus R-26.

  6. Two putative protein kinase CK2 phosphorylation sites are important for Myf-5 activity

    DEFF Research Database (Denmark)

    Winter, B; Kautzner, I; Issinger, O G;

    1997-01-01

    Myf-5, a member of a family of muscle-specific transcription factors, is important for myogenic cell determination and differentiation. Here, we report that Myf-5 protein constitutes a substrate for phosphorylation in vitro by protein kinase CK2. We identified two potential phosphorylation sites ...

  7. Identification of salt-inducible peptide with putative kinase activity in halophilic bacterium Virgibacillus halodenitrificans.

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    Rafiee, Mahmoud-Reza; Sokhansanj, Ashrafaddin; Yoosefi, Mitra; Naghizadeh, Mohammad-Ali

    2007-09-01

    Strain XII, a moderately halophilic bacterium, expressed a peptide in response to saline media. This peptide was designated as salt-inducible factor (Sif-A). The purpose of this study is to describe Sif-A, which might be involved in the osmoresistance mechanism of strain XII. The complete sequence of sif-A was determined using PCR. sif-A codes for a polypeptide of 20.518 kDa. The polypeptide has a putative signal peptide of 27 amino acids (2.667 kDa) preceding the mature protein (17.869 kDa). Motif analysis of the deduced amino acid sequence indicated that there is a p-loop NTPase domain on the C-terminal of the peptide, which might correlate with its function. The sequence of the 16S rRNA gene was analyzed phylogenetically to classify strain XII. This organism was found to have the closest association with Virgibacillus halodenitrificans, which was proven by its phenotypic characteristics.

  8. Sphingosine Kinase: A Novel Putative Target for the Prevention of Infection-Triggered Preterm Birth

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    Vibhuti Vyas

    2013-01-01

    Full Text Available Preterm birth is defined as any delivery before 37 complete weeks of gestation. It is a universal challenge in the field of obstetrics owing to its high rate of mortality, long-term morbidity, associated human suffering and economic burden. In the United States, about 12.18% deliveries in 2009 were preterm, producing an exorbitant cost of $5.8 billion. Infection-associated premature rupture of membranes (PROM accounts for 40% of extremely preterm births (<28 weeks of gestation. Major research efforts are directed towards improving the understanding of the pathophysiology of preterm birth and ways to prevent or at least postpone delivery. Endothelin-1 (ET-1 is a potent vasoconstrictor that plays a significant role in infection-triggered preterm birth. Its involvement in a number of pathological mechanisms and its elevation in preterm delivered amniotic fluid samples implicate it in preterm birth. Sphingosine kinase (SphK is a ubiquitous enzyme responsible for the production of sphingosine-1-phosphate (S1P. S1P acts as second messenger in a number of cell proliferation and survival pathways. SphK is found to play a key role in ET-1 mediated myometrial contraction. This review highlights SphK as a prospective target with great potential to prevent preterm birth.

  9. Involvement of a Gene Encoding Putative Acetate Kinase in Magnetosome Synthesis in Magnetospirillum magneticum AMB-1

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    ARIS TRI WAHYUDI

    2006-03-01

    Full Text Available A nonmagnetic mutant of Magnetospirillum magneticum AMB-1, designated NMA40, was constructed by mini-Tn5 transposon mutagenesis to identify genes involved in magnetosome synthesis. Transposon delivery was carried out through conjugation between M. magneticum AMB-1 as a recipient and Escherichia coli S17-1 (λ pir carrying pUTmini-Tn5Km1 as a donor strain. NAM40 did not respond to the magnetic fields and completely lacked of magnetosome in the cell. DNA sequence/gen interrupted by transposon (called flanking DNA was isolated by inverse PCR and cloned into pGEM-T Easy. Alignment of the DNA sequence of the flanking DNA allowed the isolation of an open reading frame (ORF2 within an operon consisting of three genes. The amino acid sequence deduced from ORF2 showed homology with acetate kinase from Sinorhizobium meliloti (50% identity and 67% similarity, which function for acetate metabolism. Further analysis revealed that upstream of ORF2 is ORF1, had homology with phosphotransacetylase of S. meliloti (67% identity, 77% similarity, and ORF3 located downstream of ORF2, had homology with hypothetical protein of Thermotoga maritima (30% identity, 60% similarity. ORF2 was subsequently isolated, cloned, and overexpressed in Escherichia coli BL21 (DE3 pLysS as an ORF2-Histag fusion polypeptide.

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

    Science.gov (United States)

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

    2016-09-20

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

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

    Science.gov (United States)

    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

  12. Schizosaccharomyces pombe cell division cycle under limited glucose requires Ssp1 kinase, the putative CaMKK, and Sds23, a PP2A-related phosphatase inhibitor.

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    Hanyu, Yuichiro; Imai, Kumiko K; Kawasaki, Yosuke; Nakamura, Takahiro; Nakaseko, Yukinobu; Nagao, Koji; Kokubu, Aya; Ebe, Masahiro; Fujisawa, Asuka; Hayashi, Takeshi; Obuse, Chikashi; Yanagida, Mitsuhiro

    2009-05-01

    Calcium/calmodulin-dependent protein kinase (CaMK) is required for diverse cellular functions, and similar kinases exist in fungi. Although mammalian CaMK kinase (CaMKK) activates CaMK and also evolutionarily-conserved AMP-activated protein kinase (AMPK), CaMKK is yet to be established in yeast. We here report that the fission yeast Schizosaccharomyces pombe Ssp1 kinase, which controls G2/M transition and response to stress, is the putative CaMKK. Ssp1 has a CaM binding domain (CBD) and associates with 14-3-3 proteins as mammalian CaMKK does. Temperature-sensitive ssp1 mutants isolated are defective in the tolerance to limited glucose, and this tolerance requires the conserved stretch present between the kinase domain and CBD. Sds23, multi-copy suppressor for mutants defective in type 1 phosphatase and APC/cyclosome, also suppresses the ssp1 phenotype, and is required for the tolerance to limited glucose. We demonstrate that Sds23 binds to type 2A protein phosphatases (PP2A) and PP2A-related phosphatase Ppe1, and that Sds23 inhibits Ppe1 phosphatase activity. Ssp1 and Ppe1 thus seem to antagonize in utilizing limited glucose. We also show that Ppk9 and Ssp2 are the catalytic subunits of AMPK and AMPK-related kinases, respectively, which bind to common beta-(Amk2) and gamma-(Cbs2) subunits.

  13. Monoclonal antibody against brain calmodulin-dependent protein kinase type II detects putative conformational changes induced by Ca/sup 2 +/-calmodulin

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    LeVine, H. III; Su, J.L.; Sahyoun, N.E.

    1988-08-23

    A mouse monoclonal IgG1 antibody has been generated against the soluble form of the calmodulin-dependent protein kinase type II. This antibody recognizes both the soluble and cytoskeletal forms of the enzyme, requiring Ca/sup 2 +/ for the interaction. Other divalent cations such as Zn/sup 2 +/, Mn/sup 2 +/, Cd/sup 2 +/, Co/sup 2 +/, and Ni/sup 2 +/ will substitute for Ca/sup 2 +/, while Mg/sup 2 +/ and Ba/sup 2 +/ will not. The antibody reacts with both the ..cap alpha..- and ..beta..-subunits on Western blots in a similar Ca/sup 2 +/-dependent fashion but with a lower sensitivity. The affinity of the antibody for the kinase is 0.13 nM determined by displacement of /sup 125/I Bolton-Hunter-labeled kinase with unlabeled enzyme. Calmodulin and antibody reciprocally potentiate each other's interaction with the enzyme. This is illustrated both by direct binding studies and by a decrease of the K/sub m app/ for calmodulin and an increase in the V/sub max/ for the autophosphorylation reaction of the enzyme. The antibody thus appears to recognize and stabilize a conformation of the kinase which favors calmodulin binding although it does not itself activate the kinase in the absence of calmodulin. Since the M/sub r/ 30,000 catalytic fragment of the kinase is not immunoreactive, either the antibody combining site of the kinase must be present in the noncatalytic portion of the protein along with the calmodulin binding site or proteolysis interferes with the putative Ca/sup 2 +/-dependent conformational change. Thus, monoclonal antibodies can be useful tools in elucidating the mechanism by which Ca/sup 2 +/ and calmodulin act on the kinase molecule.

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

    Energy Technology Data Exchange (ETDEWEB)

    1993-01-01

    The serine/threonine protein kinase (SRK) protein was predicted to be similar to the growth factor receptor tyrosine kinases in animals but its amino acid sequence of the catalytic domain is more similar to that of the catalytic domains of protein serine/threonine kinases than to protein tyrosine kinases. We have shown that the SRK protein has intrinsic scrine/threonine kinase activity. We subcloned the protein kinase-homologous domain of the SRK[sub 6] cDNA into the bacterial expression vector pGEX-3X and we have constructed a second plasmid identical to the first except that it carried a conservative mutation that substituted Arg for the Lys[sup 524] codon of SRK6 This lysine corresponds to the ATP-binding site, is essential in protein kinases, and is a common target for site-directed mutagenesis as a means to obtain kinase-defective proteins. Cultures bearing the wild-type and mutant SRK catalytic domains each produced an approximately 64 kD protein that reacted with anti-SRK6 antibodies. Following pulse-labeling with [sup 32]P we found that the wild-type SRK6 protein but not the mutant form was detectably phosphorylated. Phosphoamino acid analysis of the affinity purified [sup 32]p-labeled GST-SRK6 fusion protein demonstrated that SRK was phosphorylated predominantly on semine and to a lesser extent on threonine, but not on tyrosine. Thus, SRK6 is a functional serine/threonine protein kinase.

  15. The putative effector-binding site of Leishmania mexicana pyruvate kinase studied by site-directed mutagenesis.

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    Hannaert, Véronique; Yernaux, Cédric; Rigden, Daniel J; Fothergill-Gilmore, Linda A; Opperdoes, Fred R; Michels, Paul A M

    2002-03-13

    The activity of pyruvate kinase of Leishmania mexicana is allosterically regulated by fructose 2,6-bisphosphate (F-2,6-P(2)), contrary to the pyruvate kinases from other eukaryotes that are usually stimulated by fructose 1,6-bisphosphate (F-1,6-P(2)). Based on the comparison of the three-dimensional structure of Saccharomyces cerevisiae pyruvate kinase crystallized with F-1,6-P(2) present at the effector site (R-state) and the L. mexicana enzyme crystallized in the T-state, two residues (Lys453 and His480) were proposed to bind the 2-phospho group of the effector. This hypothesis was tested by site-directed mutagenesis. The allosteric activation by F-2,6-P(2) appeared to be entirely abrogated in the mutated enzymes confirming our predictions.

  16. Characterization of a putative S-locus encoded receptor protein kinase and its role in self-incompatibility. Progress report

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    Nasrallah, J.B.

    1994-05-01

    The major results of our research effort include the determination of the S-Receptor Kinase (SRK) gene structure, the demonstration of S-haplotype-associated SRK polymorphisms and possible co-evolution of SRK and SLG, the characterization of the temporal and spatial expression patterns of SRK, and the demonstration that SRK has intrinsic serine/threonine kinase activity. Our results have indicated that SLG originated from an SRK-like gene by a gene duplication event and suggested a possible molecular basis for leaky S haplotypes. The data have allowed us to develop a model of self-incompatibility based on the interaction of SRK and SLG and the activation of SRK in response to self-pollination. More generally, the information that we have obtained is potentially relevant to understanding mechanisms of signalling inplants. Thus, the interaction of membrane-based receptor protein kinases with secreted forms of their extracellular domains may represent a generalized mechanism by which receptors signal across the plant cell wall.

  17. Putative histidine kinase inhibitors with antibacterial effect against multi-drug resistant clinical isolates identified by in vitro and in silico screens

    Science.gov (United States)

    Velikova, Nadya; Fulle, Simone; Manso, Ana Sousa; Mechkarska, Milena; Finn, Paul; Conlon, J. Michael; Oggioni, Marco Rinaldo; Wells, Jerry M.; Marina, Alberto

    2016-05-01

    Novel antibacterials are urgently needed to address the growing problem of bacterial resistance to conventional antibiotics. Two-component systems (TCS) are widely used by bacteria to regulate gene expression in response to various environmental stimuli and physiological stress and have been previously proposed as promising antibacterial targets. TCS consist of a sensor histidine kinase (HK) and an effector response regulator. The HK component contains a highly conserved ATP-binding site that is considered to be a promising target for broad-spectrum antibacterial drugs. Here, we describe the identification of putative HK autophosphorylation inhibitors following two independent experimental approaches: in vitro fragment-based screen via differential scanning fluorimetry and in silico structure-based screening, each followed up by the exploration of analogue compounds as identified by ligand-based similarity searches. Nine of the tested compounds showed antibacterial effect against multi-drug resistant clinical isolates of bacterial pathogens and include three novel scaffolds, which have not been explored so far in other antibacterial compounds. Overall, putative HK autophosphorylation inhibitors were found that together provide a promising starting point for further optimization as antibacterials.

  18. Regulation of protein kinase Cmu by basic peptides and heparin. Putative role of an acidic domain in the activation of the kinase.

    Science.gov (United States)

    Gschwendt, M; Johannes, F J; Kittstein, W; Marks, F

    1997-08-15

    Protein kinase Cmu is a novel member of the protein kinase C (PKC) family that differs from the other isoenzymes in structural and enzymatic properties. No substrate proteins of PKCmu have been identified as yet. Moreover, the regulation of PKCmu activity remains obscure, since a structural region corresponding to the pseudosubstrate domains of other PKC isoenzymes has not been found for PKCmu. Here we show that aldolase is phosphorylated by PKCmu in vitro. Phosphorylation of aldolase and of two substrate peptides by PKCmu is inhibited by various proteins and peptides, including typical PKC substrates such as histone H1, myelin basic protein, and p53. This inhibitory activity seems to depend on clusters of basic amino acids in the protein/peptide structures. Moreover, in contrast to other PKC isoenzymes PKCmu is activated by heparin and dextran sulfate. Maximal activation by heparin is about twice and that by dextran sulfate four times as effective as maximal activation by phosphatidylserine plus 12-O-tetradecanoylphorbol-13-acetate, the conventional activators of c- and nPKC isoforms. We postulate that PKCmu contains an acidic domain, which is involved in the formation and stabilization of an active state and which, in the inactive enzyme, is blocked by an intramolecular interaction with a basic domain. This intramolecular block is thought to be released by heparin and possibly also by 12-O-tetradecanoylphorbol-13-acetate/phosphatidylserine, whereas basic peptides and proteins inhibit PKCmu activity by binding to the acidic domain of the active enzyme.

  19. A putative bifunctional histidine kinase/phosphatase of the HWE family exerts positive and negative control on the Sinorhizobium meliloti general stress response.

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    Sauviac, Laurent; Bruand, Claude

    2014-07-01

    The EcfG-type sigma factor RpoE2 is the regulator of the general stress response in Sinorhizobium meliloti. RpoE2 activity is negatively regulated by two NepR-type anti-sigma factors (RsiA1/A2), themselves under the control of two anti-anti-sigma factors (RsiB1/B2) belonging to the PhyR family of response regulators. The current model of RpoE2 activation suggests that in response to stress, RsiB1/B2 are activated by phosphorylation of an aspartate residue in their receiver domain. Once activated, RsiB1/B2 become able to interact with the anti-sigma factors and release RpoE2, which can then associate with the RNA polymerase to transcribe its target genes. The purpose of this work was to identify and characterize proteins involved in controlling the phosphorylation status of RsiB1/B2. Using in vivo approaches, we show that the putative histidine kinase encoded by the rsiC gene (SMc01507), located downstream from rpoE2, is able to both positively and negatively regulate the general stress response. In addition, our data suggest that the negative action of RsiC results from inhibition of RsiB1/B2 phosphorylation. From these observations, we propose that RsiC is a bifunctional histidine kinase/phosphatase responsible for RsiB1/B2 phosphorylation or dephosphorylation in the presence or absence of stress, respectively. Two proteins were previously proposed to control PhyR phosphorylation in Caulobacter crescentus and Sphingomonas sp. strain FR1. However, these proteins contain a Pfam:HisKA_2 domain of dimerization and histidine phosphotransfer, whereas S. meliloti RsiC harbors a Pfam:HWE_HK domain instead. Therefore, this is the first report of an HWE_HK-containing protein controlling the general stress response in Alphaproteobacteria.

  20. Fibroblast Biomarkers of Sporadic Parkinson's Disease and LRRK2 Kinase Inhibition.

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    Smith, G A; Jansson, J; Rocha, E M; Osborn, T; Hallett, P J; Isacson, O

    2016-10-01

    It has been uncertain whether specific disease-relevant biomarker phenotypes can be found using sporadic Parkinson's disease (PD) patient-derived samples, as it has been proposed that there may be a plethora of underlying causes and pathological mechanisms. Fibroblasts derived from familial PD patients harboring leucine-rich repeat kinase 2 (LRRK2), PTEN-induced putative kinase 1 (PINK1), and Parkin mutations show clear disease-relevant mitochondrial phenotypes, which are exacerbated under conditions of pharmacological stress. We utilized fibroblasts derived from non-familial sporadic PD patients (without LRRK2 mutations) or LRRK2 mutation carriers to directly compare the cellular phenotypes during and after mitochondrial stress. We then determined the effects of pharmacological LRRK2 kinase inhibition using LRRK2-in-1. We found that there were two distinct populations of sporadic PD patient-derived fibroblast lines. One group of sporadic PD lines was highly susceptible to valinomycin-induced mitochondrial depolarization, emulating the mutant LRRK2 phenotype. These lines showed elevated mitochondrial superoxide/ nitric oxide levels, displayed increased mitochondrial and lysosome co-localization, and an increased rate of mitochondrial collapse, which corresponded with changes in mitochondrial fission and fusion proteins. The application of LRRK2-in-1 reversed decreased levels of mitochondrial and lysosome co-localization and partially restored mitochondrial network associated proteins and the mitochondrial membrane potential in the fibroblasts. This study identifies novel mitochondrial biomarkers in sporadic PD patient-derived fibroblast lines, which could be used as preclinical tools in which to test novel and known neuroprotective compounds.

  1. E3 Ligase Subunit Fbxo15 and PINK1 Kinase Regulate Cardiolipin Synthase 1 Stability and Mitochondrial Function in Pneumonia

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    Bill B. Chen

    2014-04-01

    Full Text Available Acute lung injury (ALI is linked to mitochondrial injury, resulting in impaired cellular oxygen utilization; however, it is unknown how these events are linked on the molecular level. Cardiolipin, a mitochondrial-specific lipid, is generated by cardiolipin synthase (CLS1. Here, we show that S. aureus activates a ubiquitin E3 ligase component, Fbxo15, that is sufficient to mediate proteasomal degradation of CLS1 in epithelia, resulting in decreased cardiolipin availability and disrupted mitochondrial function. CLS1 is destabilized by the phosphatase and tensin homolog (PTEN-induced putative kinase 1 (PINK1, which binds CLS1 to phosphorylate and regulates CLS1 disposal. Like Fbxo15, PINK1 interacts with and regulates levels of CLS1 through a mechanism dependent upon Thr219. S. aureus infection upregulates this Fbxo15-PINK1 pathway to impair mitochondrial integrity, and Pink1 knockout mice are less prone to S. aureus-induced ALI. Thus, ALI-associated disruption of cellular bioenergetics involves bioeffectors that utilize a phosphodegron to elicit ubiquitin-mediated disposal of a key mitochondrial enzyme.

  2. Curcumin exposure induces expression of the Parkinson's disease-associated leucine-rich repeat kinase 2 (LRRK2) in rat mesencephalic cells.

    Science.gov (United States)

    Ortiz-Ortiz, Miguel A; Morán, José M; Ruiz-Mesa, Luz M; Niso-Santano, Mireia; Bravo-SanPedro, José M; Gómez-Sánchez, Rubén; González-Polo, Rosa A; Fuentes, José M

    2010-01-04

    Turmeric (curry powder), an essential ingredient of culinary preparations of Southeast Asia, contains a major polyphenolic compound known as curcumin or diferuloylmethane. Curcumin is a widely studied phytochemical with a variety of biological activities. In addition to its anti-inflammatory and antimicrobial/antiviral properties, curcumin is considered as a cancer chemopreventive agent as well as a modulator of gene expression and a potent antioxidant. Since oxidative stress has been implicated in the degeneration of dopaminergic neurons in the substantia nigra in Parkinson's disease (PD), curcumin has been proposed to have potential therapeutic value for the treatment of neurodegenerative diseases such as PD. Following age, a family history of PD is the most commonly reported risk factor, suggesting a genetic component of the disease in a subgroup of patients. The LRRK2 gene has emerged as the gene most commonly associated with both familial and sporadic PD. Here, we report that exposure of rat mesencephalic cells to curcumin induces the expression of LRRK2 mRNA and protein in a time-dependent manner. The expression of other PD-related genes, such alpha-synuclein and parkin, was not affected by exposure to curcumin, and PTEN-induced putative kinase 1 (PINK1) was not expressed in rat mesencephalic cells. As LRRK2 overexpression is strongly associated with the pathological inclusions found in several neurodegenerative disorders, further studies are needed to evaluate the effects of curcumin as a therapeutic agent for neurodegenerative diseases.

  3. [Characterization of a putative S locus encoded receptor protein kinase and its role in self-incompatibility]. Progress report, January 1993

    Energy Technology Data Exchange (ETDEWEB)

    1993-06-01

    The serine/threonine protein kinase (SRK) protein was predicted to be similar to the growth factor receptor tyrosine kinases in animals but its amino acid sequence of the catalytic domain is more similar to that of the catalytic domains of protein serine/threonine kinases than to protein tyrosine kinases. We have shown that the SRK protein has intrinsic scrine/threonine kinase activity. We subcloned the protein kinase-homologous domain of the SRK{sub 6} cDNA into the bacterial expression vector pGEX-3X and we have constructed a second plasmid identical to the first except that it carried a conservative mutation that substituted Arg for the Lys{sup 524} codon of SRK6 This lysine corresponds to the ATP-binding site, is essential in protein kinases, and is a common target for site-directed mutagenesis as a means to obtain kinase-defective proteins. Cultures bearing the wild-type and mutant SRK catalytic domains each produced an approximately 64 kD protein that reacted with anti-SRK6 antibodies. Following pulse-labeling with {sup 32}P we found that the wild-type SRK6 protein but not the mutant form was detectably phosphorylated. Phosphoamino acid analysis of the affinity purified {sup 32}p-labeled GST-SRK6 fusion protein demonstrated that SRK was phosphorylated predominantly on semine and to a lesser extent on threonine, but not on tyrosine. Thus, SRK6 is a functional serine/threonine protein kinase.

  4. Mutations in CG8878, a Novel Putative Protein Kinase, Enhance P Element Dependent Silencing (PDS) and Position Effect Variegation (PEV) in Drosophila melanogaster

    Science.gov (United States)

    McCracken, Allen; Locke, John

    2014-01-01

    Genes in multicellular organisms are expressed as part of a developmental program that is largely dependent on self-perpetuating higher-order chromatin states. The mechanism of establishing and maintaining these epigenetic events is well studied in Drosophila. The first known example of an epigenetic effect was that of (PEV) in Drosophila, which has been shown to be due to gene silencing via heterochromatin formation. We are investigating a process similar to Position Effect Variegation (PEV) using a mini-w transgene, called Pci, inserted in the upstream regulatory region of ci. The mini-white+ transgene in Pci is expressed throughout the adult eye; however, when other P or KP elements are present, a variegated eye phenotype results indicating random w+ silencing during development. This P element dependent silencing (PDS) can be modified by the haplo-suppressors/triplo-enhancers, Su(var)205 and Su(var)3–7, indicating that these heterochromatic modifiers also act dose dependently in PDS. Here we use a spontaneous derivative mutation of Pci called PciE1 (E1) that variegates like PDS in the absence of P elements, presumably due to an adjacent gypsy element insertion, to screen for second-site modifier mutations that enhance variable silencing of white+ in E1. We isolated 7 mutations in CG8878, an essential gene, that enhance the E1 variegated phenotype. CG8878, a previously uncharacterized gene, potentially encodes a serine/threonine kinase whose closest Drosophila paralogue, ballchen (nhk-1), phosphorylates histones. These mutant alleles enhance both PDS at E1 and Position Effect Variegation (PEV) at wm4, indicating a previously unknown common silencing mechanism between the two. PMID:24614804

  5. Mutations in CG8878, a novel putative protein kinase, enhance P element dependent silencing (PDS and position effect variegation (PEV in Drosophila melanogaster.

    Directory of Open Access Journals (Sweden)

    Allen McCracken

    Full Text Available Genes in multicellular organisms are expressed as part of a developmental program that is largely dependent on self-perpetuating higher-order chromatin states. The mechanism of establishing and maintaining these epigenetic events is well studied in Drosophila. The first known example of an epigenetic effect was that of (PEV in Drosophila, which has been shown to be due to gene silencing via heterochromatin formation. We are investigating a process similar to Position Effect Variegation (PEV using a mini-w transgene, called Pci, inserted in the upstream regulatory region of ci. The mini-white+ transgene in Pci is expressed throughout the adult eye; however, when other P or KP elements are present, a variegated eye phenotype results indicating random w+ silencing during development. This P element dependent silencing (PDS can be modified by the haplo-suppressors/triplo-enhancers, Su(var205 and Su(var3-7, indicating that these heterochromatic modifiers also act dose dependently in PDS. Here we use a spontaneous derivative mutation of Pci called PciE1 (E1 that variegates like PDS in the absence of P elements, presumably due to an adjacent gypsy element insertion, to screen for second-site modifier mutations that enhance variable silencing of white+ in E1. We isolated 7 mutations in CG8878, an essential gene, that enhance the E1 variegated phenotype. CG8878, a previously uncharacterized gene, potentially encodes a serine/threonine kinase whose closest Drosophila paralogue, ballchen (nhk-1, phosphorylates histones. These mutant alleles enhance both PDS at E1 and Position Effect Variegation (PEV at w(m4, indicating a previously unknown common silencing mechanism between the two.

  6. Impact of the putative cancer stem cell markers and growth factor receptor expression on the sensitivity of ovarian cancer cells to treatment with various forms of small molecule tyrosine kinase inhibitors and cytotoxic drugs

    Science.gov (United States)

    Puvanenthiran, Soozana; Essapen, Sharadah; Seddon, Alan M.; Modjtahedi, Helmout

    2016-01-01

    Increased expression and activation of human epidermal growth factor receptor (EGFR) and HER-2 have been reported in numerous cancers. The aim of this study was to determine the sensitivity of a large panel of human ovarian cancer cell lines (OCCLs) to treatment with various forms of small molecule tyrosine kinase inhibitors (TKIs) and cytotoxic drugs. The aim was to see if there was any association between the protein expression of various biomarkers including three putative ovarian cancer stem cell (CSC) markers (CD24, CD44, CD117/c-Kit), P-glycoprotein (P-gp), and HER family members and response to treatment with these agents. The sensitivity of 10 ovarian tumour cell lines to the treatment with various forms of HER TKIs (gefitinib, erlotinib, lapatinib, sapitinib, afatinib, canertinib, neratinib), as well as other TKIs (dasatinib, imatinib, NVP-AEW541, crizotinib) and cytotoxic agents (paclitaxel, cisplatin and doxorubicin), as single agents or in combination, was determined by SRB assay. The effect on these agents on the cell cycle distribution, and downstream signaling molecules and tumour migration were determined using flow cytometry, western blotting, and the IncuCyte Clear View cell migration assay respectively. Of the HER inhibitors, the irreversible pan-TKIs (canertinib, neratinib and afatinib) were the most effective TKIs for inhibiting the growth of all ovarian cancer cells, and for blocking the phosphorylation of EGFR, HER-2, AKT and MAPK in SKOV3 cells. Interestingly, while the majority of cancer cells were highly sensitive to treatment with dasatinib, they were relatively resistant to treatment with imatinib (i.e., IC50 >10 μM). Of the cytotoxic agents, paclitaxel was the most effective for inhibiting the growth of OCCLs, and of various combinations of these drugs, only treatment with a combination of NVP-AEW541 and paclitaxel produced a synergistic or additive anti-proliferative effect in all three cell lines examined (i.e., SKOV3, Caov3, ES2

  7. Depletion of PINK1 affects mitochondrial metabolism, calcium homeostasis and energy maintenance

    NARCIS (Netherlands)

    Heeman, B.; Haute, C. Van den; Aelvoet, S.A.; Valsecchi, F.; Rodenburg, R.J.T.; Reumers, V.; Debyser, Z.; Callewaert, G.; Koopman, W.J.H.; Willems, P.H.G.M.; Baekelandt, V.

    2011-01-01

    Loss-of-function mutations in the gene encoding the mitochondrial PTEN-induced putative kinase 1 (PINK1) are a major cause of early-onset familial Parkinson's disease (PD). Recent studies have highlighted an important function for PINK1 in clearing depolarized mitochondria by mitophagy. However, the

  8. The human PINK1 locus is regulated in vivo by a non-coding natural antisense RNA during modulation of mitochondrial function

    DEFF Research Database (Denmark)

    Scheele, Camilla; Petrovic, Natasa; Faghihi, Mohammad A

    2007-01-01

    BACKGROUND: Mutations in the PTEN induced putative kinase 1 (PINK1) are implicated in early-onset Parkinson's disease. PINK1 is expressed abundantly in mitochondria rich tissues, such as skeletal muscle, where it plays a critical role determining mitochondrial structural integrity in Drosophila...

  9. Sequence analysis of a 13.4 kbp fragment from the left arm of chromosome XV reveals a malate dehydrogenase gene, a putative Ser/Thr protein kinase, the ribosomal L25 gene and four new open reading frames.

    Science.gov (United States)

    Casamayor, A; Khalid, H; Balcells, L; Aldea, M; Casas, C; Herrero, E; Ariño, J

    1996-09-01

    A 13421 bp fragment located near the left telomere of chromosome XV (cosmid pEOA461) has been sequenced. Seven non-overlapping open reading frames (ORFs) encoding polypeptides longer than 100 residues have been found (AOB859, AOC184, AOE375, AOX142i, AOE423, AOA476 and AOE433). An additional ORF (AOE131) is found within AOA476. Three of them (AOC184, AOA476 and AOE433) show no remarkable identity with proteins deposited in the data banks. ORF AOB859 is quite similar to a hypothetical yeast protein of similar size located in chromosome VI, particularly within the C-terminal half. AOE375 encodes a new member of the glycogen synthase kinase-3 subfamily of Ser/Thr protein kinases. AOX142i is the gene encoding the previously described ribosomal protein L25. AOE423 codes for a protein virtually identical to the MDH2 malate dehydrogenase isozyme. However, our DNA sequence shows a single one-base insertion upstream of the reported initiating codon. This would produce a larger ORF by extending 46 residues the N-terminus of the protein. The existence of this insertion has been confirmed in three different yeast strains, including FY1679.

  10. Comparison of Peptide Array Substrate Phosphorylation of c-Raf and Mitogen Activated Protein Kinase Kinase Kinase 8

    NARCIS (Netherlands)

    Parikh, Kaushal; Diks, Sander H.; Tuynman, Jurriaan H. B.; Verhaar, Auke; Lowenberg, Mark; Hommes, Daan W.; Joore, Jos; Pandey, Akhilesh; Peppelenbosch, Maikel P.

    2009-01-01

    Kinases are pivotal regulators of cellular physiology. The human genome contains more than 500 putative kinases, which exert their action via the phosphorylation of specific substrates. The determinants of this specificity are still only partly understood and as a consequence it is difficult to pred

  11. 下调PTEN基因对偏头痛大鼠三叉神经节CREB的调节作用%Down-Regulation of PTEN Induced The Modulation of CREB in Trigeminal Ganglia of Migraine Rats

    Institute of Scientific and Technical Information of China (English)

    桂蓓; 申崇标; 陈力学; 周冀英; 谭戈

    2010-01-01

    本研究利用RNAi重组腺病毒(AdR-siPTEN)下调偏头痛大鼠三叉神经节的PTEN(phosphatase and tensin homolog deleted on chromosome ten)基因,探讨其对偏头痛大鼠行为学的影响,以及经Akt(serine-threonine kinase)信号途径对CREB(cAMP response element-binding protein)的调控情况.实验采用健康雄性SD大鼠,随机分为假手术组(Sham)、硝酸甘油模型组(GTN)、Ad-RFP非特异siRNA处理空载体对照组(Vehicle+GTN)、AdR-siPTEN下调组(AdR-siPTEN+GTN).用AdR-siPTEN重组腺病毒对大鼠进行预处理,然后通过硝酸甘油(glyceryl trinitrate,GTN)法建立大鼠偏头痛模型,进行大鼠挠头和爬笼次数的检测,并用RT-PCR和Western-blot法进行相关基因的mRNA和蛋白检测.结果表明,当PTEN基因表达下调时,有效缓解了偏头痛导致的挠头和爬笼行为,并激活Akt信号途径,增加其下游作用因子CREB的表达,进而可能经"PTEN/Akt/CREB"信号通路影响神经突触可塑性,参与了偏头痛的发病机制.

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

    Science.gov (United States)

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

    1998-09-04

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

  13. Control of meristem development by CLAVATA1 receptor kinase and kinase-associated protein phosphatase interactions

    Energy Technology Data Exchange (ETDEWEB)

    Stone, J.M.; Walker, J.C. [Univ. of Missouri, Columbia, MO (United States). Div. of Biological Sciences; Trotochaud, A.E.; Clark, S.E. [Univ. of Michigan, Ann Arbor, MI (United States). Dept. of Biology

    1998-08-01

    The CLAVATA1 (CLV1) gene encodes a putative receptor kinase required for the proper balance between cell proliferation and differentiation in Arabidopsis shoot and flower meristems. Impaired CLV1 signaling results in masses of undifferentiated cells at the shoot and floral meristems. Although many putative receptor kinases have been identified in plants, the mechanism of signal transduction mediated by plant receptor-like kinases is largely unknown. One potential effector of receptor kinase signaling is kinase-associated protein phosphatase (KAPP), a protein that binds to multiple plant receptor-like kinases in a phosphorylation-dependent manner. To examine a possible role for KAPP in CLV1-dependent plant development, the interaction of CLV1 and KAPP was investigated in vitro and in vivo. KAPP binds directly to autophosphorylated CLV1 in vitro and co-immunoprecipitates with CLV1 in plant extracts derived from meristematic tissue. Reduction of KAPP transcript accumulation in an intermediate clv1 mutant suppresses the mutant phenotype, and the degree of suppression is inversely correlated with KAPP mRNA levels. These data suggest that KAPP functions as a negative regulator of CLV1 signaling in plant development. This may represent a general model for the interaction of KAPP with receptor kinases.

  14. Casein kinases

    DEFF Research Database (Denmark)

    Issinger, O G

    1993-01-01

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

  15. Nucleoside analogues are activated by bacterial deoxyribonucleoside kinases in a species-specific manner

    DEFF Research Database (Denmark)

    Sandrini, Michael; Clausen, Anders; On, Stephen L. W.

    2007-01-01

    bactericidal activity against several clinical bacterial isolates and type strains. We identified and subcloned the genes coding for putative deoxyribonucleoside kinases in Escherichia coli, Pasteurella multocida, Salmonella enterica, Yersinia enterocolitica, Bacillus cereus, Clostridium perfringens...

  16. Mitogen-activated protein kinase cascades in Vitis vinifera

    Science.gov (United States)

    Çakır, Birsen; Kılıçkaya, Ozan

    2015-01-01

    Protein phosphorylation is one of the most important mechanisms to control cellular functions in response to external and endogenous signals. Mitogen-activated protein kinases (MAPK) are universal signaling molecules in eukaryotes that mediate the intracellular transmission of extracellular signals resulting in the induction of appropriate cellular responses. MAPK cascades are composed of four protein kinase modules: MAPKKK kinases (MAPKKKKs), MAPKK kinases (MAPKKKs), MAPK kinases (MAPKKs), and MAPKs. In plants, MAPKs are activated in response to abiotic stresses, wounding, and hormones, and during plant pathogen interactions and cell division. In this report, we performed a complete inventory of MAPK cascades genes in Vitis vinifera, the whole genome of which has been sequenced. By comparison with MAPK, MAPK kinases, MAPK kinase kinases and MAPK kinase kinase kinase kinase members of Arabidopsis thaliana, we revealed the existence of 14 MAPKs, 5 MAPKKs, 62 MAPKKKs, and 7 MAPKKKKs in Vitis vinifera. We identified orthologs of V. vinifera putative MAPKs in different species, and ESTs corresponding to members of MAPK cascades in various tissues. This work represents the first complete inventory of MAPK cascades in V. vinifera and could help elucidate the biological and physiological functions of these proteins in V. vinifera. PMID:26257761

  17. The DNA sequence of a 7941 bp fragment of the left arm of chromosome VII of Saccharomyces cerevisiae contains four open reading frames including the multicopy suppressor gene of the pop2 mutation and a putative serine/threonine protein kinase gene.

    Science.gov (United States)

    Coglievina, M; Bertani, I; Klima, R; Zaccaria, P; Bruschi, C V

    1995-06-30

    We report the sequence of a 7941 bp DNA fragment from the left arm of chromosome VII of Saccharomyces cerevisiae which contains four open reading frames (ORFs) of greater than 100 amino acid residues. ORF biC834 shows 100% bp identity with the recently identified multicopy suppressor gene of the pop2 mutation (MPT5); its deduced protein product carries an eight-repeat domain region, homologous to that found in the hypothetical regulatory YGL023 protein of S. cerevisiae and the Pumilio protein of Drosophila. ORF biE560 protein exhibits patterns typical of serine/threonine protein kinases, with which it shares high degrees of homology.

  18. Interactions of protein kinase CK2beta subunit within the holoenzyme and with other proteins

    DEFF Research Database (Denmark)

    Kusk, M; Ahmed, R; Thomsen, B;

    1999-01-01

    in alpha-beta and beta-beta interactions. We also detected an intramolecular beta interaction within the amino acid stretch 132-165. Using CK2beta as a bait in a two-hybrid library screening several new putative cellular partners have been identified, among them the S6 kinase p90rsk, the putative tumor...

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

    Directory of Open Access Journals (Sweden)

    Nuzhat N. Kabir

    2011-01-01

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

  20. Cloning and expression of the heterodimeric deoxyguanosine kinase/deoxyadenosine kinase of Lactobacillus acidophilus R-26.

    Science.gov (United States)

    Ma, G T; Hong, Y S; Ives, D H

    1995-03-24

    Two uniquely paired deoxynucleoside kinases, deoxycytidine kinase/deoxyadenosine kinase (dCK/dAK) and deoxyguanosine kinase/deoxyadenosine kinase (dGK/dAK) are required, together with thymidine kinase (TK), for deoxynucleotide synthesis in Lactobacillus acidophilus R-26. Using polymerase chain reaction-generated probes based on N-terminal amino acid sequences, we have cloned tandem genes for 25- and 26-kDa polypeptides, whose derived amino acid sequences and size correspond to wild-type Lactobacillus enzyme subunits. Expression in Escherichia coli uses a single endogenous promoter and yields active dGK/dAK (approximately 3% of extracted protein) closely resembling wild-type dGK/dAK in specificity, kinetics, heterotropic activation, and end product inhibition. Alignment of cloned genes reveals 65% identity in their DNA sequences and 61% identity in derived amino acid sequences. Comparison with herpes-viral TKs reveals three conserved regions: glycine- and arginine-rich ATP-binding motifs and a D/E-R-S/H motif at the putative TK deoxynucleoside site. Greater homology, however, is seen upon multiple alignment of dGK with mammalian deoxycytidine kinases, yielding the consensus sequence-D/E-R-S-I/V-Y-x-D-.dGK also shares a sequence (-Y-D-P-T-I/L-E-D-S/Y-Y-) required for GTP hydrolysis by p21ras.

  1. Prediction of 492 human protein kinase substrate specificities.

    Science.gov (United States)

    Safaei, Javad; Maňuch, Ján; Gupta, Arvind; Stacho, Ladislav; Pelech, Steven

    2011-10-14

    Complex intracellular signaling networks monitor diverse environmental inputs to evoke appropriate and coordinated effector responses. Defective signal transduction underlies many pathologies, including cancer, diabetes, autoimmunity and about 400 other human diseases. Therefore, there is high impetus to define the composition and architecture of cellular communications networks in humans. The major components of intracellular signaling networks are protein kinases and protein phosphatases, which catalyze the reversible phosphorylation of proteins. Here, we have focused on identification of kinase-substrate interactions through prediction of the phosphorylation site specificity from knowledge of the primary amino acid sequence of the catalytic domain of each kinase. The presented method predicts 488 different kinase catalytic domain substrate specificity matrices in 478 typical and 4 atypical human kinases that rely on both positive and negative determinants for scoring individual phosphosites for their suitability as kinase substrates. This represents a marked advancement over existing methods such as those used in NetPhorest (179 kinases in 76 groups) and NetworKIN (123 kinases), which consider only positive determinants for kinase substrate prediction. Comparison of our predicted matrices with experimentally-derived matrices from about 9,000 known kinase-phosphosite substrate pairs revealed a high degree of concordance with the established preferences of about 150 well studied protein kinases. Furthermore for many of the better known kinases, the predicted optimal phosphosite sequences were more accurate than the consensus phosphosite sequences inferred by simple alignment of the phosphosites of known kinase substrates. Application of this improved kinase substrate prediction algorithm to the primary structures of over 23, 000 proteins encoded by the human genome has permitted the identification of about 650, 000 putative phosphosites, which are posted on the

  2. Putative archaeal viruses from the mesopelagic ocean.

    Science.gov (United States)

    Vik, Dean R; Roux, Simon; Brum, Jennifer R; Bolduc, Ben; Emerson, Joanne B; Padilla, Cory C; Stewart, Frank J; Sullivan, Matthew B

    2017-01-01

    Oceanic viruses that infect bacteria, or phages, are known to modulate host diversity, metabolisms, and biogeochemical cycling, while the viruses that infect marine Archaea remain understudied despite the critical ecosystem roles played by their hosts. Here we introduce "MArVD", for Metagenomic Archaeal Virus Detector, an annotation tool designed to identify putative archaeal virus contigs in metagenomic datasets. MArVD is made publicly available through the online iVirus analytical platform. Benchmarking analysis of MArVD showed it to be >99% accurate and 100% sensitive in identifying the 127 known archaeal viruses among the 12,499 viruses in the VirSorter curated dataset. Application of MArVD to 10 viral metagenomes from two depth profiles in the Eastern Tropical North Pacific (ETNP) oxygen minimum zone revealed 43 new putative archaeal virus genomes and large genome fragments ranging in size from 10 to 31 kb. Network-based classifications, which were consistent with marker gene phylogenies where available, suggested that these putative archaeal virus contigs represented six novel candidate genera. Ecological analyses, via fragment recruitment and ordination, revealed that the diversity and relative abundances of these putative archaeal viruses were correlated with oxygen concentration and temperature along two OMZ-spanning depth profiles, presumably due to structuring of the host Archaea community. Peak viral diversity and abundances were found in surface waters, where Thermoplasmata 16S rRNA genes are prevalent, suggesting these archaea as hosts in the surface habitats. Together these findings provide a baseline for identifying archaeal viruses in sequence datasets, and an initial picture of the ecology of such viruses in non-extreme environments.

  3. Ten Putative Contributors to the Obesity Epidemic

    Science.gov (United States)

    McAllister, Emily J.; Dhurandhar, Nikhil V.; Keith, Scott W.; Aronne, Louis J.; Barger, Jamie; Baskin, Monica; Benca, Ruth M.; Biggio, Joseph; Boggiano, Mary M.; Eisenmann, Joe C.; Elobeid, Mai; Fontaine, Kevin R.; Gluckman, Peter; Hanlon, Erin C.; Katzmarzyk, Peter; Pietrobelli, Angelo; Redden, David T.; Ruden, Douglas M.; Wang, Chenxi; Waterland, Robert A.; Wright, Suzanne M.; Allison, David B.

    2010-01-01

    The obesity epidemic is a global issue and shows no signs of abating, while the cause of this epidemic remains unclear. Marketing practices of energy-dense foods and institutionally-driven declines in physical activity are the alleged perpetrators for the epidemic, despite a lack of solid evidence to demonstrate their causal role. While both may contribute to obesity, we call attention to their unquestioned dominance in program funding and public efforts to reduce obesity, and propose several alternative putative contributors that would benefit from equal consideration and attention. Evidence for microorganisms, epigenetics, increasing maternal age, greater fecundity among people with higher adiposity, assortative mating, sleep debt, endocrine disruptors, pharmaceutical iatrogenesis, reduction in variability of ambient temperatures, and intrauterine and intergenerational effects, as contributing factors to the obesity epidemic are reviewed herein. While the evidence is strong for some contributors such as pharmaceutical-induced weight gain, it is still emerging for other reviewed factors. Considering the role of such putative etiological factors of obesity may lead to comprehensive, cause specific, and effective strategies for prevention and treatment of this global epidemic. PMID:19960394

  4. Preliminary X-ray crystallographic analysis of SMU.573, a putative sugar kinase from Streptococcus mutans

    Energy Technology Data Exchange (ETDEWEB)

    Zhou, Yan-Feng; Li, Lan-Fen [National Laboratory of Protein Engineering and Plant Genetic Engineering, College of Life Sciences, Peking University, Beijing 100871 (China); Yang, Cheng [National Laboratory of Protein Engineering and Plant Genetic Engineering, College of Life Sciences, Peking University, Beijing 100871 (China); Rigaku/MSC Inc., 9009 New Trails Drive, The Woodlands, TX 77381 (United States); Liang, Yu-He, E-mail: liangyh@pku.edu.cn [National Laboratory of Protein Engineering and Plant Genetic Engineering, College of Life Sciences, Peking University, Beijing 100871 (China); Su, Xiao-Dong, E-mail: liangyh@pku.edu.cn [National Laboratory of Protein Engineering and Plant Genetic Engineering, College of Life Sciences, Peking University, Beijing 100871 (China); Shenzhen Graduate School of Peking University, Shenzhen 518055 (China)

    2008-01-01

    SMU.573 from S. mutans was expressed in E. coli and crystallized. The crystals belong to space group I4 and 2.5 Å resolution diffraction data were collected at an in-house chromium radiation source. SMU.573 from Streptococcus mutans is a structurally and functionally uncharacterized protein that was selected for structural biology studies. Native and SeMet-labelled proteins were expressed with an N-His tag in Escherichia coli BL21 (DE3) and purified by Ni{sup 2+}-chelating and size-exclusion chromatography. Crystals of the SeMet-labelled protein were obtained by the hanging-drop vapour-diffusion method and a 2.5 Å resolution diffraction data set was collected using an in-house chromium radiation source. The crystals belong to space group I4, with unit-cell parameters a = b = 96.53, c = 56.26 Å, α = β = γ = 90°.

  5. Putative pyrophosphate phosphofructose 1-kinase genes identified in sugar cane may be getting energy from pyrophosphate.

    Science.gov (United States)

    Suzuki, J; Mutton, M A; Ferro, M I T; Lemos, M V F; Pizauro, J M; Mutton, M J R; Di Mauro, S M Z

    2003-12-30

    Pyrophosphate-dependent phosphofructokinase (PPi-PFK) has been detected in several types of plant cells, but the gene has not been reported in sugar cane. Using Citrus paradisi PPi-PFK gene (AF095520 and AF095521) sequences to search the sugar cane EST database, we have identified both the alpha and beta subunits of this enzyme. The deduced amino acid sequences showed 76 and 80% similarity with the corresponding alpha and beta subunits of C. paradisi. A high degree of similarity was also observed among the PFK b subunits when the alignment of the sugar cane sequences was compared to those of Ricinus communis and Solanum tuberosum. It appears that alpha and beta are two distinct subunits; they were found at different concentrations in several sugar cane tissues. It remains to be determined if the different gene expression levels have some physiological importance and how they affect sucrose synthesis, export, and storage in vacuoles. A comparison between the amino acid sequences of b PFKs from a variety of organisms allowed us to identify the two critical Asp residues typical of this enzyme's activity site and the other binding sites; these residues are tightly conserved in all members of this protein family. Apparently, there are catalytic residues on the b subunit of the pyrophosphate-dependent enzyme.

  6. Expression of a gibberellin-induced leucine-rich repeat receptor-like protein kinase in deepwater rice and its interaction with kinase-associated protein phosphatase

    Energy Technology Data Exchange (ETDEWEB)

    Knaap, E. van der; Sauter, M.; Kende, H. (Michigan State Univ., East Lansing, MI (United States). DOE Plant Research Lab.); Song, W.Y.; Ruan, D.L.; Ronald, P.C. (Univ. of California, Davis, CA (United States). Dept. of Plant Pathology)

    1999-06-01

    The authors identified in deepwater rice (Oryza sativa L.) a gene encoding a leucine-rich repeat receptor-like transmembrane protein kinase, OsTMK (O. sativa transmembrane kinase). The transcript levels of OsTMK increased in the rice internode in response to gibberellin. Expression of OsTMK was especially high in regions undergoing cell division and elongation. The kinase domain of OsTMK was enzymatically active autophosphorylating on serine and threonine residues. A cDNA encoding a rice ortholog of a kinase-associated type 2C protein phosphatase (OsKAPP) was cloned. KAPPs are putative downstream components in kinase-mediated signal transduction pathways. The kinase interaction domain of OsKAPP was phosphorylated in vitro by the kinase domain of OsTMK. RNA gel-blot analysis indicated that the expression of OsTMK and OsKAPP was similar in different tissues of the rice plant. In protein-binding assays, OsKAPP interacted with a receptor-like protein kinase, RLK5 of Arabidopsis, but not with the protein kinase domains of the rice and maize receptor-like protein kinases Xa21 and ZmPK1, respectively.

  7. Muscle phosphorylase kinase deficiency

    DEFF Research Database (Denmark)

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

    2012-01-01

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

  8. Putative Nitrogen Sensing Systems in Higher Plants

    Institute of Scientific and Technical Information of China (English)

    Hon-Ming Lam; Ying Ann Chiao; Man-Wah Li; Yuk-Kwong Yung; Sang Ji

    2006-01-01

    Nitrogen (N) metabolism is essential for the biosynthesis of vital biomolecules. N status thus exerts profound effects on plant growth and development, and must be closely monitored. In bacteria and fungi, a few sophisticated N sensing systems have been extensively studied. In animals, the ability to receive amino acid signals has evolved to become an integral part of the nervous coordination system. In this review, we will summarize recent developments in the search for putative N sensing systems in higher plants based on homologous systems in bacteria, fungi, and animals. Apparently, although plants have separated and diversified from other organisms during the evolution process, striking similarities can be found in their N sensing systems compared with those of their counterparts; however, our understanding of these systems is still incomplete. Significant modifications of the N sensing systems (including cross-talk with other signal transduction pathways) in higher plants may be a strategy of adaptation to their unique mode of life.

  9. Putative respiratory chain of Porphyromonas gingivalis.

    Science.gov (United States)

    Meuric, Vincent; Rouillon, Astrid; Chandad, Fatiha; Bonnaure-Mallet, Martine

    2010-05-01

    The electron transfer chain in Porphyromonas gingivalis, or periodontopathogens, has not yet been characterized. P. gingivalis, a strict anaerobic bacteria and the second colonizer of the oral cavity, is considered to be a major causal agent involved in periodontal diseases. Primary colonizers create a favorable environment for P. gingivalis growth by decreasing oxygen pressure. Oxygen does not appear to be the final electron acceptor of the respiratory chain. Fumarate and cytochrome b have been implicated as major components of the respiratory activity. However, the P. gingivalis genome shows many other enzymes that could be implicated in aerobic or nitrite respiration. Using bioinformatic tools and literature studies of respiratory pathways, the ATP synthesis mechanism from the sodium cycle and nutrients metabolism, the putative respirasome of P. gingivalis has been proposed.

  10. Parkinson's disease-associated kinase PINK1 regulates Miro protein level and axonal transport of mitochondria.

    Directory of Open Access Journals (Sweden)

    Song Liu

    Full Text Available Mutations in Pten-induced kinase 1 (PINK1 are linked to early-onset familial Parkinson's disease (FPD. PINK1 has previously been implicated in mitochondrial fission/fusion dynamics, quality control, and electron transport chain function. However, it is not clear how these processes are interconnected and whether they are sufficient to explain all aspects of PINK1 pathogenesis. Here we show that PINK1 also controls mitochondrial motility. In Drosophila, downregulation of dMiro or other components of the mitochondrial transport machinery rescued dPINK1 mutant phenotypes in the muscle and dopaminergic (DA neurons, whereas dMiro overexpression alone caused DA neuron loss. dMiro protein level was increased in dPINK1 mutant but decreased in dPINK1 or dParkin overexpression conditions. In Drosophila larval motor neurons, overexpression of dPINK1 inhibited axonal mitochondria transport in both anterograde and retrograde directions, whereas dPINK1 knockdown promoted anterograde transport. In HeLa cells, overexpressed hPINK1 worked together with hParkin, another FPD gene, to regulate the ubiquitination and degradation of hMiro1 and hMiro2, apparently in a Ser-156 phosphorylation-independent manner. Also in HeLa cells, loss of hMiro promoted the perinuclear clustering of mitochondria and facilitated autophagy of damaged mitochondria, effects previously associated with activation of the PINK1/Parkin pathway. These newly identified functions of PINK1/Parkin and Miro in mitochondrial transport and mitophagy contribute to our understanding of the complex interplays in mitochondrial quality control that are critically involved in PD pathogenesis, and they may explain the peripheral neuropathy symptoms seen in some PD patients carrying particular PINK1 or Parkin mutations. Moreover, the different effects of loss of PINK1 function on Miro protein level in Drosophila and mouse cells may offer one explanation of the distinct phenotypic manifestations of PINK1

  11. Mst2 and Lats kinases regulate apoptotic function of Yes kinase-associated protein (YAP).

    Science.gov (United States)

    Oka, Tsutomu; Mazack, Virginia; Sudol, Marius

    2008-10-10

    The Hippo pathway in Drosophila controls the size and shape of organs. In the fly, activation of this pathway conveys growth-inhibitory signals and promotes apoptosis in epithelial cells. We "reconstituted" the Hippo pathway in a human epithelial cell line and showed that, in contrast to flies, the activation of this pathway results in anti-apoptotic signals. We have shown that in human embryonic kidney (HEK) 293 cells, the complex formation between transcriptional co-activators YAPs (Yes kinase-associated proteins) and Lats kinases requires the intact WW domains of YAPs, as well as intact Pro-Pro-AA-Tyr (where AA is any amino acid) motifs in Lats kinases. These kinases cooperate with the upstream Mst2 kinase to phosphorylate YAPs at Ser-127. Overexpression of YAP2 in HEK293 cells promoted apoptosis, whereas the Mst2/Lats1-induced phosphorylation of YAP partially rescued the cells from apoptotic death. Apoptotic signaling of YAP2 was mediated via stabilization of p73, which formed a complex with YAP2. All components of the Hippo pathway that we studied were localized in the cytoplasm, with the exception of YAP, which also localized in the nucleus. The localization of YAP2 in the nucleus was negatively controlled by the Lats1 kinase. Our apoptotic "readout" of the Hippo pathway in embryonic kidney cells represents a useful experimental system for the identification of the putative upstream receptor, membrane protein, or extracellular factor that initiates an entire signaling cascade and ultimately controls the size of organs.

  12. PKA Phosphorylation of NCLX Reverses Mitochondrial Calcium Overload and Depolarization, Promoting Survival of PINK1-Deficient Dopaminergic Neurons

    Directory of Open Access Journals (Sweden)

    Marko Kostic

    2015-10-01

    Full Text Available Mitochondrial Ca2+ overload is a critical, preceding event in neuronal damage encountered during neurodegenerative and ischemic insults. We found that loss of PTEN-induced putative kinase 1 (PINK1 function, implicated in Parkinson disease, inhibits the mitochondrial Na+/Ca2+ exchanger (NCLX, leading to impaired mitochondrial Ca2+ extrusion. NCLX activity was, however, fully rescued by activation of the protein kinase A (PKA pathway. We further show that PKA rescues NCLX activity by phosphorylating serine 258, a putative regulatory NCLX site. Remarkably, a constitutively active phosphomimetic mutant of NCLX (NCLXS258D prevents mitochondrial Ca2+ overload and mitochondrial depolarization in PINK1 knockout neurons, thereby enhancing neuronal survival. Our results identify an mitochondrial Ca2+ transport regulatory pathway that protects against mitochondrial Ca2+ overload. Because mitochondrial Ca2+ dyshomeostasis is a prominent feature of multiple disorders, the link between NCLX and PKA may offer a therapeutic target.

  13. A secreted tyrosine kinase acts in the extracellular environment.

    Science.gov (United States)

    Bordoli, Mattia R; Yum, Jina; Breitkopf, Susanne B; Thon, Jonathan N; Italiano, Joseph E; Xiao, Junyu; Worby, Carolyn; Wong, Swee-Kee; Lin, Grace; Edenius, Maja; Keller, Tracy L; Asara, John M; Dixon, Jack E; Yeo, Chang-Yeol; Whitman, Malcolm

    2014-08-28

    Although tyrosine phosphorylation of extracellular proteins has been reported to occur extensively in vivo, no secreted protein tyrosine kinase has been identified. As a result, investigation of the potential role of extracellular tyrosine phosphorylation in physiological and pathological tissue regulation has not been possible. Here, we show that VLK, a putative protein kinase previously shown to be essential in embryonic development, is a secreted protein kinase, with preference for tyrosine, that phosphorylates a broad range of secreted and ER-resident substrate proteins. We find that VLK is rapidly and quantitatively secreted from platelets in response to stimuli and can tyrosine phosphorylate coreleased proteins utilizing endogenous as well as exogenous ATP sources. We propose that discovery of VLK activity provides an explanation for the extensive and conserved pattern of extracellular tyrosine phosphophorylation seen in vivo, and extends the importance of regulated tyrosine phosphorylation into the extracellular environment.

  14. Structure, regulation, and putative function of the arginine deiminase system of Streptococcus suis.

    Science.gov (United States)

    Gruening, Petra; Fulde, Marcus; Valentin-Weigand, Peter; Goethe, Ralph

    2006-01-01

    Streptococcus suis is an important cause of infectious diseases in young pigs. Little is known about the virulence factors or protective antigens of S. suis. Recently, we have identified two proteins of the arginine deiminase system (ADS) of S. suis, which were temperature induced and expressed on the streptococcal surface (N. Winterhoff, R. Goethe, P. Gruening, M. Rohde, H. Kalisz, H. E. Smith, and P. Valentin-Weigand, J. Bacteriol. 184:6768-6776, 2002). In the present study, we analyzed the complete ADS of S. suis. Due to their homologies to the recently published S. gordonii ADS genes, the genes for arginine deiminase, ornithine carbamoyl-transferase, and carbamate kinase, which were previously designated adiS, octS, and ckS, respectively, were renamed arcA, arcB, and arcC, respectively. Our data revealed that arcA, arcB, and arcC of the S. suis ADS are transcribed from an operon (arcABC operon). Additionally, putative ADS-associated genes were cloned and sequenced which, however, did not belong to the arcABC operon. These were the flpS gene upstream of the arcABC operon with homology to the flp transcription regulator of S. gordonii and the arcD, arcT, arcH, and argR genes downstream of the arcABC operon with high homologies to a putative arginine-ornithine antiporter, a putative dipeptidase of S. gordonii, a putative beta-N-acetylhexosaminidase of S. pneumoniae, and a putative arginine repressor of S. gordonii, respectively. The transcriptional start point of the arcABC operon was determined, and promoter analysis provided evidence that multiple factors contribute to the regulation of the ADS. Thus, a putative binding site for a transcription regulator of the Crp/Fnr family, an ArgR-binding site, and two cis-acting catabolite response elements were identified in the promoter-operator region of the operon. Consistent with this, we could demonstrate that the ADS of S. suis is inducible by arginine and reduced O2 tension and subject to carbon catabolite

  15. Putative bronchopulmonary flagellated protozoa in immunosuppressed patients.

    Science.gov (United States)

    Kilimcioglu, Ali Ahmet; Havlucu, Yavuz; Girginkardesler, Nogay; Celik, Pınar; Yereli, Kor; Özbilgin, Ahmet

    2014-01-01

    Flagellated protozoa that cause bronchopulmonary symptoms in humans are commonly neglected. These protozoal forms which were presumed to be "flagellated protozoa" have been previously identified in immunosuppressed patients in a number of studies, but have not been certainly classified so far. Since no human cases of bronchopulmonary flagellated protozoa were reported from Turkey, we aimed to investigate these putative protozoa in immunosuppressed patients who are particularly at risk of infectious diseases. Bronchoalveolar lavage fluid samples of 110 immunosuppressed adult patients who were admitted to the Department of Chest Diseases, Hafsa Sultan Hospital of Celal Bayar University, Manisa, Turkey, were examined in terms of parasites by light microscopy. Flagellated protozoal forms were detected in nine (8.2%) of 110 cases. Metronidazole (500 mg b.i.d. for 30 days) was given to all positive cases and a second bronchoscopy was performed at the end of the treatment, which revealed no parasites. In conclusion, immunosuppressed patients with bronchopulmonary symptoms should attentively be examined with regard to flagellated protozoa which can easily be misidentified as epithelial cells.

  16. The Biogeography of Putative Microbial Antibiotic Production.

    Directory of Open Access Journals (Sweden)

    Hélène Morlon

    Full Text Available Understanding patterns in the distribution and abundance of functional traits across a landscape is of fundamental importance to ecology. Mapping these distributions is particularly challenging for species-rich groups with sparse trait measurement coverage, such as flowering plants, insects, and microorganisms. Here, we use likelihood-based character reconstruction to infer and analyze the spatial distribution of unmeasured traits. We apply this framework to a microbial dataset comprised of 11,732 ketosynthase alpha gene sequences extracted from 144 soil samples from three continents to document the spatial distribution of putative microbial polyketide antibiotic production. Antibiotic production is a key competitive strategy for soil microbial survival and performance. Additionally, novel antibiotic discovery is highly relevant to human health, making natural antibiotic production by soil microorganisms a major target for bioprospecting. Our comparison of trait-based biogeographical patterns to patterns based on taxonomy and phylogeny is relevant to our basic understanding of microbial biogeography as well as the pressing need for new antibiotics.

  17. Mechanosensory neurons, cutaneous mechanoreceptors, and putative mechanoproteins.

    Science.gov (United States)

    Del Valle, M E; Cobo, T; Cobo, J L; Vega, J A

    2012-08-01

    The mammalian skin has developed sensory structures (mechanoreceptors) that are responsible for different modalities of mechanosensitivity like touch, vibration, and pressure sensation. These specialized sensory organs are anatomically and functionally connected to a special subset of sensory neurons called mechanosensory neurons, which electrophysiologically correspond with Aβ fibers. Although mechanosensory neurons and cutaneous mechanoreceptors are rather well known, the biology of the sense of touch still remains poorly understood. Basically, the process of mechanosensitivity requires the conversion of a mechanical stimulus into an electrical signal through the activation of ion channels that gate in response to mechanical stimuli. These ion channels belong primarily to the family of the degenerin/epithelium sodium channels, especially the subfamily acid-sensing ion channels, and to the family of transient receptor potential channels. This review compiles the current knowledge on the occurrence of putative mechanoproteins in mechanosensory neurons and mechanoreceptors, as well as the involvement of these proteins on the biology of touch. Furthermore, we include a section about what the knock-out mice for mechanoproteins are teaching us. Finally, the possibilities for mechanotransduction in mechanoreceptors, and the common involvement of the ion channels, extracellular membrane, and cytoskeleton, are revisited.

  18. Studying Kinetochore Kinases

    NARCIS (Netherlands)

    Saurin, Adrian T; Kops, Geert J P L

    2016-01-01

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

  19. Protein kinase C-associated kinase (PKK) mediates Bcl10-independent NF-kappa B activation induced by phorbol ester.

    Science.gov (United States)

    Muto, Akihiro; Ruland, Jürgen; McAllister-Lucas, Linda M; Lucas, Peter C; Yamaoka, Shoji; Chen, Felicia F; Lin, Amy; Mak, Tak W; Núñez, Gabriel; Inohara, Naohiro

    2002-08-30

    Protein kinase C-associated kinase (PKK) is a recently described kinase of unknown function that was identified on the basis of its specific interaction with PKC beta. PKK contains N-terminal kinase and C-terminal ankyrin repeats domains linked to an intermediate region. Here we report that the kinase domain of PKK is highly homologous to that of two mediators of nuclear factor-kappa B (NF-kappa B) activation, RICK and RIP, but these related kinases have different C-terminal domains for binding to upstream factors. We find that expression of PKK, like RICK and RIP, induces NF-kappa B activation. Mutational analysis revealed that the kinase domain of PKK is essential for NF-kappa B activation, whereas replacement of serine residues in the putative activation loop did not affect the ability of PKK to activate NF-kappa B. A catalytic inactive PKK mutant inhibited NF-kappa B activation induced by phorbol ester and Ca(2+)-ionophore, but it did not block that mediated by tumor necrosis factor alpha, interleukin-1 beta, or Nod1. Inhibition of NF-kappa B activation by dominant negative PKK was reverted by co-expression of PKC beta I, suggesting a functional association between PKK and PKC beta I. PKK-mediated NF-kappa B activation required IKK alpha and IKK beta but not IKK gamma, the regulatory subunit of the IKK complex. Moreover, NF-kappa B activation induced by PKK was not inhibited by dominant negative Bimp1 and proceeded in the absence of Bcl10, two components of a recently described PKC signaling pathway. These results suggest that PKK is a member of the RICK/RIP family of kinases, which is involved in a PKC-activated NF-kappa B signaling pathway that is independent of Bcl10 and IKK gamma.

  20. Two-Component Signal Transduction Systems of Desulfovibrio Vulgaris: Structural and Phylogenetic Analysis and Deduction of Putative Cognate Pairs

    Energy Technology Data Exchange (ETDEWEB)

    Zhang, Weiwen; Culley, David E.; Wu, Gang; Brockman, Fred J.

    2006-01-20

    ABSTRACT-Two-component signal transduction systems (TCSTS) composed of sensory histidine kinases (HK) and response regulators (RR), constitute a key element of the mechanism by which bacteria sense and respond to changes in environments. A large number of TCSTSs including 59 putative HKs and 55 RRs were identified from the Desulfovibrio vulgaris genome, indicating their important roles in regulation of cellular metabolism. In this study, the structural and phylogenetic analysis of all putative TCSTSs in D. vulgaris was performed. The results showed D. vulgaris contained an unexpectedly large number of hybrid-type HKs, implying that multiple-step phosphorelay may be a common signal transduction mechanism in D. vulgaris. Most TCSTS components of D. vulgaris were found clustered into several subfamilies previously recognized in other bacteria and extensive co-evolution between D. vulgaris HKs and RRs was observed, suggesting that the concordance of HKs and RRs in cognate phylogenetic groups could be indicative of cognate TCSTSs...

  1. Identification of putative CLE peptide receptors involved in determinate nodulation on soybean.

    Science.gov (United States)

    Mortier, Virginie; Fenta, Berhanu Amsalu; Kunert, Karl; Holsters, Marcelle; Goormachtig, Sofie

    2011-07-01

    CLAVATA3/EMBRYO SURROUNDING REGION (CLE) peptides tightly control the balance between stem cell proliferation and differentiation in several plant developmental processes. Transmission of the CLE peptide signal has been shown to be rather complex. Despite their recent identification, little is known about the receptors by which nodulation-specific CLE peptides, which were identified in soybean, are perceived. Genetic analysis has indicated that the leucine-rich repeat receptor-like kinase NARK of soybean (Glycine max) and its orthologs in other legumes are possible candidates. However, more receptors need to be identified because CLE peptides are often detected by heteromultimeric complexes. Here, we identified two additional putative CLE peptide receptor pairs in the soybean genome with a nodulation-related expression pattern, GmRLK1-GmRLK2 and GmRLK3-GmRLK4, and discuss their role in CLE peptide perception during nodulation.

  2. Characterization of ERAS, a putative novel human oncogene, in skin and breast

    Energy Technology Data Exchange (ETDEWEB)

    Peña Avalos, B.L. de la

    2014-07-01

    Most human tumors have mutations in genes of the RAS small GTPase protein family. RAS works as a molecular switch for signaling pathways that modulate many aspects of cell behavior, including proliferation, differentiation, motility and death. Oncogenic mutations in RAS prevent GTP hydrolysis, locking RAS in a permanently active state, being the most common mutations in HRAS, KRAS and NRAS. The human RAS family consists of at least 36 different genes, many of which have been scarcely studied. One of these relatively unknown genes is ERAS (ES cell-expressed RAS), which is a constitutively active RAS protein, localized in chromosome X and expressed only in embryonic cells, being undetectable in adult tissues. New high throughput technologies have made it possible to screen complete cancer genomes for identification of mutations associated to cancer. Using the Sleeping Beauty (SB) transposon system, ERAS was identified as a putative novel oncogene in non-melanoma skin and breast cancers. The major aim of this project is to determine the general characteristics of ERAS as a putative novel human oncogene in skin and breast cells. Forced expression of ERAS results in drastic changes in cell shape, proliferation and motility. When ERAS is overexpressed in skin and breast human cells it is mainly localized in the cytoplasmic membrane. ERAS activates the phosphatidylinositol-3-OH kinase (PI3K) pathway but not the mitogen-activated protein kinase (MAPK) pathway. ERAS-expressing cells suffer spontaneous morphologic and phenotypic EMT-like changes, including cytoskeleton reorganization, vimentin and N-cadherin up-regulation and down-regulation of E-cadherin, which can be associated with increased malignancy, and invasive and metastatic potential. Our results suggest that inappropriate expression of ERAS lead to transformation of human cells. (Author)

  3. Enterococcus faecalis phosphomevalonate kinase.

    Science.gov (United States)

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

    2005-05-01

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

  4. Regulation of Arabidopsis Early Anther Development by Putative Cell-Cell Signaling Molecules and Transcriptional Regulators

    Institute of Scientific and Technical Information of China (English)

    Yu-Jin Sun; Carey LH Hord; Chang-Bin Chen; Hong Ma

    2007-01-01

    Anther development in flowering plants involves the formation of several cell types, including the tapetal and pollen mother cells. The use of genetic and molecular tools has led to the identification and characterization of genes that are critical for normal cell division and differentiation in Arabidopsis early anther development. We review here several recent studies on these genes, including the demonstration that the putative receptor protein kinases BAM1 and BAM2 together play essential roles in the control of early cell division and differentiation. In addition, we discuss the hypothesis that BAM1/2 may form a positive-negative feedback regulatory loop with a previously identified key regulator, SPOROCYTELESS (also called NOZZLE),to control the balance between sporogenous and somatic cell types in the anther. Furthermore, we summarize the isolation and functional analysis of the DYSFUNCTIONAL TAPETUM1 (DYT1) gene in promoting proper tapetal cell differentiation. Our finding that DYT1 encodes a putative transcription factor of the bHLH family, as well as relevant expression analyses, strongly supports a model that DYT1 serves as a critical link between upstream factors and downstream target genes that are critical for normal tapetum development and function. These studies, together with other recently published works, indicate that cell-cell communication and transcriptional control are key processes essential for cell fate specification in anther development.

  5. Targeting the Pim kinases in multiple myeloma.

    LENUS (Irish Health Repository)

    Keane, N A

    2015-07-17

    Multiple myeloma (MM) is a plasma cell malignancy that remains incurable. Novel treatment strategies to improve survival are urgently required. The Pims are a small family of serine\\/threonine kinases with increased expression across the hematological malignancies. Pim-2 shows highest expression in MM and constitutes a promising therapeutic target. It is upregulated by the bone marrow microenvironment to mediate proliferation and promote MM survival. Pim-2 also has a key role in the bone destruction typically seen in MM. Additional putative roles of the Pim kinases in MM include trafficking of malignant cells, promoting oncogenic signaling in the hypoxic bone marrow microenvironment and mediating resistance to therapy. A number of Pim inhibitors are now under development with lead compounds entering the clinic. The ATP-competitive Pim inhibitor LGH447 has recently been reported to have single agent activity in MM. It is anticipated that Pim inhibition will be of clinical benefit in combination with standard treatments and\\/or with novel drugs targeting other survival pathways in MM.

  6. Plant phosphatidylinositol 3-kinase

    NARCIS (Netherlands)

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

    2010-01-01

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

  7. Plant phosphatidylinositol 3-kinase

    NARCIS (Netherlands)

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

    2010-01-01

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

  8. Putative Corneal Neuralgia Responding to Vitamin D Supplementation

    Directory of Open Access Journals (Sweden)

    Eric L. Singman

    2013-09-01

    Full Text Available A patient with putative corneal neuralgia was incidentally discovered to have hypovitaminosis D. Supplementation of vitamin D appears to have led to a resolution of the patient's pain, whereas other efforts to treat the patient were unsuccessful.

  9. Substrate specificity engineering of Escherichia coli derived fructosamine 6-kinase.

    Science.gov (United States)

    Kojima, Katsuhiro; Mikami-Sakaguchi, Akane; Kameya, Miho; Miyamoto, Yusuke; Ferri, Stefano; Tsugawa, Wakako; Sode, Koji

    2013-02-01

    A three-dimensional structural model of Escherichia coli fructosamine 6-kinase (FN6K), an enzyme that phosphorylates fructosamines at C6 and catalyzes the production of the fructosamine 6-phosphate stable intermediate, was generated using the crystal structure of 2-keto-3-deoxygluconate kinase isolated from Thermus thermophilus as template. The putative active site region was then investigated by site-directed mutagenesis to reveal several amino acid residues that likely play important roles in the enzyme reaction. Met220 was identified as a residue that plays a role in substrate recognition when compared to Bacillus subtilis derived FN6K, which shows different substrate specificity from the E. coli FN6K. Among the various Met220-substituted mutant enzymes, Met220Leu, which corresponded to the B. subtilis residue, resulted in an increased activity of fructosyl-valine and decreased activity of fructosyl-lysine, thus increasing the specificity for fructosyl-valine by 40-fold.

  10. The kinase activation loop is the key to mixed lineage kinase-3 activation via both autophosphorylation and hematopoietic progenitor kinase 1 phosphorylation.

    Science.gov (United States)

    Leung, I W; Lassam, N

    2001-01-19

    We have demonstrated previously that Cdc42 induced MLK-3 homodimerization leads to both autophosphorylation and activation of MLK-3 and postulated that autophosphorylation is an intermediate step of MLK-3 activation following its dimerization. In this report we sought to refine further the mechanism of MLK-3 activation and study the role of the putative kinase activation loop in MLK-3 activation. First we mutated the three potential phosphorylation sites in MLK-3 putative activation loop to alanine in an effort to abrogate MLK-3 autophosphorylation. Mutant T277A displayed almost no autophosphorylation activity and was nearly nonfunctional; mutant S281A, that displayed a low level of autophosphorylation, only slightly activated its downstream targets, whereas the T278A mutant, that exhibited autophosphorylation comparable to that of the wild type, was almost fully functional. Thus, these residues within the activation loop are critical for MLK-3 autophosphorylation and activation. In addition, when the Thr277 and Ser281 residues were mutated to negatively charged glutamic acid to mimic phosphorylated serine/threonine residues, the resulting mutants were fully functional, implying that these two residues may serve as the autophosphorylation sites. Interestingly, HPK1 also phosphorylated MLK-3 activation loop in vitro, and Ser281 was found to be the major phosphorylation site, indicating that HPK1 also activates MLK-3 via phosphorylation of the kinase activation loop.

  11. Tyrosine kinases in rheumatoid arthritis

    Directory of Open Access Journals (Sweden)

    Kobayashi Akiko

    2011-08-01

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

  12. Molecular cloning and characterization of a novel human kinase gene, PDIK1L

    Indian Academy of Sciences (India)

    Lingchen Guo; Chaoneng Ji; Shaohua Gu; Kang Ying; Haipeng Cheng; Xiaoghua Ni; Jianping Liu; Yi Xie; Yumin Mao

    2003-04-01

    We isolated a 4301-bp cDNA from a human foetal brain cDNA library by high-throughput cDNA sequencing. It encodes a protein of 341 amino acids, which shows 69% identity with the human kinase CLIK1 (AAL99353), which was suggested to be the CLP-36 interacting kinase. Bioinformatics analysis suggests that the putative kinase may interact with PDZ and LIM domain proteins. Therefore the protein and its cDNA were named ‘PDLIM1 interacting kinase 1 like’ (PDIK1L; nomenclature approved by the HUGO Gene Nomenclature Committee). Ensembl Genome Browser located PDIK1L to human chromosome 1p35.3. It spans about 13.7 kb and consists of four exons and three introns. Multiple-tissue cDNA panel PCR revealed that the gene is expressed widely in human tissues: liver, kidney, pancreas, spleen, thymus and prostate. The protein appears to be localized to the nucleus.

  13. Oncoprotein protein kinase

    Energy Technology Data Exchange (ETDEWEB)

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

    2003-02-04

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

  14. Enterococcus faecalis phosphomevalonate kinase

    OpenAIRE

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

    2005-01-01

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

  15. Regulation of basal resistance by a powdery mildew-induced cysteine-rich receptor-like protein kinase in barley

    DEFF Research Database (Denmark)

    Rayapuram, Cbgowda; Jensen, Michael K; Maiser, Fabian

    2012-01-01

    , followed by a rather short 17-amino-acid transmembrane domain, which includes an AAA motif, two features characteristic of endoplasmic reticulum (ER)-targeted proteins and, finally, a characteristic putative protein kinase domain in the C-terminus. The HvCRK1 transcript was isolated from leaves inoculated...

  16. Uncoupling anaphase-promoting complex/cyclosome activity from spindle assembly checkpoint control by deregulating polo-like kinase 1

    NARCIS (Netherlands)

    Weerdt, B.C.M. van de; Vugt, M.A.T.M. van; Lindon, C.; Kauw, J.J.W.; Rozendaal, M.J.; Klompmaker, R.; Wolthuis, R.M.F.; Medema, R.H.

    2005-01-01

    Polo-like kinase 1 (Plk1) plays a role in numerous events in mitosis, but how the multiple functions of Plk1 are separated is poorly understood. We studied regulation of Plk1 through two putative phosphorylation residues, Ser-137 and Thr-210. Using phospho-specific antibodies, we found that Thr-210

  17. Uncoupling anaphase-promoting complex/cyclosome activity from spindle assembly checkpoint control by deregulating polo-like kinase 1

    NARCIS (Netherlands)

    van de Weerdt, BCM; van Vugt, MATM; Lindon, C; Kauw, JJW; Rozendaal, MJ; Klompmaker, R; Wolthuis, RMF; Medema, RH

    2005-01-01

    Polo-like kinase 1 (Plk1) plays a role in numerous events in mitosis, but how the multiple functions of Plk1 are separated is poorly understood. We studied regulation of Plkl through two putative phosphorylation residues, Ser-137 and Thr-210. Using phospho-specific antibodies, we found that Thr-210

  18. Regulation of Autophagy by Kinases

    Energy Technology Data Exchange (ETDEWEB)

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

    2011-06-09

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

  19. The mitogen-activated protein kinase kinase kinase BcOs4 is required for vegetative differentiation and pathogenicity in Botrytis cinerea.

    Science.gov (United States)

    Yang, Qianqian; Yan, Leiyan; Gu, Qin; Ma, Zhonghua

    2012-10-01

    The high-osmolarity glycerol signal pathway plays an important role in the response of fungi to various environmental stresses. In this study, we characterized a mitogen-activated protein kinase kinase kinase gene BcOS4 in Botrytis cinerea, which is homologous to Saccharomyces cerevisiae SSK2/SSK22. The BcOS4 deletion mutant was significantly impaired in vegetative growth and conidial formation. The mutant exhibited increased sensitivity to the osmotic, oxidative stresses and to the fungicides iprodione and fludioxonil. Western blot analysis showed that BcSak1, a putative downstream component of BcOs4, was not phosphorylated in the mutant. In addition, the BcOS4 mutant was unable to infect leaves of rapeseed and cucumber, and grape fruits, although it can cause disease on apple fruits. All the defects were restored by genetic complementation of the BcOS4 deletion mutant with the wild-type BcOS4 gene. The data of this study indicate that BcOS4 is involved in vegetative differentiation, virulence, adaption to hyperosmotic and oxidative stresses, and to fungicides in B. cinerea.

  20. Adenoviral transduction of PTEN induces apoptosis of cultured hepatic stellate cells

    Institute of Scientific and Technical Information of China (English)

    HAO Li-sen; ZHANG Xiao-lan; AN Jun-yan; YAO Dong-mei; Justin Karlin; FANG Shu-ming; JIANG Hui-qing; BAI Wen-yuan; CHEN Shuang

    2009-01-01

    @@ Hepatic fibrosis is the liver's wound healing response to virtually all forms of chronic liver injury: toxic insult, viral infection, immunological conditions and metabolic diseases. Uncontrolled liver fibrosis eventually results in cirrhosis and associated complications, such as cancer and liver failure.

  1. Exploring the function of protein kinases in schistosomes: perspectives from the laboratory and from comparative genomics

    Directory of Open Access Journals (Sweden)

    Anthony John Walker

    2014-07-01

    Full Text Available Eukaryotic protein kinases are well conserved through evolution. The genome of Schistosoma mansoni, which causes intestinal schistosomiasis, encodes over 250 putative protein kinases with all of the main eukaryotic groups represented. However, unraveling functional roles for these kinases is a considerable endeavour, particularly as protein kinases regulate multiple and sometimes overlapping cell and tissue functions in organisms. In this article, elucidating protein kinase signal transduction and function in schistosomes is considered from the perspective of the state-of-the-art methodologies used and comparative organismal biology, with a focus on current advances and future directions. Using the free-living nematode Caenorhabditis elegans as a comparator we predict roles for various schistosome protein kinases in processes vital for host invasion and successful parasitism such as sensory behaviour, growth and development. It is anticipated that the characterization of schistosome protein kinases in the context of parasite function will catalyze cutting edge research into host-parasite interactions and will reveal new targets for developing drug interventions against human schistosomiasis.

  2. Phosphatidylinositol 3-kinase in myogenesis.

    Science.gov (United States)

    Kaliman, P; Zorzano, A

    1997-08-01

    Phosphatidylinositol 3-kinase (PI 3-kinase) has been cloned and characterized in a wide range of organisms. PI 3-kinases are activated by a diversity of extracellular stimuli and are involved in multiple cell processes such as cell proliferation, protein trafficking, cell motility, differentiation, regulation of cytoskeletal structure, and apoptosis. It has recently been shown that PI 3-kinase is a crucial second messenger in the signaling of myogenesis. Two structurally unrelated highly specific inhibitors of PI 3-kinase-wortmannin and LY294002-block the morphological and biochemical differentiation program of different skeletal-muscle cell models. Moreover, L6E9 myoblasts overexpressing a dominant-negative mutant of PI 3-kinase p85 regulatory subunit (Δp85) are unable to differentiate. Furthermore, PI 3-kinase is specifically involved in the insulinlike growth factor (IGF)-dependent myogenic pathway. Indeed, the ability of IGF-I, des-1,3-IGF-I, and IGF-II to promote cell fusion and muscle-specific protein expression is impaired after treatment with PI 3-kinase inhibitors or in cells overexpressing Δp85. The identification of additional key downstream elements of the IGF/PI 3-kinase myogenic cascade is crucial to a detailed understanding of the process of muscle differentiation and may generate new tools for skeletal and cardiac muscle regeneration therapies. (Trends Cardiovasc Med 1997;7:198-202). © 1997, Elsevier Science Inc.

  3. A putative viral defence mechanism in archaeal cells

    DEFF Research Database (Denmark)

    Lillestøl, Reidun K; Redder, Peter; Garrett, Roger Antony

    2006-01-01

    in cells, and that both the mode of inhibition of viral propagation and the mechanism of adding spacer-repeat units to clusters, are dependent on RNAs transcribed from the clusters. Moreover, the putative inhibitory apparatus (piRNA-based) may be evolutionarily related to the interference RNA systems (si...

  4. Putative Lineage of Novel African Usutu Virus, Central Europe

    Centers for Disease Control (CDC) Podcasts

    2015-10-15

    Sarah Gregory reads an abridged version of "Putative Lineage of Novel African Usutu Virus, Central Europe.".  Created: 10/15/2015 by National Center for Emerging and Zoonotic Infectious Diseases (NCEZID).   Date Released: 10/15/2015.

  5. Putative golden proportions as predictors of facial esthetics in adolescents.

    NARCIS (Netherlands)

    Kiekens, R.M.A.; Kuijpers-Jagtman, A.M.; Hof, M.A. van 't; Hof, BE van 't; Maltha, J.C.

    2008-01-01

    INTRODUCTION: In orthodontics, facial esthetics is assumed to be related to golden proportions apparent in the ideal human face. The aim of the study was to analyze the putative relationship between facial esthetics and golden proportions in white adolescents. METHODS: Seventy-six adult laypeople

  6. Identification of putative drug targets of Listeria monocytogenes F2365 by subtractive genomics approach

    Directory of Open Access Journals (Sweden)

    Md. Musharaf Hossain

    2013-01-01

    Full Text Available The prolonged and uncontrolled use of antibiotics in treatment against many pathogens causes the multiple drug resistance. The drug resistance of Listeria monocytogenes F2365 has been evolved, which cause a major disease listeriosis. The drug dose limit against that pathogen was also increased for currently prescribed antibiotics and more often combinational therapy was preferred. Therefore, identification of an extensive novel drug target, unique and essential to the microorganism and subjected to its validation and drug development is imperative. Availability of the total proteome of L. monocytogenes F2365 enabled in silico identification of putative common drug targets and their subcellular localization by subtractive genomics approach. In the present work subtractive genomics approach is used to identify vaccine and drug targets of L. monocytogenes F2365 to speed up the rational drug and vaccine design. It has revealed that out of 2821 reference sequences of the pathogen, 744 represent essential proteins and among them 274 are human non-homolog proteins. Besides, all predicted human non-homologs were then analyzed by subcellular localization servers, in which 46 proteins were identified as surface exposed proteins and can be considered as potential drug and vaccine targets for the pathogen. The 3D structure of two human non-homolog putative drug targets, pantothenate kinase (LmPK and holliday junction resolvase-like protein (LmHJR of L. monocytogenes F2365 were generated by homology modeling program Easymodeller 4.0; a GUI version of modeller. Generated structures were also validated by several online servers. The overall stereochemical quality of the model was assessed by Ramachandran plot analysis that was provided by PROCHECK. ProQ, ERRAT, Pro-SA web and VERIFY 3D of SAVES programs were also used to compute several validation parameters during the evaluation of the model. This protein structure information is important in structure

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

    Directory of Open Access Journals (Sweden)

    Agarwala Usha

    2011-06-01

    a companion diagnostic biomarker and a putative functional site for kinase-unrelated activities of Cdk4.

  8. Cellular reprogramming through mitogen-activated protein kinases

    Directory of Open Access Journals (Sweden)

    Justin eLee

    2015-10-01

    Full Text Available Mitogen-activated protein kinase (MAPK cascades are conserved eukaryote signaling modules where MAPKs, as the final kinases in the cascade, phosphorylate protein substrates to regulate cellular processes. While some progress in the identification of MAPK substrates has been made in plants, the knowledge on the spectrum of substrates and their mechanistic action is still fragmentary. In this focused review, we discuss the biological implications of the data in our original paper (Sustained mitogen-activated protein kinase activation reprograms defense metabolism and phosphoprotein profile in Arabidopsis thaliana; Frontiers in Plant Science 5: 554 in the context of related research. In our work, we mimicked in vivo activation of two stress-activated MAPKs, MPK3 and MPK6, through transgenic manipulation of Arabidopsis thaliana and used phosphoproteomics analysis to identify potential novel MAPK substrates. Here, we plotted the identified putative MAPK substrates (and downstream phosphoproteins as a global protein clustering network. Based on a highly stringent selection confidence level, the core networks highlighted a MAPK-induced cellular reprogramming at multiple levels of gene and protein expression – including transcriptional, post-transcriptional, translational, post-translational (such as protein modification, folding and degradation steps, and also protein re-compartmentalization. Additionally, the increase in putative substrates/phosphoproteins of energy metabolism and various secondary metabolite biosynthesis pathways coincides with the observed accumulation of defense antimicrobial substances as detected by metabolome analysis. Furthermore, detection of protein networks in phospholipid or redox elements suggests activation of downstream signaling events. Taken in context with other studies, MAPKs are key regulators that reprogram cellular events to orchestrate defense signaling in eukaryotes.

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

    Science.gov (United States)

    Lin, Xiaozeng; Yan, Judy; Tang, Damu

    2013-12-01

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

  10. Putative melatonin receptors in a human biological clock

    Energy Technology Data Exchange (ETDEWEB)

    Reppert, S.M.; Weaver, D.R.; Rivkees, S.A.; Stopa, E.G.

    1988-10-07

    In vitro autoradiography with /sup 125/I-labeled melatonin was used to examine melatonin binding sites in human hypothalamus. Specific /sup 125/I-labeled melatonin binding was localized to the suprachiasmatic nuclei, the site of a putative biological clock, and was not apparent in other hypothalamic regions. Specific /sup 125/I-labeled melatonin binding was consistently found in the suprachiasmatic nuclei of hypothalami from adults and fetuses. Densitometric analysis of competition experiments with varying concentrations of melatonin showed monophasic competition curves, with comparable half-maximal inhibition values for the suprachiasmatic nuclei of adults (150 picomolar) and fetuses (110 picomolar). Micromolar concentrations of the melatonin agonist 6-chloromelatonin completely inhibited specific /sup 125/I-labeled melatonin binding, whereas the same concentrations of serotonin and norepinephrine caused only a partial reduction in specific binding. The results suggest that putative melatonin receptors are located in a human biological clock.

  11. Trypanosoma brucei: a putative RNA polymerase II promoter.

    Science.gov (United States)

    Bayele, Henry K

    2009-12-01

    RNA polymerase II (pol II) promoters are rare in the African trypanosome Trypanosoma brucei because gene regulation in the parasite is complex and polycistronic. Here, we describe a putative pol II promoter and its structure-function relationship. The promoter has features of an archetypal eukaryotic pol II promoter including putative canonical CCAAT and TATA boxes, and an initiator element. However, the spatial arrangement of these elements is only similar to yeast pol II promoters. Deletion mapping and transcription assays enabled delineation of a minimal promoter that could drive orientation-independent reporter gene expression suggesting that it may be a bidirectional promoter. In vitro transcription in a heterologous nuclear extract revealed that the promoter can be recognized by the basal eukaryotic transcription complex. This suggests that the transcription machinery in the parasite may be very similar to those of other eukaryotes.

  12. Chromosomal Abnormalities and Putative Susceptibility Genes in Autism Spectrum Disorders

    DEFF Research Database (Denmark)

    Nielsen, Mette Gilling

    Autism spectrum disorders (ASDs) is a heterogeneous group of neurodevelopmental disorders with a significant genetic component as shown by family and twin studies. However, only a few genes have repeatedly been shown to be involved in the development of ASDs. The aim of this study has been...... to identify possible ASD susceptibility genes. Genome screens in ASD patients suggest possible susceptibility gene regions on almost every chromosome. We identified four ASD patients with chromosomal rearrangements, two of which were familial rearrangements involving one of these putative susceptibility gene......) was performed for all four patients. By combination of these methods we identified several putative susceptibility genes for ASDs. Expression patterns were established for several of these genes by Quantitative PCR (Q-PCR) or in situ hybridization and one gene was sequenced in 157 ASD patients. Our results...

  13. High-Resolution Genomic and Expression Profiling Reveals 105 Putative Amplification Target Genes in Pancreatic Cancer

    Directory of Open Access Journals (Sweden)

    Eija H. Mahlamaki

    2004-09-01

    Full Text Available Comparative genomic hybridization (CGH studies have provided a wealth of information on common copy number aberrations in pancreatic cancer, but the genes affected by these aberrations are largely unknown. To identify putative amplification target genes in pancreatic cancer, we performed a parallel copy number and expression survey in 13 pancreatic cancer cell lines using a 12,232-clone cDNA microarray, providing an average resolution of 300 kb throughout the human genome. CGH on cDNA microarray allowed highly accurate mapping of copy number increases and resulted in identification of 24 independent amplicons, ranging in size from 130 kb to 11 Mb. Statistical evaluation of gene copy number and expression data across all 13 cell lines revealed a set of 105 genes whose elevated expression levels were directly attributable to increased copy number. These included genes previously reported to be amplified in cancer as well as several novel targets for copy number alterations, such as p21-activated kinase 4 (PAK4, which was previously shown to be involved in cell migration, cell adhesion, and anchorage-independent growth. In conclusion, our results implicate a set of 105 genes that is likely to be actively involved in the development and progression of pancreatic cancer.

  14. MstX and a putative potassium channel facilitate biofilm formation in Bacillus subtilis.

    Directory of Open Access Journals (Sweden)

    Matthew E Lundberg

    Full Text Available Biofilms constitute the predominant form of microbial life and a potent reservoir for innate antibiotic resistance in systemic infections. In the spore-forming bacterium Bacillus subtilis, the transition from a planktonic to sessile state is mediated by mutually exclusive regulatory pathways controlling the expression of genes required for flagellum or biofilm formation. Here, we identify mstX and yugO as novel regulators of biofilm formation in B. subtilis. We show that expression of mstX and the downstream putative K+ efflux channel, yugO, is necessary for biofilm development in B. subtilis, and that overexpression of mstX induces biofilm assembly. Transcription of the mstX-yugO operon is under the negative regulation of SinR, a transcription factor that governs the switch between planktonic and sessile states. Furthermore, mstX regulates the activity of Spo0A through a positive autoregulatory loop involving KinC, a histidine kinase that is activated by potassium leakage. The addition of potassium abrogated mstX-mediated biofilm formation. Our findings expand the role of Spo0A and potassium homeostasis in the regulation of bacterial development.

  15. Cloning of partial putative gonadotropin hormone receptor sequence from fish

    Indian Academy of Sciences (India)

    G Kumaresan; T Venugopal; A Vikas; T J Pandian; S M Athavan

    2000-03-01

    A search for the presence of mariner-like elements in the Labeo rohita genome by polymerase chain reaction led to the amplification of a partial DNA sequence coding for a putative transmembrane domain of gonadotropin hormone receptor. The amplified DNA sequence shows a high degree of homology to the available turkey and human luteinizing and follicle stimulating hormone receptor coding sequences. This is the first report on cloning such sequences of piscine origin.

  16. A putative role for apelin in the etiology of obesity.

    Science.gov (United States)

    Rayalam, Srujana; Della-Fera, Mary Anne; Krieg, Paul A; Cox, Christopher M; Robins, Allan; Baile, Clifton A

    2008-04-11

    Apelin, the endogenous ligand of the G protein-coupled APJ receptor has been shown to promote tumor angiogenesis. However, the effect of apelin on inducing angiogenesis in adipose tissue has not been investigated. In this review, we propose a putative role for apelin in promoting angiogenesis in adipose tissue. We further propose that targeting adipose tissue vasculature by blocking apelin signaling with anti-apelin antibodies will lead not only to inhibition of angiogenesis in adipose tissue but also to decreased adiposity.

  17. DSTYK kinase domain ablation impaired the mice capabilities of learning and memory in water maze test.

    Science.gov (United States)

    Li, Kui; Liu, Ji-Wei; Zhu, Zhi-Chuan; Wang, Hong-Tao; Zu, Yong; Liu, Yong-Jie; Yang, Yan-Hong; Xiong, Zhi-Qi; Shen, Xu; Chen, Rui; Zheng, Jing; Hu, Ze-Lan

    2014-01-01

    DSTYK (Dual serine/threonine and tyrosine protein kinase) is a putative dual Ser/Thr and Tyr protein kinase with unique structural features. It is proposed that DSTYK may play important roles in brain because of its high expression in most brain areas. In the present study, a DSTYK knockout (KO) mouse line with the ablation of C-terminal of DSTYK including the kinase domain was generated to study the physiological function of DSTYK. The DSTYK KO mice are fertile and have no significant morphological defects revealed by Nissl staining compared with wildtype mice. Open field test and rotarod test showed there is no obvious difference in basic motor and balance capacity between the DSTYK homozygous KO mice and DSTYK heterozygous KO mice. In water maze test, however, the DSTYK homozygous KO mice show impaired capabilities of learning and memory compared with the DSTYK heterozygous KO mice.

  18. A kinome wide screen identifies novel kinases involved in regulation of monoamine transporter function

    DEFF Research Database (Denmark)

    Vuorenpää, Anne Elina; Ammendrup-Johnsen, Ina; Jorgensen, Trine N.

    2016-01-01

    cells (CAD) and rat chromocytoma (PC12) cells. Whereas SIK3 likely transcriptionally regulated expression of the three transfected transporters, depletion of PKA C-α was shown to decrease SERT function. Depletion of PrKX caused decreased surface expression and function of DAT without changing protein...... levels, suggesting that PrKX stabilizes the transporter at the cell surface. Summarized, our data provide novel insight into kinome regulation of the monoamine transporters and identifies PrKX as a yet unappreciated possible regulator of monoamine transporter function....... in regulation of monoamine transporter function and surface expression. A primary screen in HEK 293 cells stably expressing DAT or SERT with siRNAs against 573 human kinases revealed 93 kinases putatively regulating transporter function. All 93 hits, which also included kinases previously implicated...

  19. Mutation of serine 1333 in the ATR HEAT repeats creates a hyperactive kinase.

    Directory of Open Access Journals (Sweden)

    Jessica W Luzwick

    Full Text Available Subcellular localization, protein interactions, and post-translational modifications regulate the DNA damage response kinases ATR, ATM, and DNA-PK. During an analysis of putative ATR phosphorylation sites, we found that a single mutation at S1333 creates a hyperactive kinase. In vitro and in cells, mutation of S1333 to alanine (S1333A-ATR causes elevated levels of kinase activity with and without the addition of the protein activator TOPBP1. S1333 mutations to glycine, arginine, or lysine also create a hyperactive kinase, while mutation to aspartic acid decreases ATR activity. S1333A-ATR maintains the G2 checkpoint and promotes completion of DNA replication after transient exposure to replication stress but the less active kinase, S1333D-ATR, has modest defects in both of these functions. While we find no evidence that S1333 is phosphorylated in cultured cells, our data indicate that small changes in the HEAT repeats can have large effects on kinase activity. These mutants may serve as useful tools for future studies of the ATR pathway.

  20. Integrin-linked kinase is an adaptor with essential functions during mouse development.

    Science.gov (United States)

    Lange, Anika; Wickström, Sara A; Jakobson, Madis; Zent, Roy; Sainio, Kirsi; Fässler, Reinhard

    2009-10-15

    The development of multicellular organisms requires integrin-mediated interactions between cells and their extracellular environment. Integrin binding to extracellular matrix catalyses assembly of multiprotein complexes, which transduce mechanical and chemical signals that regulate many aspects of cell physiology. Integrin-linked kinase (Ilk) is a multifunctional protein that binds beta-integrin cytoplasmic domains and regulates actin dynamics by recruiting actin binding regulatory proteins such as alpha- and beta-parvin. Ilk has also been shown to possess serine/threonine kinase activity and to phosphorylate signalling proteins such as Akt1 and glycogen synthase kinase 3beta (Gsk3beta) in mammalian cells; however, these functions have been shown by genetic studies not to occur in flies and worms. Here we show that mice carrying point mutations in the proposed autophosphorylation site of the putative kinase domain and in the pleckstrin homology domain are normal. In contrast, mice with point mutations in the conserved lysine residue of the potential ATP-binding site of the kinase domain, which mediates Ilk binding to alpha-parvin, die owing to renal agenesis. Similar renal defects occur in alpha-parvin-null mice. Thus, we provide genetic evidence that the kinase activity of Ilk is dispensable for mammalian development; however, an interaction between Ilk and alpha-parvin is critical for kidney development.

  1. W55a Encodes a Novel Protein Kinase That Is Involved in Multiple Stress Responses

    Institute of Scientific and Technical Information of China (English)

    Zhao-Shi Xu; Li Liu; Zhi-Yong Ni; Pei Liu; Ming Chen; Lian-Cheng Li; Yao-Feng Chen; You-Zhi Ma

    2009-01-01

    Protein kinases play crucial roles In response to external environment stress signals. A putative protein kinase, W55a, belonging to SNF1-related protein kinase 2 (SnRK2) subfamily, was isolated from a cDNA library of drought-treated wheat seedlings. The entire length of W55a was obtained using rapid amplification of 5' cDNA ends (5'-RACE) and reverse transcription-polymerase chain reaction(RT-PCR). It contains a 1029-bp open reading frame (ORF) encoding 342 amino acids. The deduced amino acid sequence of W55a had eleven conserved catalytic subdomains and one Ser/Thr protein kinase active-site that characterize Ser/Thr protein kinases. Phylogenetic analysis showed that W55a was 90.38% homologous with rice SAPK1, a member of the SnRK2 family. Using nullisomic-tetrasomic and ditelocentric lines of Chinese Spring, W55a was located on chromosome 2BS. Expression pattern analysis revealed that W55a was upregulated by drought and salt, exogenous abscisic acid, salicylic acid, ethylene and methyl jasmonata, but was not responsive to cold stress. In addition, W55a transcripts were abundant in leaves, but not in roots or stems, under environmental stresses. Transgenic Arabidopsis plants overexprassing W55a exhibited higher tolerance to drought. Based on these findings, W55a encodes a novel dehydration-responsive protein kinase that is involved in multiple stress signal transductions.

  2. MAP Kinases in Immune Responses

    Institute of Scientific and Technical Information of China (English)

    Yongliang Zhang; Chen Dong

    2005-01-01

    MAP kinases are evolutionarily conserved signaling regulators from budding yeast to mammals and play essential roles in both innate and adaptive immune responses. There are three main families of MAPKs in mammals. Each of them has its own activators, inactivators, substrates and scaffolds, which altogether form a fine signaling network in response to different extracellular or intracellular stimulation. In this review, we summarize recent advances in understanding of the regulation of MAP kinases and the roles of MAP kinases in innate and adaptive immune responses.

  3. MAP Kinases in Immune Responses

    Institute of Scientific and Technical Information of China (English)

    YongliangZhang; ChenDong

    2005-01-01

    MAP kinases are evolutionarily conserved signaling regulators from budding yeast to mammals and play essential roles in both innate and adaptive immune responses. There are three main families of MAPKs in mammals. Each of them has its own activators, inactivators, substrates and scaffolds, which altogether form a fine signaling network in response to different extracellular or intracellular stimulation. In this review, we summarize recent advances in understanding of the regulation of MAP kinases and the roles of MAP kinases in innate and adaptive immune responses. Cellular & Molecular Immunology. 2005;2(1):20-27.

  4. Isolation and Identification of Putative Oral Cancer Stem Cells

    Institute of Scientific and Technical Information of China (English)

    ZHANG Min; ZHAO Yan-Hua; TANG Xiao-Fei

    2011-01-01

    Objective: To isolate and characterize putative cancer stem cells in Tea8113 oral squmous cell carcinoma cell line. Methods: Putative cancer stem cells were isolated by limited dilution assay in Tea8113 cell line. Biological features of putative cancer stem cells were detected by MTT assay, flow cytometry, immunofluorescence, Colony Forming Efficiency assays, cell motility assay and in vivo tumor formation experiment. Results: Compared with untreated Tea8113 cells, the putative cancer stem cells proliferated more quickly and showed heteroploid cell cycle,higher G0/G1-arrested cells, higher CFE and higher expression levels of ABCG2 belonged to tumor stem cell phenotypes. The putative cancer stem cells had stronger capacity to generate tumors in vivo. Conclusion: The holoclone cells have higher proliferation and self-renewal abilities, which may be cancer stem cells existed in Tea8113 oral squmous cell carcinoma cell line.%目的:分离鉴定口腔鳞癌细胞系Tca8113中的肿瘤干细胞.方法:利用有限稀释的方法分离Tca8113细胞系中的肿瘤干细胞.通过MTT法、流式细胞技术、细胞免疫荧光、克隆形成率分析、细胞迁移能力检测和裸鼠皮下成瘤实验确定分离得到的肿瘤干细胞的生物学特点.结果:分离得到的紧密型克隆肿瘤细胞表现为异倍体样细胞周期,大部分细胞处于G0/G1期,增殖能力、克隆形成率和体外迁移能力都明显高于未分离的肿瘤细胞.紧密型克隆肿瘤细胞肿瘤干细胞标记物ABCG2表达也高于未分离的肿瘤细胞,并且具有更强的裸鼠皮下成瘤能力.结论:我们分离得到的紧密型克隆细胞具有较强的细胞增殖和自我更新能力,可能就是口腔鳞癌细胞系Tca8113中的肿瘤干细胞.

  5. Phosphoproteomic analysis of protein kinase C signaling in Saccharomyces cerevisiae reveals Slt2 mitogen-activated protein kinase (MAPK)-dependent phosphorylation of eisosome core components.

    Science.gov (United States)

    Mascaraque, Victoria; Hernáez, María Luisa; Jiménez-Sánchez, María; Hansen, Rasmus; Gil, Concha; Martín, Humberto; Cid, Víctor J; Molina, María

    2013-03-01

    The cell wall integrity (CWI) pathway of the model organism Saccharomyces cerevisiae has been thoroughly studied as a paradigm of the mitogen-activated protein kinase (MAPK) pathway. It consists of a classic MAPK module comprising the Bck1 MAPK kinase kinase, two redundant MAPK kinases (Mkk1 and Mkk2), and the Slt2 MAPK. This module is activated under a variety of stimuli related to cell wall homeostasis by Pkc1, the only member of the protein kinase C family in budding yeast. Quantitative phosphoproteomics based on stable isotope labeling of amino acids in cell culture is a powerful tool for globally studying protein phosphorylation. Here we report an analysis of the yeast phosphoproteome upon overexpression of a PKC1 hyperactive allele that specifically activates CWI MAPK signaling in the absence of external stimuli. We found 82 phosphopeptides originating from 43 proteins that showed enhanced phosphorylation in these conditions. The MAPK S/T-P target motif was significantly overrepresented in these phosphopeptides. Hyperphosphorylated proteins provide putative novel targets of the Pkc1-cell wall integrity pathway involved in diverse functions such as the control of gene expression, protein synthesis, cytoskeleton maintenance, DNA repair, and metabolism. Remarkably, five components of the plasma-membrane-associated protein complex known as eisosomes were found among the up-regulated proteins. We show here that Pkc1-induced phosphorylation of the eisosome core components Pil1 and Lsp1 was not exerted directly by Pkc1, but involved signaling through the Slt2 MAPK module.

  6. Cloning and characterization of the gsk gene encoding guanosine kinase of Escherichia coli

    DEFF Research Database (Denmark)

    Harlow, Kenneth W.; Nygaard, Per; Hove-Jensen, Bjarne

    1995-01-01

    The Escherichia coli gsk gene encoding guanosine kinase was cloned from the Kohara gene library by complementation of the E. coli gsk-1 mutant allele. The cloned DNA fragment was sequenced and shown to encode a putative polypeptide of 433 amino acids with a molecular mass of 48,113 Da. Minicell...

  7. Proteolytic susceptibility of creatine kinase isozymes and arginine kinase.

    Science.gov (United States)

    Ercan, Altan; Grossman, Steven H

    2003-07-11

    The time course and dose-response to proteolysis of three dimeric isozymes of creatine kinase, CK-MM (muscle), CK-BB (brain), and CK-MB (heart) and the homologous monomer, arginine kinase were compared. Chymotrypsin and trypsin cause a rapid and significant loss of intact CK-BB, but limited hydrolysis of CK-MM. After 1h of hydrolysis by chymotrypsin, 80% of CK-MM is intact as judged by quantification of monomers after electrophoresis in sodium dodecyl sulfate. While 50% of the intact monomers of CK-MB remain under these conditions, no CK-BB monomers are detected. These results indicate that treatment with chymotrypsin leads to a CK-MB devoid of the B-subunit. When treated with trypsin for 1h, CK-MM is totally resistant to hydrolysis and all CK-BB is highly degraded. However, CK-MB exhibits approximately 90% intact monomers, indicating survival of intact B-subunit in CK-MB. This suggests that heterodimerization of a B-subunit with an M-subunit may have a protective effect against hydrolysis by trypsin. In view of the considerably larger number of potentially tryptic sensitive sites on the muscle isozyme, the resistance of CK-MM and susceptibility of CK-BB dimers to trypsin implies that differences in subunit tertiary structure are a factor in proteolysis of the homodimeric isozymes. Arginine kinase is rapidly degraded by trypsin, but is minimally affected by chymotrypsin. The finding that both a monomeric (arginine kinase) and dimeric (CK-BB) phosphagen kinase are highly susceptible to proteolysis by trypsin indicates that quaternary structure is not, in and of itself, an advantage in resistance to proteolysis. Since both arginine kinase and muscle creatine kinase are resistant to chymotryptic hydrolysis, it seems unlikely that in general, the increased packing density, which may result from dimerization can account for the stability of CK-MM towards trypsin.

  8. Molecular genetics: DNA analysis of a putative dog clone.

    Science.gov (United States)

    Parker, Heidi G; Kruglyak, Leonid; Ostrander, Elaine A

    2006-03-09

    In August 2005, Lee et al. reported the first cloning of a domestic dog from adult somatic cells. This putative dog clone was the result of somatic-cell nuclear transfer from a fibroblast cell of a three-year-old male Afghan hound into a donor oocyte provided by a dog of mixed breed. In light of recent concerns regarding the creation of cloned human cell lines from the same institution, we have undertaken an independent test to determine the validity of the claims made by Lee et al..

  9. Citron kinase - renaissance of a neglected mitotic kinase.

    Science.gov (United States)

    D'Avino, Pier Paolo

    2017-05-15

    Cell division controls the faithful segregation of genomic and cytoplasmic materials between the two nascent daughter cells. Members of the Aurora, Polo and cyclin-dependent (Cdk) kinase families are known to regulate multiple events throughout cell division, whereas another kinase, citron kinase (CIT-K), for a long time has been considered to function solely during cytokinesis, the last phase of cell division. CIT-K was originally proposed to regulate the ingression of the cleavage furrow that forms at the equatorial cortex of the dividing cell after chromosome segregation. However, studies in the last decade have clarified that this kinase is, instead, required for the organization of the midbody in late cytokinesis, and also revealed novel functions of CIT-K earlier in mitosis and in DNA damage control. Moreover, CIT-K mutations have recently been linked to the development of human microcephaly, and CIT-K has been identified as a potential target in cancer therapy. In this Commentary, I describe and re-evaluate the functions and regulation of CIT-K during cell division and its involvement in human disease. Finally, I offer my perspectives on the open questions and future challenges that are necessary to address, in order to fully understand this important and yet unjustly neglected mitotic kinase. © 2017. Published by The Company of Biologists Ltd.

  10. An S/T-Q cluster domain census unveils new putative targets under Tel1/Mec1 control

    Directory of Open Access Journals (Sweden)

    Cheung Hannah C

    2012-11-01

    Full Text Available Abstract Background The cellular response to DNA damage is immediate and highly coordinated in order to maintain genome integrity and proper cell division. During the DNA damage response (DDR, the sensor kinases Tel1 and Mec1 in Saccharomyces cerevisiae and ATM and ATR in human, phosphorylate multiple mediators which activate effector proteins to initiate cell cycle checkpoints and DNA repair. A subset of kinase substrates are recognized by the S/T-Q cluster domain (SCD, which contains motifs of serine (S or threonine (T followed by a glutamine (Q. However, the full repertoire of proteins and pathways controlled by Tel1 and Mec1 is unknown. Results To identify all putative SCD-containing proteins, we analyzed the distribution of S/T-Q motifs within verified Tel1/Mec1 targets and arrived at a unifying SCD definition of at least 3 S/T-Q within a stretch of 50 residues. This new SCD definition was used in a custom bioinformatics pipeline to generate a census of SCD-containing proteins in both yeast and human. In yeast, 436 proteins were identified, a significantly larger number of hits than were expected by chance. These SCD-containing proteins did not distribute equally across GO-ontology terms, but were significantly enriched for those involved in processes related to the DDR. We also found a significant enrichment of proteins involved in telophase and cytokinesis, protein transport and endocytosis suggesting possible novel Tel1/Mec1 targets in these pathways. In the human proteome, a wide range of similar proteins were identified, including homologs of some SCD-containing proteins found in yeast. This list also included high concentrations of proteins in the Mediator, spindle pole body/centrosome and actin cytoskeleton complexes. Conclusions Using a bioinformatic approach, we have generated a census of SCD-containing proteins that are involved not only in known DDR pathways but several other pathways under Tel1/Mec1 control suggesting new

  11. Putative cryptoendolithic life in Devonian pillow basalt, Rheinisches Schiefergebirge, Germany.

    Science.gov (United States)

    Peckmann, J; Bach, W; Behrens, K; Reitner, J

    2008-03-01

    Middle Devonian (Givetian) pillow basalt and inter-pillow breccia from the Rheinisches Schiefergebirge in Germany were found to contain putative biogenic filaments that indicate that life once proliferated within these volcanic rocks. Mineralized filaments are found in carbonate amygdules (vesicles filled by carbonate cement) in the volcanic rock, where they started to form on the internal surface of the once water-filled vesicles. Biogenicity of the filaments is indicated by (1) their size and shape resembling modern microorganisms including a constant diameter along the length of curved filaments, (2) their independence of crystal faces or cleavage planes, (3) branching patterns reminiscent of modern microorganisms, and (4) their spatial clustering and preferential occurrence close to the margin of pillows and in the inter-pillow breccias. A time lag between the deposition of pillow basalt and the activity of endoliths is revealed by the sequence of carbonate cements filling the amygdules. The putative filamentous microorganisms thrived after the formation of early fibrous rim cement, but before later equant calcite spar filled most of the remaining porosity. Microbial clay authigenesis analogous to the encrustation of prokaryotes in modern iron-rich environments led to the preservation of filaments. The filaments predominantly consist of the clay minerals chamosite and illite. Having dwelled in water-filled vesicles, the Devonian basalt-hosted filaments apparently represent cryptoendoliths. This finding suggests that a previously unrecognized niche for life exists within volcanic rock.

  12. Putative golden proportions as predictors of facial esthetics in adolescents.

    Science.gov (United States)

    Kiekens, Rosemie M A; Kuijpers-Jagtman, Anne Marie; van 't Hof, Martin A; van 't Hof, Bep E; Maltha, Jaap C

    2008-10-01

    In orthodontics, facial esthetics is assumed to be related to golden proportions apparent in the ideal human face. The aim of the study was to analyze the putative relationship between facial esthetics and golden proportions in white adolescents. Seventy-six adult laypeople evaluated sets of photographs of 64 adolescents on a visual analog scale (VAS) from 0 to 100. The facial esthetic value of each subject was calculated as a mean VAS score. Three observers recorded the position of 13 facial landmarks included in 19 putative golden proportions, based on the golden proportions as defined by Ricketts. The proportions and each proportion's deviation from the golden target (1.618) were calculated. This deviation was then related to the VAS scores. Only 4 of the 19 proportions had a significant negative correlation with the VAS scores, indicating that beautiful faces showed less deviation from the golden standard than less beautiful faces. Together, these variables explained only 16% of the variance. Few golden proportions have a significant relationship with facial esthetics in adolescents. The explained variance of these variables is too small to be of clinical importance.

  13. CRYSTAL STRUCTURE ANALYSIS OF A PUTATIVE OXIDOREDUCTASE FROM KLEBSIELLA PNEUMONIAE

    Energy Technology Data Exchange (ETDEWEB)

    Baig, M.; Brown, A.; Eswaramoorthy, S.; Swaminathan, S.

    2009-01-01

    Klebsiella pneumoniae, a gram-negative enteric bacterium, is found in nosocomial infections which are acquired during hospital stays for about 10% of hospital patients in the United States. The crystal structure of a putative oxidoreductase from K. pneumoniae has been determined. The structural information of this K. pneumoniae protein was used to understand its function. Crystals of the putative oxidoreductase enzyme were obtained by the sitting drop vapor diffusion method using Polyethylene glycol (PEG) 3350, Bis-Tris buffer, pH 5.5 as precipitant. These crystals were used to collect X-ray data at beam line X12C of the National Synchrotron Light Source (NSLS) at Brookhaven National Laboratory (BNL). The crystal structure was determined using the SHELX program and refi ned with CNS 1.1. This protein, which is involved in the catalysis of an oxidation-reduction (redox) reaction, has an alpha/beta structure. It utilizes nicotinamide adenine dinucleotide phosphate (NADP) or nicotine adenine dinucleotide (NAD) to perform its function. This structure could be used to determine the active and co-factor binding sites of the protein, information that could help pharmaceutical companies in drug design and in determining the protein’s relationship to disease treatment such as that for pneumonia and other related pathologies.

  14. WNK kinases and essential hypertension.

    Science.gov (United States)

    Huang, Chou-Long; Kuo, Elizabeth; Toto, Robert D

    2008-03-01

    The present review summarizes recent literature and discusses the potential roles of WNKs in the pathogenesis of essential hypertension. WNKs (with-no-lysine [K]) are a recently discovered family of serine-threonine protein kinases with unusual protein kinase domains. The role of WNK kinases in the control of blood pressure was first revealed by the findings that mutations of two members, WNK1 and WNK4, cause Gordon's syndrome. Laboratory studies have revealed that WNK kinases play important roles in the regulation of sodium and potassium transport. Animal models have been created to unravel the pathophysiology of sodium transport disorders caused by mutations of the WNK4 gene. Potassium deficiency causes sodium retention and increases hypertension prevalence. The expression of WNK1 is upregulated by potassium deficiency, raising the possibility that WNK1 may contribute to salt-sensitive essential hypertension associated with potassium deficiency. Associations of polymorphisms of WNK genes with essential hypertension in the general population have been reported. Mutations of WNK1 and WNK4 cause hypertension at least partly by increasing renal sodium retention. The role of WNK kinases in salt-sensitive hypertension within general hypertension is suggested, but future work is required to firmly establish the connection.

  15. Central Role of Pyruvate Kinase in Carbon Co-catabolism of Mycobacterium tuberculosis.

    Science.gov (United States)

    Noy, Tahel; Vergnolle, Olivia; Hartman, Travis E; Rhee, Kyu Y; Jacobs, William R; Berney, Michael; Blanchard, John S

    2016-03-25

    Mycobacterium tuberculosis (Mtb) displays a high degree of metabolic plasticity to adapt to challenging host environments. Genetic evidence suggests thatMtbrelies mainly on fatty acid catabolism in the host. However,Mtbalso maintains a functional glycolytic pathway and its role in the cellular metabolism ofMtbhas yet to be understood. Pyruvate kinase catalyzes the last and rate-limiting step in glycolysis and theMtbgenome harbors one putative pyruvate kinase (pykA, Rv1617). Here we show thatpykAencodes an active pyruvate kinase that is allosterically activated by glucose 6-phosphate (Glc-6-P) and adenosine monophosphate (AMP). Deletion ofpykApreventsMtbgrowth in the presence of fermentable carbon sources and has a cidal effect in the presence of glucose that correlates with elevated levels of the toxic catabolite methylglyoxal. Growth attenuation was also observed in media containing a combination of short chain fatty acids and glucose and surprisingly, in media containing odd and even chain fatty acids alone. Untargeted high sensitivity metabolomics revealed that inactivation of pyruvate kinase leads to accumulation of phosphoenolpyruvate (P-enolpyruvate), citrate, and aconitate, which was consistent with allosteric inhibition of isocitrate dehydrogenase by P-enolpyruvate. This metabolic block could be relieved by addition of the α-ketoglutarate precursor glutamate. Taken together, our study identifies an essential role of pyruvate kinase in preventing metabolic block during carbon co-catabolism inMtb.

  16. The systematic functional analysis of plasmodium protein kinases identifies essential regulators of mosquito transmission

    KAUST Repository

    Tewari, Rita

    2010-10-21

    Although eukaryotic protein kinases (ePKs) contribute to many cellular processes, only three Plasmodium falciparum ePKs have thus far been identified as essential for parasite asexual blood stage development. To identify pathways essential for parasite transmission between their mammalian host and mosquito vector, we undertook a systematic functional analysis of ePKs in the genetically tractable rodent parasite Plasmodium berghei. Modeling domain signatures of conventional ePKs identified 66 putative Plasmodium ePKs. Kinomes are highly conserved between Plasmodium species. Using reverse genetics, we show that 23 ePKs are redundant for asexual erythrocytic parasite development in mice. Phenotyping mutants at four life cycle stages in Anopheles stephensi mosquitoes revealed functional clusters of kinases required for sexual development and sporogony. Roles for a putative SR protein kinase (SRPK) in microgamete formation, a conserved regulator of clathrin uncoating (GAK) in ookinete formation, and a likely regulator of energy metabolism (SNF1/KIN) in sporozoite development were identified. 2010 Elsevier Inc.

  17. Exceptional error minimization in putative primordial genetic codes

    Directory of Open Access Journals (Sweden)

    Koonin Eugene V

    2009-11-01

    Full Text Available Abstract Background The standard genetic code is redundant and has a highly non-random structure. Codons for the same amino acids typically differ only by the nucleotide in the third position, whereas similar amino acids are encoded, mostly, by codon series that differ by a single base substitution in the third or the first position. As a result, the code is highly albeit not optimally robust to errors of translation, a property that has been interpreted either as a product of selection directed at the minimization of errors or as a non-adaptive by-product of evolution of the code driven by other forces. Results We investigated the error-minimization properties of putative primordial codes that consisted of 16 supercodons, with the third base being completely redundant, using a previously derived cost function and the error minimization percentage as the measure of a code's robustness to mistranslation. It is shown that, when the 16-supercodon table is populated with 10 putative primordial amino acids, inferred from the results of abiotic synthesis experiments and other evidence independent of the code's evolution, and with minimal assumptions used to assign the remaining supercodons, the resulting 2-letter codes are nearly optimal in terms of the error minimization level. Conclusion The results of the computational experiments with putative primordial genetic codes that contained only two meaningful letters in all codons and encoded 10 to 16 amino acids indicate that such codes are likely to have been nearly optimal with respect to the minimization of translation errors. This near-optimality could be the outcome of extensive early selection during the co-evolution of the code with the primordial, error-prone translation system, or a result of a unique, accidental event. Under this hypothesis, the subsequent expansion of the code resulted in a decrease of the error minimization level that became sustainable owing to the evolution of a high

  18. ESTs analysis reveals putative genes involved in symbiotic seed germination in Dendrobium officinale.

    Directory of Open Access Journals (Sweden)

    Ming-Ming Zhao

    Full Text Available Dendrobiumofficinale (Orchidaceae is one of the world's most endangered plants with great medicinal value. In nature, D. officinale seeds must establish symbiotic relationships with fungi to germinate. However, the molecular events involved in the interaction between fungus and plant during this process are poorly understood. To isolate the genes involved in symbiotic germination, a suppression subtractive hybridization (SSH cDNA library of symbiotically germinated D. officinale seeds was constructed. From this library, 1437 expressed sequence tags (ESTs were clustered to 1074 Unigenes (including 902 singletons and 172 contigs, which were searched against the NCBI non-redundant (NR protein database (E-value cutoff, e(-5. Based on sequence similarity with known proteins, 579 differentially expressed genes in D. officinale were identified and classified into different functional categories by Gene Ontology (GO, Clusters of orthologous Groups of proteins (COGs and Kyoto Encyclopedia of Genes and Genomes (KEGG pathways. The expression levels of 15 selected genes emblematic of symbiotic germination were confirmed via real-time quantitative PCR. These genes were classified into various categories, including defense and stress response, metabolism, transcriptional regulation, transport process and signal transduction pathways. All transcripts were upregulated in the symbiotically germinated seeds (SGS. The functions of these genes in symbiotic germination were predicted. Furthermore, two fungus-induced calcium-dependent protein kinases (CDPKs, which were upregulated 6.76- and 26.69-fold in SGS compared with un-germinated seeds (UGS, were cloned from D. officinale and characterized for the first time. This study provides the first global overview of genes putatively involved in D. officinale symbiotic seed germination and provides a foundation for further functional research regarding symbiotic relationships in orchids.

  19. Epidermal growth factor receptor in the prawn Macrobrachium rosenbergii: function and putative signaling cascade.

    Science.gov (United States)

    Sharabi, Omri; Ventura, Tomer; Manor, Rivka; Aflalo, Eliahu D; Sagi, Amir

    2013-09-01

    Epidermal growth factor receptors (EGFRs) are highly conserved members of the tyrosine kinase receptor superfamily found in metazoans and plants. In arthropods, EGFRs are vital for the proper development of embryos and of adult limbs, gonads, and eyes as well as affecting body size. In searching for genes involved in the growth and development of our model organism, the decapod crustacean (Macrobrachium rosenbergii), a comprehensive transcript library was established using next-generation sequencing. Using this library, the expression of several genes assigned to the signal transduction pathways mediated by EGFRs was observed, including a transcript encoding M. rosenbergii EGFR (Mr-EGFR), several potential ligands upstream to the receptor, and most of the putative downstream signal transducer genes. The deduced protein encoded by Mr-EGFR, representing the first such receptor reported thus far in crustaceans, shows sequence similarity to other arthropod EGFRs. The M. rosenbergii gene is expressed in most tested tissues. The role of Mr-EGFR was revealed by temporarily silencing the transcript through weekly injections of double-stranded Mr-EGFR RNA. Such treatment resulted in a significant reduction in growth and a delay in the appearance of a male secondary sexual characteristic, namely the appendix masculina. An additional function of Mr-EGFR was revealed with respect to eye development. Although the optic ganglion appeared to have retained its normal morphology, Mr-EGFR-silenced individuals developed abnormal eyes that presented irregular organization of the ommatidia, reflected by unorganized receptor cells occupying large areas of the dioptric portion and by a shortened crystalline tract layer.

  20. Identification and temporal expression of putative circadian clock transcripts in the amphipod crustacean Talitrus saltator.

    Science.gov (United States)

    O'Grady, Joseph F; Hoelters, Laura S; Swain, Martin T; Wilcockson, David C

    2016-01-01

    Talitrus saltator is an amphipod crustacean that inhabits the supralittoral zone on sandy beaches in the Northeast Atlantic and Mediterranean. T. saltator exhibits endogenous locomotor activity rhythms and time-compensated sun and moon orientation, both of which necessitate at least one chronometric mechanism. Whilst their behaviour is well studied, currently there are no descriptions of the underlying molecular components of a biological clock in this animal, and very few in other crustacean species. We harvested brain tissue from animals expressing robust circadian activity rhythms and used homology cloning and Illumina RNAseq approaches to sequence and identify the core circadian clock and clock-related genes in these samples. We assessed the temporal expression of these genes in time-course samples from rhythmic animals using RNAseq. We identified a comprehensive suite of circadian clock gene homologues in T. saltator including the 'core' clock genes period (Talper), cryptochrome 2 (Talcry2), timeless (Taltim), clock (Talclk), and bmal1 (Talbmal1). In addition we describe the sequence and putative structures of 23 clock-associated genes including two unusual, extended isoforms of pigment dispersing hormone (Talpdh). We examined time-course RNAseq expression data, derived from tissues harvested from behaviourally rhythmic animals, to reveal rhythmic expression of these genes with approximately circadian period in Talper and Talbmal1. Of the clock-related genes, casein kinase IIβ (TalckIIβ), ebony (Talebony), jetlag (Taljetlag), pigment dispensing hormone (Talpdh), protein phosphatase 1 (Talpp1), shaggy (Talshaggy), sirt1 (Talsirt1), sirt7 (Talsirt7) and supernumerary limbs (Talslimb) show temporal changes in expression. We report the sequences of principle genes that comprise the circadian clock of T. saltator and highlight the conserved structural and functional domains of their deduced cognate proteins. Our sequencing data contribute to the growing inventory

  1. Cloning and Characterization of a Putative CTR1 Gene from Wheat

    Institute of Scientific and Technical Information of China (English)

    BI Cai-li; WEN Xiao-jie; ZHANG Xue-yong; LIU Xu

    2010-01-01

    CTR1 is a key negative regulator in ethylene signal transduction.A salt-induced CTR1 like gene(TaCTR1)was cloned from wheat,its expression under abiotie stresses,subcellular localization and the effect of overexpression of TaCTR1 on salt tolerance in tobacco was studied.A putative CTR1 gene was cloned and characterized from wheat via rapid amplification of cDNA ends(RACE)and RT-PCR.TaCTR1 expression under stresses was analyzed using semi-quantitative RT-PCR and the effect of overexpression of TaCTR1 on salt tolerance was conducted in tobacco.The full-length cDNA of TaCTR1is 2635 bp which codes for a polypeptide of 759 amino acids.There is a conserved serine/threonine protein kinase domain at the carboxyl terminus containing an ATP-binding site.Southern blot analysis revealed that TaCTR1 consisted of a gene family in wheat.The amino acid homologies of CTR1 among different organisms share higher similarities.Expression analysis revealed that TaCTR1 was induced by NaCl and drought stress but inhibited by ABA treatment.Transient expression of TaCTR1-GFP in the onion epidermal cells indicated that TaCTR1 was probably targeted to the plasma membrane.Overexpression of TaCTR1 decreased salt tolerance in transgenic tobacco(Nicotiana tabacum L.)plants compared with the control.To our knowledge,TaCTR1 is the first CTR1 gene cloned in wheat and may be involved in various abiotic stresses.Overexpression of TaCTR1 decreased the salt tolerance in tobacco suggested that TaCTR1 may act as a negative regulator of salt stress in plants.

  2. Linkage mapping of putative regulator genes of barley grain development characterized by expression profiling

    Directory of Open Access Journals (Sweden)

    Wobus Ulrich

    2009-01-01

    Full Text Available Abstract Background Barley (Hordeum vulgare L. seed development is a highly regulated process with fine-tuned interaction of various tissues controlling distinct physiological events during prestorage, storage and dessication phase. As potential regulators involved within this process we studied 172 transcription factors and 204 kinases for their expression behaviour and anchored a subset of them to the barley linkage map to promote marker-assisted studies on barley grains. Results By a hierachical clustering of the expression profiles of 376 potential regulatory genes expressed in 37 different tissues, we found 50 regulators preferentially expressed in one of the three grain tissue fractions pericarp, endosperm and embryo during seed development. In addition, 27 regulators found to be expressed during both seed development and germination and 32 additional regulators are characteristically expressed in multiple tissues undergoing cell differentiation events during barley plant ontogeny. Another 96 regulators were, beside in the developing seed, ubiquitously expressed among all tissues of germinating seedlings as well as in reproductive tissues. SNP-marker development for those regulators resulted in anchoring 61 markers on the genetic linkage map of barley and the chromosomal assignment of another 12 loci by using wheat-barley addition lines. The SNP frequency ranged from 0.5 to 1.0 SNP/kb in the parents of the various mapping populations and was 2.3 SNP/kb over all eight lines tested. Exploration of macrosynteny to rice revealed that the chromosomal orders of the mapped putative regulatory factors were predominantly conserved during evolution. Conclusion We identified expression patterns of major transcription factors and signaling related genes expressed during barley ontogeny and further assigned possible functions based on likely orthologs functionally well characterized in model plant species. The combined linkage map and reference

  3. Evolutionary Ancestry of Eukaryotic Protein Kinases and Choline Kinases*

    Science.gov (United States)

    Lai, Shenshen; Safaei, Javad

    2016-01-01

    The reversible phosphorylation of proteins catalyzed by protein kinases in eukaryotes supports an important role for eukaryotic protein kinases (ePKs) in the emergence of nucleated cells in the third superkingdom of life. Choline kinases (ChKs) could also be critical in the early evolution of eukaryotes, because of their function in the biosynthesis of phosphatidylcholine, which is unique to eukaryotic membranes. However, the genomic origins of ePKs and ChKs are unclear. The high degeneracy of protein sequences and broad expansion of ePK families have made this fundamental question difficult to answer. In this study, we identified two class-I aminoacyl-tRNA synthetases with high similarities to consensus amino acid sequences of human protein-serine/threonine kinases. Comparisons of primary and tertiary structures supported that ePKs and ChKs evolved from a common ancestor related to glutaminyl aminoacyl-tRNA synthetases, which may have been one of the key factors in the successful of emergence of ancient eukaryotic cells from bacterial colonies. PMID:26742849

  4. Basal ganglia calcification as a putative cause for cognitive decline

    Directory of Open Access Journals (Sweden)

    João Ricardo Mendes de Oliveira

    Full Text Available ABSTRACT Basal ganglia calcifications (BGC may be present in various medical conditions, such as infections, metabolic, psychiatric and neurological diseases, associated with different etiologies and clinical outcomes, including parkinsonism, psychosis, mood swings and dementia. A literature review was performed highlighting the main neuropsychological findings of BGC, with particular attention to clinical reports of cognitive decline. Neuroimaging studies combined with neuropsychological analysis show that some patients have shown progressive disturbances of selective attention, declarative memory and verbal perseveration. Therefore, the calcification process might represent a putative cause for dementia syndromes, suggesting a probable link among calcinosis, the aging process and eventually with neuronal death. The increasing number of reports available will foster a necessary discussion about cerebral calcinosis and its role in determining symptomatology in dementia patients

  5. Probing the putative active site of YjdL

    DEFF Research Database (Denmark)

    Jensen, Johanne Mørch; Ismat, Fouzia; Szakonyi, Gerda;

    2012-01-01

    YjdL from E. coli is an unusual proton-coupled oligopeptide transporter (POT). Unlike prototypical POTs, dipeptides are preferred over tripeptides, in particular dipeptides with a positively charged C-terminal residue. To further understand this difference in peptide specificity, the sequences...... of YjdL and YdgR, a prototypical E. coli POT, were compared in light of the crystal structure of a POT from Shewanella oneidensis. Several residues found in the putative active site were mutated and the activities of the mutated variants were assessed in terms of substrate uptake assays, and changes...... pocket that opens towards the extracellular space. The C-terminal side chain faces in the opposite direction into a sub pocket that faces the cytoplasm. These data indicated a stabilizing effect on a bulky N-terminal residue by an Ala281Phe variant and on the dipeptide backbone by Trp278...

  6. Novel putative mechanisms to link circadian clocks to healthy aging.

    Science.gov (United States)

    Popa-Wagner, Aurel; Catalin, Bogdan; Buga, Ana-Maria

    2015-08-01

    The circadian clock coordinates the internal physiology to increase the homeostatic capacity thereby providing both a survival advantage to the system and an optimization of energy budgeting. Multiple-oscillator circadian mechanisms are likely to play a role in regulating human health and may contribute to the aging process. Our aim is to give an overview of how the central clock in the hypothalamus and peripheral clocks relate to aging and metabolic disorders, including hyperlipidemia and hyperglycemia. In particular, we unravel novel putative mechanisms to link circadian clocks to healthy aging. This review may lead to the design of large-scale interventions to help people stay healthy as they age by adjusting daily activities, such as feeding behavior, and or adaptation to age-related changes in individual circadian rhythms.

  7. Ballistic gelatin as a putative substrate for EEG phantom devices

    CERN Document Server

    Hairston, W David; Yu, Alfred B

    2016-01-01

    Phantom devices allow the human variable to be controlled for in order to allow clear comparison and validation of biomedical imaging hardware and software. There is currently no standard phantom for electroencephalography (EEG). To be useful, such a device would need to: (a) accurately recreate the real and imaginary components of scalp electrical impedance, (b) contain internal emitters to create electrical dipoles, and (c) be easily replicable across various labs and research groups. Cost-effective materials, which are conductive, repeatable, and easily formed are a missing key enabler for EEG phantoms. Here, we explore the use of ballistics gelatin, an inexpensive, easily-formable and repeatable material, as a putative substrate by examining its electrical properties and physical stability over time. We show that varied concentrations of NaCl salt relative to gelatin powder shifts the phase/frequency response profile, allowing for selective tuning of the material electrical properties.

  8. Putative benefits of microalgal astaxanthin on exercise and human health

    Directory of Open Access Journals (Sweden)

    Marcelo P. Barros

    2011-04-01

    Full Text Available Astaxanthin (ASTA is a pinkish-orange carotenoid produced by microalgae, but also commonly found in shrimp, lobster and salmon, which accumulate ASTA from the aquatic food chain. Numerous studies have addressed the benefits of ASTA for human health, including the inhibition of LDL oxidation, UV-photoprotection and prophylaxis of bacterial stomach ulcers. ASTA is recognized as a powerful scavenger of reactive oxygen species (ROS, especially those involved in lipid peroxidation. Both aerobic and anaerobic exercise are closely related to overproduction of ROS in muscle tissue. Post-exercise inflammatory processes can even exacerbate the oxidative stress imposed by exercise. Thus, ASTA is suggested here as a putative nutritional alternative/coadjutant for antioxidant therapy to afford additional protection to muscle tissues against oxidative damage induced by exercise, as well as for an (overall integrative redox re-balance and general human health.

  9. Cryptic species in putative ancient asexual darwinulids (Crustacea, Ostracoda.

    Directory of Open Access Journals (Sweden)

    Isa Schön

    Full Text Available BACKGROUND: Fully asexually reproducing taxa lack outcrossing. Hence, the classic Biological Species Concept cannot be applied. METHODOLOGY/PRINCIPAL FINDINGS: We used DNA sequences from the mitochondrial COI gene and the nuclear ITS2 region to check species boundaries according to the evolutionary genetic (EG species concept in five morphospecies in the putative ancient asexual ostracod genera, Penthesilenula and Darwinula, from different continents. We applied two methods for detecting cryptic species, namely the K/θ method and the General Mixed Yule Coalescent model (GMYC. We could confirm the existence of species in all five darwinulid morphospecies and additional cryptic diversity in three morphospecies, namely in Penthesilenula brasiliensis, Darwinula stevensoni and in P. aotearoa. The number of cryptic species within one morphospecies varied between seven (P. brasiliensis, five to six (D. stevensoni and two (P. aotearoa, respectively, depending on the method used. Cryptic species mainly followed continental distributions. We also found evidence for coexistence at the local scale for Brazilian cryptic species of P. brasiliensis and P. aotearoa. Our ITS2 data confirmed that species exist in darwinulids but detected far less EG species, namely two to three cryptic species in P. brasiliensis and no cryptic species at all in the other darwinulid morphospecies. CONCLUSIONS/SIGNIFICANCE: Our results clearly demonstrate that both species and cryptic diversity can be recognized in putative ancient asexual ostracods using the EG species concept, and that COI data are more suitable than ITS2 for this purpose. The discovery of up to eight cryptic species within a single morphospecies will significantly increase estimates of biodiversity in this asexual ostracod group. Which factors, other than long-term geographic isolation, are important for speciation processes in these ancient asexuals remains to be investigated.

  10. Putative regulatory factors associated with intramuscular fat content.

    Directory of Open Access Journals (Sweden)

    Aline S M Cesar

    Full Text Available Intramuscular fat (IMF content is related to insulin resistance, which is an important prediction factor for disorders, such as cardiovascular disease, obesity and type 2 diabetes in human. At the same time, it is an economically important trait, which influences the sensorial and nutritional value of meat. The deposition of IMF is influenced by many factors such as sex, age, nutrition, and genetics. In this study Nellore steers (Bos taurus indicus subspecies were used to better understand the molecular mechanisms involved in IMF content. This was accomplished by identifying differentially expressed genes (DEG, biological pathways and putative regulatory factors. Animals included in this study had extreme genomic estimated breeding value (GEBV for IMF. RNA-seq analysis, gene set enrichment analysis (GSEA and co-expression network methods, such as partial correlation coefficient with information theory (PCIT, regulatory impact factor (RIF and phenotypic impact factor (PIF were utilized to better understand intramuscular adipogenesis. A total of 16,101 genes were analyzed in both groups (high (H and low (L GEBV and 77 DEG (FDR 10% were identified between the two groups. Pathway Studio software identified 13 significantly over-represented pathways, functional classes and small molecule signaling pathways within the DEG list. PCIT analyses identified genes with a difference in the number of gene-gene correlations between H and L group and detected putative regulatory factors involved in IMF content. Candidate genes identified by PCIT include: ANKRD26, HOXC5 and PPAPDC2. RIF and PIF analyses identified several candidate genes: GLI2 and IGF2 (RIF1, MPC1 and UBL5 (RIF2 and a host of small RNAs, including miR-1281 (PIF. These findings contribute to a better understanding of the molecular mechanisms that underlie fat content and energy balance in muscle and provide important information for the production of healthier beef for human consumption.

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

    Science.gov (United States)

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

    2002-12-06

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

  12. Predikin and PredikinDB: a computational framework for the prediction of protein kinase peptide specificity and an associated database of phosphorylation sites

    Directory of Open Access Journals (Sweden)

    Kemp Bruce E

    2008-05-01

    Full Text Available Abstract Background We have previously described an approach to predicting the substrate specificity of serine-threonine protein kinases. The method, named Predikin, identifies key conserved substrate-determining residues in the kinase catalytic domain that contact the substrate in the region of the phosphorylation site and so determine the sequence surrounding the phosphorylation site. Predikin was implemented originally as a web application written in Javascript. Results Here, we describe a new version of Predikin, completely revised and rewritten as a modular framework that provides multiple enhancements compared with the original. Predikin now consists of two components: (i PredikinDB, a database of phosphorylation sites that links substrates to kinase sequences and (ii a Perl module, which provides methods to classify protein kinases, reliably identify substrate-determining residues, generate scoring matrices and score putative phosphorylation sites in query sequences. The performance of Predikin as measured using receiver operator characteristic (ROC graph analysis equals or surpasses that of existing comparable methods. The Predikin website has been redesigned to incorporate the new features. Conclusion New features in Predikin include the use of SQL queries to PredikinDB to generate predictions, scoring of predictions, more reliable identification of substrate-determining residues and putative phosphorylation sites, extended options to handle protein kinase and substrate data and an improved web interface. The new features significantly enhance the ability of Predikin to analyse protein kinases and their substrates. Predikin is available at http://predikin.biosci.uq.edu.au.

  13. Reciprocal regulation of protein kinase and pyruvate kinase activities of pyruvate kinase M2 by growth signals.

    Science.gov (United States)

    Gao, Xueliang; Wang, Haizhen; Yang, Jenny J; Chen, Jing; Jie, Jiang; Li, Liangwei; Zhang, Yinwei; Liu, Zhi-Ren

    2013-05-31

    Pyruvate kinase isoform M2 (PKM2) is an enzyme-catalyzing conversion of phosphoenolpyruvate to pyruvate in the glycolysis pathway. It was demonstrated that PKM2 interacts with tyrosine phosphopeptide, and the interaction with the tyrosine phosphopeptide affects the pyruvate kinase activity of PKM2. Our experiments suggest that PKM2 is also an active protein kinase (Gao, X., Wang, H., Yang, J. J., Liu, X., and Liu, Z. R. (2012) Mol. Cell 45, 598-609). We report here that growth signals reciprocally regulate the pyruvate kinase and protein kinase activities of PKM2 by different mechanisms. On the one hand, growth signals induce protein tyrosine phosphorylations. The tyrosine-phosphorylated protein(s) regulates the conversion of pyruvate kinase and protein kinase of PKM2 by directly interacting with PKM2. Binding of the tyrosyl-phosphorylated proteins at the fructose 1,6-bisphosphate-binding site converts the tetrameric PKM2 to a dimer. On the other hand, growth stimulations also lead to PKM2 phosphorylation, which consequently regulates the conversion of protein kinase and pyruvate kinase activities. Growth factor stimulations significantly increase the dimer/tetramer PKM2 ratio in cells and consequently activate the protein kinase activity of PKM2. Our study suggests that the conversion between the pyruvate kinase and protein kinase activities of PKM2 may be an important mechanism mediating the effects of growth signals in promoting cell proliferation.

  14. Bacterial Protein-Tyrosine Kinases

    DEFF Research Database (Denmark)

    Shi, Lei; Kobir, Ahasanul; Jers, Carsten

    2010-01-01

    in exopolysaccharide production, virulence, DNA metabolism, stress response and other key functions of the bacterial cell. BY-kinases act through autophosphorylation (mainly in exopolysaccharide production) and phosphorylation of other proteins, which have in most cases been shown to be activated by tyrosine...

  15. Renal targeting of kinase inhibitors

    NARCIS (Netherlands)

    Dolman, M. E. M.; Fretz, M. M.; Segers, Gj. W.; Lacombe, M.; Prakash, J.; Storm, G.; Hennink, W. E.; Kok, R. J.

    2008-01-01

    Activation of proximal tubular cells by fibrotic and inflammatory mediators is an important hallmark of chronic kidney disease. We have developed a novel strategy to intervene in renal fibrosis, by means of locally delivered kinase inhibitors. Such compounds will display enhanced activity within tub

  16. Inhibitors of protein kinase C

    Institute of Scientific and Technical Information of China (English)

    LIU Shiying; JIANG Yuyang; CAO Jian; LIU Feng; MA Li; ZHAO Yufen

    2005-01-01

    Protein kinase catalyzes the transfer of the γ-phosphoryl group from ATP to the hydroxyl groups of protein side chains, which plays critical roles in signal transduction pathways by transmitting extracellular signals across the plasma membrane and nuclear membrane to the destination sites in the cytoplasm and the nucleus. Protein kinase C (PKC) is a superfamily of phospholipid-dependent Ser/Thr kinase. There are at least 12 isozymes in PKC family. They are distributed in different tissues and play different roles in physiological processes. On account of their concern with a variety of pathophysiologic states, such as cancer, inflammatory conditions, autoimmune disorder, and cardiac diseases, the inhibitors, which can inhibit the activity of PKC and the interaction of cytokine with receptor, and interfere signal transduction pathway, may be candidates of therapeutic drugs. Therefore, intense efforts have been made to develop specific protein kinase inhibitors as biological tools and therapeutic agents. This article reviews the recent development of some of PKC inhibitors based on their interaction with different conserved domains and different inhibition mechanisms.

  17. Renal targeting of kinase inhibitors

    NARCIS (Netherlands)

    Dolman, M. E. M.; Fretz, M. M.; Segers, Gj. W.; Lacombe, M.; Prakash, J.; Storm, G.; Hennink, W. E.; Kok, R. J.

    2008-01-01

    Activation of proximal tubular cells by fibrotic and inflammatory mediators is an important hallmark of chronic kidney disease. We have developed a novel strategy to intervene in renal fibrosis, by means of locally delivered kinase inhibitors. Such compounds will display enhanced activity within

  18. Deferasirox in pyruvate kinase deficiency

    OpenAIRE

    Deeren, Dries

    2008-01-01

    Deferasirox in pyruvate kinase deficiency phone: +32-51-237437 (Deeren, Dries) (Deeren, Dries) Department of Haematology, Heilig-Hartziekenhuis Roeselare-Menen vzw - Wilgenstraat 2 - B-8800 - Roeselare - BELGIUM (Deeren, Dries) BELGIUM Registration: 2008-09-10 Received: 2008-09-05 Accepted: 2008-09-10 ePublished: 2008-09-23

  19. Non-Viral Deoxyribonucleoside Kinases

    DEFF Research Database (Denmark)

    Christiansen, Louise Slot; Munch-Petersen, Birgitte; Knecht, Wolfgang

    2015-01-01

    Deoxyribonucleoside kinases (dNKs) phosphorylate deoxyribonucleosides to their corresponding monophosphate compounds. dNks also phosphorylate deoxyribonucleoside analogues that are used in the treatment of cancer or viral infections. The study of the mammalian dNKs has therefore always been of gr...

  20. Anticancer Alkaloid Lamellarins Inhibit Protein Kinases

    Directory of Open Access Journals (Sweden)

    Laurent Meijer

    2008-10-01

    Full Text Available Lamellarins, a family of hexacyclic pyrrole alkaloids originally isolated from marine invertebrates, display promising anti-tumor activity. They induce apoptotic cell death through multi-target mechanisms, including inhibition of topoisomerase I, interaction with DNA and direct effects on mitochondria. We here report that lamellarins inhibit several protein kinases relevant to cancer such as cyclin-dependent kinases, dualspecificity tyrosine phosphorylation activated kinase 1A, casein kinase 1, glycogen synthase kinase-3 and PIM-1. A good correlation is observed between the effects of lamellarins on protein kinases and their action on cell death, suggesting that inhibition of specific kinases may contribute to the cytotoxicity of lamellarins. Structure/activity relationship suggests several paths for the optimization of lamellarins as kinase inhibitors.

  1. Monoclonal Antibodies Against Xenopus Greatwall Kinase

    OpenAIRE

    WANG Ling; Fisher, Laura A.; Wahl, James K.; Peng, Aimin

    2011-01-01

    Mitosis is known to be regulated by protein kinases, including MPF, Plk1, Aurora kinases, and so on, which become active in M-phase and phosphorylate a wide range of substrates to control multiple aspects of mitotic entry, progression, and exit. Mechanistic investigations of these kinases not only provide key insights into cell cycle regulation, but also hold great promise for cancer therapy. Recent studies, largely in Xenopus, characterized a new mitotic kinase named Greatwall (Gwl) that pla...

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

    Science.gov (United States)

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

    2003-10-09

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

  3. M-CSF receptor mutations in hereditary diffuse leukoencephalopathy with spheroids impair not only kinase activity but also surface expression

    Energy Technology Data Exchange (ETDEWEB)

    Hiyoshi, Masateru; Hashimoto, Michihiro; Yukihara, Mamiko; Bhuyan, Farzana; Suzu, Shinya, E-mail: ssuzu06@kumamoto-u.ac.jp

    2013-11-01

    Highlights: •Many mutations were identified in Fms as a putative genetic cause of HDLS. •All of the mutations tested severely impair the kinase activity. •Most of the mutations also impair the trafficking to the cell surface. •These defects further suggest that HDLS is caused by a loss of Fms function. -- Abstract: The tyrosine kinase Fms, the cell surface receptor for M-CSF and IL-34, is critical for microglial proliferation and differentiation in the brain. Recently, a number of mutations have been identified in Fms as a putative genetic cause of hereditary diffuse leukoencephalopathy with spheroids (HDLS), implying an important role of microglial dysfunction in HDLS pathogenesis. In this study, we initially confirmed that 11 mutations, which reside within the ATP-binding or major tyrosine kinase domain, caused a severe impairment of ligand-induced Fms auto-phosphorylation. Intriguingly, we found that 10 of the 11 mutants also showed a weak cell surface expression, which was associated with a concomitant increase in the low molecular weight hypo-N-glycosylated immature gp130Fms-like species. Indeed, the mutant proteins heavily accumulated to the Golgi-like perinuclear regions. These results indicate that all of the Fms mutations tested severely impair the kinase activity and most of the mutations also impair the trafficking to the cell surface, further suggesting that HDLS is caused by the loss of Fms function.

  4. Cardiac protein kinases: the cardiomyocyte kinome and differential kinase expression in human failing hearts

    OpenAIRE

    Fuller, Stephen J.; Osborne, Sally A.; Leonard, Sam J.; Hardyman, Michelle A.; Vaniotis, George; Allen, Bruce G.; Sugden, Peter H.; Clerk, Angela

    2015-01-01

    Aims. Protein kinases are potential therapeutic targets for heart failure, but most studies of cardiac protein kinases derive from other systems, an approach that fails to account for specific kinases expressed in the heart and the contractile cardiomyocytes. We aimed to define the cardiomyocyte kinome (i.e. the protein kinases expressed in cardiomyocytes) and identify kinases with altered expression in human failing hearts. Methods and Results. Expression profiling (Affymetrix microarrays) d...

  5. Molecular cloning of a novel glucuronokinase/putative pyrophosphorylase from zebrafish acting in an UDP-glucuronic acid salvage pathway.

    Directory of Open Access Journals (Sweden)

    Roman Gangl

    Full Text Available In animals, the main precursor for glycosaminoglycan and furthermore proteoglycan biosynthesis, like hyaluronic acid, is UDP-glucuronic acid, which is synthesized via the nucleotide sugar oxidation pathway. Mutations in this pathway cause severe developmental defects (deficiency in the initiation of heart valve formation. In plants, UDP-glucuronic acid is synthesized via two independent pathways. Beside the nucleotide sugar oxidation pathway, a second minor route to UDP-glucuronic acid exist termed the myo-inositol oxygenation pathway. Within this myo-inositol is ring cleaved into glucuronic acid, which is subsequently converted to UDP-glucuronic acid by glucuronokinase and UDP-sugar pyrophosphorylase. Here we report on a similar, but bifunctional enzyme from zebrafish (Danio rerio which has glucuronokinase/putative pyrophosphorylase activity. The enzyme can convert glucuronic acid into UDP-glucuronic acid, required for completion of the alternative pathway to UDP-glucuronic acid via myo-inositol and thus establishes a so far unknown second route to UDP-glucuronic acid in animals. Glucuronokinase from zebrafish is a member of the GHMP-kinase superfamily having unique substrate specificity for glucuronic acid with a Km of 31 ± 8 µM and accepting ATP as the only phosphate donor (Km: 59 ± 9 µM. UDP-glucuronic acid pyrophosphorylase from zebrafish has homology to bacterial nucleotidyltransferases and requires UTP as nucleosid diphosphate donor. Genes for bifunctional glucuronokinase and putative UDP-glucuronic acid pyrophosphorylase are conserved among some groups of lower animals, including fishes, frogs, tunicates, and polychaeta, but are absent from mammals. The existence of a second pathway for UDP-glucuronic acid biosynthesis in zebrafish likely explains some previous contradictory finding in jekyll/ugdh zebrafish developmental mutants, which showed residual glycosaminoglycans and proteoglycans in knockout mutants of UDP

  6. Cloning, Expression, and Purification of Nucleoside Diphosphate Kinase from Acinetobacter baumannii

    Directory of Open Access Journals (Sweden)

    Juhi Sikarwar

    2013-01-01

    Full Text Available Acinetobacter baumannii is a multidrug resistant pathogenic bacteria associated with hospital acquired infections. This bacterium possesses a variety of resistance mechanisms which makes it more difficult to control the bacterium with conventional drugs, and, so far no effective drug treatment is available against it. Nucleoside diphosphate kinase is an important enzyme, which maintains the total nucleotide triphosphate pool inside the cell by the transfer of γ-phosphate from NTPs to NDPs. The role of nucleoside diphosphate kinase (Ndk has also been observed in pathogenesis in other organisms. However, intensive studies are needed to decipher its other putative roles in Acinetobacter baumannii. In the present study, we have successfully cloned the gene encoding Ndk and achieved overexpression in bacterial host BL-21 (DE3. The overexpressed protein is further purified by nickel-nitrilotriacetic acid (Ni-NTA chromatography.

  7. Cloning, Expression, and Purification of Nucleoside Diphosphate Kinase from Acinetobacter baumannii

    Science.gov (United States)

    Sikarwar, Juhi; Kaushik, Sanket; Sinha, Mau; Kaur, Punit; Sharma, Sujata; Singh, Tej P.

    2013-01-01

    Acinetobacter baumannii is a multidrug resistant pathogenic bacteria associated with hospital acquired infections. This bacterium possesses a variety of resistance mechanisms which makes it more difficult to control the bacterium with conventional drugs, and, so far no effective drug treatment is available against it. Nucleoside diphosphate kinase is an important enzyme, which maintains the total nucleotide triphosphate pool inside the cell by the transfer of γ-phosphate from NTPs to NDPs. The role of nucleoside diphosphate kinase (Ndk) has also been observed in pathogenesis in other organisms. However, intensive studies are needed to decipher its other putative roles in Acinetobacter baumannii. In the present study, we have successfully cloned the gene encoding Ndk and achieved overexpression in bacterial host BL-21 (DE3). The overexpressed protein is further purified by nickel-nitrilotriacetic acid (Ni-NTA) chromatography. PMID:23662205

  8. Putative uremic encephalopathy in horses: five cases (1978-1998).

    Science.gov (United States)

    Frye, M A; Johnson, J S; Traub-Dargatz, J L; Savage, C J; Fettman, M J; Gould, D H

    2001-02-15

    To determine historical, physical examination, clinicopathologic, and postmortem findings in horses with putative uremic encephalopathy. Design-Retrospective study. Animals-5 horses with renal failure and neurologic disease not attributable to abnormalities in any other organ system. Medical records from 1978 to 1998 were examined for horses with renal disease and neurologic signs not attributable to primary neurologic, hepatic, or other diseases. Signalment, history, physical examination findings, clinicopathologic data, renal ultrasonographic findings, and postmortem data were reviewed. Of 332 horses with renal disease, 5 met selection criteria. Historical findings, physical examination findings, clinicopathologic data, ultrasonographic data, and postmortem findings were consistent with chronic renal failure. Swollen astrocytes were detected in all 4 horses examined at necropsy. A single criterion was not determined to be pathognomonic for uremic encephalopathy in horses. Uremic encephalopathy should be considered as a differential diagnosis in horses with evidence of chronic renal failure and encephalopathic neurologic sign not attributable to other causes. Astrocyte swelling, which was common to all 4 horses examined at necropsy, may serve as a microscopic indicator of uremic encephalopathy in horses.

  9. Phytophthora infestans specific phosphorylation patterns and new putative control targets.

    Science.gov (United States)

    Frades, Itziar; Andreasson, Erik

    2016-04-01

    In this study we applied biomathematical searches of gene regulatory mechanisms to learn more about oomycete biology and to identify new putative targets for pesticides or biological control against Phytophthora infestans. First, oomycete phylum-specific phosphorylation motifs were found by discriminative n-gram analysis. We found 11.600 P. infestans specific n-grams, mapping 642 phosphoproteins. The most abundant group among these related to phosphatidylinositol metabolism. Due to the large number of possible targets found and our hypothesis that multi-level control is a sign of usefulness as targets for intervention, we identified overlapping targets with a second screen. This was performed to identify proteins dually regulated by small RNA and phosphorylation. We found 164 proteins to be regulated by both sRNA and phosphorylation and the dominating functions where phosphatidylinositol signalling/metabolism, endocytosis, and autophagy. Furthermore we performed a similar regulatory study and discriminative n-gram analysis of proteins with no clear orthologs in other species and proteins that are known to be unique to P. infestans such as the RxLR effectors, Crinkler (CRN) proteins and elicitins. We identified CRN proteins with specific phospho-motifs present in all life stages. PITG_12626, PITG_14042 and PITG_23175 are CRN proteins that have species-specific phosphorylation motifs and are subject to dual regulation.

  10. Rapid Discrimination Among Putative Mechanistic Models of Biochemical Systems.

    Science.gov (United States)

    Lomnitz, Jason G; Savageau, Michael A

    2016-08-31

    An overarching goal in molecular biology is to gain an understanding of the mechanistic basis underlying biochemical systems. Success is critical if we are to predict effectively the outcome of drug treatments and the development of abnormal phenotypes. However, data from most experimental studies is typically noisy and sparse. This allows multiple potential mechanisms to account for experimental observations, and often devising experiments to test each is not feasible. Here, we introduce a novel strategy that discriminates among putative models based on their repertoire of qualitatively distinct phenotypes, without relying on knowledge of specific values for rate constants and binding constants. As an illustration, we apply this strategy to two synthetic gene circuits exhibiting anomalous behaviors. Our results show that the conventional models, based on their well-characterized components, cannot account for the experimental observations. We examine a total of 40 alternative hypotheses and show that only 5 have the potential to reproduce the experimental data, and one can do so with biologically relevant parameter values.

  11. Putative role of Tat-Env interaction in HIV infection.

    Science.gov (United States)

    Poon, Selina; Moscoso, Carlos G; Xing, Li; Kan, Elaine; Sun, Yide; Kolatkar, Prasanna R; Vahlne, Anders G; Srivastava, Indresh K; Barnett, Susan W; Cheng, R Holland

    2013-09-24

    To study the complex formed between Tat protein and Env soluble trimeric immunogen, and compare with previously determined structures of Env native trimers and Env-CD4m complexes. The soluble Env trimer was used to mimic the spike glycoprotein on the virus surface for the study. To overcome limitations of other structural determination methods, cryoelectron microscopy was employed to image the complex, and single particle reconstruction was utilized to reconstruct the structure of the complex from collected micrographs. Molecular modeling of gp120-Tat was performed to provide atomic coordinates for docking. Images were preprocessed by multivariate statistical analysis to identify principal components of variation then submitted for reconstruction. Reconstructed structures were docked with modeled gp120-Tat atomic coordinates to study the positions of crucial epitopes. Analysis of the Env-Tat complex demonstrated an intermediate structure between Env native trimers and Env-CD4m structures. Docking results indicate that the CD4-binding site and the V3 loop are exposed in the Env-Tat complex. The integrin-binding sequence in Tat was also exposed in Env-Tat docking. The intermediate structure induced by Tat-interaction with Env could potentially provide an explanation for increased virus infection in the presence of Tat protein. Consequently, exposure of CD4-binding sites and a putative integrin-binding sequence on Tat in the complex may provide a new avenue for rational design of an effective HIV vaccine. © 2013 Wolters Kluwer Health | Lippincott Williams & Wilkins

  12. Phosphoglycerate Dehydrogenase: Potential Therapeutic Target and Putative Metabolic Oncogene

    Directory of Open Access Journals (Sweden)

    Cheryl K. Zogg

    2014-01-01

    Full Text Available Exemplified by cancer cells’ preference for glycolysis, for example, the Warburg effect, altered metabolism in tumorigenesis has emerged as an important aspect of cancer in the past 10–20 years. Whether due to changes in regulatory tumor suppressors/oncogenes or by acting as metabolic oncogenes themselves, enzymes involved in the complex network of metabolic pathways are being studied to understand their role and assess their utility as therapeutic targets. Conversion of glycolytic intermediate 3-phosphoglycerate into phosphohydroxypyruvate by the enzyme phosphoglycerate dehydrogenase (PHGDH—a rate-limiting step in the conversion of 3-phosphoglycerate to serine—represents one such mechanism. Forgotten since classic animal studies in the 1980s, the role of PHGDH as a potential therapeutic target and putative metabolic oncogene has recently reemerged following publication of two prominent papers near-simultaneously in 2011. Since that time, numerous studies and a host of metabolic explanations have been put forward in an attempt to understand the results observed. In this paper, I review the historic progression of our understanding of the role of PHGDH in cancer from the early work by Snell through its reemergence and rise to prominence, culminating in an assessment of subsequent work and what it means for the future of PHGDH.

  13. Small intestinal mucosa expression of putative chaperone fls485

    Directory of Open Access Journals (Sweden)

    Raupach Kerstin

    2010-03-01

    Full Text Available Abstract Background Maturation of enterocytes along the small intestinal crypt-villus axis is associated with significant changes in gene expression profiles. fls485 coding a putative chaperone protein has been recently suggested as a gene involved in this process. The aim of the present study was to analyze fls485 expression in human small intestinal mucosa. Methods fls485 expression in purified normal or intestinal mucosa affected with celiac disease was investigated with a molecular approach including qRT-PCR, Western blotting, and expression strategies. Molecular data were corroborated with several in situ techniques and usage of newly synthesized mouse monoclonal antibodies. Results fls485 mRNA expression was preferentially found in enterocytes and chromaffine cells of human intestinal mucosa as well as in several cell lines including Rko, Lovo, and CaCo2 cells. Western blot analysis with our new anti-fls485 antibodies revealed at least two fls485 proteins. In a functional CaCo2 model, an increase in fls485 expression was paralleled by cellular maturation stage. Immunohistochemistry demonstrated fls485 as a cytosolic protein with a slightly increasing expression gradient along the crypt-villus axis which was impaired in celiac disease Marsh IIIa-c. Conclusions Expression and synthesis of fls485 are found in surface lining epithelia of normal human intestinal mucosa and deriving epithelial cell lines. An interdependence of enterocyte differentiation along the crypt-villus axis and fls485 chaperone activity might be possible.

  14. Small intestinal mucosa expression of putative chaperone fls485.

    Science.gov (United States)

    Reinartz, Andrea; Ehling, Josef; Franz, Susanne; Simon, Verena; Bravo, Ignacio G; Tessmer, Claudia; Zentgraf, Hanswalter; Lyer, Stefan; Schneider, Ursula; Köster, Jan; Raupach, Kerstin; Kämmerer, Elke; Klaus, Christina; Tischendorf, Jens J W; Kopitz, Jürgen; Alonso, Angel; Gassler, Nikolaus

    2010-03-07

    Maturation of enterocytes along the small intestinal crypt-villus axis is associated with significant changes in gene expression profiles. fls485 coding a putative chaperone protein has been recently suggested as a gene involved in this process. The aim of the present study was to analyze fls485 expression in human small intestinal mucosa. fls485 expression in purified normal or intestinal mucosa affected with celiac disease was investigated with a molecular approach including qRT-PCR, Western blotting, and expression strategies. Molecular data were corroborated with several in situ techniques and usage of newly synthesized mouse monoclonal antibodies. fls485 mRNA expression was preferentially found in enterocytes and chromaffine cells of human intestinal mucosa as well as in several cell lines including Rko, Lovo, and CaCo2 cells. Western blot analysis with our new anti-fls485 antibodies revealed at least two fls485 proteins. In a functional CaCo2 model, an increase in fls485 expression was paralleled by cellular maturation stage. Immunohistochemistry demonstrated fls485 as a cytosolic protein with a slightly increasing expression gradient along the crypt-villus axis which was impaired in celiac disease Marsh IIIa-c. Expression and synthesis of fls485 are found in surface lining epithelia of normal human intestinal mucosa and deriving epithelial cell lines. An interdependence of enterocyte differentiation along the crypt-villus axis and fls485 chaperone activity might be possible.

  15. Putative impact of RNA editing on drug discovery.

    Science.gov (United States)

    Decher, Niels; Netter, Michael F; Streit, Anne K

    2013-01-01

    Virtually all organisms use RNA editing as a powerful post-transcriptional mechanism to recode genomic information and to increase functional protein diversity. The enzymatic editing of pre-mRNA by ADARs and CDARs is known to change the functional properties of neuronal receptors and ion channels regulating cellular excitability. However, RNA editing is also an important mechanism for genes expressed outside the brain. The fact that RNA editing breaks the 'one gene encodes one protein' hypothesis is daunting for scientists and a probable drawback for drug development, as scientists might search for drugs targeting the 'wrong' protein. This possible difficulty for drug discovery and development became more evident from recent publications, describing that RNA editing events have profound impact on the pharmacology of some common drug targets. These recent studies highlight that RNA editing can cause massive discrepancies between the in vitro and in vivo pharmacology. Here, we review the putative impact of RNA editing on drug discovery, as RNA editing has to be considered before using high-throughput screens, rational drug design or choosing the right model organism for target validation.

  16. Epigenetic regulation of putative tumor suppressor TGFBI in human leukemias

    Institute of Scientific and Technical Information of China (English)

    Fang Hongbo; Liu Jing; Guo Dan; Liu Peixiang; Zhao Yongliang

    2014-01-01

    Background Both in vitro and in vivo data have demonstrated the TGFBI gene functions as a putative tumor suppressor and is frequently downregulated in human tumors of different histological types.The hypermethylation of the TGFBI promoter,as one of the main regulatory mechanisms,is associated with TGFBI silencing.In this study,we used a methylation-specific PCR (MSP) method to evaluate the methylation status of the TGFBI promoter in human leukemias.Methods Real-time RT-PCR and methylation-specific PCR approaches were performed to define the TGFBI expression and promoter methylation in human leukemia call lines and clinical samples.Genomic DNA was isolated from peripheral blood mononuclear cells from leukemia patients,bisulfite-converted,and analyzed by the MSP method.Results Hypermethylation of the TGFBI promoter occurred in leukemia cell lines and demethylation treatment reexpressed TGFBI at a substantially increased level in most of leukemia cell lines tested.Furthermore,a much higher level of CpG island methylation and a significantly lower TGFBI expression were also identified in clinical leukemia samples.Conclusion The results suggest an important role of promoter methylation in regulating TGFBI expression in leukemia,which provides a useful diagnostic marker for clinical management of human leukemias.

  17. Expression and characterization of rice putative PAUSED gene

    Institute of Scientific and Technical Information of China (English)

    Chengguo Yao; Liangfa Ge; Wei Li; Botao Zhao; Chaoqun Li; Kangcheng Ruan; Hongxuan Lin; Youxin Jin

    2008-01-01

    In Arab idopsis, PA USED ( PSD ) encodes the ortholog of loslp/ exportin-t, which mediates the nuclear export of transfer RNA (tRNA) in yeast and mammals. However, in monocot plants such as rice, knowledge of the corresponding ortholog is limited, and its effects on growth development and productivity remain unknown. In this study, we verified a rice transfer-DNA insertional mutantpsd line and analyzed its phenotypes;the mutant displayed severe morphological defects including retarded development and low fertility compared with wild-type rice. Examining intronless tRNA-Tyr and intron-containing pre-tRNA-Ala expression levels in cytoplasmic and nuclear fraction with Northern blot analysis between wild -type and mutant leaf tissue suggested that rice PSD might be involved in tRNA export from the nucleus to the cytoplasm.Additionally, reverse transcription-polymerase chain reaction analysis revealed that PSD transcript was expressed throughout normal rice plant development, and subcellular localization assays showed that rice PSD protein was present in both the nucleus and cytoplasm. In summary, our data implied that the putative PSD gene might be indispensable for normal rice development and its function might be the same as that ofArabidopsis PSD.

  18. Conformational study of a putative HLTV-1 retroviral protease inhibitor.

    Science.gov (United States)

    Llido, S; d'Estaintot, B L; Dautant, A; Geoffre, S; Picard, P; Precigoux, G

    1993-05-01

    The crystal structure of prolyl-glutaminyl-valyl-statyl-alanyl-leucine (Pro-Gln-Val-Sta-Ala-Leu, C(32)H(57)N(7)0(9).5H(2)0, M(r) = 683.9 + 90.1), a putative HTLV-1 protease inhibitor based on one of the consensus retroviral protease cleavage sequences, and containing the statine residue [(4S,3S)-4-amino-3-hydroxy-6-methylheptanoic acid], has been determined by X-ray diffraction. The same molecule has been modelled in the active site of the HTLV-1 protease and both conformations have been compared. The peptide crystallizes as a pentahydrate in space group P2(1) with a = 10.874(2), b = 9.501(2), c = 21.062(5) A, beta = 103.68 (1) degrees, Z = 2, V= 2114.3 A(3), D(x) = 1.21 g cm(-3), micro = 8.02 cm(-1), T= 293 K, lambda(Cu Kalpha) = 1.5418 A. The structure has been refined to an R value of 0.070 for 2152 observed reflections. The peptide main chain can be described as extended and adopts the usual zigzag conformation from the prolyl to the statyl residue. The main difference in conformation between the individual observed and modelled molecules is located on the Sta, Ala and Leu residues with the main chain of the modelled molecule rotated by about 180 degrees as compared to the observed conformation in the crystal state.

  19. A new putative sigma factor of Myxococcus xanthus.

    Science.gov (United States)

    Apelian, D; Inouye, S

    1993-06-01

    A third putative sigma factor gene, sigC, has been isolated from Myxococcus xanthus by using the sigA gene (formerly rpoD of M. xanthus) as a probe. The nucleotide sequence of sigC has been determined, and an open reading frame of 295 residues (M(r) = 33,430) has been identified. The deduced amino acid sequence of sigC exhibits the features which are characteristic of other bacterial sigma factors. The characterization of a sigC-lacZ strain has demonstrated that sigC expression is induced immediately after cells enter into the developmental cycle and is dramatically reduced at the onset of sporulation. A deletion mutant of sigC grows normally in vegetative culture and is able to develop normally. However, in contrast to the wild-type cells, the sigC deletion mutant cells became capable of forming fruiting bodies and myxospores on semirich agar plates. This suggests that sigC may play a role in expression of genes involved in negatively regulating the initiation of fruiting body formation.

  20. Endocytosis of Receptor Tyrosine Kinases

    Science.gov (United States)

    Goh, Lai Kuan

    2013-01-01

    Endocytosis is the major regulator of signaling from receptor tyrosine kinases (RTKs). The canonical model of RTK endocytosis involves rapid internalization of an RTK activated by ligand binding at the cell surface and subsequent sorting of internalized ligand-RTK complexes to lysosomes for degradation. Activation of the intrinsic tyrosine kinase activity of RTKs results in autophosphorylation, which is mechanistically coupled to the recruitment of adaptor proteins and conjugation of ubiquitin to RTKs. Ubiquitination serves to mediate interactions of RTKs with sorting machineries both at the cell surface and on endosomes. The pathways and kinetics of RTK endocytic trafficking, molecular mechanisms underlying sorting processes, and examples of deviations from the standard trafficking itinerary in the RTK family are discussed in this work. PMID:23637288

  1. Receptor tyrosine kinases in carcinogenesis.

    Science.gov (United States)

    Zhang, Xiao-Ying; Zhang, Pei-Ying

    2016-11-01

    Receptor tyrosine kinases (RTKs) are cell surface glycoproteins with enzymatic activity involved in the regulation of various important functions. In all-important physiological functions including differentiation, cell-cell interactions, survival, proliferation, metabolism, migration and signaling these receptors are the key players of regulation. Additionally, mutations of RTKs or their overexpression have been described in many human cancers and are being explored as a novel avenue for a new therapeutic approach. Some of the deregulated RTKs observed to be significantly affected in cancers included vascular endothelial growth factor receptor, epidermal growth factor receptor, fibroblast growth factor receptor, RTK-like orphan receptor 1 (ROR1) and the platelet-derived growth factor receptor. These deregulated RTKs offer attractive possibilities for the new anticancer therapeutic approach involving specific targeting by monoclonal antibodies as well as kinase. The present review aimed to highlight recent perspectives of RTK ROR1 in cancer.

  2. Genome-Wide Identification and Evolutionary Analysis of Sarcocystis neurona Protein Kinases

    Directory of Open Access Journals (Sweden)

    Edwin K. Murungi

    2017-03-01

    Full Text Available The apicomplexan parasite Sarcocystis neurona causes equine protozoal myeloencephalitis (EPM, a degenerative neurological disease of horses. Due to its host range expansion, S. neurona is an emerging threat that requires close monitoring. In apicomplexans, protein kinases (PKs have been implicated in a myriad of critical functions, such as host cell invasion, cell cycle progression and host immune response evasion. Here, we used various bioinformatics methods to define the kinome of S. neurona and phylogenetic relatedness of its PKs to other apicomplexans. We identified 97 putative PKs clustering within the various eukaryotic kinase groups. Although containing the universally-conserved PKA (AGC group, S. neurona kinome was devoid of PKB and PKC. Moreover, the kinome contains the six-conserved apicomplexan CDPKs (CAMK group. Several OPK atypical kinases, including ROPKs 19A, 27, 30, 33, 35 and 37 were identified. Notably, S. neurona is devoid of the virulence-associated ROPKs 5, 6, 18 and 38, as well as the Alpha and RIO kinases. Two out of the three S. neurona CK1 enzymes had high sequence similarities to Toxoplasma gondii TgCK1-α and TgCK1-β and the Plasmodium PfCK1. Further experimental studies on the S. neurona putative PKs identified in this study are required to validate the functional roles of the PKs and to understand their involvement in mechanisms that regulate various cellular processes and host-parasite interactions. Given the essentiality of apicomplexan PKs in the survival of apicomplexans, the current study offers a platform for future development of novel therapeutics for EPM, for instance via application of PK inhibitors to block parasite invasion and development in their host.

  3. Genome-Wide Identification and Evolutionary Analysis of Sarcocystis neurona Protein Kinases.

    Science.gov (United States)

    Murungi, Edwin K; Kariithi, Henry M

    2017-03-21

    The apicomplexan parasite Sarcocystis neurona causes equine protozoal myeloencephalitis (EPM), a degenerative neurological disease of horses. Due to its host range expansion, S. neurona is an emerging threat that requires close monitoring. In apicomplexans, protein kinases (PKs) have been implicated in a myriad of critical functions, such as host cell invasion, cell cycle progression and host immune response evasion. Here, we used various bioinformatics methods to define the kinome of S. neurona and phylogenetic relatedness of its PKs to other apicomplexans. We identified 97 putative PKs clustering within the various eukaryotic kinase groups. Although containing the universally-conserved PKA (AGC group), S. neurona kinome was devoid of PKB and PKC. Moreover, the kinome contains the six-conserved apicomplexan CDPKs (CAMK group). Several OPK atypical kinases, including ROPKs 19A, 27, 30, 33, 35 and 37 were identified. Notably, S. neurona is devoid of the virulence-associated ROPKs 5, 6, 18 and 38, as well as the Alpha and RIO kinases. Two out of the three S. neurona CK1 enzymes had high sequence similarities to Toxoplasma gondii TgCK1-α and TgCK1-β and the Plasmodium PfCK1. Further experimental studies on the S. neurona putative PKs identified in this study are required to validate the functional roles of the PKs and to understand their involvement in mechanisms that regulate various cellular processes and host-parasite interactions. Given the essentiality of apicomplexan PKs in the survival of apicomplexans, the current study offers a platform for future development of novel therapeutics for EPM, for instance via application of PK inhibitors to block parasite invasion and development in their host.

  4. Biochemical examination of the potential eukaryotic-type protein kinase genes in the complete genome of the unicellular Cyanobacterium synechocystis sp. PCC 6803.

    Science.gov (United States)

    Kamei, Ayako; Yuasa, Takashi; Geng, Xiaoxing; Ikeuchi, Masahiko

    2002-06-30

    The complete genome of the unicellular motile cyanobacterium Synechocystis sp. PCC 6803 harbors seven putative genes for a subfamily Pkn2 of the eukaryotic-type (or "Hanks-type") protein kinase. Previously, SpkA and SpkB were shown to have protein kinase activity and to be required for cell motility. Here, the other five genes were examined. These genes, except for spkG (slr0152), were successfully expressed in Escherichia coli. Eukaryotic-type protein kinase activity of the expressed SpkC (Slr0599), SpkD (S110776) and SpkF (Slr1225) was demonstrated as autophosphorylation and phosphorylation of the general substrate proteins. SpkE (Slr1443) did not show any activity, a finding consistent with its lack of several key amino acid residues in its kinase motif. Gene-disrupted mutants showed no discernible defect in phenotype except that spkD was apparently essential for survival.

  5. Oncoprotein protein kinase antibody kit

    Science.gov (United States)

    Karin, Michael; Hibi, Masahiko; Lin, Anning

    2008-12-23

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

  6. Thymidine kinase diversity in bacteria

    DEFF Research Database (Denmark)

    Sandrini, Michael; Clausen, A.R.; Munch-Petersen, B.

    2006-01-01

    Thymidine kinases (TKs) appear to be almost ubiquitous and are found in nearly all prokaryotes, eukaryotes, and several viruses. They are the key enzymes in thymidine salvage and activation of several anti-cancer and antiviral drugs. We show that bacterial TKs can be subdivided into 2 groups. The....... The TKs from Gram-positive bacteria are more closely related to the eukaryotic TK1 enzymes than are TKs from Gram-negative bacteria....

  7. Thymidine kinase diversity in bacteria

    DEFF Research Database (Denmark)

    Sandrini, Michael; Clausen, A.R.; Munch-Petersen, B.

    2006-01-01

    Thymidine kinases (TKs) appear to be almost ubiquitous and are found in nearly all prokaryotes, eukaryotes, and several viruses. They are the key enzymes in thymidine salvage and activation of several anti-cancer and antiviral drugs. We show that bacterial TKs can be subdivided into 2 groups. The....... The TKs from Gram-positive bacteria are more closely related to the eukaryotic TK1 enzymes than are TKs from Gram-negative bacteria....

  8. RIP Kinases Initiate Programmed Necrosis

    Institute of Scientific and Technical Information of China (English)

    Lorenzo Galluzzi; Oliver Kepp; Guido Kroemer

    2009-01-01

    Some lethal stimuli can induce either apoptosis or necrosis, depending on the cell type and/or experimental setting. Until recently,the molecular bases of this phenomenon were largely unknown. Now, two members of the receptor-interacting serine-threonine kinase (RIP) family, RIP1 and RIP3, have been demonstrated to control the switch between apoptotic and necrotic cell death.Some mechanistic details, however, remain controversial.

  9. Regulation and function of TPL-2,an IκB kinase-regulated MAP kinase kinase kinase

    Institute of Scientific and Technical Information of China (English)

    Thorsten Gantke; Srividya Sriskantharajah; Steven C Ley

    2011-01-01

    The IκB kinase(IKK)complex plays a well-documented role in innate and adaptive immunity.This function has been widely attributed to its role as the central activator of the NF-κB family of transcription factors.However,another important consequence of IKK activation is the regulation of TPL-2,a MEK kinase that is required for activation of ERK-1/2 MAP kinases in myeioid cells following Toll-like receptor and TNF receptor stimulation.In unstimulated cells,TPL-2 is stoichiometrically complexed with the NF-κB inhibitory protein NF-κB1 p105,which blocks TPL-2 access to its substrate MEK,and the ubiquitin-binding protein ABIN-2(A20-binding inhibitor of NF-κB 2),both of which are required to maintain TPL-2 protein stability.Following agonist stimulation,the IKK complex phosphorylates p105,triggering its K48-1inked ubiquitination and degradation by the proteasome.This releases TPL-2 from p105-mediated inhibition,facilitating activation of MEK,in addition to modulating NF-κB activation by liberating associated Rel subunits for translocation into the nucleus.IKK-induced proteolysis of 0105,therefore,can directly regulate both NF-κB and ERK MAP kinase activation via NF-κB1 p105.TPL-2 is critical for production of the proinflammatory cytokine TNF during inflammatory responses.Consequently,there has been considerable interest in the pharmaceutical industry to develop selective TPL-2 inhibitors as drugs for the treatment of TNF-dependent inflammatory,diseases,such as rheumatoid arthritis and inflammatory bowel disease.This review summarizes our current understanding of the regulation of TPL-2 signaling function,and also the complex positive and negative roles of TPL-2 in immune and inflammatory responses.

  10. DMPD: Bruton's tyrosine kinase (Btk)-the critical tyrosine kinase in LPS signalling? [Dynamic Macrophage Pathway CSML Database

    Lifescience Database Archive (English)

    Full Text Available 15081522 Bruton's tyrosine kinase (Btk)-the critical tyrosine kinase in LPS signall...ruton's tyrosine kinase (Btk)-the critical tyrosine kinase in LPS signalling? PubmedID 15081522 Title Bruton...'s tyrosine kinase (Btk)-the critical tyrosine kinase in LPS signalling? Authors

  11. Oxidized Form of Creatine Kinase

    Institute of Scientific and Technical Information of China (English)

    王希成; 王帆; 邹晓明; 周海梦

    1994-01-01

    The purified rabbit muscle creatine kinase (R-CK) was previously considered homogeneousand without disulfide bonds.By the method of NR/R two-dimensional diagonal SDS-PAGE,two forms of R-CK,designated respectively "oxidized form" of creatine kinase which contained intrachain disulfide bondsand "reduced form" of creatine kinase which did not have any —S—S— bridges,were for the first time sepa-rated.They were found to be the same in amino acid composition,in subunit molecular Weight and in isoelec-tric point,and were almost identical in enzyme activities.Thus it is hard to isolate one from the other bycommon biochemical methods.More extensive studies show that the oxidized form of CK also contains a pair of reactive thiol groupswhich are essential to the enzyme activity,and it has one intrachain disulfide bond per subunit.In the nativestate,this —S—S— bond cannot be reduced by DTT,but by treating the reduced form of CK with some ox-idants,these —S—S— bonds can be formed in vitro.Thus it is presumed that the disulfide bonds are cross-linked through the oxidization of two shallowly buried —SH groups.

  12. TEC protein tyrosine kinase is involved in the Erk signaling pathway induced by HGF

    Energy Technology Data Exchange (ETDEWEB)

    Li, Feifei; Jiang, Yinan [Department of Pathophysiology, Anhui Medical University, Hefei 230032 (China); Zheng, Qiping [Department of Anatomy and Cell Biology, Rush University Medical Center, Chicago, IL 60612 (United States); Yang, Xiaoming [Institute of Radiation Medicine, Academy of Military Medical Sciences, Beijing 100850 (China); Wang, Siying, E-mail: sywang@ahmu.edu.cn [Department of Pathophysiology, Anhui Medical University, Hefei 230032 (China)

    2011-01-07

    Research highlights: {yields} TEC is rapidly tyrosine-phosphorylated and activated by HGF-stimulation in vivo or after partial hepatectomy in mice. {yields} TEC enhances the activity of Elk and serum response element (SRE) in HGF signaling pathway in hepatocyte. {yields} TEC promotes hepatocyte proliferation through the Erk-MAPK pathway. -- Abstract: Background/aims: TEC, a member of the TEC family of non-receptor type protein tyrosine kinases, has recently been suggested to play a role in hepatocyte proliferation and liver regeneration. This study aims to investigate the putative mechanisms of TEC kinase regulation of hepatocyte differentiation, i.e. to explore which signaling pathway TEC is involved in, and how TEC is activated in hepatocyte after hepatectomy and hepatocyte growth factor (HGF) stimulation. Methods: We performed immunoprecipitation (IP) and immunoblotting (IB) to examine TEC tyrosine phosphorylation after partial hepatectomy in mice and HGF stimulation in WB F-344 hepatic cells. The TEC kinase activity was determined by in vitro kinase assay. Reporter gene assay, antisense oligonucleotide and TEC dominant negative mutant (TEC{sup KM}) were used to examine the possible signaling pathways in which TEC is involved. The cell proliferation rate was evaluated by {sup 3}H-TdR incorporation. Results: TEC phosphorylation and kinase activity were increased in 1 h after hepatectomy or HGF treatment. TEC enhanced the activity of Elk and serum response element (SRE). Inhibition of MEK1 suppressed TEC phosphorylation. Blocking TEC activity dramatically decreased the activation of Erk. Reduced TEC kinase activity also suppressed the proliferation of WB F-344 cells. These results suggest TEC is involved in the Ras-MAPK pathway and acts between MEK1 and Erk. Conclusions: TEC promotes hepatocyte proliferation and regeneration and is involved in HGF-induced Erk signaling pathway.

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

    Directory of Open Access Journals (Sweden)

    Mutsuki Amano

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

  14. The aerosols' fate in a putative ammonia ocean on Titan

    Science.gov (United States)

    Ramírez, S. I.; Coll, P.; Buch, A.; Brassé, C.; Poch, O.; Raulin, F.

    2010-04-01

    A laboratory study on the chemical transformation of Titan's aerosol analogues placed under putative surface conditions of the satellite was performed. The surface of Titan was one of the targets of the Cassini-Huygens mission and of several of the Cassini orbiter instruments, especially ISS, VIMS and Radar. The first images revealed an interesting solid surface with features that suggest aeolian, tectonic, fluvial processes and even an impact structure[1]. Since then, more detailed descriptions of dunes, channels, lakes, impact craters and cryovolcanic structures have been documented[2]. The existence of an internal liquid water ocean, containing a few percent ammonia has been proposed[2, 3]. It has also been proposed that ammonia-water mixtures can erupt from the putative subsurface ocean leading to cryovolcanism[4]. The Cassini Titan Radar Mapper obtained Synthetic Aperture Radar (SAR) images during 2004 and 2005 that revealed a highly complex geology occurring at Titan's surface[5], among which cryovolcanic features play a central role. The composition of the cryomagma is mainly proposed to be a mixture of water ice and ammonia[6, 7, 8], although ammonia has not been directly detected on Titan, but suggested by recent Cassini-VIMS observations[9]. In order to understand the role that ammonia may play on the chemical transformation of atmospheric aerosols once they reach the surface, we designed the following protocol: laboratory analogues of Titan's aerosols were synthesized from a N2:CH4 (98:2) mixture irradiated under a continuous flow regime of 845 sccm inside which, a cold plasma of 180 W was established. The synthesized analogues were recovered and partitioned in several 10.0 mg samples that were placed in 4.0 mL-volume of aqueous ammonia solutions (25.00, 12.50, 6.25 and 3.125%) at different temperatures (298, 277, 253 and 93 K) for 10 weeks. After a derivatization process performed to the aerosols' refractory phase with N

  15. Molecular diagnosis of putative Stargardt disease probands by exome sequencing

    Directory of Open Access Journals (Sweden)

    Strom Samuel P

    2012-08-01

    Full Text Available Abstract Background The commonest genetic form of juvenile or early adult onset macular degeneration is Stargardt Disease (STGD caused by recessive mutations in the gene ABCA4. However, high phenotypic and allelic heterogeneity and a small but non-trivial amount of locus heterogeneity currently impede conclusive molecular diagnosis in a significant proportion of cases. Methods We performed whole exome sequencing (WES of nine putative Stargardt Disease probands and searched for potentially disease-causing genetic variants in previously identified retinal or macular dystrophy genes. Follow-up dideoxy sequencing was performed for confirmation and to screen for mutations in an additional set of affected individuals lacking a definitive molecular diagnosis. Results Whole exome sequencing revealed seven likely disease-causing variants across four genes, providing a confident genetic diagnosis in six previously uncharacterized participants. We identified four previously missed mutations in ABCA4 across three individuals. Likely disease-causing mutations in RDS/PRPH2, ELOVL, and CRB1 were also identified. Conclusions Our findings highlight the enormous potential of whole exome sequencing in Stargardt Disease molecular diagnosis and research. WES adequately assayed all coding sequences and canonical splice sites of ABCA4 in this study. Additionally, WES enables the identification of disease-related alleles in other genes. This work highlights the importance of collecting parental genetic material for WES testing as the current knowledge of human genome variation limits the determination of causality between identified variants and disease. While larger sample sizes are required to establish the precision and accuracy of this type of testing, this study supports WES for inherited early onset macular degeneration disorders as an alternative to standard mutation screening techniques.

  16. A putatively novel form of spontaneous coordination in neural activity.

    Science.gov (United States)

    Hermer-Vazquez, Raymond; Hermer-Vazquez, Linda; Srinivasan, Sridhar

    2009-04-06

    We simultaneously recorded local field potentials from three sites along the olfactory-entorhinal axis in rats lightly anesthetized with isoflurane, as part of another experiment. While analyzing the initial data from that experiment with spectrograms, we discovered a potentially novel form of correlated neural activity, with near-simultaneous occurrence across the three widely separated brain sites. After validating their existence further, we named these events Synchronous Frequency Bursts (SFBs). Here we report our initial investigations into their properties and their potential functional significance. In Experiment 1, we found that SFBs have highly regular properties, consisting of brief (approximately 250 ms), high amplitude bursts of LFP energy spanning frequency ranges from the delta band (1-4 Hz) to at least the low gamma band (30-50 Hz). SFBs occurred almost simultaneously across recording sites, usually with onsets sites. While the SFBs had fairly typical, exponentially decaying power spectral density plots, their coherence structure was unusual, with high peaks in several narrow frequency ranges and little coherence in other bands. In Experiment 2, we found that SFBs occurred far more often under light anesthesia than deeper anesthetic states, and were especially prevalent as the animals regained consciousness. Finally, in Experiment 3 we showed that SFBs occur simultaneously at a significant rate across brain sites from putatively different functional subsystems--olfactory versus motor pathways. We suggest that SFBs do not carry information per se, but rather, play a role in coordinating activity in different frequency bands, potentially brain-wide, as animals progress from sleep or anesthesia toward full consciousness.

  17. Tissue factor residues that putatively interact with membrane phospholipids.

    Directory of Open Access Journals (Sweden)

    Ke Ke

    Full Text Available Blood clotting is initiated by the two-subunit enzyme consisting of the plasma protease, factor VIIa (the catalytic subunit, bound to the integral membrane protein, tissue factor (the regulatory subunit. Molecular dynamics simulations have predicted that certain residues in the tissue factor ectodomain interact with phosphatidylserine headgroups to ensure optimal positioning of the tissue factor/factor VIIa complex relative to its membrane-bound protein substrates, factors IX and X. In this study, we individually mutated to alanine all the putative phosphatidylserine-interactive residues in the tissue factor ectodomain and measured their effects on tissue factor cofactor function (activation of factors IX and X by tissue factor/factor VIIa, and clotting of plasma. Some tissue factor mutants exhibited decreased activity in all three assays, with the most profound defects observed from mutations in or near the flexible loop from Lys159 to Gly164. The decreased activity of all of these tissue factor mutants could be partially or completely overcome by increasing the phosphatidylserine content of tissue factor-liposomes. Additionally, yeast surface display was used to screen a random library of tissue factor mutants for enhanced factor VIIa binding. Surprisingly, mutations at a single amino acid (Lys165 predominated, with the Lys165→Glu mutant exhibiting a 3-fold enhancement in factor VIIa binding affinity. Our studies reveal the functional contributions of residues in the C-terminal half of the tissue factor ectodomain that are implicated in interacting with phosphatidylserine headgroups to enhance tissue factor cofactor activity, possibly by allosterically modulating the conformation of the adjacent substrate-binding exosite region of tissue factor.

  18. A putative viral defence mechanism in archaeal cells

    Directory of Open Access Journals (Sweden)

    Reidun Lillestøl

    2006-01-01

    Full Text Available Clusters of regularly spaced direct repeats, separated by unconserved spacer sequences, are ubiquitous in archaeal chromosomes and occur in some plasmids. Some clusters constitute around 1% of chromosomal DNA. Similarly structured clusters, generally smaller, also occur in some bacterial chromosomes. Although early studies implicated these clusters in segregation/partition functions, recent evidence suggests that the spacer sequences derive from extrachromosomal elements, and, primarily, viruses. This has led to the proposal that the clusters provide a defence against viral propagation in cells, and that both the mode of inhibition of viral propagation and the mechanism of adding spacer-repeat units to clusters, are dependent on RNAs transcribed from the clusters. Moreover, the putative inhibitory apparatus (piRNA-based may be evolutionarily related to the interference RNA systems (siRNA and miRNA, which are common in eukarya. Here, we analyze all the current data on archaeal repeat clusters and provide some new insights into their diverse structures, transcriptional properties and mode of structural development. The results are consistent with larger cluster transcripts being processed at the centers of the repeat sequences and being further trimmed by exonucleases to yield a dominant, intracellular RNA species, which corresponds approximately to the size of a spacer. Furthermore, analysis of the extensive clusters of Sulfolobus solfataricus strains P1 and P2B provides support for the presence of a flanking sequence adjoining a cluster being a prerequisite for the incorporation of new spacer-repeat units, which occurs between the flanking sequence and the cluster. An archaeal database summarizing the data will be maintained at http://dac.molbio.ku.dk/dbs/SRSR/.

  19. Putative Risk Factors in Developmental Dyslexia: A Case-Control Study of Italian Children

    Science.gov (United States)

    Mascheretti, Sara; Marino, Cecilia; Simone, Daniela; Quadrelli, Ermanno; Riva, Valentina; Cellino, Maria Rosaria; Maziade, Michel; Brombin, Chiara; Battaglia, Marco

    2015-01-01

    Although dyslexia runs in families, several putative risk factors that cannot be immediately identified as genetic predict reading disability. Published studies analyzed one or a few risk factors at a time, with relatively inconsistent results. To assess the contribution of several putative risk factors to the development of dyslexia, we conducted…

  20. Putative Risk Factors in Developmental Dyslexia: A Case-Control Study of Italian Children

    Science.gov (United States)

    Mascheretti, Sara; Marino, Cecilia; Simone, Daniela; Quadrelli, Ermanno; Riva, Valentina; Cellino, Maria Rosaria; Maziade, Michel; Brombin, Chiara; Battaglia, Marco

    2015-01-01

    Although dyslexia runs in families, several putative risk factors that cannot be immediately identified as genetic predict reading disability. Published studies analyzed one or a few risk factors at a time, with relatively inconsistent results. To assess the contribution of several putative risk factors to the development of dyslexia, we conducted…

  1. Protein kinase C-associated kinase can activate NFkappaB in both a kinase-dependent and a kinase-independent manner.

    Science.gov (United States)

    Moran, Stewart T; Haider, Khaleda; Ow, Yongkai; Milton, Peter; Chen, Luojing; Pillai, Shiv

    2003-06-13

    Protein kinase C-associated kinase (PKK, also known as RIP4/DIK) activates NFkappaB when overexpressed in cell lines and is required for keratinocyte differentiation in vivo. However, very little is understood about the factors upstream of PKK or how PKK activates NFkappaB. Here we show that certain catalytically inactive mutants of PKK can activate NFkappaB, although to a lesser degree than wild type PKK. The deletion of specific domains of wild type PKK diminishes the ability of this enzyme to activate NFkappaB; the same deletions made on a catalytically inactive PKK background completely ablate NFkappaB activation. PKK may be phosphorylated by two specific mitogen-activated protein kinase kinase kinases, MEKK2 and MEKK3, and this interaction may in part be mediated through a critical activation loop residue, Thr184. Catalytically inactive PKK mutants that block phorbol ester-induced NFkappaB activation do not interfere with, but unexpectedly enhance, the activation of NFkappaB by these two mitogen-activated protein kinase kinase kinases. Taken together, these data indicate that PKK may function in both a kinase-dependent as well as a kinase-independent manner to activate NFkappaB.

  2. RASSF6; the Putative Tumor Suppressor of the RASSF Family

    Directory of Open Access Journals (Sweden)

    Hiroaki Iwasa

    2015-12-01

    Full Text Available Humans have 10 genes that belong to the Ras association (RA domain family (RASSF. Among them, RASSF7 to RASSF10 have the RA domain in the N-terminal region and are called the N-RASSF proteins. In contradistinction to them, RASSF1 to RASSF6 are referred to as the C-RASSF proteins. The C-RASSF proteins have the RA domain in the middle region and the Salvador/RASSF/Hippo domain in the C-terminal region. RASSF6 additionally harbors the PSD-95/Discs large/ZO-1 (PDZ-binding motif. Expression of RASSF6 is epigenetically suppressed in human cancers and is generally regarded as a tumor suppressor. RASSF6 induces caspase-dependent and -independent apoptosis. RASSF6 interacts with mammalian Ste20-like kinases (homologs of Drosophila Hippo and cross-talks with the Hippo pathway. RASSF6 binds MDM2 and regulates p53 expression. The interactions with Ras and Modulator of apoptosis 1 (MOAP1 are also suggested by heterologous protein-protein interaction experiments. RASSF6 regulates apoptosis and cell cycle through these protein-protein interactions, and is implicated in the NF-κB and JNK signaling pathways. We summarize our current knowledge about RASSF6 and discuss what common and different properties RASSF6 and the other C-RASSF proteins have.

  3. Pim-selective inhibitor DHPCC-9 reveals Pim kinases as potent stimulators of cancer cell migration and invasion

    Directory of Open Access Journals (Sweden)

    Prudhomme Michelle

    2010-10-01

    Full Text Available Abstract Background Pim family kinases are small constitutively active serine/threonine-specific kinases, elevated levels of which have been detected in human hematopoietic malignancies as well as in solid tumours. While we and others have previously shown that the oncogenic Pim kinases stimulate survival of hematopoietic cells, we now examined their putative role in regulating motility of adherent cancer cells. For this purpose, we inhibited Pim kinase activity using a small molecule compound, 1,10-dihydropyrrolo[2,3-a]carbazole-3-carbaldehyde (DHPCC-9, which we had recently identified as a potent and selective inhibitor for all Pim family members. Results We now demonstrate that the Pim kinase inhibitor DHPCC-9 is very effective also in cell-based assays. DHPCC-9 impairs the anti-apoptotic effects of Pim-1 in cytokine-deprived myeloid cells and inhibits intracellular phosphorylation of Pim substrates such as Bad. Moreover, DHPCC-9 slows down migration and invasion of cancer cells derived from either prostate cancer or squamocellular carcinoma patients. Silencing of Pim expression reduces cell motility, while Pim overexpression enhances it, strongly suggesting that the observed effects of DHPCC-9 are dependent on Pim kinase activity. Interestingly, DHPCC-9 also abrogates NFATc-dependent migration of cancer cells, implying that NFATc factors mediate at least part of the pro-migratory effects of Pim kinases. Conclusions Altogether, our data indicate that DHPCC-9 is not only a powerful tool to investigate physiological effects of the oncogenic Pim family kinases, but also an attractive molecule for drug development to inhibit invasiveness of Pim-overexpressing cancer cells.

  4. Characterization of four plasma membrane aquaporins in tulip petals: a putative homolog is regulated by phosphorylation.

    Science.gov (United States)

    Azad, Abul Kalam; Katsuhara, Maki; Sawa, Yoshihiro; Ishikawa, Takahiro; Shibata, Hitoshi

    2008-08-01

    We suggested previously that temperature-dependent tulip (Tulipa gesneriana) petal movement that is concomitant with water transport is regulated by reversible phosphorylation of an unidentified plasma membrane intrinsic protein (PIP). In this study, four full-length cDNAs of PIPs from tulip petals were identified and cloned. Two PIPs, namely TgPIP1;1 and TgPIP1;2, are members of the PIP1 subfamily, and the remaining two PIPs, namely TgPIP2;1 and TgPIP2;2, belong to the PIP2 subfamily of aquaporins and were named according to the nomenclature of PIP genes in plants. Of these four homologs, only TgPIP2;2 displayed significant water channel activity in the heterologous expression assay using Xenopus laevis oocytes. The water channel activity of this functional isoform was abolished by mercury and was affected by inhibitors of protein kinase and protein phosphatase. Using a site-directed mutagenesis approach to substitute several serine residues with alanine, and assessing water channel activity using the methylotrophic yeast Pichia pastoris expression assay, we showed that Ser35, Ser116 and Ser274 are the putative phosphorylation sites of TgPIP2;2. Real-time reverse transcription-PCR analysis revealed that the transcript levels of TgPIP1;1 and TgPIP1;2 in tulip petals, stems, leaves, bulbs and roots are very low when compared with those of TgPIP2;1 and TgPIP2;2. The transcript level of TgPIP2;1 is negligible in roots, and TgPIP2;2 is ubiquitously expressed in all organs with significant transcript levels. From the data reported herein, we suggest that TgPIP2;2 might be modulated by phosphorylation and dephosphorylation for regulating water channel activity, and may play a role in transcellular water transport in all tulip organs.

  5. Protein kinase C-associated kinase (PKK), a novel membrane-associated, ankyrin repeat-containing protein kinase.

    Science.gov (United States)

    Chen, L; Haider, K; Ponda, M; Cariappa, A; Rowitch, D; Pillai, S

    2001-06-15

    A novel murine membrane-associated protein kinase, PKK (protein kinase C-associated kinase), was cloned on the basis of its physical association with protein kinase Cbeta (PKCbeta). The regulated expression of PKK in mouse embryos is consistent with a role for this kinase in early embryogenesis. The human homolog of PKK has over 90% identity to its murine counterpart, has been localized to chromosome 21q22.3, and is identical to the PKCdelta-interacting kinase, DIK (Bahr, C., Rohwer, A., Stempka, L., Rincke, G., Marks, F., and Gschwendt, M. (2000) J. Biol. Chem. 275, 36350-36357). PKK comprises an N-terminal kinase domain and a C-terminal region containing 11 ankyrin repeats. PKK exhibits protein kinase activity in vitro and associates with cellular membranes. PKK exists in three discernible forms at steady state: an underphosphorylated form of 100 kDa; a soluble, cytosolic, phosphorylated form of 110 kDa; and a phosphorylated, detergent-insoluble form of 112 kDa. PKK is initially synthesized as an underphosphorylated soluble 100-kDa protein that is quantitatively converted to a detergent-soluble 110-kDa form. This conversion requires an active catalytic domain. Although PKK physically associates with PKCbeta, it does not phosphorylate this PKC isoform. However, PKK itself may be phosphorylated by PKCbeta. PKK represents a developmentally regulated protein kinase that can associate with membranes. The functional significance of its association with PKCbeta remains to be ascertained.

  6. Protein Kinase D family kinases: roads start to segregate.

    Science.gov (United States)

    Wille, Christoph; Seufferlein, Thomas; Eiseler, Tim

    2014-01-01

    Highly invasive pancreatic tumors are often recognized in late stages due to a lack of clear symptoms and pose major challenges for treatment and disease management. Broad-band Protein Kinase D (PKD) inhibitors have recently been proposed as additional treatment option for this disease. PKDs are implicated in the control of cancer cell motility, angiogenesis, proliferation and metastasis. In particular, PKD2 expression is elevated in pancreatic cancer, whereas PKD1 expression is comparably lower. In our recent study we report that both kinases control PDAC cell invasive properties in an isoform-specific, but opposing manner. PKD1 selectively mediates anti-migratory/anti-invasive features by preferential regulation of the actin-regulatory Cofilin-phosphatase Slingshot1L (SSH1L). PKD2, on the other hand enhances invasion and angiogenesis of PDAC cells in 3D-ECM cultures and chorioallantois tumor models by stimulating expression and secretion of matrix-metalloproteinase 7 and 9 (MMP7/9). MMP9 also enhances PKD2-mediated tumor angiogenesis releasing extracellular matrix-bound VEGF-A. We thus suggest high PKD2 expression and loss of PKD1 may be beneficial for tumor cells to enhance their matrix-invading abilities. In our recent study we demonstrate for the first time PKD1 and 2 isoform-selective effects on pancreatic cancer cell invasion, in-vitro and in-vivo, defining isoform-specific regulation of PKDs as a major future issue.

  7. The PPLA motif of glycogen synthase kinase 3beta is required for interaction with Fe65.

    Science.gov (United States)

    Lee, Eun Jeoung; Hyun, Sunghee; Chun, Jaesun; Shin, Sung Hwa; Lee, Kyung Eun; Yeon, Kwang Hum; Park, Tae Yoon; Kang, Sang Sun

    2008-07-31

    Glycogen synthase kinase 3beta (GSK 3 beta) is a serine/ threonine kinase that phosphorylates substrates such as beta-catenin and is involved in a variety of biological processes, including embryonic development, metabolism, tumorigenesis, and cell death. Here, we present evidence that human GSK 3beta is associated with Fe65, which has the characteristics of an adaptor protein, possessing a WW domain, and two phosphotyrosine interaction domains, PID1 and PID2. The GSK 3beta catalytic domain also contains a putative WW domain binding motif ((371)PPLA(374)), and we observed, using a pull down approach and co-immuno-precipitation, that it interacts physically with Fe65 via this motif. In addition, we detected co-localization of GSK 3beta and Fe65 by confocal microscopy, and this co-localization was disrupted by mutation of the putative WW domain binding motif of GSK 3beta.Finally, in transient transfection assays interaction of GSK 3 beta (wt) with Fe65 induced substantial cell apoptosis, whereas interaction with the GSK 3beta AALA mutant ((371)AALA(374)) did not, and we noted that phosphorylation of the Tyr 216 residue of the GSK 3beta AALA mutant was significantly reduced compared to that of GSK 3beta wild type. Thus, our observations indicate that GSK 3beta binds to Fe65 through its (371)PPLA(374) motif and that this interaction regulates apoptosis and phosphorylation of Tyr 216 of GSK 3beta.

  8. MAP kinases and histone modification

    Institute of Scientific and Technical Information of China (English)

    Tamaki Suganuma; Jerry L. Workman

    2012-01-01

    Signal transduction pathways alter the gene expression program in response to extracellular or intracellular cues.Mitogen-activated protein kinases (MAPKs) govern numerous cellular processes including cell growth,stress response,apoptosis,and differentiation.In the past decade,MAPKs have been shown to regulate the transcription machinery and associate with chromatin-modifying complexes.Moreover,recent studies demonstrate that several MAPKs bind directly to chromatin at target genes.This review highlights the recent discoveries of MAPK signaling in regard to histone modifications and chromatin regulation.Evidence suggesting that further unknown mechanisms integrate signal transduction with chromatin biology is discussed.

  9. Assessing protein kinase target similarity

    DEFF Research Database (Denmark)

    Gani, Osman A; Thakkar, Balmukund; Narayanan, Dilip

    2015-01-01

    : focussed chemical libraries, drug repurposing, polypharmacological design, to name a few. Protein kinase target similarity is easily quantified by sequence, and its relevance to ligand design includes broad classification by key binding sites, evaluation of resistance mutations, and the use of surrogate......" of sequence and crystal structure information, with statistical methods able to identify key correlates to activity but also here, "the devil is in the details." Examples from specific repurposing and polypharmacology applications illustrate these points. This article is part of a Special Issue entitled...

  10. Functional analysis of nine putative chemoreceptor proteins in Sinorhizobium meliloti.

    Science.gov (United States)

    Meier, Veronika M; Muschler, Paul; Scharf, Birgit E

    2007-03-01

    The genome of the symbiotic soil bacterium Sinorhizobium meliloti contains eight genes coding for methyl-accepting chemotaxis proteins (MCPs) McpS to McpZ and one gene coding for a transducer-like protein, IcpA. Seven of the MCPs are localized in the cytoplasmic membrane via two membrane-spanning regions, whereas McpY and IcpA lack such hydrophobic regions. The periplasmic regions of McpU, McpV, and McpX contain the small-ligand-binding domain Cache. In addition, McpU possesses the ligand-binding domain TarH. By probing gene expression with lacZ fusions, we have identified mcpU and mcpX as being highly expressed. Deletion of any one of the receptor genes caused impairments in the chemotactic response toward most organic acids, amino acids, and sugars in a swarm plate assay. The data imply that chemoreceptor proteins in S. meliloti can sense more than one class of carbon source and suggest that many or all receptors work as an ensemble. Tactic responses were virtually eliminated for a strain lacking all nine receptor genes. Capillary assays revealed three important sensors for the strong attractant proline: McpU, McpX, and McpY. Receptor deletions variously affected free-swimming speed and attractant-induced chemokinesis. Noticeably, cells lacking mcpU were swimming 9% slower than the wild-type control. We infer that McpU inhibits the kinase activity of CheA in the absence of an attractant. Cells lacking one of the two soluble receptors were impaired in chemokinetic proficiency by more than 50%. We propose that the internal sensors, IcpA and the PAS domain containing McpY, monitor the metabolic state of S. meliloti.

  11. A CHASE domain containing protein kinase OsCRL4, represents a new AtCRE1-like gene family in rice

    Institute of Scientific and Technical Information of China (English)

    韩秋敏; 姜华武; 齐晓朋; 于洁; 吴平

    2004-01-01

    AtCRE1 is known to be a cytokinin receptor in Arabidopsis. The AtCRE1 protein contains CHASE domain at the N-terminal part, followed by a transmitter (histidine kinase) domain and two receiver domains. The N-terminal CHASE domain of AtCRE1 contains putative recognition sites for cytokinin. Five CHASE domains containing proteins were found in rice, OsCRL1a, OsCRL1b, OsCRL2, OsCRL3, and OsCRL4. OsCRL1a, OsCRL1b, OsCRL2 and OsCRL3 contain the four domains existing in CRE1, whereas OsCRL4 only contains the CHASE domain and a putative Ser/Thr protein kinase domain. The authors cloned the encoding gene OsCRL4 and found that it represents a new member of the cytokinin receptor protein in rice.

  12. A CHASE domain containing protein kinase OsCRL4, represents a new AtCRE1-like gene family in rice

    Institute of Scientific and Technical Information of China (English)

    韩秋敏; 姜华武; 齐晓朋; 丁洁; 吴平

    2004-01-01

    AtCRE1 is known to be a cytokinin receptor inArabidopsis. The AtCRE1 protein contains CHASE domain at the N-terminal part, followed by a transmitter (histidine kinase) domain and two receiver domains. The N-terminal CHASE domain of AtCRE1 contains putative recognition sites for cytokinin. Five CHASE domains containing proteins were found in rice, OsCRLla, OsCRLlb, OsCRL2, OsCRL3, and OsCRL4. OsCRL1a, OsCRL1b, OsCRL2 and OsCRL3 contain the four domains existing in CRE1, whereas OsCRL4 only contains the CHASE domain and a putative Ser/Thr protein kinase domain The authors cloned the encoding gene OsCRL4 and found that it represents a new member of the cytokinin receptor protein in rice.

  13. Putative functions of extracellular matrix glycoproteins in secondary palate morphogenesis

    Science.gov (United States)

    d'Amaro, Rocca; Scheidegger, Rolf; Blumer, Susan; Pazera, Pawel; Katsaros, Christos; Graf, Daniel; Chiquet, Matthias

    2012-01-01

    Cleft palate is a common birth defect in humans. Elevation and fusion of paired palatal shelves are coordinated by growth and transcription factors, and mutations in these can cause malformations. Among the effector genes for growth factor signaling are extracellular matrix (ECM) glycoproteins. These provide substrates for cell adhesion (e.g., fibronectin, tenascins), but also regulate growth factor availability (e.g., fibrillins). Cleft palate in Bmp7 null mouse embryos is caused by a delay in palatal shelf elevation. In contrast, palatal shelves of Tgf-β3 knockout mice elevate normally, but a cleft develops due to their failure to fuse. However, nothing is known about a possible functional interaction between specific ECM proteins and Tgf-β/Bmp family members in palatogenesis. To start addressing this question, we studied the mRNA and protein distribution of relevant ECM components during secondary palate development, and compared it to growth factor expression in wildtypewild type and mutant mice. We found that fibrillin-2 (but not fibrillin-1) mRNA appeared in the mesenchyme of elevated palatal shelves adjacent to the midline epithelial cells, which were positive for Tgf-β3 mRNA. Moreover, midline epithelial cells started expressing fibronectin upon contact of the two palatal shelves. These findings support the hypothesis that fibrillin-2 and fibronectin are involved in regulating the activity of Tgf-β3 at the fusing midline. In addition, we observed that tenascin-W (but not tenascin-C) was misexpressed in palatal shelves of Bmp7-deficient mouse embryos. In contrast to tenascin-C, tenascin-W secretion was strongly induced by Bmp7 in embryonic cranial fibroblasts in vitro. These results are consistent with a putative function for tenascin-W as a target of Bmp7 signaling during palate elevation. Our results indicate that distinct ECM proteins are important for morphogenesis of the secondary palate, both as downstream effectors and as regulators of Tgf

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

    Energy Technology Data Exchange (ETDEWEB)

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

    2009-10-01

    APS kinase from Thiobacillus denitrificans contains an inactive N-terminal ATP sulfurylase domain. The structure presented unveils the first hexameric assembly for an APS kinase, and reveals that structural changes in the N-terminal domain disrupt the ATP sulfurylase active site thus prohibiting activity. The Tbd-0210 gene of the chemolithotrophic bacterium Thiobacillus denitrificans is annotated to encode a 60.5 kDa bifunctional enzyme with ATP sulfurylase and APS kinase activity. This putative bifunctional enzyme was cloned, expressed and structurally characterized. The 2.95 Å resolution X-ray crystal structure reported here revealed a hexameric assembly with D{sub 3} symmetry. Each subunit contains a large N-terminal sulfurylase-like domain and a C-terminal APS kinase domain reminiscent of the two-domain fungal ATP sulfurylases of Penicillium chrysogenum and Saccharomyces cerevisiae, which also exhibit a hexameric assembly. However, the T. denitrificans enzyme exhibits numerous structural and sequence differences in the N-terminal domain that render it inactive with respect to ATP sulfurylase activity. Surprisingly, the C-terminal domain does indeed display APS kinase activity, indicating that this gene product is a true APS kinase. Therefore, these results provide the first structural insights into a unique hexameric APS kinase that contains a nonfunctional ATP sulfurylase-like domain of unknown function.

  15. MST kinases in development and disease

    OpenAIRE

    Thompson, Barry J.; Sahai, Erik

    2015-01-01

    The mammalian MST kinase family, which is related to the Hippo kinase in Drosophila melanogaster, includes five related proteins: MST1 (also called STK4), MST2 (also called STK3), MST3 (also called STK24), MST4, and YSK1 (also called STK25 or SOK1). MST kinases are emerging as key signaling molecules that influence cell proliferation, organ size, cell migration, and cell polarity. Here we review the regulation and function of these kinases in normal physiology and pathologies, including cance...

  16. MST kinases in development and disease.

    Science.gov (United States)

    Thompson, Barry J; Sahai, Erik

    2015-09-14

    The mammalian MST kinase family, which is related to the Hippo kinase in Drosophila melanogaster, includes five related proteins: MST1 (also called STK4), MST2 (also called STK3), MST3 (also called STK24), MST4, and YSK1 (also called STK25 or SOK1). MST kinases are emerging as key signaling molecules that influence cell proliferation, organ size, cell migration, and cell polarity. Here we review the regulation and function of these kinases in normal physiology and pathologies, including cancer, endothelial malformations, and autoimmune disease.

  17. Clinical and biological significance of PIM1 kinase in osteosarcoma.

    Science.gov (United States)

    Liao, Yunfei; Feng, Yong; Shen, Jacson; Gao, Yan; Cote, Gregory; Choy, Edwin; Harmon, David; Mankin, Henry; Hornicek, Francis; Duan, Zhenfeng

    2016-07-01

    Osteosarcoma is the most prevalent histological form of primary malignant bone tumor. The majority of osteosarcoma patients have limited alternative therapeutic options and metastatic patients generally have a poor prognosis. Proto-oncogene serine/threonine-protein kinase PIM1 is associated with growth and survival of many kinds of tumor cells. However, the role of PIM1 in osteosarcoma remains largely unknown. In this study, we investigated the functional and therapeutic relevance of PIM1 as a putative target in osteosarcoma. We found PIM1 was highly expressed in various osteosarcoma cell lines and in tumor tissues from osteosarcoma patients. Tissue microarray and immunohistochemistry analysis showed that the overall and disease-free survival rate of patients with high levels of PIM1 protein expression were significantly shorter than patients with low levels. High levels of PIM1 were also associated with present metastasis and can be considered as an independent prognostic factor in osteosarcoma patients. Knockdown of PIM1 expression by synthetic siRNA or shRNA greatly inhibited cell growth, migration, and invasion. Moreover, these changes accompanied with down-regulation of anti-apoptotic protein Bcl-2. The similar results were obtained in osteosarcoma cells treated with PIM1 specific inhibitor (SMI-4a). These results suggest that PIM1 kinase is critical for the growth and metastasis of osteosarcoma cells and can be a potential therapeutic target for osteosarcoma treatment. © 2015 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 34:1185-1194, 2016. © 2015 Orthopaedic Research Society. Published by Wiley Periodicals, Inc.

  18. Prioritization of putative metabolite identifications in LC-MS/MS experiments using a computational pipeline.

    Science.gov (United States)

    Zhou, Bin; Xiao, Jun Feng; Ressom, Habtom W

    2013-01-01

    One of the major bottle-necks in current LC-MS-based metabolomic investigations is metabolite identification. An often-used approach is to first look up metabolites from databases through peak mass, followed by verification of the obtained putative identifications using MS/MS data. However, the mass-based search may provide inappropriate putative identifications when the observed peak is from isotopes, fragments, or adducts. In addition, a large fraction of peaks is often left with multiple putative identifications. To differentiate these putative identifications, manual verification of metabolites through comparison between biological samples and authentic compounds is necessary. However, such experiments are laborious, especially when multiple putative identifications are encountered. It is desirable to use computational approaches to obtain more reliable putative identifications and prioritize them before performing experimental verification of the metabolites. In this article, a computational pipeline is proposed to assist metabolite identification with improved metabolome coverage and prioritization capability. Multiple publicly available software tools and databases, along with in-house developed algorithms, are utilized to fully exploit the information acquired from LC-MS/MS experiments. The pipeline is successfully applied to identify metabolites on the basis of LC-MS as well as MS/MS data. Using accurate masses, retention time values, MS/MS spectra, and metabolic pathways/networks, more appropriate putative identifications are retrieved and prioritized to guide subsequent metabolite verification experiments.

  19. Extracellular signal-regulated kinase is essential for interleukin-1-induced and nuclear factor kappaB-mediated gene expression in insulin-producing INS-1E cells

    DEFF Research Database (Denmark)

    Larsen, Lykke; Størling, J; Darville, M;

    2005-01-01

    The beta cell destruction and insulin deficiency that characterises type 1 diabetes mellitus is partially mediated by cytokines, such as IL-1beta, and by nitric oxide (NO)-dependent and -independent effector mechanisms. IL-1beta activates mitogen-activated protein kinases (MAPKs), including extra...... by which these two pathways regulate beta cell Nos2 expression are currently unknown. Therefore, the aim of this study was to clarify the putative crosstalk between MAPK and NFkappaB activation in beta cells....

  20. Data regarding the growth of Lactobacillus acidophilus NCFM on different carbohydrates and recombinant production of elongation factor G and pyruvate kinase

    DEFF Research Database (Denmark)

    Celebioglu, Hasan Ufuk; Olesen, Sita Vaag; Prehn, Kennie

    2017-01-01

    he present study describes the growth of the very well-known probiotic bacterium Lactobacillus acidophilus NCFM on different carbohydrates. Furthermore, recombinant production of putative moonlighting proteins elongation factor G and pyruvate kinase from this bacterium is described. For further...... and detailed interpretation of the data presented here, please see the research article “Mucin- and carbohydrate-stimulated adhesion and subproteome changes of the probiotic bacterium Lactobacillus acidophilus NCFM” (Celebioglu et al., 2017) [1]....

  1. The Arabidopsis kinase-associated protein phosphatase controls internalization of the somatic embryogenesis receptor kinase 1

    NARCIS (Netherlands)

    Shah, K.; Russinova, E.; Gadella, T.W.J.; Willemse, J.; Vries, de S.C.

    2002-01-01

    The AtSERK1 protein is a plasma membrane-located LRR receptor-like serine threonine kinase that is transiently expressed during plant embryogenesis. Our results show that AtSERK1 interacts with the kinase-associated protein phosphatase (KAPP) in vitro. The kinase interaction (KI) domain of KAPP does

  2. Novel Library of Selenocompounds as Kinase Modulators

    Directory of Open Access Journals (Sweden)

    Carmen Sanmartín

    2011-07-01

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

  3. Spontaneous nisin-resistant Listeria monocytogenes mutants with increased expression of a putative penicillin-binding protein and their sensitivity to various antibiotics.

    Science.gov (United States)

    Gravesen, A; Sørensen, K; Aarestrup, F M; Knøchel, S

    2001-01-01

    A concern regarding the use of bacteriocins, as for example the lantibiotic nisin, for biopreservation of certain food products is the possibility of resistance development and potential cross-resistance to antibiotics in the target organism. The genetic basis for nisin resistance development is as yet unknown. We analyzed changes in gene expression following nisin resistance development in Listeria monocytogenes 412 by restriction fragment differential display. The mutant had increased expression of a protein with strong homology to the glycosyltransferase domain of high-molecular-weight penicillin-binding proteins (PBPs), a histidine protein kinase, a protein of unknown function, and ClpB (putative functions from homology). The three former proteins had increased expression in a total of six out of 10 independent mutants originating from five different wild-type strains, indicating a prevalent nisin resistance mechanism under the employed isolation conditions. Increased expression of the putative PBP may affect the cell wall composition and thereby alter the sensitivity to cell wall-targeting compounds. The mutants had an isolate-specific increase in sensitivity to different beta-lactams and a slight decrease in sensitivity to another lantibiotic, mersacidin. A model incorporating these observations is proposed based on current knowledge of nisin's mode of action.

  4. Epidermal growth factor promotes protein degradation of epithelial protein lost in neoplasm (EPLIN), a putative metastasis suppressor, during epithelial-mesenchymal transition.

    Science.gov (United States)

    Zhang, Shumin; Wang, Xu; Iqbal, Shareen; Wang, Yanru; Osunkoya, Adeboye O; Chen, Zhengjia; Chen, Zhuo; Shin, Dong M; Yuan, Hongwei; Wang, Yongqiang A; Zhau, Haiyen E; Chung, Leland W K; Ritenour, Chad; Kucuk, Omer; Wu, Daqing

    2013-01-18

    Aberrant expression of EGF receptors has been associated with hormone-refractory and metastatic prostate cancer (PCa). However, the molecular mechanism for EGF signaling in promoting PCa metastasis remains elusive. Using experimental models of PCa metastasis, we demonstrated that EGF could induce robust epithelial-mesenchymal transition (EMT) and increase invasiveness. Interestingly, EGF was found to be capable of promoting protein turnover of epithelial protein lost in neoplasm (EPLIN), a putative suppressor of EMT and tumor metastasis. Mechanistic study revealed that EGF could activate the phosphorylation, ubiquitination, and degradation of EPLIN through an extracellular signal-regulated kinase 1/2 (ERK1/2)-dependent signaling cascade. Pharmacological inhibition of the ERK1/2 pathway effectively antagonized EGF-induced EPLIN degradation. Two serine residues, i.e. serine 362 and serine 604, were identified as putative ERK1/2 phosphorylation sites in human EPLIN, whose point mutation rendered resistance to EGF-induced protein turnover. This study elucidated a novel molecular mechanism for EGF regulation of EMT and invasiveness in PCa cells, indicating that blockade of EGF signaling could be beneficial in preventing and retarding PCa metastasis at early stages.

  5. The Phosphorylation-Dependent Regulation of Mitochondrial Proteins in Stress Responses

    Directory of Open Access Journals (Sweden)

    Yusuke Kanamaru

    2012-01-01

    Full Text Available To maintain cellular homeostasis, cells are equipped with precise systems that trigger the appropriate stress responses. Mitochondria not only provide cellular energy but also integrate stress response signaling pathways, including those regulating cell death. Several lines of evidence suggest that the mitochondrial proteins that function in this process, such as Bcl-2 family proteins in apoptosis and phosphoglycerate mutase family member 5 (PGAM5 in necroptosis, are regulated by several kinases. It has also been suggested that the phosphorylation-dependent regulation of mitochondrial fission machinery, dynamin-related protein 1 (Drp1, facilitates appropriate cellular stress responses. However, mitochondria themselves are also damaged by various stresses. To avoid the deleterious effects exerted by damaged mitochondria, cells remove these mitochondria in a selective autophagic degradation process called mitophagy. Interestingly, several kinases, such as PTEN-induced putative kinase 1 (PINK1 in mammals and stress-responsive mitogen-activated protein (MAP kinases in yeast, have recently been shown to be involved in mitophagy. In this paper, we focus on the phosphorylation-dependent regulation of mitochondrial proteins and discuss the roles of this regulation in the mitochondrial and cellular stress responses.

  6. Abundant intergenic TAACTGA direct repeats and putative alternate RNA polymerase β´ subunits in marine Beggiatoaceae genomes: possible regulatory roles and origins

    Directory of Open Access Journals (Sweden)

    Barbara J. MacGregor

    2015-12-01

    Full Text Available The genome sequences of several giant marine sulfur-oxidizing bacteria present evidence of a possible post-transcriptional regulatory network that may have been transmitted to or from two distantly related bacteria lineages. The draft genome of a Cand. Maribeggiatoa filament from the Guaymas Basin (Gulf of California, Mexico seafloor contains 169 sets of TAACTGA direct repeats and one indirect repeat, with two to six copies per set. Related heptamers are rarely or never found as direct repeats. TAACTGA direct repeats are also found in some other Beggiatoaceae, Thiocystis violascens, a range of Cyanobacteria, and five Bacteroidetes. This phylogenetic distribution suggests they may have been transmitted horizontally, but no mechanism is evident. There is no correlation between total TAACTGA occurrences and repeats per genome. In most species the repeat units are relatively short, but longer arrays of up to 43 copies are found in several Bacteroidetes and Cyanobacteria. The majority of TAACTGA repeats in the Cand. Maribeggiatoa Orange Guaymas (BOGUAY genome are within several nucleotides upstream of a putative start codon, suggesting they may be binding sites for a post-transcriptional regulator. Candidates include members of the ribosomal protein S1, Csp (cold shock protein, and Csr (carbon storage regulator families. No pattern was evident in the predicted functions of the open reading frames (ORFs downstream of repeats, but some encode presumably essential products such as ribosomal proteins. Among these is an ORF encoding a possible alternate or modified RNA polymerase beta prime subunit, predicted to have the expected subunit interaction domains but lacking most catalytic residues. A similar ORF was found in the Thioploca ingrica draft genome, but in no others. In both species they are immediately upstream of putative sensor kinase genes with nearly identical domain structures. In the marine Beggiatoaceae, a role for the TAACTGA repeats in

  7. Extract of Reishi polysaccharides induces cytokine expression via TLR4-modulated protein kinase signaling pathways.

    Science.gov (United States)

    Hsu, Hsien-Yeh; Hua, Kuo-Feng; Lin, Chun-Cheng; Lin, Chun-Hung; Hsu, Jason; Wong, Chi-Huey

    2004-11-15

    We have demonstrated that an extract of Ganoderma lucidum (Reishi or Ling-Zhi) polysaccharides (EORP) exerts immunomodulating activities by stimulating the expression of inflammatory cytokines from mouse spleen cells. Interestingly, via responding to LPS in genetic variation of murine macrophage HeNC2 and GG2EE cell lines, and using TLR4 Ab blockage in human blood-derived monocytic macrophages, we have found that the TLR4, but not complement receptor type 3, is a putative receptor of EORP, mediating the consequent immunomodulating events associated with IL-1 gene expression. Based on our studies of reactive oxygen species production, polymyxin B inhibition, and protein tyrosine kinase (PTK) activity, we ruled out the possibility of LPS contamination in EORP. We have found that EORP differentially modulates the protein kinase (PK)-mediated signal transduction pathways associated with inflammatory cytokine IL-1. In human macrophages and murine macrophage J774A.1 cells, EORP was found to up-regulate IL-1 secretion and pro-IL-1 (precursor of IL-1) as well as IL-1-converting enzyme expression. Specifically, EORP rapidly stimulates PTK-mediated phosphorylation, followed by induction of PKs and activation of MAPKs: ERK, JNK, and p38. Using PK inhibitors in the kinase activity assays, Western blot analyses and IL-1 ELISA, we have extensively examined and dissected the role of individual PK in the regulation of pro-IL-1/IL-1. Our findings establish that EORP-mediated signaling pathways are involved in the pro-IL-1/IL-1 regulation: PTK/protein kinase C/MEK1/ERK and PTK/Rac1/p21-activated kinase/p38.

  8. Combinatory action of VEGFR2 and MAP kinase pathways maintains endothelial-cell integrity

    Institute of Scientific and Technical Information of China (English)

    Hanbing Zhong; Danyang Wang; Nan Wang; Yesenia Rios; Haigen Huang; Song Li; Xinrong Wu; Shuo Lin

    2011-01-01

    Blood vessels normally maintain stereotyped lumen diameters and their stable structures are crucial for vascular function. However, very little is known about the molecular mechanisms controlling the maintenance of vessel diameters and the integrity of endothelial cells. We investigated this issue in zebrafish embryos by a chemical genetics approach. Small molecule libraries were screened using live Tg(kdrl:GRCFP)zn1 transgenic embryos in which endothelial cells are specifically labeled with GFP. By analyzing the effects of compounds on the morphology and function of embryonic blood vessels after lumen formation, PP1, a putative Src kinase inhibitor, was identified as capable of specifically reducing vascular lumen size by interrupting endothelial-cell integrity. The inhibitory effect is not due to Src or general VEGF signaling inhibition because another Src inhibitor and Src morpholino as well as several VEGFR inhibitors failed to produce a similar phenotype. After profiling a panel of 22 representative mammalian kinases and surveying published data, we selected a few possible new candidates. Combinational analysis of these candidate kinase inhibitors established that PP1 induced endothelial collapse by inhibiting both the VEGFR2 and MAP kinase pathways. More importantly, combinatory use of two clinically approved drugs Dasatinib and Sunitinib produced the same phenotype. This is the first study to elucidate the pathways controlling maintenance of endothelial integrity using a chemical genetics approach, indicating that endothelial integrity is controlled by the combined action of the VEGFR2 and MAP kinase pathways. Our results also suggest the possible side effect of the combination of two anticancer drugs on the circulatory system.

  9. Distinct functions of the dual leucine zipper kinase depending on its subcellular localization.

    Science.gov (United States)

    Wallbach, Manuel; Duque Escobar, Jorge; Babaeikelishomi, Rohollah; Stahnke, Marie-Jeannette; Blume, Roland; Schröder, Sabine; Kruegel, Jenny; Maedler, Kathrin; Kluth, Oliver; Kehlenbach, Ralph H; Miosge, Nicolai; Oetjen, Elke

    2016-04-01

    The dual leucine zipper kinase DLK induces β-cell apoptosis by inhibiting the transcriptional activity conferred by the β-cell protective transcription factor cAMP response element binding protein CREB. This action might contribute to β-cell loss and ultimately diabetes. Within its kinase domain DLK shares high homology with the mixed lineage kinase (MLK) 3, which is activated by tumor necrosis factor (TNF) α and interleukin (IL)-1β, known prediabetic signals. In the present study, the regulation of DLK in β-cells by these cytokines was investigated. Both, TNFα and IL-1β induced the nuclear translocation of DLK. Mutations within a putative nuclear localization signal (NLS) prevented basal and cytokine-induced nuclear localization of DLK and binding to the importin receptor importin α, thereby demonstrating a functional NLS within DLK. DLK NLS mutants were catalytically active as they phosphorylated their down-stream kinase c-Jun N-terminal kinase to the same extent as DLK wild-type but did neither inhibit CREB-dependent gene transcription nor transcription conferred by the promoter of the anti-apoptotic protein BCL-xL. In addition, the β-cell apoptosis-inducing effect of DLK was severely diminished by mutation of its NLS. In a murine model of prediabetes, enhanced nuclear DLK was found. These data demonstrate that DLK exerts distinct functions, depending on its subcellular localization and thus provide a novel level of regulating DLK action. Furthermore, the prevention of the nuclear localization of DLK as induced by prediabetic signals with consecutive suppression of β-cell apoptosis might constitute a novel target in the therapy of diabetes mellitus.

  10. Regulation of the Saccharomyces cerevisiae EKI1-encoded Ethanolamine Kinase by Zinc Depletion*

    Science.gov (United States)

    Kersting, Michael C.; Carman, George M.

    2006-01-01

    Ethanolamine kinase catalyzes the committed step in the synthesis of phosphatidylethanolamine via the CDP-ethanolamine branch of the Kennedy pathway. Regulation of the EKI1-encoded ethanolamine kinase by the essential nutrient zinc was examined in Saccharomyces cerevisiae. The level of ethanolamine kinase activity increased when zinc was depleted from the growth medium. This regulation correlated with increases in the CDP-ethanolamine pathway intermediates phosphoethanolamine and CDP-ethanolamine, and an increase in the methylated derivative of phosphatidylethanolamine, phosphatidylcholine. The β-galactosidase activity driven by the PEKI1-lacZ reporter gene was elevated in zinc-depleted cells, indicating that the increase in ethanolamine kinase activity was attributed to a transcriptional mechanism. The expression level of PEKI1-lacZ reporter gene activity in the zrt1Δzrt2Δ mutant (defective in plasma membrane zinc transport) cells grown with zinc was similar to the activity expressed in wild-type cells grown without zinc. This indicated that EKI1 expression was sensitive to intracellular zinc. The zinc-mediated regulation of EKI1 expression was attenuated in the zap1Δ mutant defective in the zinc-regulated transcription factor Zap1p. Direct interactions between Zap1p and putative zinc-responsive elements in the EKI1 promoter were demonstrated by electrophoretic mobility shift assays. Mutations of these elements to a nonconsensus sequence abolished Zap1p-DNA interactions. Taken together, this work demonstrated that the zinc-mediated regulation of ethanolamine kinase and the synthesis of phospholipids via the CDP-ethanolamine branch of the Kennedy pathway were controlled in part by Zap1p. PMID:16551612

  11. Regulation of the Saccharomyces cerevisiae EKI1-encoded ethanolamine kinase by zinc depletion.

    Science.gov (United States)

    Kersting, Michael C; Carman, George M

    2006-05-12

    Ethanolamine kinase catalyzes the committed step in the synthesis of phosphatidylethanolamine via the CDP-ethanolamine branch of the Kennedy pathway. Regulation of the EKI1-encoded ethanolamine kinase by the essential nutrient zinc was examined in Saccharomyces cerevisiae. The level of ethanolamine kinase activity increased when zinc was depleted from the growth medium. This regulation correlated with increases in the CDP-ethanolamine pathway intermediates phosphoethanolamine and CDP-ethanolamine, and an increase in the methylated derivative of phosphatidylethanolamine, phosphatidylcholine. The beta-galactosidase activity driven by the P(EKI1)-lacZ reporter gene was elevated in zinc-depleted cells, indicating that the increase in ethanolamine kinase activity was attributed to a transcriptional mechanism. The expression level of P(EKI1)-lacZ reporter gene activity in the zrt1deltazrt2delta mutant (defective in plasma membrane zinc transport) cells grown with zinc was similar to the activity expressed in wild-type cells grown without zinc. This indicated that EKI1 expression was sensitive to intracellular zinc. The zinc-mediated regulation of EKI1 expression was attenuated in the zap1delta mutant defective in the zinc-regulated transcription factor Zap1p. Direct interactions between Zap1p and putative zinc-responsive elements in the EKI1 promoter were demonstrated by electrophoretic mobility shift assays. Mutations of these elements to a nonconsensus sequence abolished Zap1p-DNA interactions. Taken together, this work demonstrated that the zinc-mediated regulation of ethanolamine kinase and the synthesis of phospholipids via the CDP-ethanolamine branch of the Kennedy pathway were controlled in part by Zap1p.

  12. An atlas of human kinase regulation.

    Science.gov (United States)

    Ochoa, David; Jonikas, Mindaugas; Lawrence, Robert T; El Debs, Bachir; Selkrig, Joel; Typas, Athanasios; Villén, Judit; Santos, Silvia Dm; Beltrao, Pedro

    2016-12-01

    The coordinated regulation of protein kinases is a rapid mechanism that integrates diverse cues and swiftly determines appropriate cellular responses. However, our understanding of cellular decision-making has been limited by the small number of simultaneously monitored phospho-regulatory events. Here, we have estimated changes in activity in 215 human kinases in 399 conditions derived from a large compilation of phosphopeptide quantifications. This atlas identifies commonly regulated kinases as those that are central in the signaling network and defines the logic relationships between kinase pairs. Co-regulation along the conditions predicts kinase-complex and kinase-substrate associations. Additionally, the kinase regulation profile acts as a molecular fingerprint to identify related and opposing signaling states. Using this atlas, we identified essential mediators of stem cell differentiation, modulators of Salmonella infection, and new targets of AKT1. This provides a global view of human phosphorylation-based signaling and the necessary context to better understand kinase-driven decision-making. © 2016 The Authors. Published under the terms of the CC BY 4.0 license.

  13. A multisubstrate deoxyribonucleoside kinase from plants

    DEFF Research Database (Denmark)

    Clausen, Anders R.; Girandon, Lenart; Knecht, Wolfgang;

    2008-01-01

    and biochemical properties suggest that this deoxyribonucleoside kinase represents a living fossil resembling the progenitor of the modern animal deoxycytidine, deoxyguanosine and thymidine 2 kinases. The broad substrate specificity makes this enzyme an interesting candidate to be evaluated as a suicide gene...

  14. Mutational profiling of kinases in glioblastoma

    NARCIS (Netherlands)

    F.E. Bleeker (Fonnet); S. Lamba (Simona); C. Zanon (Carlo); R.J. Molenaar (Remco J.); T. Hulsebos (Theo); D. Troost (Dirk); A.A.G. van Tilborg (Angela); W.P. Vandertop (Peter); S. Leenstra (Sieger); C.J.F. van Noorden (Cornelis); A. Bardelli (Alberto)

    2014-01-01

    textabstractBackground: Glioblastoma is a highly malignant brain tumor for which no cure is available. To identify new therapeutic targets, we performed a mutation analysis of kinase genes in glioblastoma.Methods: Database mining and a literature search identified 76 kinases that have been found to

  15. Regulation of the interaction between protein kinase C-related protein kinase 2 (PRK2) and its upstream kinase, 3-phosphoinositide-dependent protein kinase 1 (PDK1)

    DEFF Research Database (Denmark)

    Dettori, Rosalia; Sonzogni, Silvina; Meyer, Lucas;

    2009-01-01

    The members of the AGC kinase family frequently exhibit three conserved phosphorylation sites: the activation loop, the hydrophobic motif (HM), and the zipper (Z)/turn-motif (TM) phosphorylation site. 3-Phosphoinositide-dependent protein kinase 1 (PDK1) phosphorylates the activation loop of numer...

  16. Measuring the Activity of Leucine-Rich Repeat Kinase 2: A Kinase Involved in Parkinson's Disease

    Science.gov (United States)

    Lee, Byoung Dae; Li, Xiaojie; Dawson, Ted M.; Dawson, Valina L.

    2015-01-01

    Mutations in the LRRK2 (Leucine-Rich Repeat Kinase 2) gene are the most common cause of autosomal dominant Parkinson's disease. LRRK2 has multiple functional domains including a kinase domain. The kinase activity of LRRK2 is implicated in the pathogenesis of Parkinson's disease. Developing an assay to understand the mechanisms of LRRK2 kinase activity is important for the development of pharmacologic and therapeutic applications. Here, we describe how to measure in vitro LRRK2 kinase activity and its inhibition. PMID:21960214

  17. Expression, purification and kinase activity analysis of maize ...

    African Journals Online (AJOL)

    STORAGESEVER

    2009-07-06

    Jul 6, 2009 ... lytic domain and a protein kinase ATP-binding region sig- nature. A Ser/Thr ... kinase physically interacts with and is activated by the calcium- binding ... protein kinase subfamily: Metabolic sensors of the eukaryotic cell? Annu.

  18. TYROSINE KINASE INHIBITORS AND PREGNANCY

    Directory of Open Access Journals (Sweden)

    Elisabetta Abruzzese

    2014-04-01

    Full Text Available The management of patients with chronic myeloid leukemia (CML during pregnancy has became recently a matter of continuous debate.  The introduction of the Tyrosine Kinase Inhibitors (TKIs in clinical practice has dramatically changed the prognosis of CML patients.  Patients diagnosed in chronic phase can reasonably expect many years of excellent disease control and good quality of life, as well as a normal life expectancy.  This fact has come the necessity to address issues relating to fertility and pregnancy. Physicians are not infrequently being asked for advice regarding the need for, and or the appropriateness of, stopping treatment in order to conceive. In this report we will review the data published in terms of fertility, conception, pregnancy, pregnancy outcome and illness control for all the approved TKIs, as well as suggest how to manage a planned and/or unplanned pregnancy.

  19. Immunohistochemical distribution of myotonic dystrophy kinase (DNK) in muscle

    Energy Technology Data Exchange (ETDEWEB)

    Whiting, E.J.; Tamai, K. [Univ. of Ottawa (Canada); Waring, J.D. [Chilrdren`s Hospital of Eastern Ontario, Ottawa (Canada)] [and others

    1994-09-01

    Myotonic dystrophy (DM) is the most common form of inherited neuromuscular disease in adults and is characterized by progressive muscle wasting and myotonia. The mutation responsible for DM has been identified as the expansion of a polymorphic (CTG)n repeat in the 3{prime} untranslated region of a gene encoding a putative serine/threonine kinase (DMK). We have raised a polyclonal raised a polyclonal rabbit antisera against a fusion protein encoding exons 11-15 of DMK. The antisera detects both the full length and a truncated isoform (missing amino acids corresponding to exons 13-15) of the human DMK expressed in a recombinant baculovirus system. In addition, it recognizes a 69 kDA protein on Western blots of both human and mouse myoblasts. Use of this antiserum in immunohistochemical studies of human tissue demonstrates that DMK is expressed in the cytoplasm of both skeletal and smooth muscle and is expressed postsynaptically (as determined by codistribution with acetylcholinesterase and acetylcholine receptors) within the vicinity of neuromuscular junction of skeletal muscle. Further, no obvious differences in DMK localization were observed between muscle tissues from normal and DM-affected individuals.

  20. Cloning and characterization of fructosamine-6-kinase from Arthrobacter aurescens.

    Science.gov (United States)

    Sakaguchi-Mikami, Akane; Kameya, Miho; Ferri, Stefano; Tsugawa, Wakako; Sode, Koji

    2013-06-01

    Fructosamine-6-kinases (FN6Ks) that catalyze phosphorylation of glycated amino acids, i.e., fructosyl amino acids (FAs), have been shown as a potential recognition element for glycated protein detection. However, there are only two available FN6Ks: those from Escherichia coli which is specific for ε-fructosyl lysine (ε-FK) and Bacillus subtilis which recognizes both ε-FK and α-FA as substrates. In this study, we characterized an FN6K homologue isolated from Arthrobacter, some of whose species are reported to assimilate FA. The BLAST searches of Arthrobacter genomic database, using the bacterial FN6K primary structure information, revealed the presence of an FN6K homologue in Arthrobacter aurescens TC1 strain. Indeed, enzymatic assays confirmed that the putative FN6K from A. aurescens is an FN6K that is specific for ε-FK, although the primary sequence alignments showed similarity of A. aurescens FN6Ks with FN6Ks from B. subtilis and E. coli at the same level. In this study, we describe for the first time the presence of FN6K in Arthrobacter spp. and ε-FK-specific degradation pathway from Gram-positive bacteria, providing important information for the development of FA-recognizing molecules as well as for the FA assimilation system in bacteria.

  1. Tropomyosin-1, A Putative Tumor-Suppressor and a Biomarker of Human Breast Cancer

    Science.gov (United States)

    2004-10-01

    cDNA. Lobular carcinoma - 2 A polyclonal pan-TM antibody that recognizes multiple TM Phyllodes tumor - 1 Not determined from the initial pathology...AD Award Number: DAMD17-98-1-8162 TITLE: Tropomyosin-1, A Putative Tumor -Suppressor and a Biomarker of Human Breast Cancer PRINCIPAL INVESTIGATOR...4. TITLE AND SUBTITLE 5. FUNDING NUMBERS Tropomyosin-l, A Putative Tumor -Suppressor and a Biomarker DAMD17-98-1-8162 of Human Breast Cancer 6. A UTHOR

  2. Fluoroquinolone-Resistant Haemophilus parasuis Isolates Exhibit More Putative Virulence Factors than Their Susceptible Counterparts

    OpenAIRE

    Zhang, Qiang; Liu, Jiantao; Yan, Shuxian; Yang, Yujie; Zhang, Anding; Jin, Meilin

    2013-01-01

    The prevalence of 23 putative virulence factors among fluoroquinolone-susceptible and -resistant Haemophilus parasuis isolates was analyzed. Putative hemolysin precursor, fimbrial assembly chaperone, and type I site-specific restriction modification system R subunit genes were more prevalent among fluoroquinolone-resistant H. parasuis isolates than among fluoroquinolone-susceptible H. parasuis isolates. Fluoroquinolone resistance may be associated with an increase in the presence of some viru...

  3. Gene ercA, encoding a putative iron-containing alcohol dehydrogenase, is involved in regulation of ethanol utilization in Pseudomonas aeruginosa.

    Science.gov (United States)

    Hempel, Niels; Görisch, Helmut; Mern, Demissew S

    2013-09-01

    Several two-component regulatory systems are known to be involved in the signal transduction pathway of the ethanol oxidation system in Pseudomonas aeruginosa ATCC 17933. These sensor kinases and response regulators are organized in a hierarchical manner. In addition, a cytoplasmic putative iron-containing alcohol dehydrogenase (Fe-ADH) encoded by ercA (PA1991) has been identified to play an essential role in this regulatory network. The gene ercA (PA1991) is located next to ercS, which encodes a sensor kinase. Inactivation of ercA (PA1991) by insertion of a kanamycin resistance cassette created mutant NH1. NH1 showed poor growth on various alcohols. On ethanol, NH1 grew only with an extremely extended lag phase. During the induction period on ethanol, transcription of structural genes exa and pqqABCDEH, encoding components of initial ethanol oxidation in P. aeruginosa, was drastically reduced in NH1, which indicates the regulatory function of ercA (PA1991). However, transcription in the extremely delayed logarithmic growth phase was comparable to that in the wild type. To date, the involvement of an Fe-ADH in signal transduction processes has not been reported.

  4. Prevalence and characteristics of Streptococcus pneumoniae "putative serotype 6E" isolates from Asian countries.

    Science.gov (United States)

    Baek, Jin Yang; Park, In Ho; So, Thomas Man-kit; Lalitha, M K; Shimono, Nobuyuki; Yasin, Rohani Md; Carlos, Celia C; Perera, Jennifer; Thamlikitkul, Visanu; Hsueh, Po-Ren; Van, Pham Hung; Shibl, Atef M; Song, Jae-Hoon; Ko, Kwan Soo

    2014-12-01

    The prevalence, antimicrobial susceptibility, and genotypes of Streptococcus pneumoniae “putative serotype 6E” isolates from Asian countries were investigated. A total of 244 S. pneumoniae serogroup 6 isolates obtained from 11 Asian countries were included in this study. Of the 244 serogroup 6 isolates, 101 (41.4%) were typed as "putative serotype 6E," followed by serotypes 6A, 6B, 6C, and 6D (27.0, 20.1, 5.7, and 5.7%, respectively). Multilocus sequence typing revealed that clonal complex (CC) 90, including ST90 and its variants, was the most prevalent clonal group of "putative serotype 6E" isolates (n = 63; 62.4%). CC146 and CC315 were also found frequently in some of the countries. Most of the "putative serotype 6E" isolates showed very high resistance rates against cefuroxime, erythromycin, azithromycin, clarithromycin, clindamycin, and trimethoprim/sulfamethoxazole, probably due to their highly resistant to antimicrobials clone, CC90. Our results indicate that “putative serotype 6E” is prevalent in Asian countries. The clonal dissemination of "putative serotype 6E" isolates was also identified.

  5. Characterization of putative effectors from the cereal cyst nematode Heterodera avenae.

    Science.gov (United States)

    Cui, Jiangkuan; Peng, Huan; Qiao, Fen; Wang, Gaofeng; Huang, Wenkun; Wu, Duqign; Peng, Deliang

    2017-09-20

    Few molecular details of effectors of Heterodera avenae parasitism are known. We performed a high-throughput sequencing analysis of the H. avenae transcriptome at five developmental stages. A total of 82,549 unigenes were ultimately obtained, and 747 transcripts showed best hits to genes putatively encoding carbohydrate-active enzymes in plant parasitic nematodes that play an important role in the invasion process. A total of 1480 unigenes were homologous to known phytonematode effectors, and 63 putative novel effectors were identified in the H. avenae transcriptomes. Twenty-three unigenes were analyzed by qRT-PCR and confirmed to be highly expressed during at least one developmental stage. For in situ hybridization, 17 of the 22 tested putative effectors were specifically expressed and located in the subventral gland cells, and five putative novel effectors were specifically expressed in the dorsal gland. Furthermore, 115 transcripts were found to have putative lethal RNA interference (RNAi) phenotypes. Three target genes with lethal RNAi phenotypes and two of the four tested putative effectors were associated with a decrease in the number of cysts through in vitro RNAi technology. These transcriptomic data lay a foundation for further studies of interactions of H. avenae with cereal and H. avenae parasitic control.

  6. Growth Inhibition by Bupivacaine Is Associated with Inactivation of Ribosomal Protein S6 Kinase 1

    Directory of Open Access Journals (Sweden)

    Mushtaq Ahmad Beigh

    2014-01-01

    Full Text Available Bupivacaine is an amide type long acting local anesthetic used for epidural anesthesia and nerve blockade in patients. Use of bupivacaine is associated with severe cytotoxicity and apoptosis along with inhibition of cell growth and proliferation. Although inhibition of Erk, Akt, and AMPK seemingly appears to mediate some of the bupivacaine effects, potential downstream targets that mediate its effect remain unknown. S6 kinase 1 is a common downstream effector of several growth regulatory pathways involved in cell growth and proliferation known to be affected by bupivacaine. We have accordingly attempted to relate the growth inhibitory effects of bupivacaine with the status of S6K1 activity and we present evidence that decrease in cell growth and proliferation by bupivacaine is mediated through inactivation of S6 kinase 1 in a concentration and time dependent manner. We also show that ectopic expression of constitutively active S6 kinase 1 imparts substantial protection from bupivacaine induced cytotoxicity. Inactivation of S6K1 though associated with loss of putative mTOR mediated phosphorylation did not correspond with loss of similar phosphorylations in 4EBP1 indicating that S6K1 inhibition was not mediated through inactivation of mTORC1 signaling pathway or its down regulation.

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

    Science.gov (United States)

    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

  8. The role of the Drosophila LAMMER protein kinase DOA in somatic sex determination

    Indian Academy of Sciences (India)

    Leonard Rabinow; Marie-Laure Samson

    2010-09-01

    DOA kinase, the Drosophila member of the LAMMER/Clk protein kinase family, phosphorylates SR and SR-like proteins, including TRA, TRA2 and RBP1, which are responsible for the alternative splicing of transcripts encoding the key regulator of sex-specific expression in somatic cells of the fly, DOUBLESEX. Specific Doa alleles induce somatic female-to-male sex transformations, which can be enhanced when combined with mutations in loci encoding SR and SR-like proteins. The Doa locus encodes six different kinases, of which a 69-kDa isoform is expressed solely in females. Expression of this isoform is itself under the regulation of the somatic sex determination regulatory network, thus forming a putative positive autoregulatory loop which would reinforce the choice of the female cell-fate. We speculate that this loop is part of the evolutionary ancestral sex-determination machinery, based upon evidence demonstrating the existence of an autoregulatory loop involving TRA and TRA2 in several other insect species.

  9. CLK2 Is an Oncogenic Kinase and Splicing Regulator in Breast Cancer.

    Science.gov (United States)

    Yoshida, Taku; Kim, Jee Hyun; Carver, Kristopher; Su, Ying; Weremowicz, Stanislawa; Mulvey, Laura; Yamamoto, Shoji; Brennan, Cameron; Mei, Shenglin; Long, Henry; Yao, Jun; Polyak, Kornelia

    2015-04-01

    Genetically activated kinases have been attractive therapeutic targets in cancer due to the relative ease of developing tumor-specific treatment strategies for them. To discover novel putative oncogenic kinases, we identified 26 genes commonly amplified and overexpressed in breast cancer and subjected them to a lentiviral shRNA cell viability screen in a panel of breast cancer cell lines. Here, we report that CLK2, a kinase that phosphorylates SR proteins involved in splicing, acts as an oncogene in breast cancer. Deregulated alternative splicing patterns are commonly observed in human cancers but the underlying mechanisms and functional relevance are still largely unknown. CLK2 is amplified and overexpressed in a significant fraction of breast tumors. Downregulation of CLK2 inhibits breast cancer growth in cell culture and in xenograft models and it enhances cell migration and invasion. Loss of CLK2 in luminal breast cancer cells leads to the upregulation of epithelial-to-mesenchymal transition (EMT)-related genes and a switch to mesenchymal splice variants of several genes, including ENAH (MENA). These results imply that therapeutic targeting of CLK2 may be used to modulate EMT splicing patterns and to inhibit breast tumor growth.

  10. The intact CFTR protein mediates ATPase rather than adenylate kinase activity.

    Science.gov (United States)

    Ramjeesingh, Mohabir; Ugwu, Francisca; Stratford, Fiona L L; Huan, Ling-Jun; Li, Canhui; Bear, Christine E

    2008-06-01

    The two NBDs (nucleotide-binding domains) of ABC (ATP-binding-cassette) proteins function in a complex to mediate ATPase activity and this activity has been linked to their regulated transport activity. A similar model has been proposed for CFTR (cystic fibrosis transmembrane conductance regulator), the chloride channel defective in cystic fibrosis, wherein ATP binding and hydrolysis regulate the channel gate. Recently, it was shown that the individual NBDs isolated from CFTR primarily mediate adenylate kinase activity, raising the possibility that this activity may also contribute to gating of the CFTR channel. However, this present study shows that whereas the isolated NBDs exhibit adenylate kinase activity, the full-length purified and reconstituted CFTR protein functions as an ATPase, arguing that the enzymatic activity of the NBDs is dependent on their molecular context and appropriate domain-domain assembly. As expected, the disease-causing mutant bearing a mutation in the ABC signature motif, CFTR-G551D, exhibited a markedly reduced ATPase activity. Furthermore, mutation of the putative catalytic base in CFTR caused a reduction in ATPase activity, with the CFTR-E1371Q mutant supporting a low level of residual activity. Neither of these mutants exhibited detectable adenylate kinase activity. Together, these findings support the concept that the molecular mechanism of action of CFTR is dependent on ATP binding and hydrolysis, and that the structure of prokaryotic ABC ATPases provide a useful template for understanding their mechanism of action.

  11. Molecular identification of hydroxylysine kinase and of ammoniophospholyases acting on 5-phosphohydroxy-L-lysine and phosphoethanolamine.

    Science.gov (United States)

    Veiga-da-Cunha, Maria; Hadi, Farah; Balligand, Thomas; Stroobant, Vincent; Van Schaftingen, Emile

    2012-03-02

    The purpose of the present work was to identify the catalytic activity of AGXT2L1 and AGXT2L2, two closely related, putative pyridoxal-phosphate-dependent enzymes encoded by vertebrate genomes. The existence of bacterial homologues (40-50% identity with AGXT2L1 and AGXT2L2) forming bi- or tri-functional proteins with a putative kinase belonging to the family of aminoglycoside phosphotransferases suggested that AGXT2L1 and AGXT2L2 acted on phosphorylated and aminated compounds. Vertebrate genomes were found to encode a homologue (AGPHD1) of these putative bacterial kinases, which was therefore likely to phosphorylate an amino compound bearing a hydroxyl group. These and other considerations led us to hypothesize that AGPHD1 corresponded to 5-hydroxy-L-lysine kinase and that AGXT2L1 and AGXT2L2 catalyzed the pyridoxal-phosphate-dependent breakdown of phosphoethanolamine and 5-phosphohydroxy-L-lysine. The three recombinant human proteins were produced and purified to homogeneity. AGPHD1 was indeed found to catalyze the GTP-dependent phosphorylation of 5-hydroxy-L-lysine. The phosphorylation product made by this enzyme was metabolized by AGXT2L2, which converted it to ammonia, inorganic phosphate, and 2-aminoadipate semialdehyde. AGXT2L1 catalyzed a similar reaction on phosphoethanolamine, converting it to ammonia, inorganic phosphate, and acetaldehyde. AGPHD1 and AGXT2L2 are likely to be the mutated enzymes in 5-hydroxylysinuria and 5-phosphohydroxylysinuria, respectively. The high level of expression of AGXT2L1 in human brain, as well as data in the literature linking AGXT2L1 to schizophrenia and bipolar disorders, suggest that these diseases may involve a perturbation of brain phosphoethanolamine metabolism. AGXT2L1 and AGXT2L2, the first ammoniophospholyases to be identified, belong to a family of aminotransferases acting on ω-amines.

  12. Molecular Identification of Hydroxylysine Kinase and of Ammoniophospholyases Acting on 5-Phosphohydroxy-l-lysine and Phosphoethanolamine*

    Science.gov (United States)

    Veiga-da-Cunha, Maria; Hadi, Farah; Balligand, Thomas; Stroobant, Vincent; Van Schaftingen, Emile

    2012-01-01

    The purpose of the present work was to identify the catalytic activity of AGXT2L1 and AGXT2L2, two closely related, putative pyridoxal-phosphate-dependent enzymes encoded by vertebrate genomes. The existence of bacterial homologues (40–50% identity with AGXT2L1 and AGXT2L2) forming bi- or tri-functional proteins with a putative kinase belonging to the family of aminoglycoside phosphotransferases suggested that AGXT2L1 and AGXT2L2 acted on phosphorylated and aminated compounds. Vertebrate genomes were found to encode a homologue (AGPHD1) of these putative bacterial kinases, which was therefore likely to phosphorylate an amino compound bearing a hydroxyl group. These and other considerations led us to hypothesize that AGPHD1 corresponded to 5-hydroxy-l-lysine kinase and that AGXT2L1 and AGXT2L2 catalyzed the pyridoxal-phosphate-dependent breakdown of phosphoethanolamine and 5-phosphohydroxy-l-lysine. The three recombinant human proteins were produced and purified to homogeneity. AGPHD1 was indeed found to catalyze the GTP-dependent phosphorylation of 5-hydroxy-l-lysine. The phosphorylation product made by this enzyme was metabolized by AGXT2L2, which converted it to ammonia, inorganic phosphate, and 2-aminoadipate semialdehyde. AGXT2L1 catalyzed a similar reaction on phosphoethanolamine, converting it to ammonia, inorganic phosphate, and acetaldehyde. AGPHD1 and AGXT2L2 are likely to be the mutated enzymes in 5-hydroxylysinuria and 5-phosphohydroxylysinuria, respectively. The high level of expression of AGXT2L1 in human brain, as well as data in the literature linking AGXT2L1 to schizophrenia and bipolar disorders, suggest that these diseases may involve a perturbation of brain phosphoethanolamine metabolism. AGXT2L1 and AGXT2L2, the first ammoniophospholyases to be identified, belong to a family of aminotransferases acting on ω-amines. PMID:22241472

  13. Computational Simulations to Predict Creatine Kinase-Associated Factors: Protein-Protein Interaction Studies of Brain and Muscle Types of Creatine Kinases

    Directory of Open Access Journals (Sweden)

    Wei-Jiang Hu

    2011-01-01

    Full Text Available Creatine kinase (CK; EC 2.7.3.2 is related to several skin diseases such as psoriasis and dermatomyositis. CK is important in skin energy homeostasis because it catalyzes the reversible transfer of a phosphoryl group from MgATP to creatine. In this study, we predicted CK binding proteins via the use of bioinformatic tools such as protein-protein interaction (PPI mappings and suggest the putative hub proteins for CK interactions. We obtained 123 proteins for brain type CK and 85 proteins for muscle type CK in the interaction networks. Among them, several hub proteins such as NFKB1, FHL2, MYOC, and ASB9 were predicted. Determination of the binding factors of CK can further promote our understanding of the roles of CK in physiological conditions.

  14. Effects of overexpression of Pkn2, a transmembrane protein serine/threonine kinase, on development of Myxococcus xanthus.

    OpenAIRE

    Udo, H; Inouye, M; Inouye, S.

    1996-01-01

    Pkn2 is a putative transmembrane protein serine/threonine kinase required for normal development of Myxococcus xanthus. The effect of Pkn2 overexpression on development of M. xanthus was examined by expressing pkn2 under the control of a kanamycin promoter. Pkn2 was clearly detected by Western blot (immunoblot) analysis in the overexpression strain (the PKm/pkn2 strain) but could not be detected in the wild-type strain. Overexpressed Pkn2 was located almost exclusively in the membrane fractio...

  15. Physiological roles of mitogen-activated-protein-kinase-activated p38-regulated/activated protein kinase

    Institute of Scientific and Technical Information of China (English)

    Sergiy; Kostenko; Gianina; Dumitriu; Kari; Jenssen; Lgreid; Ugo; Moens

    2011-01-01

    Mitogen-activated protein kinases(MAPKs)are a family of proteins that constitute signaling pathways involved in processes that control gene expression,cell division, cell survival,apoptosis,metabolism,differentiation and motility.The MAPK pathways can be divided into conventional and atypical MAPK pathways.The first group converts a signal into a cellular response through a relay of three consecutive phosphorylation events exerted by MAPK kinase kinases,MAPK kinase,and MAPK.Atypical MAPK pathways are not organized into this three-tiered cascade.MAPK that belongs to both conventional and atypical MAPK pathways can phosphorylate both non-protein kinase substrates and other protein kinases.The latter are referred to as MAPK-activated protein kinases.This review focuses on one such MAPK-activated protein kinase,MAPK-activated protein kinase 5(MK5)or p38-regulated/activated protein kinase(PRAK).This protein is highly conserved throughout the animal kingdom and seems to be the target of both conventional and atypical MAPK pathways.Recent findings on the regulation of the activity and subcellular localization,bona fide interaction partners and physiological roles of MK5/PRAK are discussed.

  16. Identification of a novel phosphorylation site on TBC1D4 regulated by AMP-activated protein kinase in skeletal muscle

    DEFF Research Database (Denmark)

    Treebak, Jonas Thue; Taylor, Eric B.; Witczak, Carol A.

    2010-01-01

    TBC1D4 (also known as AS160) regulates GLUT4 translocation and glucose uptake in adipocytes and skeletal muscle. Its mode of action involves phosphorylation of Serine (S)/Threonine (T) residues by upstream kinases resulting in inactivation of Rab-GAP activity leading to GLUT4 mobilization....... The majority of known phosphorylation sites on TBC1D4 lie within the Akt consensus motif and are phosphorylated by insulin stimulation. However, the 5 AMP activated protein kinase (AMPK) and other kinases may also phosphorylate TBC1D4, and therefore we hypothesized the presence of additional phosphorylation...... sites. Mouse skeletal muscles were contracted or stimulated with 5-aminoimidazole-4-carboxmide riboside (AICAR) and muscle lysates were subjected to mass spectrometry analyses resulting in identification of novel putative phosphorylation sites on TBC1D4. The surrounding amino acid sequence predicted...

  17. The mechanism of protein kinase C regulation

    Institute of Scientific and Technical Information of China (English)

    Julhash U. KAZI

    2011-01-01

    Protein kinase C (PKC) is a family ofserine/threonine protein kinases that plays a central role in transducing extracellular signals into a variety of intracellular responses ranging from cell proliferation to apoptosis.Nine PKC genes have been identified in the human genome,which encode 10 proteins.Each member of this protein kinase family displays distinct biochemical characteristics and is enriched in different cellular and subcellular locations.Activation of PKC has been implicated in the regulation of cell growth and differentiation.This review summarizes works of the past years in the field of PKC biochemistry that covers regulation and activation mechanism of different PKC isoforms.

  18. Functional analysis of anomeric sugar kinases.

    Science.gov (United States)

    Conway, Louis P; Voglmeir, Josef

    2016-09-02

    Anomeric sugar kinases perform fundamental roles in the metabolism of carbohydrates. Under- or overexpression of these enzymes, or mutations causing functional impairments can give rise to diseases such as galactosaemia and so the study of this class of kinase is of critical importance. In addition, anomeric sugar kinases which are naturally promiscuous, or have been artificially made so, may find application in the synthesis of libraries of drug candidates (for example, antibiotics), and natural or unnatural oligosaccharides and glycoconjugates. In this review, we provide an overview of the biological functions of these enzymes, the tools which have been developed to investigate them, and the current frontiers in their study.

  19. Protein Kinase A in Cancer

    Energy Technology Data Exchange (ETDEWEB)

    Caretta, Antonio; Mucignat-Caretta, Carla, E-mail: carla.mucignat@unipd.it [Department of Human Anatomy and Physiology, University of Padova, Via Marzolo 3, 35131 Padova (Italy)

    2011-02-28

    In the past, many chromosomal and genetic alterations have been examined as possible causes of cancer. However, some tumors do not display a clear molecular and/or genetic signature. Therefore, other cellular processes may be involved in carcinogenesis. Genetic alterations of proteins involved in signal transduction have been extensively studied, for example oncogenes, while modifications in intracellular compartmentalization of these molecules, or changes in the expression of unmodified genes have received less attention. Yet, epigenetic modulation of second messenger systems can deeply modify cellular functioning and in the end may cause instability of many processes, including cell mitosis. It is important to understand the functional meaning of modifications in second messenger intracellular pathways and unravel the role of downstream proteins in the initiation and growth of tumors. Within this framework, the cAMP system has been examined. cAMP is a second messenger involved in regulation of a variety of cellular functions. It acts mainly through its binding to cAMP-activated protein kinases (PKA), that were suggested to participate in the onset and progression of various tumors. PKA may represent a biomarker for tumor detection, identification and staging, and may be a potential target for pharmacological treatment of tumors.

  20. Rac-1 and Raf-1 kinases, components of distinct signaling pathways, activate myotonic dystrophy protein kinase

    Science.gov (United States)

    Shimizu, M.; Wang, W.; Walch, E. T.; Dunne, P. W.; Epstein, H. F.

    2000-01-01

    Myotonic dystrophy protein kinase (DMPK) is a serine-threonine protein kinase encoded by the myotonic dystrophy (DM) locus on human chromosome 19q13.3. It is a close relative of other kinases that interact with members of the Rho family of small GTPases. We show here that the actin cytoskeleton-linked GTPase Rac-1 binds to DMPK, and coexpression of Rac-1 and DMPK activates its transphosphorylation activity in a GTP-sensitive manner. DMPK can also bind Raf-1 kinase, the Ras-activated molecule of the MAP kinase pathway. Purified Raf-1 kinase phosphorylates and activates DMPK. The interaction of DMPK with these distinct signals suggests that it may play a role as a nexus for cross-talk between their respective pathways and may partially explain the remarkable pleiotropy of DM.

  1. CDPKs are dual-specificity protein kinases and tyrosine autophosphorylation attenuates kinase activity.

    Science.gov (United States)

    Oh, Man-Ho; Wu, Xia; Kim, Hyoung Seok; Harper, Jeffrey F; Zielinski, Raymond E; Clouse, Steven D; Huber, Steven C

    2012-11-30

    Although calcium-dependent protein kinases (CDPKs or CPKs) are classified as serine/threonine protein kinases, autophosphorylation on tyrosine residues was observed for soybean CDPKβ and several Arabidopsis isoforms (AtCPK4 and AtCPK34). We identified Ser-8, Thr-17, Tyr-24 (in the kinase domain), Ser-304, and Ser-358 as autophosphorylation sites of His(6)-GmCDPKβ. Overall autophosphorylation increased kinase activity with synthetic peptides, but autophosphorylation of Tyr-24 appears to attenuate kinase activity based on studies with the Y24F directed mutant. While much remains to be done, it is clear that several CDPKs are dual-specificity kinases, which raises the possibility that phosphotyrosine signaling may play a role in Ca(2+)/CDPK-mediated processes. Published by Elsevier B.V.

  2. Altered expression of a putative progenitor cell marker DCAMKL1 in the rat gastric mucosa in regeneration, metaplasia and dysplasia

    Directory of Open Access Journals (Sweden)

    Watanabe Hiromitsu

    2010-06-01

    Full Text Available Abstract Background Doublecortin and calcium/calmodulin-dependent protein kinase-like-1 (DCAMKL1 is a candidate marker for progenitor cells in the gastrointestinal mucosa. Lineage cells in the gastric mucosa are derived from progenitor cells, but this process can be altered after injury. Therefore, we explored DCAMKL1 expression under pathological conditions. Methods An immunohistochemical analysis was performed in rat stomach with acute superficial injury, chronic ulcer, intestinal metaplasia and dysplasia. Results DCAMKL1 was exclusively expressed in immature quiescent cells in the isthmus of normal fundic glands, where putative progenitor cells are thought to reside. DCAMKL1-positive cells and proliferating cells shed into the lumen after superficial injury and re-appeared during the regenerative process, mainly in the superficial mucosa. In the marginal mucosa around the active ulcer, parietal and chief cells diminished, foveolar hyperplasia was evident, and trefoil factor family 2 (TFF2/spasmolytic polypeptide-expressing metaplasia (SPEM emerged at the gland base. DCAMKL1 cells re-emerged in the deep mucosa juxtaposed with SPEM and proliferating cells. In the healing ulcer, the TFF2 cell population expanded and seemed to redifferentiate to chief cells, while proliferating cells and DCAMKL1 cells appeared above and below the TFF2 cells to promote healing. SPEM appeared and PCNA cells increased in the intestinalized mucosa, and DCAMKL1 was expressed in the proximity of the PCNA cells in the deep mucosa. DCAMKL1, PCNA and TFF2 were expressed in different dysplastic cells lining dilated glands near SPEM. Conclusion The ultrastructural appearance of DCAMKL1-positive cells and the expression patterns of DCAMKL1 in normal and pathological states indicate that the cells belong to a progenitor cell population. DCAMKL1 expression is closely associated with TFF2/SPEM cells after injury. DCAMKL1 cells repopulate close to proliferating, hyperplastic

  3. Inactivation of the putative ubiquitin-E3 ligase PDLIM2 in classical Hodgkin and anaplastic large cell lymphoma

    Science.gov (United States)

    Wurster, K D; Hummel, F; Richter, J; Giefing, M; Hartmann, S; Hansmann, M-L; Kreher, S; Köchert, K; Krappmann, D; Klapper, W; Hummel, M; Wenzel, S-S; Lenz, G; Janz, M; Dörken, B; Siebert, R; Mathas, S

    2017-01-01

    Apart from its unique histopathological appearance with rare tumor cells embedded in an inflammatory background of bystander cells, classical Hodgkin lymphoma (cHL) is characterized by an unusual activation of a broad range of signaling pathways involved in cellular activation. This includes constitutive high-level activity of nuclear factor-κB (NF-κB), Janus kinase/signal transducer and activator of transcription (JAK/STAT), activator protein-1 (AP-1) and interferon regulatory factor (IRF) transcription factors (TFs) that are physiologically only transiently activated. Here, we demonstrate that inactivation of the putative ubiquitin E3-ligase PDLIM2 contributes to this TF activation. PDLIM2 expression is lost at the mRNA and protein levels in the majority of cHL cell lines and Hodgkin and Reed–Sternberg (HRS) cells of nearly all cHL primary samples. This loss is associated with PDLIM2 genomic alterations, promoter methylation and altered splicing. Reconstitution of PDLIM2 in HRS cell lines inhibits proliferation, blocks NF-κB transcriptional activity and contributes to cHL-specific gene expression. In non-Hodgkin B-cell lines, small interfering RNA-mediated PDLIM2 knockdown results in superactivation of TFs NF-κB and AP-1 following phorbol 12-myristate 13-acetate (PMA) stimulation. Furthermore, expression of PDLIM2 is lost in anaplastic large cell lymphoma (ALCL) that shares key biological aspects with cHL. We conclude that inactivation of PDLIM2 is a recurrent finding in cHL and ALCL, promotes activation of inflammatory signaling pathways and thereby contributes to their pathogenesis. PMID:27538486

  4. The Crystal Structure of Cancer Osaka Thyroid Kinase Reveals an Unexpected Kinase Domain Fold*

    Science.gov (United States)

    Gutmann, Sascha; Hinniger, Alexandra; Fendrich, Gabriele; Drückes, Peter; Antz, Sylvie; Mattes, Henri; Möbitz, Henrik; Ofner, Silvio; Schmiedeberg, Niko; Stojanovic, Aleksandar; Rieffel, Sebastien; Strauss, André; Troxler, Thomas; Glatthar, Ralf; Sparrer, Helmut

    2015-01-01

    Macrophages are important cellular effectors in innate immune responses and play a major role in autoimmune diseases such as rheumatoid arthritis. Cancer Osaka thyroid (COT) kinase, also known as mitogen-activated protein kinase kinase kinase 8 (MAP3K8) and tumor progression locus 2 (Tpl-2), is a serine-threonine (ST) kinase and is a key regulator in the production of pro-inflammatory cytokines in macrophages. Due to its pivotal role in immune biology, COT kinase has been identified as an attractive target for pharmaceutical research that is directed at the discovery of orally available, selective, and potent inhibitors for the treatment of autoimmune disorders and cancer. The production of monomeric, recombinant COT kinase has proven to be very difficult, and issues with solubility and stability of the enzyme have hampered the discovery and optimization of potent and selective inhibitors. We developed a protocol for the production of recombinant human COT kinase that yields pure and highly active enzyme in sufficient yields for biochemical and structural studies. The quality of the enzyme allowed us to establish a robust in vitro phosphorylation assay for the efficient biochemical characterization of COT kinase inhibitors and to determine the x-ray co-crystal structures of the COT kinase domain in complex with two ATP-binding site inhibitors. The structures presented in this study reveal two distinct ligand binding modes and a unique kinase domain architecture that has not been observed previously. The structurally versatile active site significantly impacts the design of potent, low molecular weight COT kinase inhibitors. PMID:25918157

  5. Venus Kinase Receptors: Prospects in Signaling and Biological Functions of These Invertebrate Kinases

    OpenAIRE

    Dissous, Colette; Morel, Marion; Vanderstraete, Mathieu

    2014-01-01

    Venus kinase receptors (VKRs) form a family of invertebrate receptor tyrosine kinases (RTKs) initially discovered in the parasitic platyhelminth Schistosoma mansoni. VKRs are single transmembrane receptors that contain an extracellular venus fly trap structure similar to the ligand-binding domain of G protein-coupled receptors of class C, and an intracellular tyrosine kinase domain close to that of insulin receptors. VKRs are found in a large variety of invertebrates from cnidarians to echino...

  6. Isoprenoid biosynthesis and mevalonate kinase deficiency

    NARCIS (Netherlands)

    Henneman, L.

    2011-01-01

    Mevalonaat Kinase Deficiëntie (MKD) is een aangeboren ziekte geassocieerd met heftige koortsaanvallen die drie tot vier dagen aanhouden en gepaard gaan met koude rillingen, gewrichtsklachten, huiduitslag, hoofdpijn, duizeligheid, buikpijn, braken en diarree. De koortsaanvallen treden gemiddeld eens

  7. Kinase inhibitors for advanced medullary thyroid carcinoma

    Directory of Open Access Journals (Sweden)

    Martin Schlumberger

    2012-01-01

    Full Text Available The recent availability of molecular targeted therapies leads to a reconsideration of the treatment strategy for patients with distant metastases from medullary thyroid carcinoma. In patients with progressive disease, treatment with kinase inhibitors should be offered.

  8. Mitochondrial Stress Signalling: HTRA2 and Parkinson's Disease

    Directory of Open Access Journals (Sweden)

    Enrico Desideri

    2012-01-01

    Full Text Available Mitochondria are cellular energy generators whose activity requires a continuous supply of oxygen. Recent genetic analysis has suggested that defects in mitochondrial quality control may be key factors in the development of Parkinson’s disease (PD. Mitochondria have a crucial role in supplying energy to the brain, and their deterioration can affect the function and viability of neurons, contributing to neurodegeneration. These organelles can sow the seeds of their own demise because they generate damaging oxygen-free radicals as a byproduct of their intrinsic physiological functions. Mitochondria have therefore evolved specific molecular quality control mechanisms to compensate for the action of damaging agents such as oxygen-free radicals. PTEN-induced putative kinase 1 (PINK1 and high-temperature-regulated A2 (HTRA2, a mitochondrial protease, have recently been proposed to be key modulators of mitochondrial molecular quality control. Here, we review some of the most recent advances in our understanding of mitochondria stress-control pathways, focusing on how signalling by the p38 stress kinase pathway may regulate mitochondrial stress by modulating the activity of HTRA2 via PINK1 and cyclin-dependent kinase 5 (CDK5. We also propose how defects in this pathway may contribute to PD.

  9. Diversity of secondary endosymbionts among different putative species of the whitefly Bemisia tabaci.

    Science.gov (United States)

    Bing, Xiao-Li; Ruan, Yong-Ming; Rao, Qiong; Wang, Xiao-Wei; Liu, Shu-Sheng

    2013-04-01

    Endosymbionts are important components of arthropod biology. The whitefly Bemisia tabaci (Gennadius) (Hemiptera: Aleyrodidae) is a cryptic species complex composed of ≥ 28 putative species. In addition to the primary endosymbiont Portiera aleyrodidarum, six secondary endosymbionts (S-endosymbionts), Hamiltonella, Rickettsia, Wolbachia, Cardinium, Arsenophonus and Fritschea, have been identified in B. tabaci thus far. Here, we tested five of the six S-endosymbiont lineages (excluding Fritschea) from 340 whitely individuals representing six putative species from China. Hamiltonella was detected only in the two exotic invaders, Middle East-Asia Minor 1 (MEAM1) and Mediterranean (MED). Rickettsia was absent in Asia II 1 and MED, scarce in Asia II 3 (13%), but abundant in Asia II 7 (63.2%), China 1 (84.7%) and MEAM1 (100%). Wolbachia, Cardinium and Arsenophonus were absent in the invasive MEAM1 and MED but mostly abundant in the native putative species. Furthermore, phylogenetic analyses revealed that some S-endosymbionts have several clades and different B. tabaci putative species can harbor different clades of a given S-endosymbiont, demonstrating further the complexity of S-endosymbionts in B. tabaci. All together, our results demonstrate the variation and diversity of S-endosymbionts in different putative species of B. tabaci, especially between invasive and native whiteflies.

  10. Diversity of secondary endosymbionts among different putative species of the whitefly Bemisia tabaci

    Institute of Scientific and Technical Information of China (English)

    Xiao-Li Bing; Yong-Ming Ruan; Qiong Rao; Xiao-Wei Wang; Shu-Sheng Liu

    2013-01-01

    Endosymbionts are important components of arthropod biology.The whitefly Bemisia tabaci (Gennadius) (Hemiptera:Aleyrodidae) is a cryptic species complex composed of≥28 putative species.In addition to the primary endosymbiont Portiera aleyrodidarum,six secondary endosymbionts (S-endosymbionts),Hamiltonella,Rickettsia,Wolbachia,Cardinium,Arsenophonus and Fritschea,have been identified in B.tabaci thus far.Here,we tested five of the six S-endosymbiont lineages (excluding Fritschea) from 340 whitely individuals representing six putative species from China.Hamiltonella was detected only in the two exotic invaders,Middle East-Asia Minor 1 (MEAM 1) and Mediterranean (MED).Rickettsia was absent in Asia Ⅱ 1 and MED,scarce in Asia Ⅱ 3 (13%),but abundant in Asia Ⅱ 7 (63.2%),China 1 (84.7%) and MEAM1 (100%).Wolbachia,Cardinium and Arsenophonus were absent in the invasive MEAM 1 and MED but mostly abundant in the native putative species.Furthermore,phylogenetic analyses revealed that some S-endosymbionts have several clades and different B.tabaci putative species can harbor different clades of a given S-endosymbiont,demonstrating further the complexity of S-endosymbionts in B.tabaci.All together,our results demonstrate the variation and diversity of S-endosymbionts in different putative species ofB.tabaci,especially between invasive and native whiteflies.

  11. Down-regulation apoptosis signal-regulating kinase 1 gene reduced the Litopenaeus vannamei hemocyte apoptosis in WSSV infection.

    Science.gov (United States)

    Yuan, Feng-Hua; Chen, Yong-Gui; Zhang, Ze-Zhi; Yue, Hai-Tao; Bi, Hai-Tao; Yuan, Kai; Weng, Shao-Ping; He, Jian-Guo; Chen, Yi-Hong

    2016-03-01

    Apoptosis signal-regulating kinase 1 (ASK1), a mitogen-activated protein kinase kinase kinase, is crucial in various cellular responses. In the present study, we identified and characterized an ASK1 homolog from Litopenaeus vannamei (LvASK1). The full-length cDNA of LvASK1 was 5400 bp long, with an open reading frame encoding a putative 1420 amino acid protein. LvASK1 was highly expressed in muscle, hemocyte, eyestalk and heart. Real-time RT-PCR analysis showed that the expression of the LvASK1 was upregulated during the white spot syndrome virus (WSSV) challenge. The knocked-down expression of LvASK1 by RNA interference significantly reduced the apoptotic ratio of the hemocytes collected from WSSV-infected L. vannamei. Furthermore, the down-regulation of LvASK1 also decreased the cumulative mortality of WSSV-infected L. vannamei. These results suggested that down-regulation of LvASK1 decreased the apoptotic rate of hemocytes in WSSV-infected shrimp, and that it could contribute to the reduction of cumulative mortality in WSSV-infected L. vannamei.

  12. A novel nucleoside kinase from Burkholderia thailandensis: a member of the phosphofructokinase B-type family of enzymes.

    Science.gov (United States)

    Ota, Hiroko; Sakasegawa, Shin-Ichi; Yasuda, Yuko; Imamura, Shigeyuki; Tamura, Tomohiro

    2008-12-01

    The genome of the mesophilic Gram-negative bacterium Burkholderia thailandensis contains an open reading frame (i.e. the Bth_I1158 gene) that has been annotated as a putative ribokinase and PFK-B family member. Notably, although the deduced amino acid sequence of the gene showed only 29% similarity to the recently identified nucleoside kinase from hyperthermophilic archaea Methanocaldococcus jannaschii, 15 of 17 residues reportedly involved in the catalytic activity of M. jannaschii nucleoside kinase were conserved. The gene was cloned and functionally overexpressed in Rhodococcus erythropolis, and the purified enzyme was characterized biochemically. The substrate specificity of the enzyme was unusually broad for a bacterial PFK-B protein, and the specificity extended not only to purine and purine-analog nucleosides but also to uridine. Inosine was the most effective phosphoryl acceptor, with the highest k(cat)/K(m) value (80 s(-1).mm(-1)) being achieved when ATP served as the phosphoryl donor. By contrast, this enzyme exhibited no activity toward ribose, indicating that the recombinant enzyme was a nucleoside kinase rather than a ribokinase. To our knowledge, this is the first detailed analysis of a bacterial nucleoside kinase in the PFK-B family.

  13. A two-component histidine kinase Shk1 controls stress response, sclerotial formation and fungicide resistance in Sclerotinia sclerotiorum.

    Science.gov (United States)

    Duan, Yabing; Ge, Changyan; Liu, Shengming; Wang, Jianxin; Zhou, Mingguo

    2013-09-01

    Fungal histidine kinases (HKs) are involved in osmotic and oxidative stress responses, hyphal development, fungicide sensitivity and virulence. Members of HK class III are known to signal through the high-osmolarity glycerol mitogen-activated protein kinase (HOG MAPK). In this study, we characterized the Shk1 gene (SS1G_12694.3), which encodes a putative class III HK, from the plant pathogen Sclerotinia sclerotiorum. Disruption of Shk1 resulted in resistance to phenylpyrrole and dicarboximide fungicides and increased sensitivity to hyperosmotic stress and H2 O2 -induced oxidative stress. The Shk1 mutant showed a significant reduction in vegetative hyphal growth and was unable to produce sclerotia. Quantitative real-time polymerase chain reaction (qRT-PCR and glycerol determination assays showed that the expression of SsHOG1 (the last kinase of the Hog pathway) and glycerol accumulation were regulated by the Shk1 gene, but PAK (p21-activated kinase) was not. In addition, the Shk1 mutant showed no change in virulence. All the defects were restored by genetic complementation of the Shk1 deletion mutant with the wild-type Shk1 gene. These findings indicate that Shk1 is involved in vegetative differentiation, sclerotial formation, glycerol accumulation and adaption to hyperosmotic and oxidative stresses, and to fungicides, in S. sclerotiorum. Taken together, our results demonstrate, for the first time, the role of two-component HKs in Sclerotinia.

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

    Science.gov (United States)

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

    2011-11-25

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

  15. A Molecular Brake in the Kinase Hinge Region Regulates the Activity of Receptor Tyrosine Kinases

    Energy Technology Data Exchange (ETDEWEB)

    Chen,H.; Ma, J.; Li, W.; Eliseenkova, A.; Xu, C.; Neubert, T.; Miller, W.; Mohammadi, M.

    2007-01-01

    Activating mutations in the tyrosine kinase domain of receptor tyrosine kinases (RTKs) cause cancer and skeletal disorders. Comparison of the crystal structures of unphosphorylated and phosphorylated wild-type FGFR2 kinase domains with those of seven unphosphorylated pathogenic mutants reveals an autoinhibitory 'molecular brake' mediated by a triad of residues in the kinase hinge region of all FGFRs. Structural analysis shows that many other RTKs, including PDGFRs, VEGFRs, KIT, CSF1R, FLT3, TEK, and TIE, are also subject to regulation by this brake. Pathogenic mutations activate FGFRs and other RTKs by disengaging the brake either directly or indirectly.

  16. MAP kinase meets mitosis: A role for Raf Kinase Inhibitory Protein in spindle checkpoint regulation

    Directory of Open Access Journals (Sweden)

    Rosner Marsha

    2007-01-01

    Full Text Available Abstract Raf Kinase Inhibitory Protein (RKIP is an evolutionarily conserved protein that functions as a modulator of signaling by the MAP kinase cascade. Implicated as a metastasis suppressor, Raf Kinase Inhibitory Protein depletion correlates with poor prognosis for breast, prostate and melanoma tumors but the mechanism is unknown. Recent evidence indicates that Raf Kinase Inhibitory Protein regulates the mitotic spindle assembly checkpoint by controlling Aurora B Kinase activity, and the mechanism involves Raf/MEK/ERK signaling. In contrast to elevated MAP kinase signaling during the G1, S or G2 phases of the cell cycle that activates checkpoints and induces arrest or senescence, loss of RKIP during M phase leads to bypass of the spindle assembly checkpoint and the generation of chromosomal abnormalities. These results reveal a role for Raf Kinase Inhibitory Protein and the MAP kinase cascade in ensuring the fidelity of chromosome segregation prior to cell division. Furthermore, these data highlight the need for precise titration of the MAP kinase signal to ensure the integrity of the spindle assembly process and provide a mechanism for generating genomic instability in tumors. Finally, these results raise the possibility that RKIP status in tumors could influence the efficacy of treatments such as poisons that stimulate the Aurora B-dependent spindle assembly checkpoint.

  17. LIK1, a CERK1-interacting kinase, regulates plant immune responses in Arabidopsis.

    Directory of Open Access Journals (Sweden)

    Mi Ha Le

    Full Text Available Chitin, an integral component of the fungal cell wall, is one of the best-studied microbe-associated molecular patterns. Previous work identified a LysM receptor-like kinase (LysM-RLK1/CERK1 as the primary chitin receptor in Arabidopsis. In order to identify proteins that interact with CERK1, we conducted a yeast two-hybrid screen using the intracellular kinase domain of CERK1 as the bait. This screen identified 54 putative CERK1-interactors. Screening mutants defective in 43 of these interacting proteins identified only two, a calmodulin like protein (At3g10190 and a leucine-rich repeat receptor like kinase (At3g14840, which differed in their response to pathogen challenge. In the present work, we focused on characterizing the LRR-RLK gene where mutations altered responses to chitin elicitation. This LRR-RLK was named LysM RLK1-interacting kinase 1 (LIK1. The interaction between CERK1 and LIK1 was confirmed by co-immunoprecipitation using protoplasts and transgenic plants. In vitro experiments showed that LIK1 was directly phosphorylated by CERK1. In vivo phosphorylation assays showed that Col-0 wild-type plants have more phosphorylated LIK1 than cerk1 mutant plants, suggesting that LIK1 may be directly phosphorylated by CERK1. Lik1 mutant plants showed an enhanced response to both chitin and flagellin elicitors. In comparison to the wild-type plants, lik1 mutant plants were more resistant to the hemibiotrophic pathogen Pseudomonas syringae, but more susceptible to the necrotrophic pathogen Sclerotinia sclerotiorum. Consistent with the enhanced susceptibility to necrotrophs, lik1 mutants showed reduced expression of genes involved in jasmonic acid and ethylene signaling pathways. These data suggest that LIK1 directly interacts with CERK1 and regulates MAMP-triggered innate immunity.

  18. Modular composition predicts kinase/substrate interactions

    Directory of Open Access Journals (Sweden)

    Tozeren Aydin

    2010-06-01

    Full Text Available Abstract Background Phosphorylation events direct the flow of signals and metabolites along cellular protein networks. Current annotations of kinase-substrate binding events are far from complete. In this study, we scanned the entire human protein sequences using the PROSITE domain annotation tool to identify patterns of domain composition in kinases and their substrates. We identified statistically enriched pairs of strings of domains (signature pairs in kinase-substrate couples presented in the 2006 version of the PTM database. Results The signature pairs enriched in kinase - substrate binding interactions turned out to be highly specific to kinase subtypes. The resulting list of signature pairs predicted kinase-substrate interactions in validation dataset not used in learning with high statistical accuracy. Conclusions The method presented here produces predictions of protein phosphorylation events with high accuracy and mid-level coverage. Our method can be used in expanding the currently available drafts of cell signaling pathways and thus will be an important tool in the development of combination drug therapies targeting complex diseases.

  19. Fibronectin phosphorylation by ecto-protein kinase

    Energy Technology Data Exchange (ETDEWEB)

    Imada, Sumi; Sugiyama, Yayoi; Imada, Masaru (Meiji Institute of Health Science, Odawara (Japan))

    1988-12-01

    The presence of membrane-associated, extracellular protein kinase (ecto-protein kinase) and its substrate proteins was examined with serum-free cultures of Swiss 3T3 fibroblast. When cells were incubated with ({gamma}-{sup 32})ATP for 10 min at 37{degree}C, four proteins with apparent molecular weights between 150 and 220 kDa were prominently phosphorylated. These proteins were also radiolabeled by lactoperoxidase catalyzed iodination and were sensitive to mild tryptic digestion, suggesting that they localized on the cell surface or in the extracellular matrix. Phosphorylation of extracellular proteins with ({gamma}-{sup 32}P)ATP in intact cell culture is consistent with the existence of ecto-protein kinase. Anti-fibronectin antibody immunoprecipitated one of the phosphoproteins which comigrated with a monomer and a dimer form of fibronectin under reducing and nonreducing conditions of electrophoresis, respectively. The protein had affinity for gelatin as demonstrated by retention with gelatin-conjugated agarose. This protein substrate of ecto-protein kinase was thus concluded to be fibronectin. The sites of phosphorylation by ecto-protein kinase were compared with those of intracellularly phosphorylated fibronectin by the analysis of radiolabeled amino acids and peptides. Ecto-protein kinase phosphorylated fibronectin at serine and threonine residues which were distinct from the sites of intracellular fibronectin phosphorylation.

  20. Eliciting maltreated and nonmaltreated children's transgression disclosures: narrative practice rapport building and a putative confession.

    Science.gov (United States)

    Lyon, Thomas D; Wandrey, Lindsay; Ahern, Elizabeth; Licht, Robyn; Sim, Megan P Y; Quas, Jodi A

    2014-01-01

    This study tested the effects of narrative practice rapport building (asking open-ended questions about a neutral event) and a putative confession (telling the child an adult "told me everything that happened and he wants you to tell the truth") on 4- to 9-year-old maltreated and nonmaltreated children's reports of an interaction with a stranger who asked them to keep toy breakage a secret (n = 264). Only one third of children who received no interview manipulations disclosed breakage; in response to a putative confession, one half disclosed. Narrative practice rapport building did not affect the likelihood of disclosure. Maltreated children and nonmaltreated children responded similarly to the manipulations. Neither narrative practice rapport building nor a putative confession increased false reports.

  1. Molecular diagnosis of putative Stargardt disease by capture next generation sequencing.

    Science.gov (United States)

    Zhang, Xiao; Ge, Xianglian; Shi, Wei; Huang, Ping; Min, Qingjie; Li, Minghan; Yu, Xinping; Wu, Yaming; Zhao, Guangyu; Tong, Yi; Jin, Zi-Bing; Qu, Jia; Gu, Feng

    2014-01-01

    Stargardt Disease (STGD) is the commonest genetic form of juvenile or early adult onset macular degeneration, which is a genetically heterogeneous disease. Molecular diagnosis of STGD remains a challenge in a significant proportion of cases. To address this, seven patients from five putative STGD families were recruited. We performed capture next generation sequencing (CNGS) of the probands and searched for potentially disease-causing genetic variants in previously identified retinal or macular dystrophy genes. Seven disease-causing mutations in ABCA4 and two in PROM1 were identified by CNGS, which provides a confident genetic diagnosis in these five families. We also provided a genetic basis to explain the differences among putative STGD due to various mutations in different genes. Meanwhile, we show for the first time that compound heterozygous mutations in PROM1 gene could cause cone-rod dystrophy. Our findings support the enormous potential of CNGS in putative STGD molecular diagnosis.

  2. Putative epidermal stem cell convert into corneal epithelium-like cell under corneal tissue in vitro

    Institute of Scientific and Technical Information of China (English)

    GAO Nan; CUI GuangHui; WANG ZhiChong; HUANG Bing; GE Jian; LU Rong; ZHANG KeFei; FAN ZhiGang; LU Li; PENG Zhan

    2007-01-01

    Rhesus putative epidermal stem cells are being investigated for their potential use in regenerative corneal epithelium-like cells, which may provide a practical source of autologous seed cells for the construction of bioengineered corneas. The goal of this study was to investigate the potential of epidermal stem cells for trans-differentiation into corneal epithelium-like cells. Rhesus putative epidermal stem cells were isolated by type IV collagen attachment method. Flow cytometry analysis, immunohistology and RT-PCR were conducted to identify the expression of specific markers (β1, α6 integrin, K15, K1/K10, K3/K12 and CD71) on the isolated rapid attaching cells. The isolated cells were cocultured with human corneal limbal stroma and corneal epithelial cells. After coculture, the expression of the same specific markers was evaluated in order to identify expression difference caused by the coculture conditions. K3/K12 expression was analyzed in coculture cells on day 2, 4, 6, 8 and 10. Putative epidermal stem cells in conditioned culture media were used as control. Putative epidermal stem cells were predominant in rapid attaching cells by type IV collagen attachment isolation. Before being cocultured, the rhesus putative epidermal stem cells expressed K15, α6 and β1 integrin, but no CD71, K1/K10 and K3/K12. After coculture, these cells expressed K3/K12 (a marker of corneal epithelial cells), K15 and β 1 integrin, but no K1/K10. Cells being not coculture converted into terminally differentiated cells expressing K1/K10. These results indicate that rhesus putative epidermal stem cells can trans-differentiate into corneal epithelium-like cells and, therefore, may have potential therapeutic application as autologous seed cells for the construction of bioengineered corneas.

  3. CLONING, SEQUENCE ANALYSIS, AND CHARACTERIZATION OF PUTATIVE BETA-LACTAMASE OF STENOTROPHOMONAS MALTOPHILIA

    Directory of Open Access Journals (Sweden)

    Chong Seng Shueh

    2012-10-01

    Full Text Available The main objective of current study was to explore the function of chromosomal putative beta-lactamase gene (smlt 0115 in clinical Stenotrophomonas maltophilia. Antibiotic susceptibility test (AST screening for current antimicrobial drugs was done and Minimum Inhibitory Concentration (MIC level towards beta-lactams was determined by E-test. Putative beta-lactamase gene of S. maltophilia was amplified via PCR, with specific primers, then cloned into pET-15 expression plasmid and transformed into Escherichia coli BL21. The gene was sequenced and analyzed. The expressed protein was purified by affinity chromatography and the kinetic assay was performed. S. maltophilia ATCC 13637 was included in this experiment. Besides, a hospital strain which exhibited resistant to a series of beta-lactams including cefepime was identified via AST and MIC, hence it was named as S2 strain and was considered in this study. Sequencing result showed that putative beta-lactamase gene obtained from ATCC 13637 and S2 strains were predicted to have cephalosporinase activity by National Center for Biotechnology Information (NCBI blast program. Differences in the sequences of both ATCC 13637 and S2 strains were found via ClustalW alignment software. Kinetic assay proved a cephalosporinase characteristic produced by E. coli BL21 clone that overexpressed the putative beta-lactamase gene cloned under the control of an external promoter. Yet, expressed protein purified from S2 strain had high catalytic activity against beta-lactam antibiotics which was 14-fold higher than expressed protein purified from ATCC 13637 strain. This study represents the characterization analysis of putative beta-lactamase gene (smlt 0115 of S. maltophilia. The presence of the respective gene in the chromosome of S. maltophilia suggested that putative beta-lactamase gene (smlt 0115 of S. maltophilia plays a role in beta-lactamase resistance.

  4. Putative epidermal stem cell convert into corneal epithelium-like cell under corneal tissue in vitro

    Institute of Scientific and Technical Information of China (English)

    2007-01-01

    Rhesus putative epidermal stem cells are being investigated for their potential use in regenerative corneal epithelium-like cells, which may provide a practical source of autologous seed cells for the construction of bioengineered corneas. The goal of this study was to investigate the potential of epi-dermal stem cells for trans-differentiation into corneal epithelium-like cells. Rhesus putative epidermal stem cells were isolated by type IV collagen attachment method. Flow cytometry analysis, immuno-histology and RT-PCR were conducted to identify the expression of specific markers (β1, α6 integrin, K15, K1/K10, K3/K12 and CD71) on the isolated rapid attaching cells. The isolated cells were cocultured with human corneal limbal stroma and corneal epithelial cells. After coculture, the expression of the same specific markers was evaluated in order to identify expression difference caused by the coculture conditions. K3/K12 expression was analyzed in coculture cells on day 2, 4, 6, 8 and 10. Putative epi-dermal stem cells in conditioned culture media were used as control. Putative epidermal stem cells were predominant in rapid attaching cells by type IV collagen attachment isolation. Before being co-cultured, the rhesus putative epidermal stem cells expressed K15, α6 and β1 integrin, but no CD71, K1/K10 and K3/K12. After coculture, these cells expressed K3/K12 (a marker of corneal epithelial cells), K15 and β 1 integrin, but no K1/K10. Cells being not coculture converted into terminally differentiated cells expressing K1/K10. These results indicate that rhesus putative epidermal stem cells can trans-differentiate into corneal epithelium-like cells and, therefore, may have potential therapeutic application as autologous seed cells for the construction of bioengineered corneas.

  5. Structural basis for substrate specificities of cellular deoxyribonucleoside kinases

    DEFF Research Database (Denmark)

    Johansson, K.; Ramaswamy, S.; Ljungcrantz, C.

    2001-01-01

    kinase with ATP at the nucleoside substrate binding site. Compared to the human kinase, the Drosophila kinase has a wider substrate cleft, which may be responsible for the broad substrate specificity of this enzyme. The human deoxyguanosine kinase is highly specific for purine substrates......; this is apparently due to the presence of Arg 118, which provides favorable hydrogen bonding interactions with the substrate. The two new structures provide an explanation for the substrate specificity of cellular deoxyribonucleoside kinases....

  6. Identification of a kinase profile that predicts chromosome damage induced by small molecule kinase inhibitors.

    Directory of Open Access Journals (Sweden)

    Andrew J Olaharski

    2009-07-01

    Full Text Available Kinases are heavily pursued pharmaceutical targets because of their mechanistic role in many diseases. Small molecule kinase inhibitors (SMKIs are a compound class that includes marketed drugs and compounds in various stages of drug development. While effective, many SMKIs have been associated with toxicity including chromosomal damage. Screening for kinase-mediated toxicity as early as possible is crucial, as is a better understanding of how off-target kinase inhibition may give rise to chromosomal damage. To that end, we employed a competitive binding assay and an analytical method to predict the toxicity of SMKIs. Specifically, we developed a model based on the binding affinity of SMKIs to a panel of kinases to predict whether a compound tests positive for chromosome damage. As training data, we used the binding affinity of 113 SMKIs against a representative subset of all kinases (290 kinases, yielding a 113x290 data matrix. Additionally, these 113 SMKIs were tested for genotoxicity in an in vitro micronucleus test (MNT. Among a variety of models from our analytical toolbox, we selected using cross-validation a combination of feature selection and pattern recognition techniques: Kolmogorov-Smirnov/T-test hybrid as a univariate filter, followed by Random Forests for feature selection and Support Vector Machines (SVM for pattern recognition. Feature selection identified 21 kinases predictive of MNT. Using the corresponding binding affinities, the SVM could accurately predict MNT results with 85% accuracy (68% sensitivity, 91% specificity. This indicates that kinase inhibition profiles are predictive of SMKI genotoxicity. While in vitro testing is required for regulatory review, our analysis identified a fast and cost-efficient method for screening out compounds earlier in drug development. Equally important, by identifying a panel of kinases predictive of genotoxicity, we provide medicinal chemists a set of kinases to avoid when designing

  7. Detection of putative new mutacins by bioinformatic analysis using available web tools

    Directory of Open Access Journals (Sweden)

    Nicolas Guillaume G

    2011-07-01

    Full Text Available Abstract In order to characterise new bacteriocins produced by Streptococcus mutans we perform a complete bioinformatic analyses by scanning the genome sequence of strains UA159 and NN2025. By searching in the adjacent genomic context of the two-component signal transduction system we predicted the existence of many putative new bacteriocins' maturation pathways and some of them were only exclusive to a group of Streptococcus. Computational genomic and proteomic analysis combined to predictive functionnal analysis represent an alternative way for rapid identification of new putative bacteriocins as well as new potential antimicrobial drugs compared to the more traditional methods of drugs discovery using antagonism tests.

  8. LysM domain receptor kinases regulating rhizobial Nod factor-induced infection.

    Science.gov (United States)

    Limpens, Erik; Franken, Carolien; Smit, Patrick; Willemse, Joost; Bisseling, Ton; Geurts, René

    2003-10-24

    The rhizobial infection of legumes has the most stringent demand toward Nod factor structure of all host responses, and therefore a specific Nod factor entry receptor has been proposed. The SYM2 gene identified in certain ecotypes of pea (Pisum sativum) is a good candidate for such an entry receptor. We exploited the close phylogenetic relationship of pea and the model legume Medicago truncatula to identify genes specifically involved in rhizobial infection. The SYM2 orthologous region of M. truncatula contains 15 putative receptor-like genes, of which 7 are LysM domain-containing receptor-like kinases (LYKs). Using reverse genetics in M. truncatula, we show that two LYK genes are specifically involved in infection thread formation. This, as well as the properties of the LysM domains, strongly suggests that they are Nod factor entry receptors.

  9. Molecular and biochemical analysis of symbiotic plant receptor kinase complexes

    Energy Technology Data Exchange (ETDEWEB)

    Cook, Douglas R; Riely, Brendan K

    2010-09-01

    DE-FG02-01ER15200 was a 36-month project, initiated on Sept 1, 2005 and extended with a one-year no cost extension to August 31, 2009. During the project period we published seven manuscripts (2 in review). Including the prior project period (2002-2005) we published 12 manuscripts in journals that include Science, PNAS, The Plant Cell, Plant Journal, Plant Physiology, and MPMI. The primary focus of this work was to further elucidate the function of the Nod factor signaling pathway that is involved in initiation of the legume-rhizobium symbiosis and in particular to explore the relationship between receptor kinase-like proteins and downstream effectors of symbiotic development. During the project period we have map-base cloned two additional players in symbiotic development, including an ERF transcription factor and an ethylene pathway gene (EIN2) that negatively regulates symbiotic signaling; we have also further characterized the subcellular distribution and function of a nuclear-localized symbiosis-specific ion channel, DMI1. The major outcome of the work has been the development of systems for exploring and validating protein-protein interactions that connect symbiotic receptor-like proteins to downstream responses. In this regard, we have developed both homologous (i.e., in planta) and heterologous (i.e., in yeast) systems to test protein interactions. Using yeast 2-hybrid screens we isolated the only known interactor of the nuclear-localized calcium-responsive kinase DMI3. We have also used yeast 2-hybrid methodology to identify interactions between symbiotic signaling proteins and certain RopGTPase/RopGEF proteins that regulate root hair polar growth. More important to the long-term goals of our work, we have established a TAP tagging system that identifies in planta interactions based on co-immuno precipitation and mass spectrometry. The validity of this approach has been shown using known interactors that either co-iummnoprecipate (i.e., remorin) or co

  10. Non-degradative Ubiquitination of Protein Kinases.

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    K Aurelia Ball

    2016-06-01

    Full Text Available Growing evidence supports other regulatory roles for protein ubiquitination in addition to serving as a tag for proteasomal degradation. In contrast to other common post-translational modifications, such as phosphorylation, little is known about how non-degradative ubiquitination modulates protein structure, dynamics, and function. Due to the wealth of knowledge concerning protein kinase structure and regulation, we examined kinase ubiquitination using ubiquitin remnant immunoaffinity enrichment and quantitative mass spectrometry to identify ubiquitinated kinases and the sites of ubiquitination in Jurkat and HEK293 cells. We find that, unlike phosphorylation, ubiquitination most commonly occurs in structured domains, and on the kinase domain, ubiquitination is concentrated in regions known to be important for regulating activity. We hypothesized that ubiquitination, like other post-translational modifications, may alter the conformational equilibrium of the modified protein. We chose one human kinase, ZAP-70, to simulate using molecular dynamics with and without a monoubiquitin modification. In Jurkat cells, ZAP-70 is ubiquitinated at several sites that are not sensitive to proteasome inhibition and thus may have other regulatory roles. Our simulations show that ubiquitination influences the conformational ensemble of ZAP-70 in a site-dependent manner. When monoubiquitinated at K377, near the C-helix, the active conformation of the ZAP-70 C-helix is disrupted. In contrast, when monoubiquitinated at K476, near the kinase hinge region, an active-like ZAP-70 C-helix conformation is stabilized. These results lead to testable hypotheses that ubiquitination directly modulates kinase activity, and that ubiquitination is likely to alter structure, dynamics, and function in other protein classes as well.

  11. Non-degradative Ubiquitination of Protein Kinases.

    Science.gov (United States)

    Ball, K Aurelia; Johnson, Jeffrey R; Lewinski, Mary K; Guatelli, John; Verschueren, Erik; Krogan, Nevan J; Jacobson, Matthew P

    2016-06-01

    Growing evidence supports other regulatory roles for protein ubiquitination in addition to serving as a tag for proteasomal degradation. In contrast to other common post-translational modifications, such as phosphorylation, little is known about how non-degradative ubiquitination modulates protein structure, dynamics, and function. Due to the wealth of knowledge concerning protein kinase structure and regulation, we examined kinase ubiquitination using ubiquitin remnant immunoaffinity enrichment and quantitative mass spectrometry to identify ubiquitinated kinases and the sites of ubiquitination in Jurkat and HEK293 cells. We find that, unlike phosphorylation, ubiquitination most commonly occurs in structured domains, and on the kinase domain, ubiquitination is concentrated in regions known to be important for regulating activity. We hypothesized that ubiquitination, like other post-translational modifications, may alter the conformational equilibrium of the modified protein. We chose one human kinase, ZAP-70, to simulate using molecular dynamics with and without a monoubiquitin modification. In Jurkat cells, ZAP-70 is ubiquitinated at several sites that are not sensitive to proteasome inhibition and thus may have other regulatory roles. Our simulations show that ubiquitination influences the conformational ensemble of ZAP-70 in a site-dependent manner. When monoubiquitinated at K377, near the C-helix, the active conformation of the ZAP-70 C-helix is disrupted. In contrast, when monoubiquitinated at K476, near the kinase hinge region, an active-like ZAP-70 C-helix conformation is stabilized. These results lead to testable hypotheses that ubiquitination directly modulates kinase activity, and that ubiquitination is likely to alter structure, dynamics, and function in other protein classes as well.

  12. Crystal structure of Cryptosporidium parvum pyruvate kinase.

    Directory of Open Access Journals (Sweden)

    William J Cook

    Full Text Available Pyruvate kinase plays a critical role in cellular metabolism of glucose by serving as a major regulator of glycolysis. This tetrameric enzyme is allosterically regulated by different effector molecules, mainly phosphosugars. In response to binding of effector molecules and substrates, significant structural changes have been identified in various pyruvate kinase structures. Pyruvate kinase of Cryptosporidium parvum is exceptional among known enzymes of protozoan origin in that it exhibits no allosteric property in the presence of commonly known effector molecules. The crystal structure of pyruvate kinase from C. parvum has been solved by molecular replacement techniques and refined to 2.5 Å resolution. In the active site a glycerol molecule is located near the γ-phosphate site of ATP, and the protein structure displays a partially closed active site. However, unlike other structures where the active site is closed, the α6' helix in C. parvum pyruvate kinase unwinds and assumes an extended conformation. In the crystal structure a sulfate ion is found at a site that is occupied by a phosphate of the effector molecule in many pyruvate kinase structures. A new feature of the C. parvum pyruvate kinase structure is the presence of a disulfide bond cross-linking the two monomers in the asymmetric unit. The disulfide bond is formed between cysteine residue 26 in the short N-helix of one monomer with cysteine residue 312 in a long helix (residues 303-320 of the second monomer at the interface of these monomers. Both cysteine residues are unique to C. parvum, and the disulfide bond remained intact in a reduced environment. However, the significance of this bond, if any, remains unknown at this time.

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

    DEFF Research Database (Denmark)

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

    2009-01-01

    Within the last decade, 40 crystal structures corresponding to protein kinase CK2 (former name 'casein kinase 2'), to its catalytic subunit CK2alpha and to its regulatory subunit CK2beta were published. Together they provide a valuable, yet by far not complete basis to rationalize the biochemical...

  14. Phosphorylation of nm23/nucleoside diphosphate kinase by casein kinase 2 in vitro

    DEFF Research Database (Denmark)

    Engel, M; Issinger, O G; Lascu, I;

    1994-01-01

    We have investigated phosphorylation of human nucleoside diphosphate kinase (NDPK) and of homologous NDPK from different species by human casein kinase 2 (CK-2). The human NDPK isotypes A and B were phosphorylated by CK-2 in vitro both when the purified proteins and total lysate of HL-60 leukemia...

  15. Homo- and heterodimerization of ROCO kinases: LRRK2 kinase inhibition by the LRRK2 ROCO fragment.

    Science.gov (United States)

    Klein, Christian L; Rovelli, Giorgio; Springer, Wolfdieter; Schall, Christoph; Gasser, Thomas; Kahle, Philipp J

    2009-11-01

    Mutations in the gene encoding leucine-rich repeat kinase 2 (LRRK2) are the most common cause of autosomal-dominant familial and late-onset sporadic Parkinson's disease (PD). LRRK2 is a large multi-domain protein featuring a GTP-binding C-terminal of Ras of complex proteins (ROC) (ROCO) domain combination unique for the ROCO protein family, directly followed by a kinase domain. Dimerization is a well-established phenomenon among protein kinases. Here, we confirm LRRK2 self-interaction, and provide evidence for general homo- and heterodimerization potential among the ROCO kinase family (LRRK2, LRRK1, and death-associated protein kinase 1). The ROCO domain was critically, though not exclusively involved in dimerization, as a LRRK2 deletion mutant lacking the ROCO domain retained dimeric properties. GTP binding did not appear to influence ROCO(LRRK2) self-interaction. Interestingly, ROCO(LRRK2) fragments exerted an inhibitory effect on both wild-type and the elevated G2019S LRRK2 autophosphorylation activity. Insertion of PD mutations into ROCO(LRRK2) reduced self-interaction and led to a reduction of LRRK2 kinase inhibition. Collectively, these results suggest a functional link between ROCO interactions and kinase activity of wild-type and mutant LRRK2. Importantly, our finding of ROCO(LRRK2) fragment-mediated LRRK2 kinase inhibition offers a novel lead for drug design and thus might have important implications for new therapeutic avenues in PD.

  16. A systematic evaluation of protein kinase A-A-kinase anchoring protein interaction motifs

    NARCIS (Netherlands)

    Burgers, Pepijn P; van der Heyden, MAG; Kok, Bart; Heck, Albert J R; Scholten, Arjen

    2015-01-01

    Protein kinase A (PKA) in vertebrates is localized to specific locations in the cell via A-kinase anchoring proteins (AKAPs). The regulatory subunits of the four PKA isoforms (RIα, RIβ, RIIα, and RIIβ) each form a homodimer, and their dimerization domain interacts with a small helical region present

  17. Calcium sensor kinase activates potassium uptake systems in gland cells of Venus flytraps.

    Science.gov (United States)

    Scherzer, Sönke; Böhm, Jennifer; Krol, Elzbieta; Shabala, Lana; Kreuzer, Ines; Larisch, Christina; Bemm, Felix; Al-Rasheid, Khaled A S; Shabala, Sergey; Rennenberg, Heinz; Neher, Erwin; Hedrich, Rainer

    2015-06-09

    The Darwin plant Dionaea muscipula is able to grow on mineral-poor soil, because it gains essential nutrients from captured animal prey. Given that no nutrients remain in the trap when it opens after the consumption of an animal meal, we here asked the question of how Dionaea sequesters prey-derived potassium. We show that prey capture triggers expression of a K(+) uptake system in the Venus flytrap. In search of K(+) transporters endowed with adequate properties for this role, we screened a Dionaea expressed sequence tag (EST) database and identified DmKT1 and DmHAK5 as candidates. On insect and touch hormone stimulation, the number of transcripts of these transporters increased in flytraps. After cRNA injection of K(+)-transporter genes into Xenopus oocytes, however, both putative K(+) transporters remained silent. Assuming that calcium sensor kinases are regulating Arabidopsis K(+) transporter 1 (AKT1), we coexpressed the putative K(+) transporters with a large set of kinases and identified the CBL9-CIPK23 pair as the major activating complex for both transporters in Dionaea K(+) uptake. DmKT1 was found to be a K(+)-selective channel of voltage-dependent high capacity and low affinity, whereas DmHAK5 was identified as the first, to our knowledge, proton-driven, high-affinity potassium transporter with weak selectivity. When the Venus flytrap is processing its prey, the gland cell membrane potential is maintained around -120 mV, and the apoplast is acidified to pH 3. These conditions in the green stomach formed by the closed flytrap allow DmKT1 and DmHAK5 to acquire prey-derived K(+), reducing its concentration from millimolar levels down to trace levels.

  18. Bioinformatic survey for new physiological substrates of Cyclin-dependent kinase 5.

    Science.gov (United States)

    Bórquez, Daniel A; Olmos, Cristina; Álvarez, Sebastián; Di Genova, Alex; Maass, Alejandro; González-Billault, Christian

    2013-04-01

    Cyclin-dependent kinase 5 (Cdk5) is a proline-directed serine/threonine kinase predominantly active in the nervous system where it regulates several processes such as neuronal migration, cytoskeletal dynamics, axonal guidance, and neurotransmission. We constructed a position specific scoring matrix (PSSM) based on a dataset of sites shown to be phosphorylated both in vivo and in vitro by Cdk5. This dataset was curated manually through an exhaustive search of published experimental data. We then used this PSSM to perform a search in the mouse proteome through Scansite, a web-based tool for matching sequence patterns in large databases. Considering a stringent cut-off score of 0.5, we identified 354 new putative sites present in 291 proteins. In order to assess the robustness of our results, ten random subsets (of 80 sites each) of the original dataset were used to construct new PSSMs, which were then used as input for a new Scansite search, leading to the recovery of 81% of the 354 sites by at least 5 PSSMs. In order to reduce the number of false positives in our sequence-based approach, we evaluated which of these predicted sites were phosphorylated in vivo as determined by multiple phosphoproteomics studies carried out through mass spectrometry and available in the PhosphoSitePlus database. This step resulted in a very promising list of 132 putative phosphorylation sites for Cdk5, of which, 51 are specifically phosphorylated in brain tissue, and some are involved in functions regulated by Cdk5 such as axonal growth, synaptic plasticity and neurotransmission. Other phosphorylation sites in our list suggest that Cdk5 might regulate processes through mechanisms not previously recognized such as the control of mRNA splicing. Copyright © 2013 Elsevier Inc. All rights reserved.

  19. Cloning and molecular characterization of a gene encoding MAP kinase from maize and its expression in E. coli

    Institute of Scientific and Technical Information of China (English)

    2005-01-01

    A new MAPK gene, ZmSIMK1 (Zea mays L. salt-induced mitogen-activated protein kinase 1), is isolated from a maize cDNA library. The full-length ZmSIMK1 gene contains 1636 bp and an open reading frame of 1122 nucleotides capable of encoding 373 amino acid polypeptides with a predicted molecular mass of 42.3 kda and pI of 6.01. The putative ZmSIMK1 protein contains all 11 conserved subdomains that are characteristics of serine/threonine protein kinases and the TEY motif, which is the putative phosphorylation site. Northern blot analysis shows that ZmSIMK1 is ubiquitously expressed in roots, stems, and leaves of maize seedlings and its mRNA accumulation is observed in maize seedlings treated with 30 mmol/L PEG-6000 and 137 mmol/L NaCl, but the expression of ZmSIMK1 is not significantly affected by 4℃ treatment. The expression vector pET-ZmSIMK1 is constructed by inserting the coding region of ZmSIMK1 cDNA into pET-42a(+), and transformed into E. coli strain BL21(DE3). A 77kda fusion protein is induced by the further culture at 37℃ after addition of 1mmol/L IPTG.

  20. The Link between Protein Kinase CK2 and Atypical Kinase Rio1

    Directory of Open Access Journals (Sweden)

    Konrad Kubiński

    2017-02-01

    Full Text Available The atypical kinase Rio1 is widespread in many organisms, ranging from Archaebacteria to humans, and is an essential factor in ribosome biogenesis. Little is known about the protein substrates of the enzyme and small-molecule inhibitors of the kinase. Protein kinase CK2 was the first interaction partner of Rio1, identified in yeast cells. The enzyme from various sources undergoes CK2-mediated phosphorylation at several sites and this modification regulates the activity of Rio1. The aim of this review is to present studies of the relationship between the two different kinases, with respect to CK2-mediated phosphorylation of Rio1, regulation of Rio1 activity, and similar susceptibility of the kinases to benzimidazole inhibitors.

  1. Polyphosphate Kinase from Activated Sludge Performing Enhanced Biological Phosphorus Removal†

    Science.gov (United States)

    McMahon, Katherine D.; Dojka, Michael A.; Pace, Norman R.; Jenkins, David; Keasling, Jay D.

    2002-01-01

    A novel polyphosphate kinase (PPK) was retrieved from an uncultivated organism in activated sludge carrying out enhanced biological phosphorus removal (EBPR). Acetate-fed laboratory-scale sequencing batch reactors were used to maintain sludge with a high phosphorus content (approximately 11% of the biomass). PCR-based clone libraries of small subunit rRNA genes and fluorescent in situ hybridization (FISH) were used to verify that the sludge was enriched in Rhodocyclus-like β-Proteobacteria known to be associated with sludges carrying out EBPR. These organisms comprised approximately 80% of total bacteria in the sludge, as assessed by FISH. Degenerate PCR primers were designed to retrieve fragments of putative ppk genes from a pure culture of Rhodocyclus tenuis and from organisms in the sludge. Four novel ppk homologs were found in the sludge, and two of these (types I and II) shared a high degree of amino acid similarity with R. tenuis PPK (86 and 87% similarity, respectively). Dot blot analysis of total RNA extracted from sludge demonstrated that the Type I ppk mRNA was present, indicating that this gene is expressed during EBPR. Inverse PCR was used to obtain the full Type I sequence from sludge DNA, and a full-length PPK was cloned, overexpressed, and purified to near homogeneity. The purified PPK has a specific activity comparable to that of other PPKs, has a requirement for Mg2+, and does not appear to operate in reverse. PPK activity was found mainly in the particulate fraction of lysed sludge microorganisms. PMID:12324346

  2. Tyrosine kinase BMX phosphorylates phosphotyrosine-primed motif mediating the activation of multiple receptor tyrosine kinases.

    Science.gov (United States)

    Chen, Sen; Jiang, Xinnong; Gewinner, Christina A; Asara, John M; Simon, Nicholas I; Cai, Changmeng; Cantley, Lewis C; Balk, Steven P

    2013-05-28

    The nonreceptor tyrosine kinase BMX (bone marrow tyrosine kinase gene on chromosome X) is abundant in various cell types and activated downstream of phosphatidylinositol-3 kinase (PI3K) and the kinase Src, but its substrates are unknown. Positional scanning peptide library screening revealed a marked preference for a priming phosphorylated tyrosine (pY) in the -1 position, indicating that BMX substrates may include multiple tyrosine kinases that are fully activated by pYpY sites in the kinase domain. BMX phosphorylated focal adhesion kinase (FAK) at Tyr⁵⁷⁷ subsequent to its Src-mediated phosphorylation at Tyr⁵⁷⁶. Loss of BMX by RNA interference or by genetic deletion in mouse embryonic fibroblasts (MEFs) markedly impaired FAK activity. Phosphorylation of the insulin receptor in the kinase domain at Tyr¹¹⁸⁹ and Tyr¹¹⁹⁰, as well as Tyr¹¹⁸⁵, and downstream phosphorylation of the kinase AKT at Thr³⁰⁸ were similarly impaired by BMX deficiency. However, insulin-induced phosphorylation of AKT at Ser⁴⁷³ was not impaired in Bmx knockout MEFs or liver tissue from Bmx knockout mice, which also showed increased insulin-stimulated glucose uptake, possibly because of decreased abundance of the phosphatase PHLPP (PH domain leucine-rich repeat protein phosphatase). Thus, by identifying the pYpY motif as a substrate for BMX, our findings suggest that BMX functions as a central regulator among multiple signaling pathways mediated by tyrosine kinases.

  3. Janus kinase inhibitors: jackpot or potluck?

    Directory of Open Access Journals (Sweden)

    Pavithran Keechilat

    2012-06-01

    Full Text Available The reports of a unique mutation in the Janus kinase-2 gene (JAK2 in polycythemia vera by several independent groups in 2005 quickly spurred the development of the Janus kinase inhibitors. In one of the great victories of translational research in recent times, the first smallmolecule Janus kinase inhibitor ruxolitinib entered a phase I trial in 2007. With the approval of ruxolitinib by the US Federal Drug Administration in November 2011 for high-risk and intermediate-2 risk myelofibrosis, a change in paradigm has occurred in the management of a subset of myeloproliferative neoplasms (MPN: primary myelofibrosis, post-polycythemia vera myelofibrosis, and post-essential thrombocythemia myelofibrosis. Whereas the current evidence for ruxolitinib only covers high-risk and intermediate-2 risk myelofibrosis, inhibitors with greater potency are likely to offer better disease control and survival advantage in patients belonging to these categories, and possibly to the low-risk and intermediate-1 risk categories of MPN as well. But use of the Janus kinase inhibitors also probably has certain disadvantages, such as toxicity, resistance, withdrawal phenomenon, non-reversal of histology, and an implausible goal of disease clone eradication, some of which could offset the gains. In spite of this, Janus kinase inhibitors are here to stay, and for use in more than just myeloproliferative neoplasms.

  4. Therapeutic Innovations: Tyrosine Kinase Inhibitors in Cancer

    Directory of Open Access Journals (Sweden)

    Nikolaos Dervisis

    2016-01-01

    Full Text Available Conventional cytotoxic chemotherapy involving DNA-interacting agents and indiscriminate cell death is no longer the future of cancer management. While chemotherapy is not likely to completely disappear from the armamentarium; the use of targeted therapies in combination with conventional treatment is becoming the standard of care in human medicine. Tyrosine kinases are pivotal points of functional cellular pathways and have been implicated in malignancy, inflammatory, and immune-mediated diseases. Pharmaceutical interventions targeting aberrant tyrosine kinase signaling has exploded and is the second most important area of drug development. The “Valley of Death” between drug discovery and approval threatens to blunt the enormous strides in cancer management seen thus far. Kinase inhibitors, as targeted small molecules, hold promise in the treatment and diagnosis of cancer. However, there are still many unanswered questions regarding the use of kinase inhibitors in the interpretation and management of cancer. Comparative oncology has the potential to address restrictions and limitations in the advancement in kinase inhibitor therapy.

  5. SRC kinase regulation in progressively invasive cancer.

    Directory of Open Access Journals (Sweden)

    Weichen Xu

    Full Text Available Metastatic progression is a multistep process that involves tumor growth and survival, motility and invasion, and subsequent proliferation in an inappropriate environment. The Src protein tyrosine kinase has been implicated in many of the biochemical pathways that drive these behaviors. Although Src itself is only rarely mutated in human tumors, its aberrant activity has been noted in various cancers and suggested to serve as a barometer of metastatic potential. With these features in mind, we examined Src kinase regulation at the structural, enzymatic, and expression levels as a function of progressively invasive prostate cancer cell lines. Surprisingly, both total Src content and kinase activity decrease with increasing cell line aggressiveness, an observation that appears to be inconsistent with the well-documented role of Src in the signaling pathways that drive growth and invasion. However, we do observe a direct correlation between Src kinase specific activity (total Src kinase activity/total Src content and metastatic aggressiveness, possibly suggesting that in highly aggressive cell lines, key signaling enzymes are globally recruited to drive the cancerous phenotype. In addition, although the expected enhanced phosphorylation of Src at Tyr-416 (activation site is present in the most aggressive prostate cancer cell lines, unexpectedly high phosphorylation levels at the Tyr-527 inhibitory site are observed as well. The latter, rather than representative of inhibited enzyme, is more indicative of primed Src responsive to local phosphorylated binding partners.

  6. Mining protein kinases regulation using graphical models.

    Science.gov (United States)

    Chen, Qingfeng; Chen, Yi-Ping Phoebe

    2011-03-01

    Abnormal kinase activity is a frequent cause of diseases, which makes kinases a promising pharmacological target. Thus, it is critical to identify the characteristics of protein kinases regulation by studying the activation and inhibition of kinase subunits in response to varied stimuli. Bayesian network (BN) is a formalism for probabilistic reasoning that has been widely used for learning dependency models. However, for high-dimensional discrete random vectors the set of plausible models becomes large and a full comparison of all the posterior probabilities related to the competing models becomes infeasible. A solution to this problem is based on the Markov Chain Monte Carlo (MCMC) method. This paper proposes a BN-based framework to discover the dependency correlations of kinase regulation. Our approach is to apply the MCMC method to generate a sequence of samples from a probability distribution, by which to approximate the distribution. The frequent connections (edges) are identified from the obtained sampling graphical models. Our results point to a number of novel candidate regulation patterns that are interesting in biology and include inferred associations that were unknown.

  7. Sustained mitogen-activated protein kinase activation reprograms defense metabolism and phosphoprotein profile in Arabidopsis thaliana.

    Directory of Open Access Journals (Sweden)

    Ines eLassowskat

    2014-10-01

    Full Text Available Mitogen-activated protein kinases (MAPKs target a variety of protein substrates to regulate cellular signaling processes in eukaryotes. In plants, the number of identified MAPK substrates that control plant defense responses is still limited. Here, we generated transgenic Arabidopsis thaliana plants with an inducible system to simulate in vivo activation of two stress-activated MAPKs, MPK3 and MPK6. Metabolome analysis revealed that this artificial MPK3/6 activation (without any exposure to pathogens or other stresses is sufficient to drive the production of major defense-related metabolites, including various camalexin, indole glucosinolate and agmatine derivatives. An accompanying (phosphoproteome analysis led to detection of hundreds of potential phosphoproteins downstream of MPK3/6 activation. Besides known MAPK substrates, many candidates on this list possess typical MAPK-targeted phosphosites and in many cases, the corresponding phosphopeptides were detected by mass spectrometry. Notably, several of these putative phosphoproteins have been reported to be associated with the biosynthesis of antimicrobial defense substances (e.g. WRKY transcription factors and proteins encoded by the genes from the PEN pathway required for penetration resistance to filamentous pathogens. Thus, this work provides an inventory of candidate phosphoproteins, including putative direct MAPK substrates, for future analysis of MAPK-mediated defense control. (Proteomics data are available with the identifier PXD001252 via ProteomeXchange, http://proteomecentral.proteomexchange.org.

  8. Occurrence of Putative Pathogenicity Islands in Enterococci from Distinct Species and of Differing Origins▿ †

    OpenAIRE

    Semedo-Lemsaddek, Teresa; Barreto-Crespo, Maria Teresa; Tenreiro, Rogério

    2009-01-01

    Enterococci isolated from ewe's milk and cheese, clinical isolates of human and veterinary origins, and reference strains obtained from culture collections were screened for the occurrence of putative pathogenicity island (PAIs). Results obtained after PCR amplification and hybridization point toward PAI dissemination among enterococci of diverse origins (food/clinical) and species (Enterococcus faecalis/non-E. faecalis).

  9. Cloning and characterization of prunus serotina AGAMOUS, a putative flower homeotic gene

    Science.gov (United States)

    Xiaomei Liu; Joseph Anderson; Paula Pijut

    2010-01-01

    Members of the AGAMOUS subfamily of MADS-box transcription factors play an important role in regulating the development of reproductive organs in flowering plants. To help understand the mechanism of floral development in black cherry (Prunus serotina), PsAG (a putative flower homeotic identity gene) was isolated...

  10. A new putative alphapartitivirus recovered from the powdery mildew fungus Erysiphe palczewskii.

    Science.gov (United States)

    Xiong, Guihong; Qiu, Ping; Li, Cong; Chen, Zhuo; Islam, Saif Ul; Fang, Shouguo; Wu, Zujian; Zhang, Songbai; Du, Zhenguo

    2017-02-27

    Two double-stranded RNAs (dsRNA) likely representing the genome of a novel alphapartitivirus which we provisionally named Erysiphe palczewskii alphapartitivirus 1 (EpV1) were recovered from the powdery mildew fungus E. palczewskii infecting Sophora japonica in Jingzhou, Hubei province of China. The two dsRNAs, 1955 (dsRNA1) and 1917 (dsRNA2) bp in size, respectively, each contains a single open reading frame (ORF) encoding a 585- and 528-aa protein, respectively. The 585-aa protein contains a conserved RNA-dependent RNA polymerase (RdRp) domain and shows significant homology to RdRps of approved or putative partitiviruses, particularly those belonging to the genus Alphapartitivirus. However, it shares an aa sequence identity lower than 80% with its closest relative, the RdRp of the putative alphapartitivirus Grapevine partitivirus, and lower than 60% with the RdRps of other partitiviruses. In a phylogenetic tree constructed with RdRp aa sequences of selected partitiviruses, the putative virus EpV1 clustered with Grapevine partitivirus and formed a well-supported monophyletic clade with known or putative alphapartitiviruses.

  11. A rapid approach to evaluate putative nursery sites for penaeid prawns

    Science.gov (United States)

    Taylor, Matthew D.; Smith, James A.; Boys, Craig A.; Whitney, Hannah

    2016-08-01

    Identifying nursery habitats for an aquatic species generally requires tracing adult individuals back through time and space to the area or habitat in which they developed as juveniles. We develop and trial a study design and analytical approach to evaluate the suitability of using stable isotopes to trace emigrating prawns to putative nursery sites, and evaluate assumptions inherent in the application of the approach using two penaeid species with Type-II life cycles: Penaeus (Melicertus) plebejus and Metapenaeus macleayi. Prawns were collected in putative nursery sites within the Hunter River, Australia, and analysed as composite samples of 6 individuals to provide habitat-specific isotopic signatures. Prawns emigrating from the mouth of the river were used as a proxy for individuals recruiting to the adult population, and assigned to putative nursery sites using a probabilistic mixing model and a simple, distance-based approach. Bivariate (δ15N and δ13C) isotopic data was sufficient to distinguish prawns from different putative nursery sites, and isotopic composition correlated closely with salinity. Approximately 90% of emigrating prawns collected could be assigned to these sites using bivariate isotopic data, and both analytical approaches gave similar results. The design developed here is broadly applicable to a suite of penaeid species, but its application will be most powerful when sampling is also aimed at understanding nursery function by simultaneous monitoring of size structure/growth, density, and trophic relationships within nursery habitats.

  12. Mapping the flow of information within the putative mirror neuron system during gesture observation

    NARCIS (Netherlands)

    Schippers, Marleen B.; Keysers, Christian

    2011-01-01

    The putative mirror neuron system may either function as a strict feed-forward system or as a dynamic control system. A strict feed-forward system would predict that action observation leads to a predominantly temporal -> parietal -> premotor flow of information in which a visual representation is t

  13. Complete Genome Sequence of a Putative Densovirus of the Asian Citrus Psyllid, Diaphorina citri.

    Science.gov (United States)

    Nigg, Jared C; Nouri, Shahideh; Falk, Bryce W

    2016-07-28

    Here, we report the complete genome sequence of a putative densovirus of the Asian citrus psyllid, Diaphorina citri Diaphorina citri densovirus (DcDNV) was originally identified through metagenomics, and here, we obtained the complete nucleotide sequence using PCR-based approaches. Phylogenetic analysis places DcDNV between viruses of the Ambidensovirus and Iteradensovirus genera.

  14. Expression of putative expansin genes in phylloxera (Daktulosphaira vitifoliae Fitch) induced root galls of Vitis spp.

    Science.gov (United States)

    Lawo, N C; Griesser, M; Forneck, A

    Grape phylloxera (Daktulosphaira vitifoliae Fitch) is a serious global pest in viticulture. The insects are sedentary feeders and require a gall to feed and reproduce. The insects induce their feeding site within the meristematic zone of the root tip, where they stay attached, feeding both intra- and intercellularly, and causing damage by reducing plant vigour. Several changes in cell structure and composition, including increased cell division and tissue swelling close to the feeding site, cause an organoid gall called a nodosity to develop. Because alpha expansin genes are involved in cell enlargement and cell wall loosening in many plant tissues it may be anticipated that they are also involved in nodosity formation. To identify expansin genes in Vitis vinifera cv. Pinot noir, we mined for orthologues genes in a comparative analysis. Eleven putative expansin genes were identified and shown to be present in the rootstock Teleki 5C (V. berlandieri Planch. x V. riparia Michx.) using specific PCR followed by DNA sequencing. Expression analysis of young and mature nodosities and uninfested root tips were conducted via quantitative real time PCR (qRT-PCR). Up-regulation was measured for three putative expansin genes (VvEXPA15, -A17 and partly -A20) or down-regulation for three other putative genes (VvEXPA7, -A12, -A20) in nodosities. The present study clearly shows the involvement of putative expansin genes in the phylloxera-root interaction.

  15. A review on the putative association between beta-blockers and depression

    NARCIS (Netherlands)

    Verbeek, D.E.; van Riezen, J.; de Boer, R.A.; van Melle, J.P.; de Jonge, P.

    Several kinds of systematic studies have been conducted verifying the putative association between beta-blockers and depressive symptoms. However, many of these studies had important limitations in their design. In most of the studies, no effect of beta-blockers on depressive symptoms was seen.

  16. A review on the putative association between beta-blockers and depression

    NARCIS (Netherlands)

    Verbeek, D.E.; van Riezen, J.; de Boer, R.A.; van Melle, J.P.; de Jonge, P.

    2011-01-01

    Several kinds of systematic studies have been conducted verifying the putative association between beta-blockers and depressive symptoms. However, many of these studies had important limitations in their design. In most of the studies, no effect of beta-blockers on depressive symptoms was seen. Beca

  17. DETERMINATION OF ROCURONIUM AND ITS PUTATIVE METABOLITES IN BODY-FLUIDS AND TISSUE-HOMOGENATES

    NARCIS (Netherlands)

    KLEEF, UW; PROOST, JH; ROGGEVELD, J

    1993-01-01

    A sensitive and selective HPLC method was developed for the quantification of the neuromuscular blocking agent rocuronium and its putative metabolites (the 17-desacetyl derivative and the N-desallyl derivative of rocuronium) in plasma, urine, bile, tissue homogenates and stoma fluid. Samples were pr

  18. Search strings for the study of putative occupational determinants of disease

    NARCIS (Netherlands)

    S. Mattioli; F. Zanardi; A. Baldasseroni; F. Schaafsma; R.M.T. Cooke; G. Mancini; M. Fierro; C. Santangelo; A. Farioli; S. Fucksia; S. Curti; F.S. Violante; J. Verbeek

    2010-01-01

    Objective To identify efficient PubMed search strategies to retrieve articles regarding putative occupational determinants of conditions not generally considered to be work related. Methods Based on MeSH definitions and expert knowledge, we selected as candidate search terms the four MeSH terms desc

  19. Search strings for the study of putative occupational determinants of disease

    NARCIS (Netherlands)

    Mattioli, S.; Zanardi, F.; Baldasseroni, A.; Schaafsma, F.; Cooke, R.M.T.; Mancini, G.; Fierro, M.; Santangelo, C.; Farioli, A.; Fucksia, S.; Curti, S.; Violante, F.S.; Verbeek, J.

    2010-01-01

    Objective To identify efficient PubMed search strategies to retrieve articles regarding putative occupational determinants of conditions not generally considered to be work related. Methods Based on MeSH definitions and expert knowledge, we selected as candidate search terms the four MeSH terms

  20. Isolation of Bartonella quintana from a Woman and a Cat following Putative Bite Transmission▿

    OpenAIRE

    Breitschwerdt, Edward B; Maggi, Ricardo G; Sigmon, Betsy; Nicholson, William L.

    2006-01-01

    We report here the detection of Bartonella quintana, after putative bite transmission, in pre-enrichment blood cultures from a woman and from two feral barn cats. Prospective molecular epidemiological studies are necessary to characterize the risk of human Bartonella quintana infection following cat bites.

  1. Isolation and characterization of 17 different genes encoding putative endopolygalacturonase genes from Rhizopus oryzae

    Science.gov (United States)

    Polygalacturonase enzymes are a valuable aid in the retting of flax for production of linens and, more recently, production of biofuels from citrus wastes. In a search of the recently sequenced Rhizopus oryzae strain 99-880 genome database, 18 putative endopolygalacturonase genes were identified, w...

  2. Nemo-like kinase 1 (Nlk1) and paraxial protocadherin (PAPC) cooperatively control Xenopus gastrulation through regulation of Wnt/planar cell polarity (PCP) signaling.

    Science.gov (United States)

    Kumar, Rahul; Ciprianidis, Anja; Theiß, Susanne; Steinbeißer, Herbert; Kaufmann, Lilian T

    The Wnt/planar cell polarity (PCP) pathway directs cell migration during vertebrate gastrulation and is essential for proper embryonic development. Paraxial protocadherin (PAPC, Gene Symbol pcdh8.2) is an important activator of Wnt/PCP signaling during Xenopus gastrulation, but how PAPC activity is controlled is incompletely understood. Here we show that Nemo-like kinase 1 (Nlk1), an atypical mitogen-activated protein (MAP) kinase, physically associates with the C-terminus of PAPC. This interaction mutually stabilizes both proteins by inhibiting polyubiquitination. The Nlk1 mediated stabilization of PAPC is essential for Wnt/PCP signaling, tissue separation and gastrulation movements. We identified two conserved putative phosphorylation sites in the PAPC C-terminus that are critical for Nlk1 mediated PAPC stabilization and Wnt/PCP regulation. Intriguingly, the kinase activity of Nlk1 itself was not essential for its cooperation with PAPC, suggesting an indirect regulation for example by impeding a different kinase that promotes protein degradation. Overall these results outline a novel, kinase independent role of Nlk1, wherein Nlk1 regulates PAPC stabilization and thereby controls gastrulation movements and Wnt/PCP signaling during development. Copyright © 2016 International Society of Differentiation. Published by Elsevier B.V. All rights reserved.

  3. Crystal structure of human nicotinamide riboside kinase.

    Science.gov (United States)

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

    2007-08-01

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

  4. Crystal Structure of Human Nicotinamide Riboside Kinase

    Energy Technology Data Exchange (ETDEWEB)

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

    2007-01-01

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

  5. Protein Kinases and Parkinson’s Disease

    Science.gov (United States)

    Mehdi, Syed Jafar; Rosas-Hernandez, Hector; Cuevas, Elvis; Lantz, Susan M.; Barger, Steven W.; Sarkar, Sumit; Paule, Merle G.; Ali, Syed F.; Imam, Syed Z.

    2016-01-01

    Currently, the lack of new drug candidates for the treatment of major neurological disorders such as Parkinson’s disease has intensified the search for drugs that can be repurposed or repositioned for such treatment. Typically, the search focuses on drugs that have been approved and are used clinically for other indications. Kinase inhibitors represent a family of popular molecules for the treatment and prevention of various cancers, and have emerged as strong candidates for such repurposing because numerous serine/threonine and tyrosine kinases have been implicated in the pathobiology of Parkinson’s disease. This review focuses on various kinase-dependent pathways associated with the expression of Parkinson’s disease pathology, and evaluates how inhibitors of these pathways might play a major role as effective therapeutic molecules. PMID:27657053

  6. Exploring the scaffold universe of kinase inhibitors.

    Science.gov (United States)

    Hu, Ye; Bajorath, Jürgen

    2015-01-08

    The scaffold concept was applied to systematically determine, analyze, and compare core structures of kinase inhibitors. From publicly available inhibitors of the human kinome, scaffolds and cyclic skeletons were systematically extracted and organized taking activity data, structural relationships, and retrosynthetic criteria into account. Scaffold coverage varied greatly across the kinome, and many scaffolds representing compounds with different activity profiles were identified. The majority of kinase inhibitor scaffolds were involved in well-defined yet distinct structural relationships, which had different consequences on compound activity. Scaffolds exclusively representing highly potent compounds were identified as well as structurally analogous scaffolds with very different degrees of promiscuity. Scaffold relationships presented herein suggest a variety of hypotheses for inhibitor design. Our detailed organization of the kinase inhibitor scaffold universe with respect to different activity and structural criteria, all scaffolds, and the original compound data assembled for our analysis are made freely available.

  7. Lipid activators of protein kinase C

    Energy Technology Data Exchange (ETDEWEB)

    Chauhan, V.P.S.; Chauhan, A.; Deshmukh, D.S.; Brockerhoff, H. (New York State Institute for Basic Research in Developmental Disabilities, Staten Island, NY (USA))

    1990-01-01

    Among the many reported lipid activators of protein kinase C only those of high affinity can be considered true physiological effectors, at present the tumor promoters, e.g., phorbol esters; 1,2-diacyl-sn-glycerols; and phosphatidylinositol 4,5-bisphosphate. Many other compounds (including arachidonic acid) are activators at high, unphysiological concentrations only, and they seem to be sterically unsuited for bonding to the enzyme. Such pseudoactivators possibly act by scrambling the structure of the regulatory moiety of the kinase.

  8. Purification and kinase assay of PKN.

    Science.gov (United States)

    Mukai, Hideyuki; Ono, Yoshitaka

    2006-01-01

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

  9. Rational design of protein kinase inhibitors

    Directory of Open Access Journals (Sweden)

    Yarmoluk S. M.

    2013-07-01

    Full Text Available Modern methodological approaches to rational design of low molecular weight compounds with specific activity in relation to predetermined biomolecular targets are considered by example of development of high effective protein kinase inhibitors. The application of new computational methods that allow to significantly improve the quality of computational experiments (in, particular, accuracy of low molecular weight compounds activity prediction without increase of computational and time costs are highlighted. The effectiveness of strategy of rational design is demonstrated by examples of several own investigations devoted to development of new inhibitors that are high effective and selective towards protein kinases CK2, FGFR1 and ASK1.

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

    Science.gov (United States)

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

    1996-02-02

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

  11. The phosphoarginine energy-buffering system of trypanosoma brucei involves multiple arginine kinase isoforms with different subcellular locations.

    Directory of Open Access Journals (Sweden)

    Frank Voncken

    Full Text Available Phosphagen energy-buffering systems play an essential role in regulating the cellular energy homeostasis in periods of high-energy demand or energy supply fluctuations. Here we describe the phosphoarginine/arginine kinase system of the kinetoplastid parasite Trypanosoma brucei, consisting of three highly similar arginine kinase isoforms (TbAK1-3. Immunofluorescence microscopy using myc-tagged protein versions revealed that each isoform is located in a specific subcellular compartment: TbAK1 is exclusively found in the flagellum, TbAK2 in the glycosome, and TbAK3 in the cytosol of T. brucei. The flagellar location of TbAK1 is dependent on a 22 amino acid long N-terminal sequence, which is sufficient for targeting a GFP-fusion protein to the trypanosome flagellum. The glycosomal location of TbAK2 is in agreement with the presence of a conserved peroxisomal targeting signal, the C-terminal tripeptide 'SNL'. TbAK3 lacks any apparent targeting sequences and is accordingly located in the cytosol of the parasite. Northern blot analysis indicated that each TbAK isoform is differentially expressed in bloodstream and procyclic forms of T. brucei, while the total cellular arginine kinase activity was 3-fold higher in bloodstream form trypanosomes. These results suggest a substantial change in the temporal and spatial energy requirements during parasite differentiation. Increased arginine kinase activity improved growth of procyclic form T. brucei during oxidative challenges with hydrogen peroxide. Elimination of the total cellular arginine kinase activity by RNA interference significantly decreased growth (>90% of procyclic form T. brucei under standard culture conditions and was lethal for this life cycle stage in the presence of hydrogen peroxide. The putative physiological roles of the different TbAK isoforms in T. brucei are further discussed.

  12. Understanding the role of PknJ in Mycobacterium tuberculosis: biochemical characterization and identification of novel substrate pyruvate kinase A.

    Directory of Open Access Journals (Sweden)

    Gunjan Arora

    Full Text Available Reversible protein phosphorylation is a prevalent signaling mechanism which modulates cellular metabolism in response to changing environmental conditions. In this study, we focus on previously uncharacterized Mycobacterium tuberculosis Ser/Thr protein kinase (STPK PknJ, a putative transmembrane protein. PknJ is shown to possess autophosphorylation activity and is also found to be capable of carrying out phosphorylation on the artificial substrate myelin basic protein (MyBP. Previous studies have shown that the autophosphorylation activity of M. tuberculosis STPKs is dependent on the conserved residues in the activation loop. However, our results show that apart from the conventional conserved residues, additional residues in the activation loop may also play a crucial role in kinase activation. Further characterization of PknJ reveals that the kinase utilizes unusual ions (Ni(2+, Co(2+ as cofactors, thus hinting at a novel mechanism for PknJ activation. Additionally, as shown for other STPKs, we observe that PknJ possesses the capability to dimerize. In order to elucidate the signal transduction cascade emanating from PknJ, the M. tuberculosis membrane-associated protein fraction is treated with the active kinase and glycolytic enzyme Pyruvate kinase A (mtPykA is identified as one of the potential substrates of PknJ. The phospholabel is found to be localized on serine and threonine residue(s, with Ser(37 identified as one of the sites of phosphorylation. Since Pyk is known to catalyze the last step of glycolysis, our study shows that the fundamental pathways such as glycolysis can also be governed by STPK-mediated signaling.

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

    Science.gov (United States)

    Li, H.; Roux, S. J.

    1992-01-01

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

  14. Contribution of casein kinase 2 and spleen tyrosine kinase to CFTR trafficking and protein kinase A-induced activity.

    Science.gov (United States)

    Luz, Simão; Kongsuphol, Patthara; Mendes, Ana Isabel; Romeiras, Francisco; Sousa, Marisa; Schreiber, Rainer; Matos, Paulo; Jordan, Peter; Mehta, Anil; Amaral, Margarida D; Kunzelmann, Karl; Farinha, Carlos M

    2011-11-01

    Previously, the pleiotropic "master kinase" casein kinase 2 (CK2) was shown to interact with CFTR, the protein responsible for cystic fibrosis (CF). Moreover, CK2 inhibition abolished CFTR conductance in cell-attached membrane patches, native epithelial ducts, and Xenopus oocytes. CFTR possesses two CK2 phosphorylation sites (S422 and T1471), with unclear impact on its processing and trafficking. Here, we investigated the effects of mutating these CK2 sites on CFTR abundance, maturation, and degradation coupled to effects on ion channel activity and surface expression. We report that CK2 inhibition significantly decreased processing of wild-type (wt) CFTR, with no effect on F508del CFTR. Eliminating phosphorylation at S422 and T1471 revealed antagonistic roles in CFTR trafficking: S422 activation versus T1471 inhibition, as evidenced by a severe trafficking defect for the T1471D mutant. Notably, mutation of Y512, a consensus sequence for the spleen tyrosine kinase (SYK) possibly acting in a CK2 context adjacent to the common CF-causing defect F508del, had a strong effect on both maturation and CFTR currents, allowing the identification of this kinase as a novel regulator of CFTR. These results reinforce the importance of CK2 and the S422 and T1471 residues for regulation of CFTR and uncover a novel regulation of CFTR by SYK, a recognized controller of inflammation.

  15. Cloning, expression and characterization of a nucleoside diphosphate kinase (NDPK) gene from tobacco

    Institute of Scientific and Technical Information of China (English)

    2007-01-01

    Nucleoside diphosphate kinase (NDPK) is a housekeeping enzyme that maintains the intracellular levels of all (d)NTPs used in biosynthesis except ATP. Here we report that a full-length cDNA encoding nucleoside diphosphate kinase (NDPK) was cloned using yeast two-hybrid approach. A tobacco NDPK gene was obtained and designated as NtNDPK1 . NtNDPK1 is 704 bp in length and encodes a putative 16.2 kD protein of 148 amino acids. Phylogenic analysis showed that NtNDPK1 is highly homologous to other plant NDPK genes and identified as type Ⅰ (NDPK1). RNA-gel blot analysis showed that there was no significant difference of NtNDPK1 expression in roots, stems, leaves and buds. And expression of NtNDPK1 was induced by ABA and PEG and repressed by NaCl, but not significantly affected by Paraquat, wounding and low temperature (4℃) treatments, indicating that NtNDPK1 may play a certain role in response to abiotic stress. In vitro phosphorylation assay demonstrated that NtNDPK1 had autophosphorylation activity.

  16. Functional Divergence of Poplar Histidine-Aspartate Kinase HK1 Paralogs in Response to Osmotic Stress

    Directory of Open Access Journals (Sweden)

    François Héricourt

    2016-12-01

    Full Text Available Previous works have shown the existence of protein partnerships belonging to a MultiStep Phosphorelay (MSP in Populus putatively involved in osmosensing. This study is focused on the identification of a histidine-aspartate kinase, HK1b, paralog of HK1a. The characterization of HK1b showed its ability to homo- and hetero-dimerize and to interact with a few Histidine-containing Phosphotransfer (HPt proteins, suggesting a preferential partnership in poplar MSP linked to drought perception. Furthermore, determinants for interaction specificity between HK1a/1b and HPts were studied by mutagenesis analysis, identifying amino acids involved in this specificity. The HK1b expression analysis in different poplar organs revealed its co-expression with three HPts, reinforcing the hypothesis of partnership participation in the MSP in planta. Moreover, HK1b was shown to act as an osmosensor with kinase activity in a functional complementation assay of an osmosensor deficient yeast strain. These results revealed that HK1b showed a different behaviour for canonical phosphorylation of histidine and aspartate residues. These phosphorylation modularities of canonical amino acids could explain the improved osmosensor performances observed in yeast. As conserved duplicates reflect the selective pressures imposed by the environmental requirements on the species, our results emphasize the importance of HK1 gene duplication in poplar adaptation to drought stress.

  17. Shrimp arginine kinase being a binding protein of WSSV envelope protein VP31

    Science.gov (United States)

    Ma, Cuiyan; Gao, Qiang; Liang, Yan; Li, Chen; Liu, Chao; Huang, Jie

    2016-11-01

    Viral entry into the host is the earliest stage of infection in the viral life cycle in which attachment proteins play a key role. VP31 (WSV340/WSSV396), an envelope protein of white spot syndrome virus (WSSV), contains an Arg-Gly-Asp (RGD) peptide domain known as a cellular attachment site. At present, the process of VP31 interacting with shrimp host cells has not been explored. Therefore, the VP31 gene was cloned into pET30a (+), expressed in Escherichia coli strain BL21 and purified with immobilized metal ion affinity chromatography. Four gill cellular proteins of shrimp ( Fenneropenaeus chinensis) were pulled down by an affinity column coupled with recombinant VP31 (rVP31), and the amino acid sequences were identified with MALDI-TOF/TOF mass spectrometry. Hemocyanin, beta-actin, arginine kinase (AK), and an unknown protein were suggested as the putative VP31 receptor proteins. SDS-PAGE showed that AK is the predominant binding protein of VP31. An i n vitro binding activity experiment indicated that recombinant AK's (rAK) binding activity with rVP31 is comparable to that with the same amount of WSSV. These results suggested that AK, as a member of the phosphagen kinase family, plays a role in WSSV infection. This is the first evidence showing that AK is a binding protein of VP31. Further studies on this topic will elucidate WSSV infection mechanism in the future.

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

    Science.gov (United States)

    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.

  19. Structural Evolution of the Protein Kinase-Like Superfamily.

    Directory of Open Access Journals (Sweden)

    2005-10-01

    Full Text Available The protein kinase family is large and important, but it is only one family in a larger superfamily of homologous kinases that phosphorylate a variety of substrates and play important roles in all three superkingdoms of life. We used a carefully constructed structural alignment of selected kinases as the basis for a study of the structural evolution of the protein kinase-like superfamily. The comparison of structures revealed a "universal core" domain consisting only of regions required for ATP binding and the phosphotransfer reaction. Remarkably, even within the universal core some kinase structures display notable changes, while still retaining essential activity. Hence, the protein kinase-like superfamily has undergone substantial structural and sequence revision over long evolutionary timescales. We constructed a phylogenetic tree for the superfamily using a novel approach that allowed for the combination of sequence and structure information into a unified quantitative analysis. When considered against the backdrop of species distribution and other metrics, our tree provides a compelling scenario for the development of the various kinase families from a shared common ancestor. We propose that most of the so-called "atypical kinases" are not intermittently derived from protein kinases, but rather diverged early in evolution to form a distinct phyletic group. Within the atypical kinases, the aminoglycoside and choline kinase families appear to share the closest relationship. These two families in turn appear to be the most closely related to the protein kinase family. In addition, our analysis suggests that the actin-fragmin kinase, an atypical protein kinase, is more closely related to the phosphoinositide-3 kinase family than to the protein kinase family. The two most divergent families, alpha-kinases and phosphatidylinositol phosphate kinases (PIPKs, appear to have distinct evolutionary histories. While the PIPKs probably have an

  20. Putative and unique gene sequence utilization for the design of species specific probes as modeled by Lactobacillus plantarum

    Science.gov (United States)

    The concept of utilizing putative and unique gene sequences for the design of species specific probes was tested. The abundance profile of assigned functions within the Lactobacillus plantarum genome was used for the identification of the putative and unique gene sequence, csh. The targeted gene (cs...

  1. Phospho-ubiquitin: upending the PINK-Parkin-ubiquitin cascade.

    Science.gov (United States)

    Matsuda, Noriyuki

    2016-04-01

    Mitochondria with decreased membrane potential are characterized by defects in protein import into the matrix and impairments in high-efficiency synthesis of ATP. These low-quality mitochondria are marked with ubiquitin for selective degradation. Key factors in this mechanism are PTEN-induced putative kinase 1 (PINK1, a mitochondrial kinase) and Parkin (a ubiquitin ligase), disruption of which has been implicated in predisposition to Parkinson's disease. Previously, the clearance of damaged mitochondria had been thought to be the end result of a simple cascading reaction of PINK1-Parkin-ubiquitin. However, in the past year, several research groups including ours unexpectedly revealed that Parkin regulation is mediated by PINK1-dependent phosphorylation of ubiquitin. These results overturned the simple hierarchy that posited PINK1 and ubiquitin as the upstream and downstream factors of Parkin, respectively. Although ubiquitylation is well-known as a post-translational modification, it has recently become clear that ubiquitin itself can be modified, and that this modification unexpectedly converts ubiquitin to a factor that functions in retrograde signalling.

  2. Downregulation of Pink1 influences mitochondrial fusion–fission machinery and sensitizes to neurotoxins in dopaminergic cells

    Science.gov (United States)

    Rojas-Charry, Liliana; Cookson, Mark R.; Niño, Andrea; Arboleda, Humberto; Arboleda, Gonzalo

    2016-01-01

    It is now well established that mitochondria are organelles that, far from being static, are subject to a constant process of change. This process, which has been called mitochondrial dynamics, includes processes of both fusion and fission. Loss of Pink1 (PTEN-induced putative kinase 1) function is associated with early onset recessive Parkinson’s disease and it has been proposed that mitochondrial dynamics might be affected by loss of the mitochondrial kinase. Here, we report the effects of silencing Pink1 on mitochondrial fusion and fission events in dopaminergic neuron cell lines. Cells lacking Pink1 were more sensitive to cell death induced by C2-Ceramide, which inhibits proliferation and induces apoptosis. In the same cell lines, mitochondrial morphology was fragmented and this was enhanced by application of forskolin, which stimulates the cAMP pathway that phosphorylates Drp1 and thereby inactivates it. Cells lacking Pink1 had lower Drp1 and Mfn2 expression. Based on these data, we propose that Pink1 may exert a neuroprotective role in part by limiting mitochondrial fission. PMID:24792327

  3. Parkinson phenotype in aged PINK1-deficient mice is accompanied by progressive mitochondrial dysfunction in absence of neurodegeneration.

    Directory of Open Access Journals (Sweden)

    Suzana Gispert

    Full Text Available BACKGROUND: Parkinson's disease (PD is an adult-onset movement disorder of largely unknown etiology. We have previously shown that loss-of-function mutations of the mitochondrial protein kinase PINK1 (PTEN induced putative kinase 1 cause the recessive PARK6 variant of PD. METHODOLOGY/PRINCIPAL FINDINGS: Now we generated a PINK1 deficient mouse and observed several novel phenotypes: A progressive reduction of weight and of locomotor activity selectively for spontaneous movements occurred at old age. As in PD, abnormal dopamine levels in the aged nigrostriatal projection accompanied the reduced movements. Possibly in line with the PARK6 syndrome but in contrast to sporadic PD, a reduced lifespan, dysfunction of brainstem and sympathetic nerves, visible aggregates of alpha-synuclein within Lewy bodies or nigrostriatal neurodegeneration were not present in aged PINK1-deficient mice. However, we demonstrate PINK1 mutant mice to exhibit a progressive reduction in mitochondrial preprotein import correlating with defects of core mitochondrial functions like ATP-generation and respiration. In contrast to the strong effect of PINK1 on mitochondrial dynamics in Drosophila melanogaster and in spite of reduced expression of fission factor Mtp18, we show reduced fission and increased aggregation of mitochondria only under stress in PINK1-deficient mouse neurons. CONCLUSION: Thus, aging Pink1(-/- mice show increasing mitochondrial dysfunction resulting in impaired neural activity similar to PD, in absence of overt neuronal death.

  4. Implications of genetics on the diagnosis and care of patients with Parkinson disease.

    Science.gov (United States)

    Klein, Christine

    2006-03-01

    The identification of several monogenic forms has established Parkinson disease (PD) as a movement disorder with a considerable genetic origin in at least a subset of patients. Four of the known forms, Parkin-, PINK1 (PTEN-induced putative kinase 1)-, DJ1-, and LRRK2 (leucine-rich repeat kinase 2)-linked PD, may present clinically as "idiopathic PD" and account for at least 1% of all cases of PD. However, all known monogenic forms combined explain about only 20% of early-onset PD and less than 3% of late-onset PD at best. Although the individual clinical course cannot be predicted, overall, many cases of genetic PD will progress more slowly and respond better to treatment than patients without mutations. Genetic testing frequently yields inconclusive results, is expensive, and should be used for diagnostic purposes only after careful consideration in selected cases at specialty centers. While genetic findings have greatly advanced our understanding of the pathophysiology of PD, we are faced with many novel challenges. These include the definition of the phenotypic and genotypic spectrum of the monogenic forms, a revised terminology and classification of parkinsonian syndromes, identification of genetic susceptibility factors, and development of guidelines for genetic testing and of new treatment options for PD.

  5. The Progress of Induced Pluripotent Stem Cells as Models of Parkinson’s Disease

    Directory of Open Access Journals (Sweden)

    Ji-feng Kang

    2016-01-01

    Full Text Available In recent years, induced pluripotent stem cells (iPSCs were widely used for investigating the mechanisms of Parkinson’s disease (PD. Somatic cells from patients with SNCA (α-synuclein, LRRK2 (leucine-rich repeat kinase 2, PINK1 (PTEN induced putative kinase 1, Parkin mutations, and at-risk individuals carrying GBA (β-glucocerebrosidase mutations have been successfully induced to iPSCs and subsequently differentiated into dopaminergic (DA neurons. Importantly, some PD-related cell phenotypes, including α-synuclein aggregation, mitophagy, damaged mitochondrial DNA, and mitochondrial dysfunction, have been described in these iPSCs models, which further investigated the pathogenesis of PD. In 2007, Takahashi et al. and Vodyanik et al. generated iPSCs from human somatic cells for the first time. Since then, patients derived iPSCs were applied for disease modeling, drug discovery and screening, autologous cell replacement therapy, and other biological applications. iPSC research has now become a hot topic in a wide range of fields. This review summarizes the recent progress of PD patients derived iPSC models in pathogenic mechanism investigation and potential clinical applications, especially their promising strategy in pharmacological study and DA neurons transplantation therapy. However, the challenges of iPSC transplantation still exist, and it has a long way to go before it can be used in clinical application.

  6. Discovery of inhibitors of bacterial histidine kinases

    NARCIS (Netherlands)

    Velikova, N.R.

    2014-01-01

    Discovery of Inhibitors of Bacterial Histidine Kinases

    Summary

    The thesis is on novel antibacterial drug discovery (http://youtu.be/NRMWOGgeysM). Using structure-based and fragment-based dru

  7. Statistical analysis of protein kinase specificity determinants

    DEFF Research Database (Denmark)

    Kreegipuu, Andres; Blom, Nikolaj; Brunak, Søren;

    1998-01-01

    The site and sequence specificity of protein kinase, as well as the role of the secondary structure and surface accessibility of the phosphorylation sites on substrate proteins, was statistically analyzed. The experimental data were collected from the literature and are available on the World Wide...

  8. Gene regulation by MAP kinase cascades

    DEFF Research Database (Denmark)

    Fiil, Berthe Katrine; Petersen, Klaus; Petersen, Morten

    2009-01-01

    Mitogen-activated protein kinase (MAPK) cascades are signaling modules that transduce extracellular stimuli to a range of cellular responses. Research in yeast and metazoans has shown that MAPK-mediated phosphorylation directly or indirectly regulates the activity of transcription factors. Plant ...

  9. MAP kinases in inflammatory bowel disease

    DEFF Research Database (Denmark)

    Coskun, Mehmet; Olsen, Jørgen; Seidelin, Jakob Benedict;

    2011-01-01

    The mammalian family of mitogen-activated protein kinases (MAPKs) is activated by diverse extracellular and intracellular stimuli, and thereby they play an essential role in connecting cell-surface receptors to changes in transcriptional programs. The MAPK signaling pathways regulate a wide range...

  10. Discovery of inhibitors of bacterial histidine kinases

    NARCIS (Netherlands)

    Velikova, N.R.

    2014-01-01

    Discovery of Inhibitors of Bacterial Histidine Kinases

    Summary

    The thesis is on novel antibacterial drug discovery (http://youtu.be/NRMWOGgeysM). Using structure-based and fragment-based

  11. Periodic fever and mevalonate kinase deficiency

    NARCIS (Netherlands)

    Frenkel, Joost

    2002-01-01

    Mevalonate kinase (MK) deficiency is an autosomal recessive disorder, caused by mutations in the MVK-gene on chromosome 12q24. The affected enzyme catalyzes an early step in isoprenoid biosynthesis, the pathway that produces cholesterol and several non-sterol isoprenoids. The clinical spectrum inclu

  12. Monoclonal Antibodies Against Xenopus Greatwall Kinase

    Science.gov (United States)

    Wang, Ling; Fisher, Laura A.; Wahl, James K.

    2011-01-01

    Mitosis is known to be regulated by protein kinases, including MPF, Plk1, Aurora kinases, and so on, which become active in M-phase and phosphorylate a wide range of substrates to control multiple aspects of mitotic entry, progression, and exit. Mechanistic investigations of these kinases not only provide key insights into cell cycle regulation, but also hold great promise for cancer therapy. Recent studies, largely in Xenopus, characterized a new mitotic kinase named Greatwall (Gwl) that plays essential roles in both mitotic entry and maintenance. In this study, we generated a panel of mouse monoclonal antibodies (MAbs) specific for Xenopus Gwl and characterized these antibodies for their utility in immunoblotting, immunoprecipitation, and immunodepletion in Xenopus egg extracts. Importantly, we generated an MAb that is capable of neutralizing endogenous Gwl. The addition of this antibody into M-phase extracts results in loss of mitotic phosphorylation of Gwl, Plk1, and Cdk1 substrates. These results illustrate a new tool to study loss-of-function of Gwl, and support its essential role in mitosis. Finally, we demonstrated the usefulness of the MAb against human Gwl/MASTL. PMID:22008075

  13. Allosteric small-molecule kinase inhibitors

    DEFF Research Database (Denmark)

    Wu, Peng; Clausen, Mads Hartvig; Nielsen, Thomas E.

    2015-01-01

    current barriers of kinase inhibitors, including poor selectivity and emergence of drug resistance. In spite of the small number of identified allosteric inhibitors in comparison with that of inhibitors targeting the ATP pocket, encouraging results, such as the FDA-approval of the first small...

  14. Thrombopoietin potentiates the protein-kinase-C-mediated activation of mitogen-activated protein kinase/ERK kinases and extracellular signal-regulated kinases in human platelets.

    Science.gov (United States)

    Ezumi, Y; Uchiyama, T; Takayama, H

    1998-12-15

    The thrombopoietin (TPO) receptor is expressed in the megakaryocytic lineage from late progenitors to platelets. We investigated the effect of TPO on the extracellular signal-regulated kinase (ERK) activation pathway in human platelets. TPO by itself did not activate ERK1, ERK2 and protein kinase C (PKC), whereas TPO directly enhanced the PKC-dependent activation of ERKs induced by other agonists including thrombin and phorbol esters, without affecting the PKC activation by those agonists. TPO did not activate the mitogen-activated protein kinase/ERK kinases, MEK1 and MEK2, but activated Raf-1 and directly augmented the PKC-mediated MEK activation, suggesting that TPO primarily potentiates the ERK pathway through regulating MEKs or upstream steps of MEKs including Raf-1. The MEK inhibitor PD098059 failed to affect not only thrombin-induced or phorbol ester-induced aggregation, but also potentiation of aggregation by TPO, denying the primary involvement of ERKs and MEKs in those events. ERKs and MEKs were located mainly in the detergent-soluble/non-cytoskeletal fractions. ERKs but not MEKs were relocated to the cytoskeleton following platelet aggregation and actin polymerization. These data indicate that TPO synergizes with other agonists in the ERK activation pathway of platelets and that this synergy might affect functions of the cytoskeleton possibly regulated by ERKs.

  15. Drosophila melanogaster deoxyribonucleoside kinase activates gemcitabine

    DEFF Research Database (Denmark)

    Knecht, Wolfgang; Mikkelsen, N.E.; Clausen, A.R.

    2009-01-01

    Drosophila melanogaster multisubstrate deoxyribonucleoside kinase (Dm-dNK) can additionally sensitize human cancer cell lines towards the anti-cancer drug gemcitabine. We show that this property is based on the Dm-dNK ability to efficiently phosphorylate gemcitabine. The 2.2 angstrom resolution s...

  16. Differential expression profiles of poplar MAP kinase kinases in response to abiotic stresses and plant hormones, and overexpression of PtMKK4 improves the drought tolerance of poplar.

    Science.gov (United States)

    Wang, Lei; Su, Hongyan; Han, Liya; Wang, Chuanqi; Sun, Yanlin; Liu, Fenghong

    2014-07-15

    Mitogen-activated protein kinase (MAPK) cascades are universal signal transduction modules that play essential roles in plant growth, development and stress response. MAPK kinases (MAPKKs), which link MAPKs and MAPKK kinases (MAPKKKs), are integral in mediating various stress responses in plants. However, to date few data about the roles of poplar MAPKKs in stress signal transduction are available. In this study, we performed a systemic analysis of poplar MAPKK gene family expression profiles in response to several abiotic stresses and stress-associated hormones. Furthermore, Populus trichocarpa MAPKK4 (PtMKK4) was chosen for functional characterization. Transgenic analysis showed that overexpression of the PtMKK4 gene remarkably enhanced drought stress tolerance in the transgenic poplar plants. The PtMKK4-overexpressing plants also exhibited much lower levels of H2O2 and higher antioxidant enzyme activity after exposure to drought stress compared to the wide type lines. Besides, some drought marker genes including PtP5CS, PtSUS3, PtLTP3 and PtDREB8 exhibited higher expression levels in the transgenic lines than in the wide type under drought conditions. This study provided valuable information for understanding the putative functions of poplar MAPKKs involved in important signaling pathways under different stress conditions.

  17. Molecular cloning and characterization of taurocyamine kinase from Clonorchis sinensis: a candidate chemotherapeutic target.

    Directory of Open Access Journals (Sweden)

    Jing-Ying Xiao

    2013-11-01

    Full Text Available BACKGROUND: Adult Clonorchis sinensis lives in the bile duct and causes endemic clonorchiasis in East Asian countries. Phosphagen kinases (PK constitute a highly conserved family of enzymes, which play a role in ATP buffering in cells, and are potential targets for chemotherapeutic agents, since variants of PK are found only in invertebrate animals, including helminthic parasites. This work is conducted to characterize a PK from C. sinensis and to address further investigation for future drug development. METHODOLOGY/PRINCIPAL FINDINGS: [corrected] A cDNA clone encoding a putative polypeptide of 717 amino acids was retrieved from a C. sinensis transcriptome. This polypeptide was homologous to taurocyamine kinase (TK of the invertebrate animals and consisted of two contiguous domains. C. sinensis TK (CsTK gene was reported and found consist of 13 exons intercalated with 12 introns. This suggested an evolutionary pathway originating from an arginine kinase gene group, and distinguished annelid TK from the general CK phylogenetic group. CsTK was found not to have a homologous counterpart in sequences analysis of its mammalian hosts from public databases. Individual domains of CsTK, as well as the whole two-domain enzyme, showed enzymatic activity and specificity toward taurocyamine substrate. Of the CsTK residues, R58, I60 and Y84 of domain 1, and H60, I63 and Y87 of domain 2 were found to participate in binding taurocyamine. CsTK expression was distributed in locomotive and reproductive organs of adult C. sinensis. Developmentally, CsTK was stably expressed in both the adult and metacercariae stages. Recombinant CsTK protein was found to have low sensitivity and specificity toward C. sinensis and platyhelminth-infected human sera on ELISA. CONCLUSION: CsTK is a promising anti-C. sinensis drug target since the enzyme is found only in the C. sinensis and has a substrate specificity for taurocyamine, which is different from its mammalian counterpart

  18. Regulation of a Myb transcription factor by cyclin-dependent kinase 2 in Giardia lamblia.

    Science.gov (United States)

    Cho, Chao-Cheng; Su, Li-Hsin; Huang, Yu-Chang; Pan, Yu-Jiao; Sun, Chin-Hung

    2012-02-03

    The protozoan Giardia lamblia parasitizes the human small intestine to cause diseases. It undergoes differentiation into infectious cysts by responding to intestinal stimulation. How the activated signal transduction pathways relate to encystation stimulation remain largely unknown. During encystation, genes encoding cyst wall proteins (CWPs) are coordinately up-regulated by a Myb2 transcription factor. Because cell differentiation is linked to cell cycle regulation, we tried to understand the role of cell cycle regulators, cyclin-dependent kinases (Cdks), in encystation. We found that the recombinant Myb2 was phosphorylated by Cdk-associated complexes and the levels of phosphorylation increased significantly during encystation. We have identified a putative cdk gene (cdk2) by searching the Giardia genome database. Cdk2 was found to localize in the cytoplasm with higher expression during encystation. Interestingly, overexpression of Cdk2 resulted in a significant increase of the levels of cwp gene expression and cyst formation. In addition, the Cdk2-associated complexes can phosphorylate Myb2 and the levels of phosphorylation increased significantly during encystation. Mutations of important catalytic residues of Cdk2 resulted in a significant decrease of kinase activity and ability of inducing cyst formation. Addition of a Cdk inhibitor, purvalanol A, significantly decreased the Cdk2 kinase activity and the levels of cwp gene expression and cyst formation. Our results suggest that the Cdk2 pathway may be involved in phosphorylation of Myb2, leading to activation of the Myb2 function and up-regulation of cwp genes during encystation. The results provide insights into the use of Cdk inhibitory drugs in disruption of Giardia differentiation into cysts.

  19. Molecular cloning and characterization of taurocyamine kinase from Clonorchis sinensis: a candidate chemotherapeutic target.

    Directory of Open Access Journals (Sweden)

    Jing-Ying Xiao

    2013-11-01

    Full Text Available BACKGROUND: Adult Clonorchis sinensis lives in the bile duct and causes endemic clonorchiasis in East Asian countries. Phosphagen kinases (PK constitute a highly conserved family of enzymes, which play a role in ATP buffering in cells, and are potential targets for chemotherapeutic agents, since variants of PK are found only in invertebrate animals, including helminthic parasites. This work is conducted to characterize a PK from C. sinensis and to address further investigation for future drug development. METHODOLOGY/PRINCIPAL FINDINGS: [corrected] A cDNA clone encoding a putative polypeptide of 717 amino acids was retrieved from a C. sinensis transcriptome. This polypeptide was homologous to taurocyamine kinase (TK of the invertebrate animals and consisted of two contiguous domains. C. sinensis TK (CsTK gene was reported and found consist of 13 exons intercalated with 12 introns. This suggested an evolutionary pathway originating from an arginine kinase gene group, and distinguished annelid TK from the general CK phylogenetic group. CsTK was found not to have a homologous counterpart in sequences analysis of its mammalian hosts from public databases. Individual domains of CsTK, as well as the whole two-domain enzyme, showed enzymatic activity and specificity toward taurocyamine substrate. Of the CsTK residues, R58, I60 and Y84 of domain 1, and H60, I63 and Y87 of domain 2 were found to participate in binding taurocyamine. CsTK expression was distributed in locomotive and reproductive organs of adult C. sinensis. Developmentally, CsTK was stably expressed in both the adult and metacercariae stages. Recombinant CsTK protein was found to have low sensitivity and specificity toward C. sinensis and platyhelminth-infected human sera on ELISA. CONCLUSION: CsTK is a promising anti-C. sinensis drug target since the enzyme is found only in the C. sinensis and has a substrate specificity for taurocyamine, which is different from its mammalian counterpart

  20. Isolation and functional characterization of the C-terminus of rice phosphatidylinositol 4-kinase in vitro

    Institute of Scientific and Technical Information of China (English)

    2003-01-01

    A partial rice (Oryza sativa L.) cDNA clone, OsPI4K1c, was isolated through screening of a cDNA library constructed from tillering materials. OsPI4K1c encoded a peptide of 608 amino acids with a calculated molecular mass of 68.4 kDa. The OsPI4K1c peptide shared high homology and possessed the highly conserved domains present in most isolated cloned PI4-kinases, i.e. a lipid kinase unique (LKU) domain and a catalytic (CAT) domain. A region with similarity to pleckstrin homology (PH) domain was present in OsPI4K1c as well. Further comparison with genomic sequences in databases revealed that OsPI4K1c is located at the 3'-end of a putative rice PI 4-kinase coding gene OsPI4K1, and its coding region corresponded to the C-terminal half of OsPI4K1 protein. Twelve exons (49-562 bp in size) and 11 introns (77-974 bp in size) were identified in OsPI4K1c. The recombinant protein expressed in Escherichia coli phosphorylates phosphatidylinositol at the D4 position of the inositol ring. OsPI4K1 transcript levels were detected in a low but constitutive manner in shoot, stem, leaf, spike and root tissues and did not change upon treatment with different hormones, calcium and jasmonic acid (JA). However, treatment with salicylic acid (SA) elevated the mRNA level of the OsPI4K1 gene, which suggested the involvement of OsPI4K1 in wounding responses.

  1. 90-kDa ribosomal S6 kinase is phosphorylated and activated by 3-phosphoinositide-dependent protein kinase-1

    DEFF Research Database (Denmark)

    Jensen, Claus Antonio Juel; Buch, M B; Krag, T O;

    1999-01-01

    90-kDa ribosomal S6 kinase-2 (RSK2) belongs to a family of growth factor-activated serine/threonine kinases composed of two kinase domains connected by a regulatory linker region. The N-terminal kinase of RSK2 is involved in substrate phosphorylation. Its activation requires phosphorylation of th...... of Ser(227), Ser(369), and Ser(386). Our study extend recent findings which implicate PDK1 in the activation of protein kinases B and C and p70(S6K), suggesting that PDK1 controls several major growth factor-activated signal transduction pathways.......90-kDa ribosomal S6 kinase-2 (RSK2) belongs to a family of growth factor-activated serine/threonine kinases composed of two kinase domains connected by a regulatory linker region. The N-terminal kinase of RSK2 is involved in substrate phosphorylation. Its activation requires phosphorylation...... of the linker region at Ser(369), catalyzed by extracellular signal-regulated kinase (ERK), and at Ser(386), catalyzed by the C-terminal kinase, after its activation by ERK. In addition, the N-terminal kinase must be phosphorylated at Ser(227) in the activation loop by an as yet unidentified kinase. Here, we...

  2. Kinase activity ranking using phosphoproteomics data (KARP) quantifies the contribution of protein kinases to the regulation of cell viability.

    Science.gov (United States)

    Wilkes, Edmund H; Casado, Pedro; Rajeeve, Vinothini; Cutillas, Pedro R

    2017-09-01

    Cell survival is regulated by a signaling network driven by the activity of protein kinases; however, determining the contribution that each kinase in the network makes to such regulation remains challenging. Here, we report a computational approach that uses mass spectrometry-based phosphoproteomics data to rank protein kinases based on their contribution to cell regulation. We found that the scores returned by this algorithm, which we have termed kinase activity ranking using phosphoproteomics data (KARP), were a quantitative measure of the contribution that individual kinases make to the signaling output. Application of KARP to the analysis of eight hematological cell lines revealed that cyclin-dependent kinase (CDK) 1/2, casein kinase (CK) 2, extracellular signal-related kinase (ERK), and p21-activated kinase (PAK) were the most frequently highly ranked kinases in these cell models. The patterns of kinase activation were cell-line specific yet showed a significant association with cell viability as a function of kinase inhibitor treatment. Thus, our study exemplifies KARP as an untargeted approach to empirically and systematically identify regulatory kinases within signaling networks. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

  3. Expression of SOCS1 and the downstream targets of its putative tumor suppressor functions in prostate cancer.

    Science.gov (United States)

    Chevrier, Martin; Bobbala, Diwakar; Villalobos-Hernandez, Alberto; Khan, Md Gulam Musawwir; Ramanathan, Sheela; Saucier, Caroline; Ferbeyre, Gerardo; Geha, Sameh; Ilangumaran, Subburaj

    2017-02-24

    Suppressor of cytokine signaling 1 (SOCS1) is considered a tumor suppressor due to frequent epigenetic and micro-RNA-mediated repression of its gene expression in diverse cancers. In prostate cancer (PCa), elevated expression of miR-30d that targets SOCS1 mRNA is associated with increased risk of disease recurrence. SOCS1 can mediate its tumor suppressor functions by diverse mechanisms such as inhibiting the JAK-STAT signaling pathway, promoting the tumor suppressor functions of p53, attenuating MET receptor tyrosine kinase signaling and blocking the oncogenic potential of the cell cycle inhibitor p21(CIP1) (p21). Here, we studied the expression of SOCS1 and the downstream targets of its putative tumor suppressor functions (p53, MET and p21) in human PCa specimens to evaluate their significance as markers of disease prognosis. Tissue microarrays were constructed of 78 archived prostatectomy specimens that were grouped according to the recommendations of the International Society of Urological Pathology (ISUP) based on the Gleason patterns. SOCS1, p53, MET and p21 protein expression were evaluated by immunohistochemical staining alongside the common prostate cancer-related markers Ki67, prostein and androgen receptor. Statistical correlations between the staining intensities of these markers and ISUP grade groups, local invasion or lymph node metastasis were evaluated. SOCS1 showed diffuse staining in the prostatic epithelium. SOCS1 staining intensity correlated inversely with the ISUP grade groups (ρ = -0.4687, p <0.0001) and Ki67 (ρ = -0.2444, p = 0.031), and positively with prostein (ρ = 0.3511, p = 0.0016). Changes in SOCS1 levels did not significantly associate with those of p53, MET or p21. However, p21 positively correlated with androgen receptor expression (ρ = -0.1388, p = 0.0003). A subset of patients with regional lymph node metastasis, although small in number, showed reduced SOCS1 expression and increased expression of

  4. Two thymidine kinases and one multisubstrate deoxyribonucleoside kinase salvage DNA precursors in Arabidopsis thaliana

    DEFF Research Database (Denmark)

    Anders Ranegaard Clausen, Anders Ranegaard; Girandon, Lenart; Ali, Ashfaq

    2012-01-01

    Deoxyribonucleotides are the building blocks of DNA and can be synthesized via de novo and salvage pathways. Deoxyribonucleoside kinases (EC 2.7.1.145) salvage deoxyribonucleosides by transfer of a phosphate group to the 5' of a deoxyribonucleoside. This salvage pathway is well characterized...... in mammals, but in contrast, little is known about how plants salvage deoxyribonucleosides. We show that during salvage, deoxyribonucleosides can be phosphorylated by extracts of Arabidopsis thaliana into corresponding monophosphate compounds with an unexpected preference for purines over pyrimidines....... Deoxyribonucleoside kinase activities were present in all tissues during all growth stages. In the A. thaliana genome, we identified two types of genes that could encode enzymes which are involved in the salvage of deoxyribonucleosides. Thymidine kinase activity was encoded by two thymidine kinase 1 (EC 2...

  5. Two thymidine kinases and one multisubstrate deoxyribonucleoside kinase salvage DNA precursors in Arabidopsis thaliana

    DEFF Research Database (Denmark)

    Clausen, Anders R.; Girandon, Lenart; Ali, Ashfaq

    2012-01-01

    Deoxyribonucleotides are the building blocks of DNA and can be synthesized via de novo and salvage pathways. Deoxyribonucleoside kinases (EC 2.7.1.145) salvage deoxyribonucleosides by transfer of a phosphate group to the 5′ of a deoxyribonucleoside. This salvage pathway is well characterized...... in mammals, but in contrast, little is known about how plants salvage deoxyribonucleosides. We show that during salvage, deoxyribonucleosides can be phosphorylated by extracts of Arabidopsis thaliana into corresponding monophosphate compounds with an unexpected preference for purines over pyrimidines....... Deoxyribonucleoside kinase activities were present in all tissues during all growth stages. In the A. thaliana genome, we identified two types of genes that could encode enzymes which are involved in the salvage of deoxyribonucleosides. Thymidine kinase activity was encoded by two thymidine kinase 1 (EC 2...

  6. Pyrrolopyridine inhibitors of mitogen-activated protein kinase-activated protein kinase 2 (MK-2).

    Science.gov (United States)

    Anderson, David R; Meyers, Marvin J; Vernier, William F; Mahoney, Matthew W; Kurumbail, Ravi G; Caspers, Nicole; Poda, Gennadiy I; Schindler, John F; Reitz, David B; Mourey, Robert J

    2007-05-31

    A new class of potent kinase inhibitors selective for mitogen-activated protein kinase-activated protein kinase 2 (MAPKAP-K2 or MK-2) for the treatment of rheumatoid arthritis has been prepared and evaluated. These inhibitors have IC50 values as low as 10 nM against the target and have good selectivity profiles against a number of kinases including CDK2, ERK, JNK, and p38. These MK-2 inhibitors have been shown to suppress TNFalpha production in U397 cells and to be efficacious in an acute inflammation model. The structure-activity relationships of this series, the selectivity for MK-2 and their activity in both in vitro and in vivo models are discussed. The observed selectivity is discussed with the aid of an MK-2/inhibitor crystal structure.

  7. CK2: a protein kinase in need of control

    DEFF Research Database (Denmark)

    Guerra, B; Boldyreff, B; Sarno, S;

    1999-01-01

    Protein kinase CK2 is a heterotetrameric alpha2beta2 Ser/Thr protein kinase with some features unusual among the eukaryotic protein kinases: (1) CK2 recognizes phosphoacceptor sites specified by several acidic determinants; (2) CK2 can use both ATP and GTP as phosphoryl donors; and (3...... response to nucleotide analogs. The increasing knowledge of CK2 structure-function relationships will allow the design of highly selective inhibitors of this pleiotropic kinase with oncogenic potential....

  8. Kinase detection with gallium nitride based high electron mobility transistors.

    Science.gov (United States)

    Makowski, Matthew S; Bryan, Isaac; Sitar, Zlatko; Arellano, Consuelo; Xie, Jinqiao; Collazo, Ramon; Ivanisevic, Albena

    2013-07-01

    A label-free kinase detection system was fabricated by the adsorption of gold nanoparticles functionalized with kinase inhibitor onto AlGaN/GaN high electron mobility transistors (HEMTs). The HEMTs were operated near threshold voltage due to the greatest sensitivity in this operational region. The Au NP/HEMT biosensor system electrically detected 1 pM SRC kinase in ionic solutions. These results are pertinent to drug development applications associated with kinase sensing.

  9. Mutations in WSC genes for putative stress receptors result in sensitivity to multiple stress conditions and impairment of Rlm1-dependent gene expression in Saccharomyces cerevisiae.

    Science.gov (United States)

    Zu, T; Verna, J; Ballester, R

    2001-09-01

    Intracellular signaling by mitogen-activated protein (MAP) kinase cascades plays an essential role in the cellular response to environmental stress. In the yeast Saccharomyces cerevisiae, the PKC1-regulated, stress-activated MAP kinase pathway, the MPK1 cascade, is activated by heat and by a decrease in osmolarity. The genes WSC1, WSC2 and WSC3 encode putative receptors that maintain cell wall integrity under conditions of heat stress. Genetic studies place the function of the WSC genes upstream of the MPK1 kinase cascade. To further define the role of the WSC family in the stress response we determined whether: (1) the wscdelta mutants are sensitive to other environmental stress conditions, in addition to heat shock; (2) expression from four transcriptional control elements, known to be activated by stress, is impaired in wscdelta mutants; and (3) Wsc4, a Wsc homolog, has functions that overlap with those of the other Wsc family members. We report here that deletion of WSC and PKC1 causes hypersensitivity to ethanol, hydrogen peroxide and DNA-damaging drugs. In wscdelta mutants expression of beta-galactosidase from the AP-1 response element (ARE), the heat shock response element (HSE) or the stress response element (STRE) is not reduced. In contrast, expression of a reporter gene placed under the control of the Rlm1 (transcription factor)-dependent response element is significantly reduced in wscdelta mutants. This suggests that the lysis defect of wscdelta mutants is at least in part caused by a defect in transcriptional regulation by Rlm1. Phenotypic analysis of the effect of deleting WSC4 in a wsc1delta mutant show that, unlike WSC2 or WSC3, deletion of WSC4 does not exacerbate the lysis defect of a wsc1delta strain. In contrast, deletion of WSC4 enhances the sensitivity of the wsc1delta mutant to heat shock, ethanol, and a DNA-damaging drug, suggesting that WSC4 plays a role in the response to environmental stress but that its function may differ from those of

  10. Isolation of arginine kinase from Apis cerana cerana and its possible involvement in response to adverse stress.

    Science.gov (United States)

    Chen, Xiaobo; Yao, Pengbo; Chu, Xiaoqian; Hao, Lili; Guo, Xingqi; Xu, Baohua

    2015-01-01

    Arginine kinases (AK) in invertebrates play the same role as creatine kinases in vertebrates. Both proteins are important for energy metabolism, and previous studies on AK focused on this attribute. In this study, the arginine kinase gene was isolated from Apis cerana cerana and was named AccAK. A 5'-flanking region was also cloned and shown to contain abundant putative binding sites for transcription factors related to development and response to adverse stress. We imitated several abiotic and biotic stresses suffered by A. cerana cerana during their life, including heavy metals, pesticides, herbicides, heat, cold, oxidants, antioxidants, ecdysone, and Ascosphaera apis and then studied the expression patterns of AccAK after these treatments. AccAK was upregulated under all conditions, and, in some conditions, this response was very pronounced. Western blot and AccAK enzyme activity assays confirmed the results. In addition, a disc diffusion assay showed that overexpression of AccAK reduced the resistance of Escherichia coli cells to multiple adverse stresses. Taken together, our results indicated that AccAK may be involved of great significance in response to adverse abiotic and biotic stresses.

  11. Comprehensive Phenotyping in Multiple Sclerosis: Discovery Based Proteomics and the Current Understanding of Putative Biomarkers

    Directory of Open Access Journals (Sweden)

    Kevin C. O’Connor

    2006-01-01

    Full Text Available Currently, there is no single test for multiple sclerosis (MS. Diagnosis is confirmed through clinical evaluation, abnormalities revealed by magnetic resonance imaging (MRI, and analysis of cerebrospinal fluid (CSF chemistry. The early and accurate diagnosis of the disease, monitoring of progression, and gauging of therapeutic intervention are important but elusive elements of patient care. Moreover, a deeper understanding of the disease pathology is needed, including discovery of accurate biomarkers for MS. Herein we review putative biomarkers of MS relating to neurodegeneration and contributions to neuropathology, with particular focus on autoimmunity. In addition, novel assessments of biomarkers not driven by hypotheses are discussed, featuring our application of advanced proteomics and metabolomics for comprehensive phenotyping of CSF and blood. This strategy allows comparison of component expression levels in CSF and serum between MS and control groups. Examination of these preliminary data suggests that several CSF proteins in MS are differentially expressed, and thus, represent putative biomarkers deserving of further evaluation.

  12. Hippocampal and thalamic neuronal metabolism in a putative rat model of schizophrenia○

    Institute of Scientific and Technical Information of China (English)

    Guolin Ma; Tianbin Song; Min Chen; Yuan Fu; Yong Xu; Ensen Ma; Wu Wang; Jiang Du; Mingxiong Huang

    2013-01-01

    The transcription factor early growth response protein 3 (EGR3) is involved in schizophrenia. We developed a putative rat model of schizophrenia by transfecting lentiviral particles carrying the Egr3 gene into bilateral hippocampal dentate gyrus. We assessed spatial working memory using the Morris water maze test, and neuronal metabolite levels in bilateral hippocampus and thalamus were determined by 3.0 T proton magnetic resonance spectroscopy. Choline content was significantly greater in the hippocampus after transfection, while N-acetylaspartate and the ratio of N-acetylaspartate to creatine/phosphocreatine in the thalamus were lower than in controls. This study is the first to report evaluation of brain metabolites using 3.0 T proton magnetic resonance spectroscopy in rats transfected with Egr3, and reveals metabolic abnormalities in the hippocampus and thalamus in this putative model of schizophrenia.

  13. Functional characterization of a putative β-lactamase gene in the genome of Zymomonas mobilis.

    Science.gov (United States)

    Rajnish, K Narayanan; Asraf, Sheik Abdul Kader Sheik; Manju, Nagarajan; Gunasekaran, Paramasamy

    2011-12-01

    Zymomonas mobilis ZM4 is resistant to β-lactam antibiotics but there are no reports of a β-lactam resistance gene and its regulation. A putative β-lactamase gene sequence (ZMO0103) in the genome of Z. mobilis showed a 55% amino acid sequence identity with class C β-lactamase genes. qPCR analysis of the β-lactamase transcript indicated a higher level expression of the β-lactamase compared to the relative transcript quantities in antibiotic-susceptible bacteria. The putative β-lactamase gene was cloned, expressed in Escherichia coli BL21 and the product, AmpC, was purified to homogeneity. Its optimal activity was at pH 6 and 30 °C. Further, the β-lactamase had a higher affinity towards penicillins than cephalosporin antibiotics. © Springer Science+Business Media B.V. 2011

  14. Putative Biomarkers and Targets of Estrogen Receptor Negative Human Breast Cancer

    Directory of Open Access Journals (Sweden)

    Stephen W. Byers

    2011-07-01

    Full Text Available Breast cancer is a progressive and potentially fatal disease that affects women of all ages. Like all progressive diseases, early and reliable diagnosis is the key for successful treatment and annihilation. Biomarkers serve as indicators of pathological, physiological, or pharmacological processes. Her2/neu, CA15.3, estrogen receptor (ER, progesterone receptor (PR, and cytokeratins are biomarkers that have been approved by the Food and Drug Administration for disease diagnosis, prognosis, and therapy selection. The structural and functional complexity of protein biomarkers and the heterogeneity of the breast cancer pathology present challenges to the scientific community. Here we review estrogen receptor-related putative breast cancer biomarkers, including those of putative breast cancer stem cells, a minor population of estrogen receptor negative tumor cells that retain the stem cell property of self renewal. We also review a few promising cytoskeleton targets for ER alpha negative breast cancer.

  15. A putative ABC transporter is involved in negative regulation of biofilm formation by Listeria monocytogenes

    DEFF Research Database (Denmark)

    Zhu, Xinna; Long, Fei; Chen, Yonghui

    2008-01-01

    Listeria monocytogenes may persist for long periods in food processing environments. In some instances, this may be due to aggregation or biofilm formation. To investigate the mechanism controlling biofilm formation in the food-borne pathogen L. monocytogenes, we characterized LM-49, a mutant...... with enhanced ability of biofilm-formation generated via transposon Tn917 mutagenesis of L. monocytogenes 4b G. In this mutant, a Tn917 insertion has disrupted the coding region of the gene encoding a putative ATP binding cassette (ABC) transporter permease identical to Lmof2365_1771 (a putative ABC......-transporter permease) presented in the sequenced strain L. monocytogenes str. 4b F2365. This disrupted gene, denoted lm.G_1771, encoded a protein with 10 transmembrane helixes. The revertant, LM-49RE, was obtained by replacing lm.G_1771::Tn917 with lm.G_1771 via homologous recombination. We found that LM-49RE formed...

  16. The solution structure of ChaB, a putative membrane ion antiporter regulator from Escherichia coli

    Directory of Open Access Journals (Sweden)

    Iannuzzi Pietro

    2004-08-01

    Full Text Available Abstract Background ChaB is a putative regulator of ChaA, a Na+/H+ antiporter that also has Ca+/H+ activity in E. coli. ChaB contains a conserved 60-residue region of unknown function found in other bacteria, archaeabacteria and a series of baculoviral proteins. As part of a structural genomics project, the structure of ChaB was elucidated by NMR spectroscopy. Results The structure of ChaB is composed of 3 α-helices and a small sheet that pack tightly to form a fold that is found in the cyclin-box family of proteins. Conclusion ChaB is distinguished from its putative DNA binding sequence homologues by a highly charged flexible loop region that has weak affinity to Mg2+ and Ca2+ divalent metal ions.

  17. Exploring Universal Partnerships and Putative Marriages as Tools for Awarding Partnership Property in Contemporary Family Law

    Directory of Open Access Journals (Sweden)

    Elsje Bonthuys

    2016-12-01

    Full Text Available Following upon the Supreme Court of Appeal's judgment in Butters v Mncora 2012 4 SA 1 (SCA, which broadened the criteria and consequences of universal partnerships in cohabitation relationships, this article investigates the potential of universal partnerships and putative marriages to allocate rights to share in partnership property in other intimate relationships. It traverses several instances in which marriages are not recognised - bigamous marriages, Muslim and Hindu religious marriages and invalid customary marriages – examining whether the wives in these marriages could use universal partnerships and putative marriages to claim a share in property. It then considers the use of universal partnerships to obtain a share of property in civil marriages out of community of property. It concludes by pointing out several issues which are in need of clarification and where the common law should be developed to give effect to fundamental constitutional rights.

  18. Enrichment of putative stem cells from adipose tissue using dielectrophoretic field-flow fractionation

    Science.gov (United States)

    Vykoukal, Jody; Vykoukal, Daynene M.; Freyberg, Susanne; Alt, Eckhard U.; Gascoyne, Peter R. C.

    2009-01-01

    We have applied the microfluidic cell separation method of dielectrophoretic field-flow fractionation (DEP-FFF) to the enrichment of a putative stem cell population from an enzyme-digested adipose tissue derived cell suspension. A DEP-FFF separator device was constructed using a novel microfluidic-microelectronic hybrid flex-circuit fabrication approach that is scaleable and anticipates future low-cost volume manufacturing. We report the separation of a nucleated cell fraction from cell debris and the bulk of the erythrocyte population, with the relatively rare (<2% starting concentration) NG2-positive cell population (pericytes and/or putative progenitor cells) being enriched up to 14-fold. This work demonstrates a potential clinical application for DEP-FFF and further establishes the utility of the method for achieving label-free fractionation of cell subpopulations. PMID:18651083

  19. Composition of the Putative Prepore Complex of Bacillus thuringiensis Cry1Ab Toxin

    Science.gov (United States)

    Nair, Manoj S.; Dean, Donald H.

    2015-01-01

    Prepore formation is hypothesized to be an obligate step in the insertion of Cry1Ab toxin into insect brush border membrane vesicles. We examined the architecture of the putative prepore when isolated using the published protocols [1] [2]. Our results demonstrate that the putative prepore form of Cry1Ab is a combination of receptor proteins attached to the toxin, when purified. The results also suggest that this prepore form as prepared by the methods published is different from other membrane-extracted oligomeric forms of Cry toxins and prepore of other toxins in general. While most other known prepores are composed of multimers of a single protein, the Cry1Ab prepore, as generated, is a protein-receptor complex oligomer and monomers of Cry toxins. PMID:26702367

  20. Mating type gene homologues and putative sex pheromone-sensing pathway in arbuscular mycorrhizal fungi, a presumably asexual plant root symbiont.

    Science.gov (United States)

    Halary, Sébastien; Daubois, Laurence; Terrat, Yves; Ellenberger, Sabrina; Wöstemeyer, Johannes; Hijri, Mohamed

    2013-01-01

    The fungal kingdom displays a fascinating diversity of sex-determination systems. Recent advances in genomics provide insights into the molecular mechanisms of sex, mating type determination, and evolution of sexual reproduction in many fungal species in both ancient and modern phylogenetic lineages. All major fungal groups have evolved sexual differentiation and recombination pathways. However, sexuality is unknown in arbuscular mycorrhizal fungi (AMF) of the phylum Glomeromycota, an ecologically vital group of obligate plant root symbionts. AMF are commonly considered an ancient asexual lineage dating back to the Ordovician, approximately 460 M years ago. In this study, we used genomic and transcriptomic surveys of several AMF species to demonstrate the presence of conserved putative sex pheromone-sensing mitogen-activated protein (MAP) kinases, comparable to those described in Ascomycota and Basidiomycota. We also find genes for high mobility group (HMG) transcription factors, homologous to SexM and SexP genes in the Mucorales. The SexM genes show a remarkable sequence diversity among multiple copies in the genome, while only a single SexP sequence was detected in some isolates of Rhizophagus irregularis. In the Mucorales and Microsporidia, the sexM gene is flanked by genes for a triosephosphate transporter (TPT) and a RNA helicase, but we find no evidence for synteny in the vicinity of the Sex locus in AMF. Nonetheless, our results, together with previous observations on meiotic machinery, suggest that AMF could undergo a complete sexual reproduction cycle.

  1. Mating type gene homologues and putative sex pheromone-sensing pathway in arbuscular mycorrhizal fungi, a presumably asexual plant root symbiont.

    Directory of Open Access Journals (Sweden)

    Sébastien Halary

    Full Text Available The fungal kingdom displays a fascinating diversity of sex-determination systems. Recent advances in genomics provide insights into the molecular mechanisms of sex, mating type determination, and evolution of sexual reproduction in many fungal species in both ancient and modern phylogenetic lineages. All major fungal groups have evolved sexual differentiation and recombination pathways. However, sexuality is unknown in arbuscular mycorrhizal fungi (AMF of the phylum Glomeromycota, an ecologically vital group of obligate plant root symbionts. AMF are commonly considered an ancient asexual lineage dating back to the Ordovician, approximately 460 M years ago. In this study, we used genomic and transcriptomic surveys of several AMF species to demonstrate the presence of conserved putative sex pheromone-sensing mitogen-activated protein (MAP kinases, comparable to those described in Ascomycota and Basidiomycota. We also find genes for high mobility group (HMG transcription factors, homologous to SexM and SexP genes in the Mucorales. The SexM genes show a remarkable sequence diversity among multiple copies in the genome, while only a single SexP sequence was detected in some isolates of Rhizophagus irregularis. In the Mucorales and Microsporidia, the sexM gene is flanked by genes for a triosephosphate transporter (TPT and a RNA helicase, but we find no evidence for synteny in the vicinity of the Sex locus in AMF. Nonetheless, our results, together with previous observations on meiotic machinery, suggest that AMF could undergo a complete sexual reproduction cycle.

  2. Structure-Function Similarities between a Plant Receptor-like Kinase and the Human Interleukin-1 Receptor-associated Kinase-4*

    OpenAIRE

    2011-01-01

    Phylogenetic analysis has previously shown that plant receptor-like kinases (RLKs) are monophyletic with respect to the kinase domain and share an evolutionary origin with the animal interleukin-1 receptor-associated kinase/Pelle-soluble kinases. The lysin motif domain-containing receptor-like kinase-3 (LYK3) of the legume Medicago truncatula shows 33% amino acid sequence identity with human IRAK-4 over the kinase domain. Using the structure of this animal kinase as a template, homology model...

  3. Mnk kinase pathway: Cellular functions and biological outcomes

    Institute of Scientific and Technical Information of China (English)

    Sonali; Joshi; Leonidas; C; Platanias

    2014-01-01

    The mitogen-activated protein kinase(MAPK) interacting protein kinases 1 and 2(Mnk1 and Mnk2) play important roles in controlling signals involved in mRNA translation. In addition to the MAPKs(p38 or Erk), multiple studies suggest that the Mnk kinases can be regulated by other known kinases such as Pak2 and/or other unidentified kinases by phosphorylation of residues distinct from the sites phosphorylated by the MAPKs. Several studies have established multiple Mnk protein targets, including PSF, heterogenous nuclear ribonucleoprotein A1, Sprouty 2 and have lead to the identification of distinct biological functions and substrate specificity for the Mnk kinases. In this review we discuss the pathways regulating the Mnk kinases, their known substrates as well as the functional consequences of engagement of pathways controlled by Mnk kinases. These kinases play an important role in mRNA translation via their regulation of eukaryotic initiation factor 4E(eIF4E) and their functions have important implications in tumor biology as well as the regulation of drug resistance to anti-oncogenic therapies. Other studies have identified a role for the Mnk kinases in cap-independent mRNA translation, suggesting that the Mnk kinases can exert important functional effects independently of the phosphorylation of eIF4 E. The role of Mnk kinases in inflammation and inflammationinduced malignancies is also discussed.

  4. Heart failure-specific changes in protein kinase signalling.

    Science.gov (United States)

    Lorenz, Kristina; Stathopoulou, Konstantina; Schmid, Evelyn; Eder, Petra; Cuello, Friederike

    2014-06-01

    Among the myriad of molecular alterations occurring in heart failure development, aggravation of the disease is often attributed to global or local changes in protein kinase activity, thus making protein kinases attractive targets for therapeutic intervention. Since protein kinases do not only have maladaptive roles, but also contribute to the physiological integrity of cells, it is a challenging task to circumvent undesired inhibition of protein kinase activity. Identification of posttranslational modifications and/or protein-protein interactions that are exclusively apparent under pathophysiological conditions provides exciting information for alternative non-kinase inhibitory treatment strategies that eliminate maladaptive functions of a protein kinase, but preserve the beneficial ones. Here, we focus on the disease-specific regulation of a number of protein kinases, namely, Ca(2+)/calmodulin-dependent protein kinase II isoform δ (CaMKIIδ), G protein-coupled receptor kinase 2 (GRK2), extracellular signal-regulated kinase 1 and 2 (ERK1/2), protein kinase D (PKD) and protein kinase C isoform β2 (PKCβ2), which are embedded in complex signal transduction pathways implicated in heart failure development, and discuss potential avenues for novel treatment strategies to combat heart disease.

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

    Energy Technology Data Exchange (ETDEWEB)

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

    2011-12-31

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

  6. Asymmetric Tyrosine Kinase Arrangements in Activation or Autophosphorylation of Receptor Tyrosine Kinases

    Energy Technology Data Exchange (ETDEWEB)

    J Bae; J Schlessinger

    2011-12-31

    Receptor tyrosine kinases (RTKs) play important roles in the control of many cellular processes including cell proliferation, cell adhesion, angiogenesis, and apoptosis. Ligand-induced dimerization of RTKs leads to autophosphorylation and activation of RTKs. Structural studies have shown that while isolated ectodomains of several RTKs form symmetric dimers the isolated cytoplasmic kinase domains of epidermal growth factor receptor (EGFR) and fibroblast growth factor receptor (FGFR) form asymmetric dimers during their activation. Binding of one kinase molecule of EGFR to a second kinase molecule asymmetrically leads to stimulation of kinase activity and enhanced autophosphorylation. Furthermore, the structures of the kinase domain of FGFR1 and FGFR2 reveal the formation of asymmetric interfaces in the processes of autophosphorylation at their specific phosphotyrosine (pY) sites. Disruption of asymmetric dimer interface of EGFR leads to reduction in enzymatic activity and drastic reduction of autophosphorylation of FGFRs in ligandstimulated live cells. These studies demonstrate that asymmetric dimer formation is as a common phenomenon critical for activation and autophosphorylation of RTKs.

  7. Computational analysis of ABL kinase mutations allows predicting drug sensitivity against selective kinase inhibitors.

    Science.gov (United States)

    Kamasani, Swapna; Akula, Sravani; Sivan, Sree Kanth; Manga, Vijjulatha; Duyster, Justus; Vudem, Dashavantha Reddy; Kancha, Rama Krishna

    2017-05-01

    The ABL kinase inhibitor imatinib has been used as front-line therapy for Philadelphia-positive chronic myeloid leukemia. However, a significant proportion of imatinib-treated patients relapse due to occurrence of mutations in the ABL kinase domain. Although inhibitor sensitivity for a set of mutations was reported, the role of less frequent ABL kinase mutations in drug sensitivity/resistance is not known. Moreover, recent reports indicate distinct resistance profiles for second-generation ABL inhibitors. We thus employed a computational approach to predict drug sensitivity of 234 point mutations that were reported in chronic myeloid leukemia patients. Initial validation analysis of our approach using a panel of previously studied frequent mutations indicated that the computational data generated in this study correlated well with the published experimental/clinical data. In addition, we present drug sensitivity profiles for remaining point mutations by computational docking analysis using imatinib as well as next generation ABL inhibitors nilotinib, dasatinib, bosutinib, axitinib, and ponatinib. Our results indicate distinct drug sensitivity profiles for ABL mutants toward kinase inhibitors. In addition, drug sensitivity profiles of a set of compound mutations in ABL kinase were also presented in this study. Thus, our large scale computational study provides comprehensive sensitivity/resistance profiles of ABL mutations toward specific kinase inhibitors.

  8. Identification of putative effector genes and their transcripts in three strains related to 'Candidatus Phytoplasma aurantifolia'.

    Science.gov (United States)

    Anabestani, Ameneh; Izadpanah, Keramat; Abbà, Simona; Galetto, Luciana; Ghorbani, Abozar; Palmano, Sabrina; Siampour, Majid; Veratti, Flavio; Marzachì, Cristina

    2017-06-01

    Molecular mechanisms underlying phytoplasma interactions with host plants are largely unknown. In this study attempts were made to identify effectors of three phytoplasma strains related to 'Ca. P. aurantifolia', crotalaria phyllody (CrP), faba bean phyllody (FBP), and witches' broom disease of lime (WBDL), using information from draft genome of peanut witches' broom phytoplasma. Seven putative effectors were identified in WBDL genome (SAP11, SAP21, Eff64, Eff115, Eff197, Eff211 and EffSAP67), five (SAP11, SAP21, Eff64, Eff99 and Eff197) in CrP and two (SAP11, Eff64) in FBP. No homologs to Eff64, Eff197 and Eff211 in phytoplasmas of other phylogenetic groups were found. SAP11 and Eff64 homologs of 'Ca. P. aurantifolia' strains shared at least 95.9% identity and were detected in the three phytoplasmas, supporting their role within the group. Five of the putative effectors (SAP11, SAP21, Eff64, Eff115, and Eff99) were transcribed from total RNA extracts of periwinkle plants infected with these phytoplasmas. Transcription profiles of selected putative effectors of CrP, FBP and WBDL indicated that SAP11 transcripts were the most abundant in the three phytoplasmas. SAP21 transcript levels were comparable to those of SAP11 for CrP and not measurable for the other phytoplasmas. Eff64 had the lowest transcription level irrespective of sampling date and phytoplasma isolate. Eff115 transcript levels were the highest in WBDL infected plants. This work reports the first sequence information for 14 putative effectors in three strains related to 'Ca. P. aurantifolia', and offers novel insight into the transcription profile of five of them during infection of periwinkle. Copyright © 2017 Elsevier GmbH. All rights reserved.

  9. A novel putative auxin carrier family regulates intracellular auxin homeostasis in plants

    OpenAIRE

    2012-01-01

    The phytohormone auxin acts as a prominent signal, providing, by its local accumulation or depletion in selected cells, a spatial and temporal reference for changes in the developmental program. The distribution of auxin depends on both auxin metabolism (biosynthesis, conjugation and degradation) and cellular auxin transport. We identified in silico a novel putative auxin transport facilitator family, called PIN-LIKES (PILS). Here we illustrate that PILS proteins are required for auxin-depend...

  10. ANTIBIOTICS RESISTANCE AND PUTATIVE VIRULENCE FACTORS OF AEROMONAS HYDROPHILA ISOLATED FROM ESTUARY

    OpenAIRE

    Olumide Adedokun Odeyemi; Ahmad Asmat; Gires Usup

    2012-01-01

    This study aim to investigate antibiotics resistance profile and putative virulence factors of Aeromonas hydrophila isolated from estuary. Bacteria used for this study were isolated from water and sediment samples obtained from Sungai Melayu, Johor, Malaysia. Serially diluted 100 µL water and 1g sediment were inoculated on modified Rimler - Shott (mRS) agar. Colonies with distinct cultural characteristics were picked for further studies. Isolates were tested for biofilm productions, protease ...

  11. Bioinformatic Analysis of Putative Gene Products Encoded in SARS-HCoV Genome

    Institute of Scientific and Technical Information of China (English)

    赵心刚; 韩敬东; 宁元亨; 孟安明; 陈晔光

    2003-01-01

    The cause of severe acute respiratory syndrome (SARS) has been identified as a new coronavirus named as SARS-HCoV.Using bioinformatic methods, we have performed a detailed domain search.In addition to the viral structure proteins, we have found that several putative polypeptides share sequence similarity to known domains or proteins.This study may provide a basis for future studies on the infection and replication process of this notorious virus.

  12. Detection of putative periodontopathic bacteria in type 1 diabetic and healthy children: A comparative study

    OpenAIRE

    2013-01-01

    Aim: The aim of this study was to compare and assess the risk of periodontitis due to the presence of four putative periodontopathic bacteria (Porphyromonas gingivalis, Treponema denticola, Tannerella forsythia, and Aggregatibacter actinomycetemcomitans) in type 1 diabetic and healthy children. Materials and Methods: Fifty type 1 diabetic and 50 healthy children in the age group of 7-14 years were recruited for the study. Subgingival plaque samples collected from permanent first molars w...

  13. Immunodiagnosis of episomal Banana streak MY virus using polyclonal antibodies to an expressed putative coat protein.

    Science.gov (United States)

    Sharma, Susheel Kumar; Kumar, P Vignesh; Baranwal, Virendra Kumar

    2014-10-01

    A cryptic Badnavirus species complex, known as banana streak viruses (BSV) poses a serious threat to banana production and genetic improvement worldwide. Due to the presence of integrated BSV sequences in the banana genome, routine detection is largely based on serological and nucleo-serological diagnostic methods which require high titre specific polyclonal antiserum. Viral structural proteins like coat protein (CP) are the best target for in vitro expression, to be used as antigen for antiserum production. However, in badnaviruses precise CP sequences are not known. In this study, two putative CP coding regions (p48 and p37) of Banana streak MY virus (BSMYV) were identified in silico by comparison with caulimoviruses, retroviruses and Rice tungro bacilliform virus. The putative CP coding region (p37) was in vitro expressed in pMAL system and affinity purified. The purified fusion protein was used as antigen for raising polyclonal antiserum in rabbit. The specificity of antiserum was confirmed in Western blots, immunosorbent electron microscopy (ISEM) and antigen coated plate-enzyme linked immunosorbent assay (ACP-ELISA). The antiserum (1:2000) was successfully used in ACP-ELISA for specific detection of BSMYV infection in field and tissue culture raised banana plants. The antiserum was also utilized in immuno-capture PCR (IC-PCR) based indexing of episomal BSMYV infection. This is the first report of in silico identification of putative CP region of BSMYV, production of polyclonal antiserum against recombinant p37 and its successful use in immunodetection.

  14. Identification of Putative Coffee Rust Mycoparasites via Single-Molecule DNA Sequencing of Infected Pustules.

    Science.gov (United States)

    James, Timothy Y; Marino, John A; Perfecto, Ivette; Vandermeer, John

    2015-11-13

    The interaction of crop pests with their natural enemies is a fundament to their control. Natural enemies of fungal pathogens of crops are poorly known relative to those of insect pests, despite the diversity of fungal pathogens and their economic importance. Currently, many regions across Latin America are experiencing unprecedented epidemics of coffee rust (Hemileia vastatrix). Identification of natural enemies of coffee rust could aid in developing management strategies or in pinpointing species that could be used for biocontrol. In the present study, we characterized fungal communities associated with coffee rust lesions by single-molecule DNA sequencing of fungal rRNA gene bar codes from leaf discs (≈28 mm(2)) containing rust lesions and control discs with no rust lesions. The leaf disc communities were hyperdiverse in terms of fungi, with up to 69 operational taxonomic units (putative species) per control disc, and the diversity was only slightly reduced in rust-infected discs, with up to 63 putative species. However, geography had a greater influence on the fungal community than whether the disc was infected by coffee rust. Through comparisons between control and rust-infected leaf discs, as well as taxonomic criteria, we identified 15 putative mycoparasitic fungi. These fungi are concentrated in the fungal family Cordycipitaceae and the order Tremellales. These data emphasize the complexity of diverse fungi of unknown ecological function within a leaf that might influence plant disease epidemics or lead to the development of species for biocontrol of fungal disease.

  15. Emissions of putative isoprene oxidation products from mango branches under abiotic stress

    Science.gov (United States)

    Jardine, Kolby J.; Meyers, Kimberly; Abrell, Leif; Alves, Eliane G.; Yanez Serrano, Ana Maria; Kesselmeier, Jürgen; Karl, Thomas; Guenther, Alex; Vickers, Claudia; Chambers, Jeffrey Q.

    2013-01-01

    Although several per cent of net carbon assimilation can be re-released as isoprene emissions to the atmosphere by many tropical plants, much uncertainty remains regarding its biological significance. In a previous study, we detected emissions of isoprene and its oxidation products methyl vinyl ketone (MVK) and methacrolein (MACR) from tropical plants under high temperature/light stress, suggesting that isoprene is oxidized not only in the atmosphere but also within plants. However, a comprehensive analysis of the suite of isoprene oxidation products in plants has not been performed and production relationships with environmental stress have not been described. In this study, putative isoprene oxidation products from mango (Mangifera indica) branches under abiotic stress were first identified. High temperature/light and freeze–thaw treatments verified direct emissions of the isoprene oxidation products MVK and MACR together with the first observations of 3-methyl furan (3-MF) and 2-methyl-3-buten-2-ol (MBO) as putative novel isoprene oxidation products. Mechanical wounding also stimulated emissions of MVK and MACR. Photosynthesis under 13CO2 resulted in rapid (<30min) labelling of up to five carbon atoms of isoprene, with a similar labelling pattern observed in the putative oxidation products. These observations highlight the need to investigate further the mechanisms of isoprene oxidation within plants under stress and its biological and atmospheric significance. PMID:23881400

  16. Cloning and molecular characterization of a putative voltage-gated sodium channel gene in the crayfish.

    Science.gov (United States)

    Coskun, Cagil; Purali, Nuhan

    2016-06-01

    Voltage-gated sodium channel genes and associated proteins have been cloned and studied in many mammalian and invertebrate species. However, there is no data available about the sodium channel gene(s) in the crayfish, although the animal has frequently been used as a model to investigate various aspects of neural cellular and circuit function. In the present work, by using RNA extracts from crayfish abdominal ganglia samples, the complete open reading frame of a putative sodium channel gene has firstly been cloned and molecular properties of the associated peptide have been analyzed. The open reading frame of the gene has a length of 5793 bp that encodes for the synthesis of a peptide, with 1930 amino acids, that is 82% similar to the α-peptide of a sodium channel in a neighboring species, Cancer borealis. The transmembrane topology analysis of the crayfish peptide indicated a pattern of four folding domains with several transmembrane segments, as observed in other known voltage-gated sodium channels. Upon analysis of the obtained sequence, functional regions of the putative sodium channel responsible for the selectivity filter, inactivation gate, voltage sensor, and phosphorylation have been predicted. The expression level of the putative sodium channel gene, as defined by a qPCR method, was measured and found to be the highest in nervous tissue.

  17. Putative periodontopathic bacteria and herpesviruses in pregnant women: a case-control study

    Science.gov (United States)

    Lu, Haixia; Zhu, Ce; Li, Fei; Xu, Wei; Tao, Danying; Feng, Xiping

    2016-01-01

    Little is known about herpesvirus and putative periodontopathic bacteria in maternal chronic periodontitis. The present case-control study aimed to explore the potential relationship between putative periodontopathic bacteria and herpesviruses in maternal chronic periodontitis.Saliva samples were collected from 36 pregnant women with chronic periodontitis (cases) and 36 pregnant women with healthy periodontal status (controls). Six putative periodontopathic bacteria (Porphyromonas gingivalis [Pg], Aggregatibacer actinomycetemcomitans [Aa], Fusobacterium nucleatum [Fn], Prevotella intermedia [Pi], Tannerella forsythia [Tf], and Treponema denticola [Td]) and three herpesviruses (Epstein-Barr virus [EBV], human cytomegalovirus [HCMV], and herpes simplex virus [HSV]) were detected. Socio-demographic data and oral health related behaviors, and salivary estradiol and progesterone levels were also collected. The results showed no significant differences in socio-demographic background, oral health related behaviors, and salivary estradiol and progesterone levels between the two groups (all P > 0.05). The detection rates of included periodontopathic microorganisms were not significantly different between the two groups (all P > 0.05), but the coinfection rate of EBV and Pg was significantly higher in the case group than in the control group (P = 0.028). EBV and Pg coinfection may promote the development of chronic periodontitis among pregnant women. PMID:27301874

  18. Genomic identification of a putative circadian system in the cladoceran crustacean Daphnia pulex

    Science.gov (United States)

    Tilden, Andrea R.; McCoole, Matthew D.; Harmon, Sarah M.; Baer, Kevin N.; Christie, Andrew E.

    2011-01-01

    Essentially nothing is known about the molecular underpinnings of crustacean circadian clocks. The genome of Daphnia pulex, the only crustacean genome available for public use, provides a unique resource for identifying putative circadian proteins in this species. Here, the Daphnia genome was mined for putative circadian protein genes using Drosophila melanogaster queries. The sequences of core clock (e.g. CLOCK, CYCLE, PERIOD, TIMELESS and CRYPTOCHROME 2), clock input (CRYPTOCHROME 1) and clock output (PIGMENT DISPERSING HORMONE RECEPTOR) proteins were deduced. Structural analyses and alignment of the Daphnia proteins with their Drosophila counterparts revealed extensive sequence conservation, particularly in functional domains. Comparisons of the Daphnia proteins with other sequences showed that they are, in most cases, more similar to homologs from other species, including vertebrates, than they are to those of Drosophila. The presence of both CRYPTOCHROME 1 and 2 in Daphnia suggests the organization of its clock may be more similar to that of the butterfly Danaus plexippus than to that of Drosophila (which possesses CRYPTOCHROME 1 but not CRYPTOCHROME 2). These data represent the first description of a putative circadian system from any crustacean, and provide a foundation for future molecular, anatomical and physiological investigations of circadian signaling in Daphnia. PMID:21798832

  19. Phylogeny of algal sequences encoding carbohydrate sulfotransferases, formylglycine-dependent sulfatases and putative sulfatase modifying factors

    Directory of Open Access Journals (Sweden)

    Chai-Ling eHo

    2015-11-01

    Full Text Available Many algae are rich sources of sulfated polysaccharides with biological activities. The physicochemical/rheological properties and biological activities of sulfated polysaccharides are affected by the pattern and number of sulfate moieties. Sulfation of carbohydrates is catalyzed by carbohydrate sulfotransferases (CHSTs while modification of sulfate moieties on sulfated polysaccharides was presumably catalyzed by sulfatases including formylglycine-dependent sulfatases (FGly-SULFs. Post-translationally modification of Cys to FGly in FGly-SULFs by sulfatase modifiying factors (SUMFs is necessary for the activity of this enzyme. The aims of this study are to mine for sequences encoding algal CHSTs, FGly-SULFs and putative SUMFs from the fully sequenced algal genomes and to infer their phylogenetic relationships to their well characterized counterparts from other organisms. Algal sequences encoding CHSTs, FGly-SULFs, SUMFs and SUMF-like proteins were successfully identified from green and brown algae. However, red algal FGly-SULFs and SUMFs were not identified. In addition, a group of SUMF-like sequences with different gene structure and possibly different functions were identified for green, brown and red algae. The phylogeny of these putative genes contributes to the corpus of knowledge of an unexplored area. The analyses of these putative genes contribute towards future production of existing and new sulfated carbohydrate polymers through enzymatic synthesis and metabolic engineering.

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

    Science.gov (United States)

    Kim, Pora; Jia, Peilin; Zhao, Zhongming

    2016-12-24

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

  1. Regulators of gene expression in Enteric Neural Crest Cells are putative Hirschsprung disease genes

    NARCIS (Netherlands)

    Schriemer, Duco; Sribudiani, Yunia; IJpma, Arne; Natarajan, Dipa; MacKenzie, Katherine C.; Metzger, Marco; Binder, Ellen; Burns, Alan J.; Thapar, Nikhil; Hofstra, Robert M. W.; Eggen, Bart J. L.

    2016-01-01

    The enteric nervous system (ENS) is required for peristalsis of the gut and is derived from Enteric Neural Crest Cells (ENCCs). During ENS development, the RET receptor tyrosine kinase plays a critical role in the proliferation and survival of ENCCs, their migration along the developing gut, and dif

  2. Regulators of gene expression in Enteric Neural Crest Cells are putative Hirschsprung disease genes

    NARCIS (Netherlands)

    Schriemer, Duco; Sribudiani, Yunia; IJpma, Arne; Natarajan, Dipa; MacKenzie, Katherine C; Metzger, Marco; Binder, Ellen; Burns, Alan J; Thapar, Nikhil; Hofstra, Robert M W; Eggen, Bart J L

    2016-01-01

    The enteric nervous system (ENS) is required for peristalsis of the gut and is derived from enteric neural crest cells (ENCCs). During ENS development, the RET receptor tyrosine kinase plays a critical role in the proliferation and survival of ENCCs, their migration along the developing gut, and dif

  3. Enhanced resistance to soybean cyst nematode Heterodera glycines in transgenic soybean by silencing putative CLE receptors

    Science.gov (United States)

    CLE peptides are small extracellular proteins important in regulating plant meristematic activity through the CLE-receptor kinase-WOX signaling module. Stem cell pools in the SAM (shoot apical meristem), RAM (root apical meristem), and vascular cambium are tightly controlled by CLE signaling pathway...

  4. A retroviral-derived peptide phosphorylates protein kinase D/protein kinase Cmu involving phospholipase C and protein kinase C.

    Science.gov (United States)

    Luangwedchakarn, Voravich; Day, Noorbibi K; Hitchcock, Remi; Brown, Pam G; Lerner, Danica L; Rucker, Rajivi P; Cianciolo, George J; Good, Robert A; Haraguchi, Soichi

    2003-05-01

    CKS-17, a synthetic peptide representing a unique amino acid motif which is highly conserved in retroviral transmembrane proteins and other immunoregulatory proteins, induces selective immunomodulatory functions, both in vitro and in vivo, and activates intracellular signaling molecules such as cAMP and extracellular signal-regulated kinases. In the present study, using Jurkat T-cells, we report that CKS-17 phosphorylates protein kinase D (PKD)/protein kinase C (PKC) mu. Total cell extracts from CKS-17-stimulated Jurkat cells were immunoblotted with an anti-phospho-PKCmu antibody. The results show that CKS-17 significantly phosphorylates PKD/PKCmu in a dose- and time-dependent manner. Treatment of cells with the PKC inhibitors GF 109203X and Ro 31-8220, which do not act directly on PKD/PKCmu, attenuates CKS-17-induced phosphorylation of PKD/PKCmu. In contrast, the selective protein kinase A inhibitor H-89 does not reverse the action of CKS-17. Furthermore, a phospholipase C (PLC) selective inhibitor, U-73122, completely blocks the phosphorylation of PKD/PKCmu by CKS-17 while a negative control U-73343 does not. In addition, substitution of lysine for arginine residues in the CKS-17 sequence completely abrogates the ability of CKS-17 to phosphorylate PKD/PKCmu. These results clearly indicate that CKS-17 phosphorylates PKD/PKCmu through a PLC- and PKC-dependent mechanism and that arginine residues play an essential role in this activity of CKS-17, presenting a novel modality of the retroviral peptide CKS-17 and molecular interaction of this compound with target cells.

  5. Localization of putative carbonic anhydrases in two marine diatoms, Phaeodactylum tricornutum and Thalassiosira pseudonana.

    Science.gov (United States)

    Tachibana, Masaaki; Allen, Andrew E; Kikutani, Sae; Endo, Yuri; Bowler, Chris; Matsuda, Yusuke

    2011-09-01

    It is believed that intracellular carbonic anhydrases (CAs) are essential components of carbon concentrating mechanisms in microalgae. In this study, putative CA-encoding genes were identified in the genome sequences of the marine diatoms Phaeodactylum tricornutum and Thalassiosira pseudonana. Subsequently, the subcellular localizations of the encoded proteins were determined. Nine and thirteen CA sequences were found in the genomes of P. tricornutum and T. pseudonana, respectively. Two of the β-CA genes in P. tricornutum corresponded to ptca1 and ptca2 identified previously. Immunostaining transmission electron microscopy of a PtCA1:YFP fusion expressed in the cells of P. tricornutum clearly showed the localization of PtCA1 within the central part of the pyrenoid structure in the chloroplast. Besides these two β-CA genes, P. tricornutum likely contains five α- and two γ-CA genes, whereas T. pseudonana has three α-, five γ-, four δ-, and one ζ-CA genes. Semi-quantitative reverse transcription PCR performed on mRNA from the two diatoms grown in changing light and CO(2) conditions revealed that levels of six putative α- and γ-CA mRNAs in P. tricornutum did not change between cells grown in air-level CO(2) and 5% CO(2). However, mRNA levels of one putative α-CA gene, CA-VII in P. tricornutum, were reduced in the dark compared to that in the light. In T. pseudonana, mRNA accumulation levels of putative α-CA (CA-1), ζ-CA (CA-3) and δ-CA (CA-7) were analyzed and all levels found to be significantly reduced when cells were grown in 0.16% CO(2). Intercellular localizations of eight putative CAs were analyzed by expressing GFP fusion in P. tricornutum and T. pseudonana. In P. tricornutum, CA-I and II localized in the periplastidial compartment, CA-III, VI, VII were found in the chloroplast endoplasmic reticulum, and CA-VIII was localized in the mitochondria. On the other hand, T. pseudonana CA-1 localized in the stroma and CA-3 was found in the periplasm

  6. Entry into mitosis without Cdc2 kinase activation.

    Science.gov (United States)

    Gowdy, P M; Anderson, H J; Roberge, M

    1998-11-01

    Mouse FT210 cells at 39 degreesC cannot enter mitosis but arrest in G2 phase, because they lack Cdc2 kinase activity as a result of a temperature-sensitive lesion in the cdc2 gene. Incubation of arrested cells with the protein phosphatase 1 and 2A inhibitor okadaic acid induces morphologically normal chromosome condensation. We now show that okadaic acid also induces two other landmark events of early mitosis, nuclear lamina depolymerization and centrosome separation, in the absence of Cdc2 kinase activity. Okadaic acid-induced entry into mitosis is accompanied by partial activation of Cdc25C and may be prevented by tyrosine phosphatase inhibitors and by the protein kinase inhibitor staurosporine, suggesting that Cdc25C and kinases distinct from Cdc2 are required for these mitotic events. Using in-gel assays, we show that a 45-kDa protein kinase normally activated at mitosis is also activated by okadaic acid independently of Cdc2 kinase. The 45-kDa kinase can utilize GTP, is stimulated by spermine and is inhibited by heparin. These properties are characteristic of the kinase CK2, but immunoprecipitation studies indicate that it is not CK2. The data underline the importance of a tyrosine phosphatase, possibly Cdc25C, and of kinases other than Cdc2 in the structural changes the cell undergoes at mitosis, and indicate that entry into mitosis involves the activation of multiple kinases working in concert with Cdc2 kinase.

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

    Science.gov (United States)

    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.

  8. A Global Protein Kinase and Phosphatase Interaction Network in Yeast

    Science.gov (United States)

    Breitkreutz, Ashton; Choi, Hyungwon; Sharom, Jeffrey R.; Boucher, Lorrie; Neduva, Victor; Larsen, Brett; Lin, Zhen-Yuan; Breitkreutz, Bobby-Joe; Stark, Chris; Liu, Guomin; Ahn, Jessica; Dewar-Darch, Danielle; Reguly, Teresa; Tang, Xiaojing; Almeida, Ricardo; Qin, Zhaohui Steve; Pawson, Tony; Gingras, Anne-Claude; Nesvizhskii, Alexey I.; Tyers, Mike

    2011-01-01

    The interactions of protein kinases and phosphatases with their regulatory subunits and substrates underpin cellular regulation. We identified a kinase and phosphatase interaction (KPI) network of 1844 interactions in budding yeast by mass spectrometric analysis of protein complexes. The KPI network contained many dense local regions of interactions that suggested new functions. Notably, the cell cycle phosphatase Cdc14 associated with multiple kinases that revealed roles for Cdc14 in mitogen-activated protein kinase signaling, the DNA damage response, and metabolism, whereas interactions of the target of rapamycin complex 1 (TORC1) uncovered new effector kinases in nitrogen and carbon metabolism. An extensive backbone of kinase-kinase interactions cross-connects the proteome and may serve to coordinate diverse cellular responses. PMID:20489023

  9. Phosphatidylinositol 4-kinases: Function, structure, and inhibition

    Energy Technology Data Exchange (ETDEWEB)

    Boura, Evzen, E-mail: boura@uochb.cas.cz; Nencka, Radim, E-mail: nencka@uochb.cas.cz

    2015-10-01

    The phosphatidylinositol 4-kinases (PI4Ks) synthesize phosphatidylinositol 4-phosphate (PI4P), a key member of the phosphoinositide family. PI4P defines the membranes of Golgi and trans-Golgi network (TGN) and regulates trafficking to and from the Golgi. Humans have two type II PI4Ks (α and β) and two type III enzymes (α and β). Recently, the crystal structures were solved for both type II and type III kinase revealing atomic details of their function. Importantly, the type III PI4Ks are hijacked by +RNA viruses to create so-called membranous web, an extensively phosphorylated and modified membrane system dedicated to their replication. Therefore, selective and potent inhibitors of PI4Ks have been developed as potential antiviral agents. Here we focus on the structure and function of PI4Ks and their potential in human medicine.

  10. Bisarylmaleimides & the Corresponding Indolocarbazoles as Kinase Inhibitors

    Institute of Scientific and Technical Information of China (English)

    Zhu Guoxin; Cathy Ogg; Bharvin Patel; Richard M. Schultz; Charles D. Spencer; Beverly Teicher; Scou A. Watkins; Scott E. Conner; Zhou Xun; Chuan Shih; Li Tiechao; Harold B. Brooks; Eileen Considine; Jack A. Dempsey; Margaret M. Faul

    2004-01-01

    Cyclin dependent kinases (CDKs) have recently raised considerable attention because of their central role in the regulation of cell cycle progression. A high incidence of genetic mutation of CDK substrates and deregulaton of CDK modulators were found in a number of disease states,particularly in cancer. A novel series of unsymmetrical substituted indolocarbazoles were synthesized and their kinase inhibitory capability was evaluated in vitro. 6-Substtuted indolocarbazoles were found to be highly potent and selective D1/CDK4 inhibitors. These indolocarbazoles exhibited ATP competitive D1/CDK4 activity and inhibited tumor cell growth,arrested tumor cell at G1 phase. These molecules demonstrated potent anti-tumor activity and inhibited pRb phosphorylation at S780 in the human lung carcinoma (Calu6) and non-small cell lung carcinoma (NCI-H460) xenograft models. The results indicate that these small molecules have potential as therapeutic agents in cancer chemotherapeutc agents.

  11. Targeting checkpoint kinase 1 in cancer therapeutics.

    Science.gov (United States)

    Tse, Archie N; Carvajal, Richard; Schwartz, Gary K

    2007-04-01

    Progression through the cell cycle is monitored by surveillance mechanisms known as cell cycle checkpoints. Our knowledge of the biochemical nature of checkpoint regulation during an unperturbed cell cycle and following DNA damage has expanded tremendously over the past decade. We now know that dysfunction in cell cycle checkpoints leads to genomic instability and contributes to tumor progression, and most agents used for cancer therapy, such as cytotoxic chemotherapy and ionizing radiation, also activate cell cycle checkpoints. Understanding how checkpoints are regulated is therefore important from the points of view of both tumorigenesis and cancer treatment. In this review, we present an overview of the molecular hierarchy of the checkpoint signaling network and the emerging role of checkpoint targets, especially checkpoint kinase 1, in cancer therapy. Further, we discuss the results of recent clinical trials involving the nonspecific checkpoint kinase 1 inhibitor, UCN-01, and the challenges we face with this new therapeutic approach.

  12. Isoprenoid biosynthesis and mevalonate kinase deficiency

    OpenAIRE

    Henneman, L.

    2011-01-01

    Mevalonaat Kinase Deficiëntie (MKD) is een aangeboren ziekte geassocieerd met heftige koortsaanvallen die drie tot vier dagen aanhouden en gepaard gaan met koude rillingen, gewrichtsklachten, huiduitslag, hoofdpijn, duizeligheid, buikpijn, braken en diarree. De koortsaanvallen treden gemiddeld eens in de drie tot zes weken op zonder dat goed duidelijk is waarom. Linda Henneman toont aan dat door een tekort van bepaalde metabolieten specifieke signaaleiwitten op de verkeerde plek in de cel ter...

  13. ABL Tyrosine Kinase Stimulates PUMA Protein Expression

    OpenAIRE

    Oon, Chet K

    2016-01-01

    ABL is an ubiquitously expressed non-receptor tyrosine kinase involved in multiple cellular functions including programmed cell death. Upon DNA damage, ABL has been shown to upregulate PUMA, p53 upregulated modulator of apoptosis, and causes downstream mitochondrial intrinsic apoptotic events. However, the mechanism by which ABL regulates PUMA expression remains unknown. We have shown that ABL does not change PUMA protein subcellular localization through immunofluorescence. Through protein an...

  14. Molecular Imaging of the ATM Kinase Activity

    Energy Technology Data Exchange (ETDEWEB)

    Williams, Terence M. [Department of Radiation Oncology, Ohio State University, Columbus, Ohio (United States); Nyati, Shyam [Department of Radiation Oncology, University of Michigan Medical Center, Ann Arbor, Michigan (United States); Center for Molecular Imaging, University of Michigan Medical Center, Ann Arbor, Michigan (United States); Ross, Brian D. [Department of Radiation Oncology, University of Michigan Medical Center, Ann Arbor, Michigan (United States); Department of Radiology, University of Michigan Medical Center, Ann Arbor, Michigan (United States); Rehemtulla, Alnawaz, E-mail: alnawaz@umich.edu [Department of Radiation Oncology, University of Michigan Medical Center, Ann Arbor, Michigan (United States); Center for Molecular Imaging, University of Michigan Medical Center, Ann Arbor, Michigan (United States); Department of Radiology, University of Michigan Medical Center, Ann Arbor, Michigan (United States)

    2013-08-01

    Purpose: Ataxia telangiectasia mutated (ATM) is a serine/threonine kinase critical to the cellular DNA-damage response, including from DNA double-strand breaks. ATM activation results in the initiation of a complex cascade of events including DNA damage repair, cell cycle checkpoint control, and survival. We sought to create a bioluminescent reporter that dynamically and noninvasively measures ATM kinase activity in living cells and subjects. Methods and Materials: Using the split luciferase technology, we constructed a hybrid cDNA, ATM-reporter (ATMR), coding for a protein that quantitatively reports on changes in ATM kinase activity through changes in bioluminescence. Results: Treatment of ATMR-expressing cells with ATM inhibitors resulted in a dose-dependent increase in bioluminescence activity. In contrast, induction of ATM kinase activity upon irradiation resulted in a decrease in reporter activity that correlated with ATM and Chk2 activation by immunoblotting in a time-dependent fashion. Nuclear targeting improved ATMR sensitivity to both ATM inhibitors and radiation, whereas a mutant ATMR (lacking the target phosphorylation site) displayed a muted response. Treatment with ATM inhibitors and small interfering (si)RNA-targeted knockdown of ATM confirm the specificity of the reporter. Using reporter expressing xenografted tumors demonstrated the ability of ATMR to report in ATM activity in mouse models that correlated in a time-dependent fashion with changes in Chk2 activity. Conclusions: We describe the development and validation of a novel, specific, noninvasive bioluminescent reporter that enables monitoring of ATM activity in real time, in vitro and in vivo. Potential applications of this reporter include the identification and development of novel ATM inhibitors or ATM-interacting partners through high-throughput screens and in vivo pharmacokinetic/pharmacodynamic studies of ATM inhibitors in preclinical models.

  15. Inhibitors of p21-activated kinases (PAKs).

    Science.gov (United States)

    Rudolph, Joachim; Crawford, James J; Hoeflich, Klaus P; Wang, Weiru

    2015-01-08

    The p21-activated kinase (PAK) family of serine/threonine protein kinases plays important roles in cytoskeletal organization, cellular morphogenesis, and survival, and members of this family have been implicated in many diseases including cancer, infectious diseases, and neurological disorders. Owing to their large and flexible ATP binding cleft, PAKs, particularly group I PAKs (PAK1, -2, and -3), are difficult to drug; hence, few PAK inhibitors with satisfactory kinase selectivity and druglike properties have been reported to date. Examples are a recently discovered group II PAK (PAK4, -5, -6) selective inhibitor series based on a benzimidazole core, a group I PAK selective series based on a pyrido[2,3-d]pyrimidine-7-one core, and an allosteric dibenzodiazepine PAK1 inhibitor series. Only one compound, an aminopyrazole based pan-PAK inhibitor, entered clinical trials but did not progress beyond phase I trials. Clinical proof of concept for pan-group I, pan-group II, or PAK isoform selective inhibition has yet to be demonstrated.

  16. Implication of Ceramide Kinase in Adipogenesis

    Directory of Open Access Journals (Sweden)

    Marta Ordoñez

    2017-01-01

    Full Text Available Ceramide kinase (CerK plays a critical role in the regulation of cell growth and survival and has been implicated in proinflammatory responses. In this work, we demonstrate that CerK regulates adipocyte differentiation, a process associated with obesity, which causes chronic low-grade inflammation. CerK was upregulated during differentiation of 3T3-L1 preadipocytes into mature adipocytes. Noteworthy, knockdown of CerK using specific siRNA to silence the gene encoding this kinase resulted in substantial decrease of lipid droplet formation and potent depletion in the content of triacylglycerols in the adipocytes. Additionally, CerK knockdown caused blockade of leptin secretion, an adipokine that is crucial for regulation of energy balance in the organism and that is increased in the obese state. Moreover, CerK gene silencing decreased the expression of peroxisome proliferator-activated receptor gamma (PPARγ, which is considered the master regulator of adipogenesis. It can be concluded that CerK is a novel regulator of adipogenesis, an action that may have potential implications in the development of obesity, and that targeting this kinase may be beneficial for treatment of obesity-associated diseases.

  17. Diacylglycerol Kinase Inhibition and Vascular Function.

    Science.gov (United States)

    Choi, Hyehun; Allahdadi, Kyan J; Tostes, Rita C A; Webb, R Clinton

    2009-01-01

    Diacylglycerol kinases (DGKs), a family of lipid kinases, convert diacylglycerol (DG) to phosphatidic acid (PA). Acting as a second messenger, DG activates protein kinase C (PKC). PA, a signaling lipid, regulates diverse functions involved in physiological responses. Since DGK modulates two lipid second messengers, DG and PA, regulation of DGK could induce related cellular responses. Currently, there are 10 mammalian isoforms of DGK that are categorized into five groups based on their structural features. These diverse isoforms of DGK are considered to activate distinct cellular functions according to extracellular stimuli. Each DGK isoform is thought to play various roles inside the cell, depending on its subcellular localization (nuclear, ER, Golgi complex or cytoplasm). In vascular smooth muscle, vasoconstrictors such as angiotensin II, endothelin-1 and norepinephrine stimulate contraction by increasing inositol trisphosphate (IP(3)), calcium, DG and PKC activity. Inhibition of DGK could increase DG availability and decrease PA levels, as well as alter intracellular responses, including calcium-mediated and PKC-mediated vascular contraction. The purpose of this review is to demonstrate a role of DGK in vascular function. Selective inhibition of DGK isoforms may represent a novel therapeutic approach in vascular dysfunction.

  18. Rapamycin inhibition of baculovirus recombinant (BVr ribosomal protein S6 kinase (S6K1 is mediated by an event other than phosphorylation

    Directory of Open Access Journals (Sweden)

    Beigh Mushtaq A

    2012-03-01

    Full Text Available Abstract Background Ribosomal protein S6 kinase 1(S6K1 is an evolutionary conserved kinase that is activated in response to growth factors and viral stimuli to influence cellular growth and proliferation. This downstream effector of target of rapamycin (TOR signaling cascade is known to be directly activated by TOR- kinase mediated hydrophobic motif (HM phosphorylation at Threonine 412 (T412. Selective loss of this phosphorylation by inactivation of TOR kinase or activation/recruitment of a phosphatase has accordingly been implicated in mediating inhibition by rapamycin. Findings We present evidence that baculovirus driven expression of S6K1 in insect cells (Sf9 fails to activate the enzyme and instead renders it modestly active representing 4-6 folds less activity than its fully active mammalian counterpart. Contrary to the contention that viral infection activates TOR signaling pathway, we report that BVr enzyme fails to exhibit putative TOR dependent phosphorylation at the HM and the resultant phosphorylation at the activation loop (AL of the enzyme, correlating with the level of activity observed. Surprisingly, the BVr enzyme continued to exhibit sensitivity to rapamycin that remained unaffected by mutations compromised for TOR phosphorylation (T412A or deletions compromised for TOR binding (ΔNH 2-46/ΔCT104. Conclusions These data together with the ability of the BVr enzyme to resist inactivation by phosphatases indicate that inhibition by rapamycin is not mediated by any phosphorylation event in general and TOR dependent phosphorylation in particular.

  19. rse, a novel receptor-type tyrosine kinase with homology to Axl/Ufo, is expressed at high levels in the brain.

    Science.gov (United States)

    Mark, M R; Scadden, D T; Wang, Z; Gu, Q; Goddard, A; Godowski, P J

    1994-04-08

    We have isolated cDNA clones that encode the human and murine forms of a novel receptor-type tyrosine kinase termed Rse. Sequence analysis indicates that human Rse contains 890 amino acids, with an extracellular region composed of two immunoglobulin-like domains followed by two fibronectin type III domains. Murine Rse contains 880 amino acids and shares 90% amino acid identity with its human counterpart. Rse is structurally similar to the receptor-type tyrosine kinase Axl/Ufo, and the two proteins have 35 and 63% sequence identity in their extracellular and intracellular domains, respectively. To study the synthesis and activation of this putative receptor-type tyrosine kinase, we constructed a version of Rse (termed gD-Rse, where gD represents glycoprotein D) that contains an NH2-terminal epitope tag. NIH3T3 cells were engineered to express gD-Rse, which could be detected at the cell surface by fluorescence-activated cell sorting. Moreover, gD-Rse was rapidly phosphorylated on tyrosine residues upon incubation of the cells with an antibody directed against the epitope tag, suggesting that rse encodes an active tyrosine kinase. In the human tissues we examined, the highest level of expression of rse mRNA was observed in the brain; rse mRNA was also detected in the premegakaryocytopoietic cell lines CMK11-5 and Dami. The gene for rse was localized to human chromosome 15.

  20. Biochemical and functional characterization of a eukaryotic-type protein kinase, SpkB, in the cyanobacterium, Synechocystis sp. PCC 6803.

    Science.gov (United States)

    Kamei, Ayako; Yoshihara, Shizue; Yuasa, Takashi; Geng, Xiaoxing; Ikeuchi, Masahiko

    2003-04-01

    On the basis of the genome sequence, the unicellular motile cyanobacterium Synechocystis sp. PCC 6803 harbors seven putative genes for eukaryotic-type protein kinase belonging to Pkn2 subfamily ( spkA approximately spkG). Previously, SpkA was shown to have protein kinase activity and to be required for cell motility. Here, the role of the spkB was examined. The spkB gene was expressed in Escherichia coli as a fusion protein with His-tag, and the protein was purified by Ni(2+) affinity chromatography. The eukaryotic-type protein kinase activity of the expressed SpkB was demonstrated as autophosphorylation to itself and phosphorylation of the general substrate proteins. SpkB showed autophosphorylation activity in the presence of both Mg(2+) and Mn(2+), but not in Ca(2+). Phenotype analysis of spkB disruptant of Synechocystis revealed that spkB is required for cell motility, but not for phototaxis. These results suggest that SpkB is the eukaryotic-type protein kinase, which regulates cellular motility via protein phosphorylation like SpkA.

  1. Alternaric acid stimulates phosphorylation of His-tagged RiCDPK2, a calcium-dependent protein kinase in potato plants.

    Science.gov (United States)

    Hassan, A; Okuta, T; Kato, M; Hatsugai, N; Sano, Y; Ishimori, T; Okazaki, K; Doullah, M A; Shah, M M

    2012-08-16

    Calcium-dependent protein kinases (CDPK) are an essential component of plant defense mechanisms against pathogens. We investigated the effect of alternaric acid, a host-specific toxin produced by the plant fungal pathogen Alternaria solani (Pleosporaceae), on a putative plasma membrane and cytosolic kinase RiCDPK2 of potato (Solanum tuberosum) and on hypersensitive cell death of host potato cells. Alternaric acid, in the presence of Ca²⁺ and Mg²⁺, stimulated in vitro phosphorylation of His-tagged RiCDPK2, a Ca²⁺-dependent protein kinase found in potato plants. We concluded that Ca²⁺ and Mg²⁺ play an important role in the interaction between alternaric acid and RiCDPK2. Based on our observations, alternaric acid regulates RiCDPK2 kinase during the infection process in an interaction between host and A. solani, leading to the inhibition of hypersensitive cell death in the host. We suggest that alternaric acid is a primary determinant by which A. solani stimulates CDPK activity in the host, suppressing hypersensitive cell death.

  2. Hexose-6-kinases in germinating honey locust cotyledons: substrate specificity of D-fructo-6-kinase.

    Science.gov (United States)

    Myers, D; Matheson, N K

    1994-11-01

    Extracts of the cotyledons of germinated honey locust (Gleditsia triacanthos) seeds, which contain galactomannan as a reserve polysaccharide in the endosperm, were fractionated by chromatography and the fractions examined for the presence of a specific manno-6-kinase which could phosphorylate the D-mannose released by hydrolysis of galactomannan. One particulate hexokinase (the major hexose-6-kinase fraction) and two soluble hexokinase fractions (the minor portion), as well as a soluble fructo-6-kinase fraction, were initially separated. From chromatography, electrophoresis and kinetic studies, no evidence for a specific manno-kinase was obtained. This and the level and kinetic behaviour of the particulate hexokinase implicated it as the enzyme catalysing the phosphorylation of released D-mannose. The fructo-kinase activity was further separated into three fractions. Kinetic studies on one of these with native and synthetic substrates indicated that the structural requirements for the monosaccharide substrate were a beta-D-anomeric 2-OH in the furanose ring, a 4-OH trans to the D-5-CH2OH and a -CH2OH substituent on C2 (trans to the 5-CH2OH) which could be modified. The orientation of the hydroxyl on C-3 had only a limited effect.

  3. Conservation and early expression of zebrafish tyrosine kinases support the utility of zebrafish as a model for tyrosine kinase biology.

    Science.gov (United States)

    Challa, Anil Kumar; Chatti, Kiranam

    2013-09-01

    Tyrosine kinases have significant roles in cell growth, apoptosis, development, and disease. To explore the use of zebrafish as a vertebrate model for tyrosine kinase signaling and to better understand their roles, we have identified all of the tyrosine kinases encoded in the zebrafish genome and quantified RNA expression of selected tyrosine kinases during early development. Using profile hidden Markov model analysis, we identified 122 zebrafish tyrosine kinase genes and proposed unambiguous gene names where needed. We found them to be organized into 39 nonreceptor and 83 receptor type, and 30 families consistent with human tyrosine kinase family assignments. We found five human tyrosine kinase genes (epha1, bmx, fgr, srm, and insrr) with no identifiable zebrafish ortholog, and one zebrafish gene (yrk) with no identifiable human ortholog. We also found that receptor tyrosine kinase genes were duplicated more often than nonreceptor tyrosine kinase genes in zebrafish. We profiled expression levels of 30 tyrosine kinases representing all families using direct digital detection at different stages during the first 24 hours of development. The profiling experiments clearly indicate regulated expression of tyrosine kinases in the zebrafish, suggesting their role during early embryonic development. In summary, our study has resulted in the first comprehensive description of the zebrafish tyrosine kinome.

  4. Phosphoinositide 3-kinase regulates crosstalk between Trk A tyrosine kinase and p75(NTR)-dependent sphingolipid signaling pathways.

    Science.gov (United States)

    Bilderback, T R; Gazula, V R; Dobrowsky, R T

    2001-03-01

    The mechanism of crosstalk between signaling pathways coupled to the Trk A and p75(NTR) neurotrophin receptors in PC12 cells was examined. In response to nerve growth factor (NGF), Trk A activation inhibited p75(NTR)-dependent sphingomyelin (SM) hydrolysis. The phosphoinositide 3-kinase (PI 3-kinase) inhibitor, LY294002, reversed this inhibition suggesting that Trk A activation of PI 3-kinase is necessary to inhibit sphingolipid signaling by p75(NTR). In contrast, SM hydrolysis induced by neurotrophin-3 (NT-3), which did not activate PI-3 kinase, was uneffected by LY294002. However, transient expression of a constituitively active PI 3-kinase inhibited p75(NTR)-dependent SM hydrolysis by both NGF and NT-3. Intriguingly, NGF induced an association of activated PI 3-kinase with acid sphingomyelinase (SMase). This interaction localized to caveolae-related domains and correlated with a 50% decrease in immunoprecipitated acid SMase activity. NGF-stimulated PI 3-kinase activity was necessary for inhibition of acid SMase but was not required for ligand-induced association of the p85 subunit of PI 3-kinase with the phospholipase. Finally, this interaction was specific for NGF since EGF did not induce an association of PI 3-kinase with acid SMase. In summary, our data suggest that PI 3-kinase regulates the inhibitory crosstalk between Trk A tyrosine kinase and p75(NTR)-dependent sphingolipid signaling pathways and that this interaction localizes to caveolae-related domains.

  5. A Hypothesis for the Abiotic and Non-Martian Origins of Putative Signs of Ancient Martian Life in ALH84001

    Science.gov (United States)

    Treiman, Allan H.

    2001-01-01

    Putative evidence of martian life in ALH84001 can be explained by abiotic and non-martian processes consistent with the meteorite's geological history. Additional information is contained in the original extended abstract.

  6. Haemolytic and cytotoxic activities of the Tween 80-extracted putative haemolysin of Pasteurella multocida B:2.

    Science.gov (United States)

    Chachra, D; Coote, J G; Parton, R; Jand, S K

    2011-06-02

    The objective of this study was to investigate the haemolytic and cytotoxic activity of Pasteurella multocida B:2 strains, originally from cases of haemorrhagic septicaemia in cattle. All six P. multocida B:2 strains were non-haemolytic on sheep blood agar (SBA) and horse blood agar (HBA) when grown aerobically and on SBA anaerobically but they were haemolytic on HBA when grown anaerobically. No haemolytic activity against horse red blood cells was detected in culture supernates from aerobically or anaerobically grown cultures and only very weak haemolytic activity was obtained in supernates or pellet fractions from sonicated cells. However, after repeated extraction of sonicated cells with Tween 80, haemolytic activity was found in various cell fractions, both Tween-soluble and -insoluble. The Tween-extracted putative haemolysin and other bacterial fractions were also cytotoxic for mouse macrophage-like J774.2 cells. Further characterisation of the putative haemolysin revealed it to be a heat-labile, non-pore-forming protein of molecular weight >10 kDa whose activity was completely destroyed by trypsin and greatly reduced with protease and proteinase K treatment. Congo red also reduced the haemolytic activity. Non-denaturing gel-electrophoresis and RBC agar overlay revealed clear haemolytic zones but suggested that Tween was bound to some component of the P. multocida B:2 fractions and was responsible, to some extent, for the haemolytic activity observed. However, the effect of heat and other reagents on the Tween-extracted fractions and the lack of haemolytic activity in different Tween-extracted cell fractions of organisms other than P. multocida suggested that some proteinaceous component of the organism could indeed act as a haemolysin. This putative haemolysin may be one of the virulence attributes of P. multocida, but its characterisation and role in pathogenesis require further study.

  7. Isolation and characterization of two mitoviruses and a putative alphapartitivirus from Fusarium spp.

    Science.gov (United States)

    Osaki, Hideki; Sasaki, Atsuko; Nomiyama, Koji; Sekiguchi, Hiroyuki; Tomioka, Keisuke; Takehara, Toshiaki

    2015-06-01

    The filamentous fungus Fusarium spp. includes several important plant pathogens. We attempted to reveal presence of double-stranded (ds) RNAs in the genus. Thirty-seven Fusarium spp. at the MAFF collection were analyzed. In the strains of Fusarium coeruleum, Fusarium globosum and Fusarium solani f. sp. pisi, single dsRNA bands were detected. The strains of F. coeruleum and F. solani f. sp. pisi cause potato dry rot and mulberry twig blight, respectively. Sequence analyses revealed that dsRNAs in F. coeruleum and F. globosum consisted of 2423 and 2414 bp, respectively. Using the fungal mitochondrial translation table, the positive strands of these cDNAs were found to contain single open reading frames with the potential to encode a protein of putative 757 and 717 amino acids (molecular mass 88.5 and 84.0 kDa, respectively), similar to RNA-dependent RNA polymerases of members of the genus Mitovirus. These dsRNAs in F. coeruleum and F. globosum were assigned to the genus Mitovirus (family Narnaviridae), and these two mitoviruses were designated as Fusarium coeruleum mitovirus 1 and Fusarium globosum mitovirus 1. On the other hand, a positive strand of cDNA (1950 bp) from dsRNA in F. solani f. sp. pisi contained an ORF potentially encoding a putative RdRp of 608 amino acids (72.0 kDa). The putative RdRp was shown to be related to those of members of the genus of Alphapartitivirus (family Partitiviridae). We coined the name Fusarium solani partitivirus 2 for dsRNA in F. solani f. sp. pisi.

  8. The intracellular mobility of NPY and a putative mitochondrial form of NPY in neuronal cells.

    Science.gov (United States)

    Kaipio, Katja; Pesonen, Ullamari

    2009-01-30

    Preproneuropeptide Y is a precursor peptide to mature neuropeptide Y (NPY), which is a universally expressed peptide in the central and peripheral nervous system. NPY is normally routed to endoplasmic reticulum and secretory vesicles in cells, which secrete NPY. In our previous studies, we found a functional Leucine7 to Proline7 (L7P) polymorphism in the signal peptide sequence of preproNPY. This polymorphism affects the secretion of NPY and causes multiple physiological effects in humans. The sequence of NPY mRNA contains two in frame kozak sequences that allow translation initiation to shift, and translation of two proteins. In addition to mature NPY(1-36) also a putative truncated NPY(17-36) with mitochondrial targeting signal is produced. The purpose of this study was to investigate the protein mobility of the putative mitochondrial fragment and the effect of the L7P polymorphism on the cellular level using GFP tagged constructs. The mobility was studied with fluorescence recovery after photobleaching technique in a neuronal cell line. We found that the mobility of the secretory vesicles with NPY(1-36) in cells with L7P genotype was increased in comparison to vesicle mobility in cells with the more abundant L7L genotype. The mobility in the cells with the putative mitochondrial construct was found to be very low. According to the results of the present study, the mitochondrial truncated peptide stays in the mitochondrion. It can be hypothesized that this could be one of the factors affecting energy balance of the membranes of the mitochondrion.

  9. Identification and characterization of a gene encoding a putative lysophosphatidyl acyltransferase from Arachis hypogaea

    Indian Academy of Sciences (India)

    Si-Long Chen; Jia-Quan Huang; Lei Yong; Yue-Ting Zhang; Xiao-Ping Ren; Yu-Ning Chen; Hui-Fang Jiang; Li-Ying Yan; Yu-Rong Li; Bo-Shou Liao

    2012-12-01

    Lysophosphatidyl acyltransferase (LPAT) is the important enzyme responsible for the acylation of lysophosphatidic acid (LPA), leading to the generation of phosphatidic acid (PA) in plant. Its encoding gene is an essential candidate for oil crops to improve oil composition and increase seed oil content through genetic engineering. In this study, a full-length AhLPAT4 gene was isolated via cDNA library screening and rapid amplification of cDNA ends (RACE); our data demonstrated that AhLPAT4 had 1631 nucleotides, encoding a putative 43.8 kDa protein with 383 amino acid residues. The deduced protein included a conserved acyltransferase domain and four motifs (I–IV) with putative LPA and acyl-CoA catalytic and binding sites. Bioinformatic analysis indicated that AhLPAT4 contained four transmembrane domains (TMDs), localized to the endoplasmic reticulum (ER) membrane; detailed analysis indicated that motif I and motifs II–III in AhLPAT4 were separated by the third TMD, which located on cytosolic and ER luminal side respectively, and hydrophobic residues on the surface of AhLPAT4 protein fold to form a hydrophobic tunnel to accommodate the acyl chain. Subcellular localization analysis confirmed that AhLPAT4 was a cytoplasm protein. Phylogenetic analysis revealed that AhLPAT4 had a high homology (63.7–78.3%) with putative LPAT4 proteins from Glycine max, Arabidopsis thaliana and Ricinus communis. AhLPAT4 was ubiquitously expressed in diverse tissues except in flower, which is almost undetectable. The expression analysis in different developmental stages in peanut seeds indicated that AhLPAT4 did not coincide with oil accumulation.

  10. Identification of putative rhamnogalacturonan-II specific glycosyltransferases in Arabidopsis using a combination of bioinformatics approaches.

    Science.gov (United States)

    Voxeur, Aline; André, Aurélie; Breton, Christelle; Lerouge, Patrice

    2012-01-01

    Rhamnogalacturonan-II (RG-II) is a complex plant cell wall polysaccharide that is composed of an α(1,4)-linked homogalacturonan backbone substituted with four side chains. It exists in the cell wall in the form of a dimer that is cross-linked by a borate di-ester. Despite its highly complex structure, RG-II is evolutionarily conserved in the plant kingdom suggesting that this polymer has fundamental functions in the primary wall organisation. In this study, we have set up a bioinformatics strategy aimed at identifying putative glycosyltransferases (GTs) involved in RG-II biosynthesis. This strategy is based on the selection of candidate genes encoding type II membrane proteins that are tightly coexpressed in both rice and Arabidopsis with previously characterised genes encoding enzymes involved in the synthesis of RG-II and exhibiting an up-regulation upon isoxaben treatment. This study results in the final selection of 26 putative Arabidopsis GTs, including 10 sequences already classified in the CAZy database. Among these CAZy sequences, the screening protocol allowed the selection of α-galacturonosyltransferases involved in the synthesis of α4-GalA oligogalacturonides present in both homogalacturonans and RG-II, and two sialyltransferase-like sequences previously proposed to be involved in the transfer of Kdo and/or Dha on the pectic backbone of RG-II. In addition, 16 non-CAZy GT sequences were retrieved in the present study. Four of them exhibited a GT-A fold. The remaining sequences harbored a GT-B like fold and a fucosyltransferase signature. Based on homologies with glycosyltransferases of known functions, putative roles in the RG-II biosynthesis are proposed for some GT candidates.

  11. Novel mutations in the GH gene (GH1) uncover putative splicing regulatory elements.

    Science.gov (United States)

    Babu, Deepak; Mellone, Simona; Fusco, Ileana; Petri, Antonella; Walker, Gillian E; Bellone, Simonetta; Prodam, Flavia; Momigliano-Richiardi, Patricia; Bona, Gianni; Giordano, Mara

    2014-05-01

    Mutations affecting exon 3 splicing are the main cause of autosomal dominant Isolated GH Deficiency II (IGHDII) by increasing the level of exon 3-skipped mRNA encoding the functionally inactive dominant-negative 17.5-kDa isoform. The exons and introns of the gene encoding GH (GH1) were screened for the presence of mutations in 103 sporadic isolated GH deficiency cases. Four different variations within exon 3 were identified in 3 patients. One carried c.261C>T (p.Pro87Pro) and c.272A>T (p.Glu91Val), the second c.255G>A (p.Pro85Pro) and c.261 C>T, and the third c.246G>C (p.Glu82Asp). All the variants were likely generated by gene conversion from an homologous gene in the GH1 cluster. In silico analysis predicted that positions c.255 and c.272 were included within 2 putative novel exon splicing enhancers (ESEs). Their effect on splicing was confirmed in vitro. Constructs bearing these 2 variants induced consistently higher levels both of transcript and protein corresponding to the 17.5-kDa isoform. When c.255 and c.272 were combined in cis with the c.261 variant, as in our patients, their effect was weaker. In conclusion, we identified 2 variations, c.255G>A and c.272A>T, located in 2 novel putative exon splicing enhancers and affecting GH1 splicing in vitro by increasing the production of alternatively spliced isoforms. The amount of aberrant isoforms is further regulated by the presence in cis of the c.261 variant. Thus, our results evidenced novel putative splicing regulatory elements within exon 3, confirming the crucial role of this exon in mRNA processing.

  12. A Proteomics Approach to Identify New Putative Cardiac Intercalated Disk Proteins.

    Directory of Open Access Journals (Sweden)

    Siddarth Soni

    Full Text Available Synchronous beating of the heart is dependent on the efficient functioning of the cardiac intercalated disk (ID. The ID is composed of a complex protein network enabling electrical continuity and chemical communication between individual cardiomyocytes. Recently, several different studies have shed light on increasingly prevalent cardiac diseases involving the ID. Insufficient knowledge of its composition makes it difficult to study these disease mechanisms in more detail and therefore here we aim expand the ID proteome. Here, using a combination of general membrane enrichment, in-depth quantitative proteomics and an intracellular location driven bioinformatics approach, we aim to discover new putative ID proteins in rat ventricular tissue.General membrane isolation, enriched amongst others also with ID proteins as based on presence of the established markers connexin-43 and n-cadherin, was performed using centrifugation. By mass spectrometry, we quantitatively evaluated the level of 3455 proteins in the enriched membrane fraction (EMF and its counterpart, the soluble cytoplasmic fraction. These data were stringently filtered to generate a final set of 97 enriched, putative ID proteins. These included Cx43 and n-cadherin, but also many interesting novel candidates. We selected 4 candidates (Flotillin-2 (FLOT2, Nexilin (NEXN, Popeye-domain-containg-protein 2 (POPDC2 and thioredoxin-related-transmembrane-protein 2 (TMX2 and confirmed their co-localization with n-cadherin in the ID of human and rat heart cryo-sections, and isolated dog cardiomyocytes.The presented proteomics dataset of putative new ID proteins is a valuable resource for future research into this important molecular intersection of the heart.

  13. Two putative-aquaporin genes are differentially expressed during arbuscular mycorrhizal symbiosis in Lotus japonicus

    Science.gov (United States)

    2012-01-01

    Background Arbuscular mycorrhizas (AM) are widespread symbioses that provide great advantages to the plant, improving its nutritional status and allowing the fungus to complete its life cycle. Nevertheless, molecular mechanisms that lead to the development of AM symbiosis are not yet fully deciphered. Here, we have focused on two putative aquaporin genes, LjNIP1 and LjXIP1, which resulted to be upregulated in a transcriptomic analysis performed on mycorrhizal roots of Lotus japonicus. Results A phylogenetic analysis has shown that the two putative aquaporins belong to different functional families: NIPs and XIPs. Transcriptomic experiments have shown the independence of their expression from their nutritional status but also a close correlation with mycorrhizal and rhizobial interaction. Further transcript quantification has revealed a good correlation between the expression of one of them, LjNIP1, and LjPT4, the phosphate transporter which is considered a marker gene for mycorrhizal functionality. By using laser microdissection, we have demonstrated that one of the two genes, LjNIP1, is expressed exclusively in arbuscule-containing cells. LjNIP1, in agreement with its putative role as an aquaporin, is capable of transferring water when expressed in yeast protoplasts. Confocal analysis have demonstrated that eGFP-LjNIP1, under its endogenous promoter, accumulates in the inner membrane system of arbusculated cells. Conclusions Overall, the results have shown different functionality and expression specificity of two mycorrhiza-inducible aquaporins in L. japonicus. One of them, LjNIP1 can be considered a novel molecular marker of mycorrhizal status at different developmental stages of the arbuscule. At the same time, LjXIP1 results to be the first XIP family aquaporin to be transcriptionally regulated during symbiosis. PMID:23046713

  14. Enrichment of putative human epidermal stem cells based on cell size and collagen type IV adhesiveness

    Institute of Scientific and Technical Information of China (English)

    Juxue Li; Enkui Duan; Chenglin Miao; Weixiang Guo; Liwei Jia; Jiaxi Zhou; Baohua Ma; Sha Peng; Shuang Liu; Yujing Cao

    2008-01-01

    The enrichment and identification of human epidermal stem cells (EpSCs) are of paramount importance for both basic research and clinical application. Although several approaches for the enrichment of EpSCs have been established, enriching a pure population of viable EpSCs is still a challenging task. An improved approach is worth developing to enhance the purity and viability of EpSCs. Here we report that cell size combined with collagen type IV adhesiveness can be used in an improved approach to enrich pure and viable human EpSCs. We separated the rapidly adherent keratinocytes into three populations that range in size from 5-7 μm (population A), to 7-9 μm (population B), to >9 μm (population C) in diameter, and found that human putative EpSCs could be further enriched in population A with the smallest size. Among the three populations, population A displayed the highest density of βl-integrin receptor, contained the highest percentage of cells in G0/G1 phase, showed the highest nucleus to cytoplasm ratio, and possessed the highest colony formation efficiency (CFE). When injected into murine blastocysts, these cells participated in multi-tissue formation. More significantly, compared with a previous approach that sorted putative EpSCs according to pl-integrin antibody staining, the viability of the EpSCs enriched by the improved approach was significantly enhanced. Our results provide a putative strategy for the enrichment of human EpSCs, and encourage further study into the role of cell size in stem cell biology.

  15. Two putative-aquaporin genes are differentially expressed during arbuscular mycorrhizal symbiosis in Lotus japonicus

    Directory of Open Access Journals (Sweden)

    Giovannetti Marco

    2012-10-01

    Full Text Available Abstract Background Arbuscular mycorrhizas (AM are widespread symbioses that provide great advantages to the plant, improving its nutritional status and allowing the fungus to complete its life cycle. Nevertheless, molecular mechanisms that lead to the development of AM symbiosis are not yet fully deciphered. Here, we have focused on two putative aquaporin genes, LjNIP1 and LjXIP1, which resulted to be upregulated in a transcriptomic analysis performed on mycorrhizal roots of Lotus japonicus. Results A phylogenetic analysis has shown that the two putative aquaporins belong to different functional families: NIPs and XIPs. Transcriptomic experiments have shown the independence of their expression from their nutritional status but also a close correlation with mycorrhizal and rhizobial interaction. Further transcript quantification has revealed a good correlation between the expression of one of them, LjNIP1, and LjPT4, the phosphate transporter which is considered a marker gene for mycorrhizal functionality. By using laser microdissection, we have demonstrated that one of the two genes, LjNIP1, is expressed exclusively in arbuscule-containing cells. LjNIP1, in agreement with its putative role as an aquaporin, is capable of transferring water when expressed in yeast protoplasts. Confocal analysis have demonstrated that eGFP-LjNIP1, under its endogenous promoter, accumulates in the inner membrane system of arbusculated cells. Conclusions Overall, the results have shown different functionality and expression specificity of two mycorrhiza-inducible aquaporins in L. japonicus. One of them, LjNIP1 can be considered a novel molecular marker of mycorrhizal status at different developmental stages of the arbuscule. At the same time, LjXIP1 results to be the first XIP family aquaporin to be transcriptionally regulated during symbiosis.

  16. Temporal dynamics and decay of putatively allochthonous and autochthonous viral genotypes in contrasting freshwater lakes.

    Science.gov (United States)

    Hewson, Ian; Barbosa, Jorge G; Brown, Julia M; Donelan, Ryan P; Eaglesham, James B; Eggleston, Erin M; LaBarre, Brenna A

    2012-09-01

    Aquatic viruses play important roles in the biogeochemistry and ecology of lacustrine ecosystems; however, their composition, dynamics, and interactions with viruses of terrestrial origin are less extensively studied. We used a viral shotgun metagenomic approach to elucidate candidate autochthonous (i.e., produced within the lake) and allochthonous (i.e., washed in from other habitats) viral genotypes for a comparative study of their dynamics in lake waters. Based on shotgun metagenomes prepared from catchment soil and freshwater samples from two contrasting lakes (Cayuga Lake and Fayetteville Green Lake), we selected two putatively autochthonous viral genotypes (phycodnaviruses likely infecting algae and cyanomyoviruses likely infecting picocyanobacteria) and two putatively allochthonous viral genotypes (geminiviruses likely infecting terrestrial plants and circoviruses infecting unknown hosts but common in soil libraries) for analysis by genotype-specific quantitative PCR (TaqMan) applied to DNAs from viruses in the viral size fraction of lake plankton, i.e., 0.2 μm > virus > 0.02 μm. The abundance of autochthonous genotypes largely reflected expected host abundance, while the abundance of allochthonous genotypes corresponded with rainfall and storm events in the respective catchments, suggesting that viruses with these genotypes may have been transported to the lake in runoff. The decay rates of allochthonous and autochthonous genotypes, assessed in incubations where all potential hosts were killed, were generally lower (0.13 to 1.50% h(-1)) than those reported for marine virioplankton but similar to those for freshwater virioplankton. Both allochthonous and autochthonous viral genotypes were detected at higher concentrations in subsurface sediments than at the water-sediment interface. Our data indicate that putatively allochthonous viruses are present in lake plankton and sediments, where their temporal dynamics reflect active transport to the lake during

  17. Further insight into reproductive incompatibility between putative cryptic species of the Bemisia tabaci whitefly complex.

    Science.gov (United States)

    Qin, Li; Pan, Li-Long; Liu, Shu-Sheng

    2016-04-01

    The whitefly Bemisia tabaci (Gennadius) (Hemiptera: Aleyrodidae), with its global distribution and extensive genetic diversity, is now known to be a complex of over 35 cryptic species. However, a satisfactory resolution of the systematics of this species complex is yet to be achieved. Here, we designed experiments to examine reproductive compatibility among species with different levels of mitochondrial cytochrome oxidase I (mtCOI) divergence. The data show that putative species with mtCOI divergence of >8% between them consistently exhibited complete reproductive isolation. However, two of the putative species, Asia II 9 and Asia II 3, with mtCOI divergence of 4.47% between them, exhibited near complete reproductive compatibility in one direction of their cross, and partial reproductive compatibility in the other direction. Together with some recent reports on this topic from the literature, our data indicates that, while divergence in the mtCOI sequences provides a valid molecular marker for species delimitation in most clades, more genetic markers and more sophisticated molecular phylogeny will be required to achieve adequate delimitation of all species in this whitefly complex. While many attempts have been made to examine the reproductive compatibility among genetic groups of the B. tabaci complex, our study represents the first effort to conduct crossing experiments with putative species that were chosen with considerations of their genetic divergence. In light of the new data, we discuss the best strategy and protocols to conduct further molecular phylogenetic analysis and crossing trials, in order to reveal the overall pattern of reproductive incompatibility among species of this whitefly complex.

  18. Tank binding kinase 1 is a centrosome-associated kinase necessary for microtubule dynamics and mitosis.

    Science.gov (United States)

    Pillai, Smitha; Nguyen, Jonathan; Johnson, Joseph; Haura, Eric; Coppola, Domenico; Chellappan, Srikumar

    2015-12-10

    TANK Binding Kinase 1 (TBK1) is a non-canonical IκB kinase that contributes to KRAS-driven lung cancer. Here we report that TBK1 plays essential roles in mammalian cell division. Specifically, levels of active phospho-TBK1 increase during mitosis and localize to centrosomes, mitotic spindles and midbody, and selective inhibition or silencing of TBK1 triggers defects in spindle assembly and prevents mitotic progression. TBK1 binds to the centrosomal protein CEP170 and to the mitotic apparatus protein NuMA, and both CEP170 and NuMA are TBK1 substrates. Further, TBK1 is necessary for CEP170 centrosomal localization and binding to the microtubule depolymerase Kif2b, and for NuMA binding to dynein. Finally, selective disruption of the TBK1-CEP170 complex augments microtubule stability and triggers defects in mitosis, suggesting that TBK1 functions as a mitotic kinase necessary for microtubule dynamics and mitosis.

  19. Targeting Spleen Tyrosine Kinase-Bruton's Tyrosine Kinase Axis for Immunologically Mediated Glomerulonephritis

    Science.gov (United States)

    Chen, Jin-Shuen; Chang, Li-Chien; Huang, Shyh-Jer

    2014-01-01

    The importance of B-cell activation and immune complex-mediated Fc-receptor activation in the pathogenesis of immunologically mediated glomerulonephritis has long been recognized. The two nonreceptor tyrosine kinases, spleen tyrosine kinase (Syk) and Bruton's tyrosine kinase (Btk), are primarily expressed by hematopoietic cells, and participate in B-cell-receptor- and Fc-receptor-mediated activation. Pharmacological inhibitors of Syk or Btk are undergoing preclinical development and clinical trials for several immune diseases; and Syk inhibitors have been shown to reduce disease activity in rheumatoid arthritis patients. However, the clinical therapeutic efficacies of these inhibitors in glomerulonephritis have not been evaluated. Herein, we review recent studies of Syk and Btk inhibitors in several experimental primary and secondary glomerulonephritis models. These inhibitors suppressed development of glomerular injury, and also ameliorated established kidney disease. Thus, targeting Syk and Btk signaling pathways is a potential therapeutic strategy for glomerulonephritis, and further evaluation is recommended. PMID:24795896

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

    Science.gov (United States)

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

    2005-01-01

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

  1. Evaluation of two putative susceptibility loci for oral clefts in the Danish population

    DEFF Research Database (Denmark)

    Mitchell, L E; Murray, J C; O'Brien, S;

    2001-01-01

    Previous studies suggest that the risk of nonsyndromic cleft lip with or without cleft palate (CL+/-P) and isolated cleft palate (CP) is influenced by genetic variation at several loci and that the relation between specific genetic variants and disease risk may be modified by environmental factors....... The present study evaluated potential associations between CL+/-P and CP and two putative clefting susceptibility loci, MSX1 and TGFB3, using data from a nationwide case-control study conducted in Denmark from 1991 to 1994. The potential effects of interactions between these genes and two common environmental...

  2. Role of putative neurotransmitters in the central gastric antisecretory effect of prostaglandin E2 in rats.

    OpenAIRE

    Puurunen, J.

    1985-01-01

    The role of putative neurotransmitters of the central nervous system in the central gastric antisecretory effect of prostaglandin E2 (PGE2) was investigated in pylorus-ligated rats. Pretreatment of the rats with an intracerebroventricular (i.c.v.) injection of 6-hydroxydopamine (6-OHDA) prevented the antisecretory effect of the i.c.v. administration of PGE2, whereas pretreatment with 5,6-dihydroxytryptamine (5,6-DHT) plus p-chlorophenylalanine (PCPA) had no effect. I.c.v.-administered phentol...

  3. A putative, novel coli surface antigen 8B (CS8B) of enterotoxigenic Escherichia coli.

    Science.gov (United States)

    Njoroge, Samuel M; Boinett, Christine J; Madé, Laure F; Ouko, Tom T; Fèvre, Eric M; Thomson, Nicholas R; Kariuki, Samuel

    2015-10-01

    Enterotoxigenic Escherichia coli (ETEC) strains harbor multiple fimbriae and pili to mediate host colonization, including the type IVb pilus, colonization factor antigen III (CFA/III). Not all colonization factors are well characterized or known in toxin positive ETEC isolates, which may have an impact identifying ETEC isolates based on molecular screening of these biomarkers. We describe a novel coli surface antigen (CS) 8 subtype B (CS8B), a family of CFA/III pilus, in a toxin producing ETEC isolate from a Kenyan collection. In highlighting the existence of this putative CS, we provide the sequence and specific primers, which can be used alongside other ETEC primers previously described.

  4. A specimen of Rhamphorhynchus with soft tissue preservation, stomach contents and a putative coprolite

    Directory of Open Access Journals (Sweden)

    David Hone

    2015-08-01

    Full Text Available Despite being known for nearly two centuries, new specimens of the derived non-pterodactyloid pterosaur Rhamphorhynchus continue to be discovered and reveal new information about their anatomy and palaeobiology. Here we describe a specimen held in the collections of the Royal Tyrrell Museum of Palaeontology, Alberta, Canada that shows both preservation and impressions of soft tissues, and also preserves material interpreted as stomach contents of vertebrate remains and, uniquely, a putative coprolite. The specimen also preserves additional evidence for fibers in the uropatagium.

  5. A putative transglycosylase encoded by SCO4132 influences morphological differentiation and actinorhodin production in Streptomyces coelicolor

    Institute of Scientific and Technical Information of China (English)

    Pengfei Xie; Ana Zeng; Xiaoting Lv; Qiuxiang Cheng; Zhongjun Qin

    2013-01-01

    Here we report that tgdA,a novel gene encoding a putative transglycosylase,affects both the morphological differentiation and the yield of blue-pigmented compound actinorhodin in Streptomyces coelicolor.The tgdA null mutant displays sparse aerial hyphae and irregular spore chains frequently lacking chromosomal DNA.Elevated actinorhodin production coincides with the overexpression of actⅡ-orf4 in mutant.tgdA expression is temporally and developmentally regulated.The tgdA orthologs in Streptomyces avermilitis and Streptomyces lividans also affect differentiation.

  6. Prevalence of Clinical Periodontitis and Putative Periodontal Pathogens among South Indian Pregnant Women

    Directory of Open Access Journals (Sweden)

    Chaitanya Tellapragada

    2014-01-01

    Full Text Available In view of recent understanding of the association of periodontal infections and adverse pregnancy outcomes, the present investigation was undertaken to study the periodontal infections among 390 asymptomatic pregnant women and to find an association of bacterial etiologies with the disease. Prevalence of gingivitis was 38% and clinical periodontitis was 10% among the study population. Subgingival plaque specimens were subjected to multiplex PCR targeting ten putative periodontopathogenic bacteria. Among the periodontitis group, high detection rates of Porphyromonas gingivalis (56%, Prevotella nigrescens (44%, Treponema denticola (32%, and Prevotella intermedius (24% were noted along with significant association with the disease (P<0.05.

  7. A specimen of Rhamphorhynchus with soft tissue preservation, stomach contents and a putative coprolite.

    Science.gov (United States)

    Hone, David; Henderson, Donald M; Therrien, François; Habib, Michael B

    2015-01-01

    Despite being known for nearly two centuries, new specimens of the derived non-pterodactyloid pterosaur Rhamphorhynchus continue to be discovered and reveal new information about their anatomy and palaeobiology. Here we describe a specimen held in the collections of the Royal Tyrrell Museum of Palaeontology, Alberta, Canada that shows both preservation and impressions of soft tissues, and also preserves material interpreted as stomach contents of vertebrate remains and, uniquely, a putative coprolite. The specimen also preserves additional evidence for fibers in the uropatagium.

  8. A comparison of the effects of two putative 5-hydroxytryptamine renal prodrugs in normal man.

    OpenAIRE

    LI KAM WA, T. C.; Freestone, S.; Samson, R. R.; JOHNSTON, N. R.; Lee, M.R.

    1993-01-01

    1. The effects of 1 h intravenous infusions of equimolar amounts of two putative 5-hydroxytryptamine (5-HT) renal prodrugs, 5-hydroxy-L-tryptophan (5-HTP, 10 micrograms kg-1 min-1) and gamma-L-glutamyl-5-hydroxy-L-tryptophan (glu-5-HTP, 16.6 micrograms kg-1 min-1) were examined in five healthy male volunteers in a randomised, placebo-controlled, cross-over study. 2. Both compounds increased urinary excretion of 5-HT and there was greater extra-renal formation of 5-HT following 5-HTP administr...

  9. Targeting the phosphoinositide 3-kinase pathway in hematologic malignancies

    Science.gov (United States)

    Jabbour, Elias; Ottmann, Oliver G.; Deininger, Michael; Hochhaus, Andreas

    2014-01-01

    The phosphoinositide 3-kinase pathway represents an important anticancer target because it has been implicated in cancer cell growth, survival, and motility. Recent studies show that PI3K may also play a role in the development of resistance to currently available therapies. In a broad range of cancers, various components of the phosphoinositide 3-kinase signaling axis are genetically modified, and the pathway can be activated through many different mechanisms. The frequency of genetic alterations in the phosphoinositide 3-kinase pathway, coupled with the impact in oncogenesis and disease progression, make this signaling axis an attractive target in anticancer therapy. A better understanding of the critical function of the phosphoinositide 3-kinase pathway in leukemias and lymphomas has led to the clinical evaluation of novel rationally designed inhibitors in this setting. Three main categories of phosphoinositide 3-kinase inhibitors have been developed so far: agents that target phosphoinositide 3-kinase and mammalian target of rapamycin (dual inhibitors), pan-phosphoinositide 3-kinase inhibitors that target all class I isoforms, and isoform-specific inhibitors that selectively target the α, -β, -γ, or -δ isoforms. Emerging data highlight the promise of phosphoinositide 3-kinase inhibitors in combination with other therapies for the treatment of patients with hematologic malignancies. Further evaluation of phosphoinositide 3-kinase inhibitors in first-line or subsequent regimens may improve clinical outcomes. This article reviews the role of phosphoinositide 3-kinase signaling in hematologic malignancies and the potential clinical utility of inhibitors that target this pathway. PMID:24425689

  10. The Structure of Lombricine Kinase: Implications for Phosphagen Conformational Changes

    Energy Technology Data Exchange (ETDEWEB)

    Bush, D. Jeffrey; Kirillova, Olga; Clark, Shawn A.; Davulcu, Omar; Fabiola, Felcy; Xie, Qing; Somasundaram, Thayumanasamy; Ellington, W. Ross; Chapman, Michael S. (Oregon HSU); (FSU)

    2012-05-29

    Lombricine kinase is a member of the phosphagen kinase family and a homolog of creatine and arginine kinases, enzymes responsible for buffering cellular ATP levels. Structures of lombricine kinase from the marine worm Urechis caupo were determined by x-ray crystallography. One form was crystallized as a nucleotide complex, and the other was substrate-free. The two structures are similar to each other and more similar to the substrate-free forms of homologs than to the substrate-bound forms of the other phosphagen kinases. Active site specificity loop 309-317, which is disordered in substrate-free structures of homologs and is known from the NMR of arginine kinase to be inherently dynamic, is resolved in both lombricine kinase structures, providing an improved basis for understanding the loop dynamics. Phosphagen kinases undergo a segmented closing on substrate binding, but the lombricine kinase ADP complex is in the open form more typical of substrate-free homologs. Through a comparison with prior complexes of intermediate structure, a correlation was revealed between the overall enzyme conformation and the substrate interactions of His{sup 178}. Comparative modeling provides a rationale for the more relaxed specificity of these kinases, of which the natural substrates are among the largest of the phosphagen substrates.

  11. The role of Y84 on domain 1 and Y87 on domain 2 of Paragonimus westermani taurocyamine kinase: Insights on the substrate binding mechanism of a trematode phosphagen kinase.

    Science.gov (United States)

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

    2013-12-01

    The two-domain taurocyamine kinase (TK) from Paragonimus westermani was suggested to have a unique substrate binding mechanism. We performed site-directed mutagenesis on each domain of this TK and compared the kinetic parameters Km(Tc) and Vmax with that of the wild-type to determine putative amino acids involved in substrate recognition and binding. Replacement of Y84 on domain 1 and Y87 on domain 2 with R resulted in the loss of activity for the substrate taurocyamine. Y84E mutant has a dramatic decrease in affinity and activity for taurocyamine while Y87E has completely lost catalytic activity. Substituting H and I on the said positions also resulted in significant changes in activity. Mutation of the residues A59 on the GS region of domain 1 also caused significant decrease in affinity and activity while mutation on the equivalent position on domain 2 resulted in complete loss of activity.

  12. Protein kinase C-associated kinase regulates NF-κB activation through inducing IKK activation.

    Science.gov (United States)

    Kim, Sang-Woo; Schifano, Matthew; Oleksyn, David; Jordan, Craig T; Ryan, Daniel; Insel, Richard; Zhao, Jiyong; Chen, Luojing

    2014-10-01

    Activation of the transcription factor NF-κB induced by extracellular stimuli requires IKKα and IKKβ kinase activity. How IKKα and IKKβ are activated by various upstream signaling molecules is not fully understood. We previously showed that protein kinase C-associated kinase (PKK, also known as DIK/RIP4), which belongs to the receptor-interacting protein (RIP) kinase family, mediates the B cell activating factor of the TNF family (BAFF)-induced NF-κB activation in diffuse large B cell lymphoma (DLBCL) cell lines. Here we have investigated the mechanism underlying NF-κB activation regulated by PKK. Our results suggest that PKK can activate both the classical and the alternative NF-κB activation pathways. PKK associates with IKKα and IKKβ in mammalian cells and induces activation of both IKKα and IKKβ via phosphorylation of their serine residues 176/180 and 177/181, respectively. Unlike other members of the RIP family that activate NF-κB through a kinase-independent pathway, PKK appears to activate IKK and NF-κB mainly in a kinase-dependent manner. Suppression of PKK expression by RNA interference inhibits phosphorylation of IKKα and IKKβ as well as activation of NF-κB in human cancer cell lines. Thus, PKK regulates NF-κB activation by modulating activation of IKKα and IKKβ in mammalian cells. We propose that PKK may provide a critical link between IKK activation and various upstream signaling cascades, and may represent a potential target for inhibiting abnormal NF-κB activation in human cancers.

  13. Dimerization via tandem leucine zippers is essential for the activation of the mitogen-activated protein kinase kinase kinase, MLK-3.

    Science.gov (United States)

    Leung, I W; Lassam, N

    1998-12-04

    Mixed lineage kinase-3 (MLK-3) is a mitogen-activated kinase kinase kinase that mediates stress-activating protein kinase (SAPK)/c-Jun NH2-terminal kinase activation. MLK-3 and other MLK family kinases are characterized by the presence of multiple protein-protein interaction domains including a tandem leucine/isoleucine zipper (LZs) motif. Leucine zippers are known to mediate protein dimerization raising the possibility that the tandem leucine/isoleucine zippers may function as a dimerization motif of MLK-3. Using both co-immunoprecipitation and nonreducing SDS-polyacrylamide gel electrophoresis, we demonstrated that MLK-3 forms disulfide bridged homo-dimers and that the LZs motif is sufficient for MLK-3 homodimerization. We next asked whether MLK-3 utilizes a dimerization-based activation mechanism analogous to that of receptor tyrosine kinases. We found that dimerization via the LZs motif is a prerequisite for MLK-3 autophosphorylation. We then demonstrated that co-expression of Cdc42 lead to a substantial increase in MLK-3 dimerization, indicating that binding by this GTPase may induce MLK-3 dimerization. Moreover, the LZs minus form of MLK-3 failed to activate the downstream target SAPK, and expression of a MLK-3 LZs polypeptide was found to block SAPK activation by wild type MLK-3. Taken together, these findings indicate that dimerization plays a pivotal role in MLK-3 activation.

  14. Protein (Viridiplantae): 15227263 [PGDBj - Ortholog DB

    Lifescience Database Archive (English)

    Full Text Available 93 putative protein kinase Arabidopsis thaliana MKLVLEGVDSFETLRVVGTFNCIDPDYVGSKRVTKKADVYAFEVILMELITGRKANYETLSVDEQNLVMWLRPKIKISTFLNLVDGTIATDKETIKRIKKIAKLAEYCTSQEVESRPLRASRTKSGNEVTSED ...

  15. Protein kinase Cθ gene expression is oppositely regulated by GCN5 and EBF1 in immature B cells.

    Science.gov (United States)

    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.

  16. Transcriptional regulation of human polo-like kinases and early mitotic inhibitors

    Institute of Scientific and Technical Information of China (English)

    Moe Tategu; Hiroki Nakagawa; Kaori Sasaki; Rieko Yamauchi; Sota Sekimachi; Yuka Suita; Naoko Watanabe; Kenichi Yoshida

    2008-01-01

    Human polo-like kinases (PLK1-PLK4) have been implicated in mitotic regulation and carcinogenesis.PLK1 phosphorylates early mitotic inhibitor 1 (Emil) to ensure mitosis entry,whereas Emi2 plays a key role during the meiotic cell cycle.Transcription factor E2F is primarily considered to regulate the G1/S transition of the cell cycle but its involvement in the regulation of mitosis has also been recently suggested.A gap still exists between the molecular basis of E2F and mitotic regulation.The present study was designed to characterize the transcriptional regulation of human PLK and Emi genes.Adenoviral overexpression of E2F1 increased PLK1 and PLK3 mRNA levels in A549 cells.A reporter gene assay revealed that the putative promoter regions of PLK1,PLK3,and PLK4 genes were responsive to ac-tivators E2F,E2F1-E2F3.We further characterized the putative promoter regions of Emil and Emi2 genes,and these could be regulated by activators E2F and E2F1-E2F4,respectively.Finally,PLK1-PLK4,Emil,and Emi2 mRNA expression levels in human adult,fetal tis-sues,and several cell lines indicated that each gene has a unique expression pattern but is uniquely expressed in common tissues and cells such as the testes and thymus.Collectively,these results indicate that E2F can integrate G1/S and G2/Mto oscillate the cell cycle by regu-lating mitotic genes PLK and Emi,leading to determination of the cell fate.

  17. Macro creatine kinase type 1: a cause of spuriously elevated serum creatine kinase associated with leukoencephalopathy in a child.

    Science.gov (United States)

    Bodensteiner, John B

    2014-07-01

    Macro creatine kinase type 1 is a complex formed by the creatine kinase isoenzyme BB and monoclonal IgG and occurs in about 1% of patients studied. First identified as a cause of spurious elevation of the total serum creatine kinase in patients suspected of myocardial infarction, the test has been largely replaced by the measurement of troponin levels. We present a child with delayed milestones and persistently elevated total serum creatine kinase measurements (∼ 1000-4000 IU) normal electromyogram and brisk myotatic reflexes. Creatine kinase isoenzymes and brain imaging showed the presence of macro creatine kinase type 1 and extensive signal abnormality of the cerebral white matter. Macro creatine kinase type 1 has been associated with several conditions though it has not been described in association with leukoencephalopathy or in patients this young. Macro creatine kinase type 1 can be a cause of elevated total creatine kinase in patients without primary muscle disease. The significance of the relationship of the macro creatine kinase to the leukoencephalopathy in this patient is unknown.

  18. Phosphatidylinositol 3-Kinase and Protein Kinase C Contribute to the Inhibition by Interleukin 6 of Phosphoenolpyruvate Carboxykinase Gene Expression in Cultured Rat Hepatocytes

    DEFF Research Database (Denmark)

    Christ, Bruno; Yazici, Emine; Nath, Annegret

    2000-01-01

    Gluconeogenesis, hepatocytes, interleukin 6, liver, phosphoinositide 3-kinase, phosphoenolpyruvate carboxykinase......Gluconeogenesis, hepatocytes, interleukin 6, liver, phosphoinositide 3-kinase, phosphoenolpyruvate carboxykinase...

  19. Stimulation of receptor-associated kinase, tyrosine kinase, and MAP kinase is required for prolactin-mediated macromolecular biosynthesis and mitogenesis in Nb2 lymphoma.

    Science.gov (United States)

    Carey, G B; Liberti, J P

    1995-01-10

    Lactogens [prolactin (Prl) and growth hormone] stimulate phosphorylation of the 40S ribosomal protein, S6, in Nb2 cells by mechanisms that do not involve participation of cAMP or protein kinase A, protein kinase C, or cGMP-dependent protein kinase. However, inhibition of tyrosine kinase (TK) abrogates Prl-mediated macromolecular biosynthesis. Inasmuch as lactogen signaling may involve sequential activation of protein kinases, the effect of Prl on the well-characterized mitogen-activated protein kinase (MAPK) and S6 kinase (S6K), the enzyme responsible for S6 phosphorylation in vivo, and their relationship to Nb2 macromolecular biosynthesis and mitogenesis were investigated. The results show that MAPK stimulation is transient (peak activity, 30 min) and precedes that of S6K, which reaches a maximum at 1.5-2 h, and slowly returns towards control levels at 6 h. Both staurosporine which inhibits GH receptor-associated kinase (JAK2) and genistein (GEN), an inhibitor of membrane-associated and cytoplasmic TKs, abrogate Prl-stimulated TK, MAPK, and S6K. Rapamycin (RAP), a specific inhibitor of p70S6K, completely blocks S6K but does not affect TK and MAPK. TK and MAPK activity correlates with Prl-stimulated anabolism, i.e., protein and DNA synthesis and mitogenesis. Thus, concentrations of STR and GEN which abrogate TK and MAPK inhibit anabolism virtually 100%. However, RAP, which inhibits S6K (ca. 100%) but not TK or MAPK, only delays Prl-mediated anabolism. These results indicate that Prl signaling in Nb2 cells involves a protein kinase cascade and that regulation of receptor-associated kinase, TK, and MAPK correlates with anabolism. The role of S6K (and S6 phosphorylation) appears to be ancillary.

  20. Putative cholesterol-binding sites in human immunodeficiency virus (HIV) coreceptors CXCR4 and CCR5.

    Science.gov (United States)

    Zhukovsky, Mikhail A; Lee, Po-Hsien; Ott, Albrecht; Helms, Volkhard

    2013-04-01

    Using molecular docking, we identified a cholesterol-binding site in the groove between transmembrane helices 1 and 7 near the inner membrane-water interface of the G protein-coupled receptor CXCR4, a coreceptor for HIV entry into cells. In this docking pose, the amino group of lysine K67 establishes a hydrogen bond with the hydroxyl group of cholesterol, whereas tyrosine Y302 stacks with cholesterol by its aromatic side chain, and a number of residues form hydrophobic contacts with cholesterol. Sequence alignment showed that a similar putative cholesterol-binding site is also present in CCR5, another HIV coreceptor. We suggest that the interaction of cholesterol with these putative cholesterol-binding sites in CXCR4 and CCR5 is responsible for the presence of these receptors in lipid rafts, for the effect of cholesterol on their conformational stability and function, and for the role that cell cholesterol plays in the cell entry of HIV strains that use these membrane proteins as coreceptors. We propose that mutations of residues that are involved in cholesterol binding will make CXCR4 and CCR5 insensitive to membrane cholesterol content. Cholesterol-binding sites in HIV coreceptors are potential targets for steroid drugs that bind to CXCR4 and CCR5 with higher binding affinity than cholesterol, but do not stabilize the native conformation of these proteins.

  1. On the Origin of the Supergiant HI Shell and Putative Companion in NGC 6822

    CERN Document Server

    Cannon, John M; Weisz, Daniel R; Skillman, Evan D; Dolphin, Andrew E; Bigiel, Frank; Cole, Andrew A; de Blok, W J G; Walter, Fabian

    2012-01-01

    We present new Hubble Space Telescope Advanced Camera for Surveys imaging of six positions spanning 5.8 kpc of the HI major axis of the Local Group dIrr NGC 6822, including both the putative companion galaxy and the large HI hole. The resulting deep color magnitude diagrams show that NGC 6822 has formed >50% of its stars in the last ~5 Gyr. The star formation histories of all six positions are similar over the most recent 500 Myr, including low-level star formation throughout this interval and a weak increase in star formation rate during the most recent 50 Myr. Stellar feedback can create the giant HI hole, assuming that the lifetime of the structure is longer than 500 Myr; such long-lived structures have now been observed in multiple systems and may be the norm in galaxies with solid-body rotation. The old stellar populations (red giants and red clump stars) of the putative companion are consistent with those of the extended halo of NGC 6822; this argues against the interpretation of this structure as a bon...

  2. ON THE ORIGIN OF THE SUPERGIANT H I SHELL AND PUTATIVE COMPANION IN NGC 6822

    Energy Technology Data Exchange (ETDEWEB)

    Cannon, John M.; O' Leary, Erin M. [Department of Physics and Astronomy, Macalester College, 1600 Grand Avenue, Saint Paul, MN 55105 (United States); Weisz, Daniel R. [Department of Astronomy, University of Washington, Seattle, WA 98195 (United States); Skillman, Evan D. [Minnesota Institute for Astrophysics, University of Minnesota, Minneapolis, MN 55455 (United States); Dolphin, Andrew E. [Raytheon Company, 1151 East Hermans Road, Tucson, AZ 85706 (United States); Bigiel, Frank [Institut fuer Theoretische Astrophysik, Universitaet Heidelberg, Albert-Ueberle-Str. 2, 69120 Heidelberg (Germany); Cole, Andrew A.; Walter, Fabian [School of Mathematics and Physics, University of Tasmania, Private Bag 37, Hobart 7001, Tasmania (Australia); De Blok, W.J.G., E-mail: jcannon@macalester.edu, E-mail: eoleary@macalester.edu, E-mail: dweisz@astro.washington.edu, E-mail: skillman@astro.umn.edu, E-mail: adolphin@raytheon.com, E-mail: bigiel@uni-heidelberg.de, E-mail: andrew.cole@utas.edu.au, E-mail: edeblok@ast.uct.ac.za, E-mail: walter@mpia.de [Department of Astronomy, University of Cape Town, Rondebosch 7700 (South Africa)

    2012-03-10

    We present new Hubble Space Telescope Advanced Camera for Surveys imaging of six positions spanning 5.8 kpc of the H I major axis of the Local Group dIrr NGC 6822, including both the putative companion galaxy and the large H I hole. The resulting deep color-magnitude diagrams show that NGC 6822 has formed >50% of its stars in the last {approx}5 Gyr. The star formation histories of all six positions are similar over the most recent 500 Myr, including low-level star formation throughout this interval and a weak increase in star formation rate during the most recent 50 Myr. Stellar feedback can create the giant H I hole, assuming that the lifetime of the structure is longer than 500 Myr; such long-lived structures have now been observed in multiple systems and may be the norm in galaxies with solid-body rotation. The old stellar populations (red giants and red clump stars) of the putative companion are consistent with those of the extended halo of NGC 6822; this argues against the interpretation of this structure as a bona fide interacting companion galaxy and against its being linked to the formation of the H I hole via an interaction. Since there is no evidence in the stellar population of a companion galaxy, the most likely explanation of the extended H I structure in NGC 6822 is a warped disk inclined to the line of sight.

  3. Transcriptome-Based Examination of Putative Pollen Allergens of Rice(Oryza sativa ssp.japonica)

    Institute of Scientific and Technical Information of China (English)

    Scott D.Russell; Prem L Bhalla; Mohan B.Singh

    2008-01-01

    Pollen allergens are among the most abundantly transcribed and translated products in the Iife history of plants,and particularly grasses.To identify different pollen allergens in rice,putative allergens were identified in the rice genome and their expression characterized using the Affymetrix 57K rice GeneChip microarray.Among the most abundant pollen-specific candidate transcripts were Ory s 1 beta-expansin.Ory s 2,Ory s 7 EFhand,Ory s 11,Ory s 12 profilin A,Ory s 23,glycosyl hydrolase family 28(polygalacturonase).and FAD binding proteins.Highly expressed pollen proteins are frequently present in multiple copy numbers,sometimes with mirror images Iocated on nearby regions of the opposite DNA strand.Many of these are intronless and inserted as copies that retain nearly exact copies of their regulatory elements.Ory s 23 reflects low variability and high copy number,suggesting recent gene amplification.Some copies contain pseudogenes,which may reflect their origin through activity of retrotransposition;some putative allergenic sequences bear fusion products with repeat sequences of transposable elements(LTRs).The abundance of nearby repetitive sequences,activation of transposable elements.and high production of mRNA transcripts appear to coincide in pollen and may contribute to a syndrome in which highly transcribed proteins may be copied and inserted with streamlined features for translation,including grouping and removaI of introns.

  4. Identification of putative DnaN-binding motifs in plasmid replication initiation proteins.

    Science.gov (United States)

    Dalrymple, Brian P; Kongsuwan, Kritaya; Wijffels, Gene

    2007-01-01

    Recently the plasmid RK2 replication initiation protein, TrfA, has been shown to bind to the beta subunit of DNA Polymerase III (DnaN) via a short pentapeptide with the consensus QL[S/D]LF. A second consensus peptide, the hexapeptide QLxLxL, has also been demonstrated to mediate binding to DnaN. Here we describe the results of a comprehensive survey of replication initiation proteins encoded by bacterial plasmids to identify putative DnaN-binding sites. Both pentapeptide and hexapeptide motifs have been identified in a number of families of replication initiation proteins. The distribution of sites is sporadic and closely related families of proteins may differ in the presence, location, or type of putative DnaN-binding motif. Neither motif has been identified in replication initiation proteins encoded by plasmids that replicate via rolling circles or strand displacement. The results suggest that the recruitment of DnaN to the origin of replication of a replisome by plasmid replication initiation proteins is not generally required for plasmid replication, but that in some cases it may be beneficial for efficiency of replication initiation.

  5. Uncovering the Putative B-Star Binary Companion of the SN 1993J Progenitor

    CERN Document Server

    Fox, Ori D; Van Dyk, Schuyler D; Filippenko, Alexei V; Fransson, Claes; Matheson, Thomas; Cenko, S Bradley; Chandra, Poonam; Dwarkadas, Vikram; Li, Weidong; Parker, Alex H; Smith, Nathan

    2014-01-01

    The Type IIb supernova (SN) 1993J is one of only a few stripped-envelope supernovae with a progenitor star identified in pre-explosion images. SN IIb models typically invoke H envelope stripping by mass transfer in a binary system. For the case of SN 1993J, the models suggest that the companion grew to 22 M_solar and became a source of ultraviolet (UV) excess. Located in M81, at a distance of only 3.6 Mpc, SN 1993J offers one of the best opportunities to detect the putative companion and test the progenitor model. Previously published near-UV spectra in 2004 showed evidence for absorption lines consistent with a hot (B2 Ia) star, but the field was crowded and dominated by flux from the SN. Here we present Hubble Space Telescope (HST) Cosmic Origins Spectrograph (COS) and Wide-Field Camera 3 (WFC3) observations of SN 1993J from 2012, at which point the flux from the SN had faded sufficiently to potentially measure the UV continuum properties from the putative companion. The resulting UV spectrum is consistent ...

  6. Evolution and structural diversification of PILS putative auxin carriers in plants

    Directory of Open Access Journals (Sweden)

    Elena eFeraru

    2012-10-01

    Full Text Available The phytohormone auxin contributes to virtually every aspect of the plant development. The spatiotemporal distribution of auxin depends on a complex interplay between auxin metabolism and intercellular auxin transport. Intracellular auxin compartmentalization provides another link between auxin transport processes and auxin metabolism. The PIN-LIKES (PILS putative auxin carriers localize to the endoplasmic reticulum (ER and contribute to cellular auxin homeostasis. PILS proteins regulate intracellular auxin accumulation, the rate of auxin conjugation and subsequently, affect nuclear auxin signalling. Here, we investigate sequence diversification of the PILS family in Arabidopsis thaliana and provide insights into the evolution of these novel putative auxin carriers in plants. Our data suggest that PILS proteins are conserved throughout the plant lineage and expanded during higher plant evolution. PILS proteins diversified early during plant evolution into three clades. Besides the ancient Clade I encompassing non-land plant species, PILS proteins evolved into two clades. The diversification of Clade II and Clade III occurred already at the level of non-vascular plant evolution and, hence, both clades contain vascular and non-vascular plant species. Nevertheless, Clade III contains fewer non- and increased numbers of vascular plants, indicating higher importance of Clade III for vascular plant evolution. Notably, PILS proteins are distinct and appear evolutionarily older than the prominent PIN-FORMED auxin carriers. Moreover, we revealed particular PILS sequence divergence in Arabidopsis and assume that these alterations could contribute to distinct gene regulations and protein functions.

  7. Characterization of putative multidrug resistance transporters of the major facilitator-superfamily expressed in Salmonella Typhi.

    Science.gov (United States)

    Shaheen, Aqsa; Ismat, Fouzia; Iqbal, Mazhar; Haque, Abdul; De Zorzi, Rita; Mirza, Osman; Walz, Thomas; Rahman, Moazur

    2015-05-01

    Multidrug resistance mediated by efflux pumps is a well-known phenomenon in infectious bacteria. Although much work has been carried out to characterize multidrug efflux pumps in Gram-negative and Gram-positive bacteria, such information is still lacking for many deadly pathogens. The aim of this study was to gain insight into the substrate specificity of previously uncharacterized transporters of Salmonella Typhi to identify their role in the development of multidrug resistance. S. Typhi genes encoding putative members of the major facilitator superfamily were cloned and expressed in the drug-hypersensitive Escherichia coli strain KAM42, and tested for transport of 25 antibacterial compounds, including representative antibiotics of various classes, antiseptics, dyes and detergents. Of the 15 tested putative transporters, STY0901, STY2458 and STY4874 exhibited a drug-resistance phenotype. Among these, STY4874 conferred resistance to at least ten of the tested antimicrobials: ciprofloxacin, norfloxacin, levofloxacin, kanamycin, streptomycin, gentamycin, nalidixic acid, chloramphenicol, ethidium bromide, and acriflavine, including fluoroquinolone antibiotics, which were drugs of choice to treat S. Typhi infections. Cell-based functional studies using ethidium bromide and acriflavine showed that STY4874 functions as a H(+)-dependent exporter. These results suggest that STY4874 may be an important drug target, which can now be tested by studying the susceptibility of a STY4874-deficient S. Typhi strain to antimicrobials.

  8. Mutation analysis of novel human liver-related putative tumor suppressor gene in hepatocellular carcinoma

    Institute of Scientific and Technical Information of China (English)

    Cheng Liao; Tsai-Ping Li; Mu-Jun Zhao; Jing Zhao; Hai Song; Pascal Pineau; Agnès Marchio; Anne Dejean; Pierre Tiollais; Hong-Yang Wang

    2003-01-01

    AIM: To find the point mutations meaningful for inactivationof liver-related putative tumor suppressor gene (LPTS) gene,a human novel liver-related putative tumor suppressor geneand telomerase inhibitor in hepatocellular carcinoma.METHODS: The entire coding sequence of LPTS genewas examined for mutations by single strand conformationpolymorphism (SSCP) assay and PCR products directsequencing in 56 liver cancer cell lines, 7 ovarian cancerand 7 head & neck tumor cell lines and 70 pairs of HCCtissues samples. The cDNA fragment coding for the mostfrequent mutant protein was subcloned into GST fusionexpression vector. The product was expressed in E. coliand purified by glutathione-agarose column. Telomericrepeat amplification protocol (TRAP) assays wereperformed to study the effect of point mutation totelomerase inhibitory activity.RESULTS: SSCP gels showed the abnormal shifting bandsand DNA sequencing found that there were 5 differentmutations and/or polymorphisms in 12 tumor cell lineslocated at exon2, exon5 and exon7. The main alterationswere A(778)A/G and A(880)T in exon7. The change in siteof 778 could not be found in HCC tissue samples, while themutation in position 880 was seen in 7 (10 %) cases. Themutation in the site of 880 had no effect on telomeraseinhibitory activity.CONCLUSION: Alterations identified in this study arepolymorphisms of LPTS gene. LPTS mutations occur in HCCbut are infrequent and of little effect on the telomeraseinhibitory function of the protein. Epigenetics, such asmethylation, acetylation, may play the key role in inactivationof LPTS.

  9. Expression of putative virulence factors in the potato pathogen Clavibacter michiganensis subsp. sepedonicus during infection.

    Science.gov (United States)

    Holtsmark, Ingrid; Takle, Gunnhild W; Brurberg, May Bente

    2008-02-01

    The Gram-positive bacterium Clavibacter michiganensis subsp. sepedonicus is the causal agent of bacterial wilt and ring rot of potato. So far, only two proteins have been shown to be essential for virulence, namely a plasmid-encoded cellulase CelA and a hypersensitive response-inducing protein. We have examined the relative expression of CelA and eight putative virulence factors during infection of potato and in liquid culture, using quantitative real-time PCR. The examined putative virulence genes were celB, a cellulase-encoding gene and genes encoding a pectate lyase, a xylanase and five homologues of the Clavibacter michiganensis subsp. michiganensis pathogenicity factor Pat-1 thought to encode a serine protease. Six of the nine assayed genes were up-regulated during infection of potato, including celA, celB, the xylanase gene, and two of the pat genes. The pectate lyase gene showed only slightly elevated expression, whereas three of the five examined pat genes were down-regulated during infection in potato. Interestingly, the two up-regulated pat genes showed a noticeable sequence difference compared to the three down-regulated pat genes. These results reveal several new proteins that are likely to be involved in Clavibacter michiganensis subsp. sepedonicus pathogenicity.

  10. Transcriptome of Aphanomyces euteiches: new oomycete putative pathogenicity factors and metabolic pathways.

    Directory of Open Access Journals (Sweden)

    Elodie Gaulin

    Full Text Available Aphanomyces euteiches is an oomycete pathogen that causes seedling blight and root rot of legumes, such as alfalfa and pea. The genus Aphanomyces is phylogenically distinct from well-studied oomycetes such as Phytophthora sp., and contains species pathogenic on plants and aquatic animals. To provide the first foray into gene diversity of A. euteiches, two cDNA libraries were constructed using mRNA extracted from mycelium grown in an artificial liquid medium or in contact to plant roots. A unigene set of 7,977 sequences was obtained from 18,864 high-quality expressed sequenced tags (ESTs and characterized for potential functions. Comparisons with oomycete proteomes revealed major differences between the gene content of A. euteiches and those of Phytophthora species, leading to the identification of biosynthetic pathways absent in Phytophthora, of new putative pathogenicity genes and of expansion of gene families encoding extracellular proteins, notably different classes of proteases. Among the genes specific of A. euteiches are members of a new family of extracellular proteins putatively involved in adhesion, containing up to four protein domains similar to fungal cellulose binding domains. Comparison of A. euteiches sequences with proteomes of fully sequenced eukaryotic pathogens, including fungi, apicomplexa and trypanosomatids, allowed the identification of A. euteiches genes with close orthologs in these microorganisms but absent in other oomycetes sequenced so far, notably transporters and non-ribosomal peptide synthetases, and suggests the presence of a defense mechanism against oxidative stress which was initially characterized in the pathogenic trypanosomatids.

  11. Characterization of Putative cis-Regulatory Elements in Genes Preferentially Expressed in Arabidopsis Male Meiocytes

    Directory of Open Access Journals (Sweden)

    Junhua Li

    2014-01-01

    Full Text Available Meiosis is essential for plant reproduction because it is the process during which homologous chromosome pairing, synapsis, and meiotic recombination occur. The meiotic transcriptome is difficult to investigate because of the size of meiocytes and the confines of anther lobes. The recent development of isolation techniques has enabled the characterization of transcriptional profiles in male meiocytes of Arabidopsis. Gene expression in male meiocytes shows unique features. The direct interaction of transcription factors (TFs with DNA regulatory sequences forms the basis for the specificity of transcriptional regulation. Here, we identified putative cis-regulatory elements (CREs associated with male meiocyte-expressed genes using in silico tools. The upstream regions (1 kb of the top 50 genes preferentially expressed in Arabidopsis meiocytes possessed conserved motifs. These motifs are putative binding sites of TFs, some of which share common functions, such as roles in cell division. In combination with cell-type-specific analysis, our findings could be a substantial aid for the identification and experimental verification of the protein-DNA interactions for the specific TFs that drive gene expression in meiocytes.

  12. Identification and site of action of the remaining four putative pseudouridine synthases in Escherichia coli.

    Science.gov (United States)

    Del Campo, M; Kaya, Y; Ofengand, J

    2001-11-01

    There are 10 known putative pseudouridine synthase genes in Escherichia coli. The products of six have been previously assigned, one to formation of the single pseudouridine in 16S RNA, three to the formation of seven pseudouridines in 23S RNA, and three to the formation of three pseudouridines in tRNA (one synthase makes pseudouridine in 23S RNA and tRNA). Here we show that the remaining four putative synthase genes make bona fide pseudouridine synthases and identify which pseudouridines they make. RluB (formerly YciL) and RluE (formerly YmfC) make pseudouridine2605 and pseudouridine2457, respectively, in 23S RNA. RluF (formerly YjbC) makes the newly discovered pseudouridine2604 in 23S RNA, and TruC (formerly YqcB) makes pseudouridine65 in tRNA(Ile1) and tRNA(Asp). Deletion of each of these synthase genes individually had no effect on exponential growth in rich media at 25 degrees C, 37 degrees C, or 42 degrees C. A strain lacking RluB and RluF also showed no growth defect under these conditions. Mutation of a conserved aspartate in a common sequence motif, previously shown to be essential for the other six E. coli pseudouridine synthases and several yeast pseudouridine synthases, also caused a loss of in vivo activity in all four of the synthases studied in this work.

  13. Transcriptome Analysis Reveals Putative Genes Involved in Iridoid Biosynthesis in Rehmannia glutinosa

    Directory of Open Access Journals (Sweden)

    Xianen Li

    2012-10-01

    Full Text Available Rehmannia glutinosa, one of the most widely used herbal medicines in the Orient, is rich in biologically active iridoids. Despite their medicinal importance, no molecular information about the iridoid biosynthesis in this plant is presently available. To explore the transcriptome of R. glutinosa and investigate genes involved in iridoid biosynthesis, we used massively parallel pyrosequencing on the 454 GS FLX Titanium platform to generate a substantial EST dataset. Based on sequence similarity searches against the public sequence databases, the sequences were first annotated and then subjected to Gene Ontology (GO and Kyoto Encyclopedia of Genes and Genomes (KEGG based analysis. Bioinformatic analysis indicated that the 454 assembly contained a set of genes putatively involved in iridoid biosynthesis. Significantly, homologues of the secoiridoid pathway genes that were only identified in terpenoid indole alkaloid producing plants were also identified, whose presence implied that route II iridoids and route I iridoids share common enzyme steps in the early stage of biosynthesis. The gene expression patterns of four prenyltransferase transcripts were analyzed using qRT-PCR, which shed light on their putative functions in tissues of R. glutinosa. The data explored in this study will provide valuable information for further studies concerning iridoid biosynthesis.

  14. Mapping the flow of information within the putative mirror neuron system during gesture observation.

    Science.gov (United States)

    Schippers, Marleen B; Keysers, Christian

    2011-07-01

    The putative mirror neuron system may either function as a strict feed-forward system or as a dynamic control system. A strict feed-forward system would predict that action observation leads to a predominantly temporal→parietal→premotor flow of information in which a visual representation is transformed into motor-programs which contribute to action understanding. Instead, a dynamic feedback control system would predict that the reverse direction of information flow predominates because of a combination of inhibitory forward and excitatory inverse models. Here we test which of these conflicting predictions best matches the information flow within the putative mirror neuron system (pMNS) and between the pMNS and the rest of the brain during the observation of comparatively long naturalistic stretches of communicative gestures. We used Granger causality to test the dominant direction of influence. Our results fit the predictions of the dynamic feedback control system: we found predominantly an information flow within the pMNS from premotor to parietal and middle temporal cortices. This is more pronounced during an active guessing task than while passively reviewing the same gestures. In particular, the ventral premotor cortex sends significantly more information to other pMNS areas than it receives during active guessing than during passive observation.

  15. Identification and functional analysis of Penicillium digitatum genes putatively involved in virulence towards citrus fruit.

    Science.gov (United States)

    López-Pérez, Mario; Ballester, Ana-Rosa; González-Candelas, Luis

    2015-04-01

    The fungus Penicillium digitatum, the causal agent of green mould rot, is the most destructive post-harvest pathogen of citrus fruit in Mediterranean regions. In order to identify P. digitatum genes up-regulated during the infection of oranges that may constitute putative virulence factors, we followed a polymerase chain reaction (PCR)-based suppression subtractive hybridization and cDNA macroarray hybridization approach. The origin of expressed sequence tags (ESTs) was determined by comparison against the available genome sequences of both organisms. Genes coding for fungal proteases and plant cell wall-degrading enzymes represent the largest categories in the subtracted cDNA library. Northern blot analysis of a selection of P. digitatum genes, including those coding for proteases, cell wall-related enzymes, redox homoeostasis and detoxification processes, confirmed their up-regulation at varying time points during the infection process. Agrobacterium tumefaciens-mediated transformation was used to generate knockout mutants for two genes encoding a pectin lyase (Pnl1) and a naphthalene dioxygenase (Ndo1). Two independent P. digitatum Δndo1 mutants were as virulent as the wild-type. However, the two Δpnl1 mutants analysed were less virulent than the parental strain or an ectopic transformant. Together, these results provide a significant advance in our understanding of the putative determinants of the virulence mechanisms of P. digitatum.

  16. Unprecedented diversity of catalytic domains in the first four modules of the putative pederin polyketide synthase.

    Science.gov (United States)

    Piel, Jörn; Wen, Gaiping; Platzer, Matthias; Hui, Dequan

    2004-01-03

    Polyketides of the pederin group are highly potent antitumor compounds found in terrestrial beetles and marine sponges. Pederin is used by beetles of the genera Paederus and Paederidus as a chemical defense. We have recently identified a group of putative pederin biosynthesis genes and localized them to the genome of an as yet unculturable Pseudomonas sp. symbiont, the likely true pederin producer. However, this polyketide synthase cluster lacks several genes expected for pederin production. Here we report an additional polyketide synthase encoded on a separate region of the symbiont genome. It contains at least three novel catalytic domains that are predicted to be involved in pederin chain initiation and the formation of an unusual exomethylene bond. The region is bordered by mobility pseudogenes; this suggests that gene transposition led to the disjointed cluster organization. With this work, all putative pederin genes have been identified. Their heterologous expression in a culturable bacterium will provide important insights into how sustainable sources of invertebrate-derived drug candidates can be created.

  17. Biochemical Characterization of Putative Adenylate Dimethylallyltransferase and Cytokinin Dehydrogenase from Nostoc sp. PCC 7120.

    Science.gov (United States)

    Frébortová, Jitka; Greplová, Marta; Seidl, Michael F; Heyl, Alexander; Frébort, Ivo

    2015-01-01

    Cytokinins, a class of phytohormones, are adenine derivatives common to many different organisms. In plants, these play a crucial role as regulators of plant development and the reaction to abiotic and biotic stress. Key enzymes in the cytokinin synthesis and degradation in modern land plants are the isopentyl transferases and the cytokinin dehydrogenases, respectively. Their encoding genes have been probably introduced into the plant lineage during the primary endosymbiosis. To shed light on the evolution of these proteins, the genes homologous to plant adenylate isopentenyl transferase and cytokinin dehydrogenase were amplified from the genomic DNA of cyanobacterium Nostoc sp. PCC 7120 and expressed in Escherichia coli. The putative isopentenyl transferase was shown to be functional in a biochemical assay. In contrast, no enzymatic activity was detected for the putative cytokinin dehydrogenase, even though the principal domains necessary for its function are present. Several mutant variants, in which conserved amino acids in land plant cytokinin dehydrogenases had been restored, were inactive. A combination of experimental data with phylogenetic analysis indicates that adenylate-type isopentenyl transferases might have evolved several times independently. While the Nostoc genome contains a gene coding for protein with characteristics of cytokinin dehydrogenase, the organism is not able to break down cytokinins in the way shown for land plants.

  18. TiD: Standalone software for mining putative drug targets from bacterial proteome.

    Science.gov (United States)

    Gupta, Reena; Pradhan, Dibyabhaba; Jain, Arun Kumar; Rai, Chandra Shekhar

    2017-01-01

    TiD is a standalone application, which relies on basic assumption that a protein must be essential for pathogens survival and non-homologous with host to qualify as putative target. With an input bacterial proteome, TiD removes paralogous proteins, picks essential ones, and excludes proteins homologous with host organisms. The targets illustrate non-homology with at least 40 out of 84 gut microbes, considered safe for human. TiD classifies proposed targets as known, novel and virulent. Users can perform pathway analysis, choke point analysis, interactome analysis, subcellular localization and functional annotations through web servers cross-referenced with the application. Drug targets identified by TiD for Listeria monocytogenes, Bacillus anthracis and Pseudomonas aeruginosa have revealed significant overlaps with previous studies. TiD takes <2h to scan putative targets from a bacterial proteome with ~5000 proteins; hence, we propose it as a useful tool for rational drug design. TiD is available at http://bmicnip.in/TiD/. Copyright © 2016 Elsevier Inc. All rights reserved.

  19. VenomKB, a new knowledge base for facilitating the validation of putative venom therapies.

    Science.gov (United States)

    Romano, Joseph D; Tatonetti, Nicholas P

    2015-11-24

    Animal venoms have been used for therapeutic purposes since the dawn of recorded history. Only a small fraction, however, have been tested for pharmaceutical utility. Modern computational methods enable the systematic exploration of novel therapeutic uses for venom compounds. Unfortunately, there is currently no comprehensive resource describing the clinical effects of venoms to support this computational analysis. We present VenomKB, a new publicly accessible knowledge base and website that aims to act as a repository for emerging and putative venom therapies. Presently, it consists of three database tables: (1) Manually curated records of putative venom therapies supported by scientific literature, (2) automatically parsed MEDLINE articles describing compounds that may be venom derived, and their effects on the human body, and (3) automatically retrieved records from the new Semantic Medline resource that describe the effects of venom compounds on mammalian anatomy. Data from VenomKB may be selectively retrieved in a variety of popular data formats, are open-source, and will be continually updated as venom therapies become better understood.

  20. Small-fragment genomic libraries for the display of putative epitopes from clinically significant pathogens.

    Science.gov (United States)

    Henics, T; Winkler, B; Pfeifer, U; Gill, S R; Buschle, M; von Gabain, A; Meinke, A L

    2003-07-01

    Taking advantage of whole genome sequences of bacterial pathogens in many thriving diseases with global impact, we developed a comprehensive screening procedure for the identification of putative vaccine candidate antigens. Importantly, this procedure relies on highly representative small-fragment genomic libraries that are expressed to display frame-selected epitope-size peptides on a bacterial cell surface and to interact directly with carefully selected disease-relevant high-titer sera. Here we describe the generation of small-fragment genomic libraries of Gram-positive and Gram-negative clinically significant pathogens, including Staphylococcus aureus and Staphylococcus epidermidis, Streptococcus pyogenes, Streptococcus agalactiae, and Streptococcus pneumoniae, Enterococcus faecalis, Helicobacter pylori, Chlamydia pneumoniae, the enterotoxigenic Escherichia coli, and Campylobacter jejuni. Large-scale sequencing revealed that the libraries, which provide an average of 20-fold coverage, were random and, as demonstrated with two S. aureus libraries, highly representative. Consistent with the comprehensive nature of this approach is the identification of epitopes that reside in both annotated and putatively novel open reading frames. The use of these libraries therefore allows for the rapid and direct identification of immunogenic epitopes with no apparent bias or difficulty that often associate with conventional expression methods.

  1. Five putative nucleoside triphosphate diphosphohydrolase genes are expressed in Trichomonas vaginalis.

    Science.gov (United States)

    Frasson, Amanda Piccoli; Dos Santos, Odelta; Meirelles, Lúcia Collares; Macedo, Alexandre José; Tasca, Tiana

    2016-01-01

    Trichomonas vaginalis is a protozoan that parasitizes the human urogenital tract causing trichomoniasis, the most common non-viral sexually transmitted disease. The parasite has unique genomic characteristics such as a large genome size and expanded gene families. Ectonucleoside triphosphate diphosphohydrolase (E-NTPDase) is an enzyme responsible for hydrolyzing nucleoside tri- and diphosphates and has already been biochemically characterized in T. vaginalis. Considering the important role of this enzyme in the production of extracellular adenosine for parasite uptake, we evaluated the gene expression of five putative NTPDases in T. vaginalis. We showed that all five putative TvNTPDase genes (TvNTPDase1-5) were expressed by both fresh clinical and long-term grown isolates. The amino acid alignment predicted the presence of the five crucial apyrase conserved regions, transmembrane domains, signal peptides, phosphorylation and catalytic sites. Moreover, a phylogenetic analysis showed that TvNTPDase sequences make up a clade with NTPDases intracellularly located. Biochemical NTPDase activity (ATP and ADP hydrolysis) is responsive to the serum-restrictive conditions and the gene expression of TvNTPDases was mostly increased, mainly TvNTPDase2 and TvNTPDase4, although there was not a clear pattern of expression among them. In summary, the present report demonstrates the gene expression patterns of predicted NTPDases in T. vaginalis.

  2. Functional characterization of PaLAX1, a putative auxin permease, in heterologous plant systems.

    Science.gov (United States)

    Hoyerová, Klára; Perry, Lucie; Hand, Paul; Lanková, Martina; Kocábek, Tomás; May, Sean; Kottová, Jana; Paces, Jan; Napier, Richard; Zazímalová, Eva

    2008-03-01

    We have isolated the cDNA of the gene PaLAX1 from a wild cherry tree (Prunus avium). The gene and its product are highly similar in sequences to both the cDNAs and the corresponding protein products of AUX/LAX-type genes, coding for putative auxin influx carriers. We have prepared and characterized transformed Nicotiana tabacum and Arabidopsis thaliana plants carrying the gene PaLAX1. We have proved that constitutive overexpression of PaLAX1 is accompanied by changes in the content and distribution of free indole-3-acetic acid, the major endogenous auxin. The increase in free indole-3-acetic acid content in transgenic plants resulted in various phenotype changes, typical for the auxin-overproducing plants. The uptake of synthetic auxin, 2,4-dichlorophenoxyacetic acid, was 3 times higher in transgenic lines compared to the wild-type lines and the treatment with the auxin uptake inhibitor 1-naphthoxyacetic acid reverted the changes caused by the expression of PaLAX1. Moreover, the agravitropic response could be restored by expression of PaLAX1 in the mutant aux1 plants, which are deficient in auxin influx carrier activity. Based on our data, we have concluded that the product of the gene PaLAX1 promotes the uptake of auxin into cells, and, as a putative auxin influx carrier, it affects the content and distribution of free endogenous auxin in transgenic plants.

  3. Sensitivity analysis of the relationship between disease occurrence and distance from a putative source of pollution

    Directory of Open Access Journals (Sweden)

    Emanuela Dreassi

    2008-05-01

    Full Text Available The relation between disease risk and a point source of pollution is usually investigated using distance from the source as a proxy of exposure. The analysis may be based on case-control data or on aggregated data. The definition of the function relating risk of disease and distance is critical, both in a classical and in a Bayesian framework, because the likelihood is usually very flat, even with large amounts of data. In this paper we investigate how the specification of the function relating risk of disease with distance from the source and of the prior distributions on the parameters of the function affects the results when case-control data and Bayesian methods are used. We consider different popular parametric models for the risk distance function in a Bayesian approach, comparing estimates with those derived by maximum likelihood. As an example we have analyzed the relationship between a putative source of environmental pollution (an asbestos cement plant and the occurrence of pleural malignant mesothelioma in the area of Casale Monferrato (Italy in 1987-1993. Risk of pleural malignant mesothelioma turns out to be strongly related to distance from the asbestos cement plant. However, as the models appeared to be sensitive to modeling choices, we suggest that any analysis of disease risk around a putative source should be integrated with a careful sensitivity analysis and possibly with prior knowledge. The choice of prior distribution is extremely important and should be based on epidemiological considerations.

  4. Putative monofunctional type I polyketide synthase units: a dinoflagellate-specific feature?

    Directory of Open Access Journals (Sweden)

    Karsten Eichholz

    Full Text Available Marine dinoflagellates (alveolata are microalgae of which some cause harmful algal blooms and produce a broad variety of most likely polyketide synthesis derived phycotoxins. Recently, novel polyketide synthesase (PKS transcripts have been described from the Florida red tide dinoflagellate Karenia brevis (gymnodiniales which are evolutionarily related to Type I PKS but were apparently expressed as monofunctional proteins, a feature typical of Type II PKS. Here, we investigated expression units of PKS I-like sequences in Alexandrium ostenfeldii (gonyaulacales and Heterocapsa triquetra (peridiniales at the transcript and protein level. The five full length transcripts we obtained were all characterized by polyadenylation, a 3' UTR and the dinoflagellate specific spliced leader sequence at the 5'end. Each of the five transcripts encoded a single ketoacylsynthase (KS domain showing high similarity to K. brevis KS sequences. The monofunctional structure was also confirmed using dinoflagellate specific KS antibodies in Western Blots. In a maximum likelihood phylogenetic analysis of KS domains from diverse PKSs, dinoflagellate KSs formed a clade placed well within the protist Type I PKS clade between apicomplexa, haptophytes and chlorophytes. These findings indicate that the atypical PKS I structure, i.e., expression as putative monofunctional units, might be a dinoflagellate specific feature. In addition, the sequenced transcripts harbored a previously unknown, apparently dinoflagellate specific conserved N-terminal domain. We discuss the implications of this novel region with regard to the putative monofunctional organization of Type I PKS in dinoflagellates.

  5. Interaction of Medicago truncatula lysin motif receptor-like kinases, NFP and LYK3, produced in Nicotiana benthamiana induces defence-like responses.

    Directory of Open Access Journals (Sweden)

    Anna Pietraszewska-Bogiel

    Full Text Available Receptor(-like kinases with Lysin Motif (LysM domains in their extracellular region play crucial roles during plant interactions with microorganisms; e.g. Arabidopsis thaliana CERK1 activates innate immunity upon perception of fungal chitin/chitooligosaccharides, whereas Medicago truncatula NFP and LYK3 mediate signalling upon perception of bacterial lipo-chitooligosaccharides, termed Nod factors, during the establishment of mutualism with nitrogen-fixing rhizobia. However, little is still known about the exact activation and signalling mechanisms of MtNFP and MtLYK3. We aimed at investigating putative molecular interactions of MtNFP and MtLYK3 produced in Nicotiana benthamiana. Surprisingly, heterologous co-production of these proteins resulted in an induction of defence-like responses, which included defence-related gene expression, accumulation of phenolic compounds, and cell death. Similar defence-like responses were observed upon production of AtCERK1 in N. benthamiana leaves. Production of either MtNFP or MtLYK3 alone or their co-production with other unrelated receptor(-like kinases did not induce cell death in N. benthamiana, indicating that a functional interaction between these LysM receptor-like kinases is required for triggering this response. Importantly, structure-function studies revealed that the MtNFP intracellular region, specific features of the MtLYK3 intracellular region (including several putative phosphorylation sites, and MtLYK3 and AtCERK1 kinase activity were indispensable for cell death induction, thereby mimicking the structural requirements of nodulation or chitin-induced signalling. The observed similarity of N. benthamiana response to MtNFP and MtLYK3 co-production and AtCERK1 production suggests the existence of parallels between Nod factor-induced and chitin-induced signalling mediated by the respective LysM receptor(-like kinases. Notably, the conserved structural requirements for MtNFP and MtLYK3 biological

  6. The secret life of kinases: functions beyond catalysis

    Directory of Open Access Journals (Sweden)

    Romano David

    2011-10-01

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

  7. Extended-spectrum antiprotozoal bumped kinase inhibitors: A review.

    Science.gov (United States)

    Van Voorhis, Wesley C; Doggett, J Stone; Parsons, Marilyn; Hulverson, Matthew A; Choi, Ryan; Arnold, Samuel L M; Riggs, Michael W; Hemphill, Andrew; Howe, Daniel K; Mealey, Robert H; Lau, Audrey O T; Merritt, Ethan A; Maly, Dustin J; Fan, Erkang; Ojo, Kayode K

    2017-09-01

    Many life-cycle processes in parasites are regulated by protein phosphorylation. Hence, disruption of essential protein kinase function has been explored for therapy of parasitic diseases. However, the difficulty of inhibiting parasite protein kinases to the exclusion of host orthologues poses a practical challenge. A possible path around this difficulty is the use of bumped kinase inhibitors for targeting calcium-dependent protein kinases that contain atypically small gatekeeper residues and are crucial for pathogenic apicomplexan parasites' survival and proliferation. In this article, we review efficacy against the kinase target, parasite growth in vitro, and in animal infection models, as well as the relevant pharmacokinetic and safety parameters of bumped kinase inhibitors. Copyright © 2017 Elsevier Inc. All rights reserved.

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

    LENUS (Irish Health Repository)

    Rauch, Jens

    2011-10-28

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

  9. Toward understanding the functional role of Ss-RIOK-1, a RIO protein kinase-encoding gene of Strongyloides stercoralis.

    Directory of Open Access Journals (Sweden)

    Wang Yuan

    2014-08-01

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

  10. Symbiotic rhizobia bacteria trigger a change in localization and dynamics of the Medicago truncatula receptor kinase LYK3.

    Science.gov (United States)

    Haney, Cara H; Riely, Brendan K; Tricoli, David M; Cook, Doug R; Ehrhardt, David W; Long, Sharon R

    2011-07-01

    To form nitrogen-fixing symbioses, legume plants recognize a bacterial signal, Nod Factor (NF). The legume Medicago truncatula has two predicted NF receptors that direct separate downstream responses to its symbiont Sinorhizobium meliloti. NOD FACTOR PERCEPTION encodes a putative low-stringency receptor that is responsible for calcium spiking and transcriptional responses. LYSIN MOTIF RECEPTOR-LIKE KINASE3 (LYK3) encodes a putative high-stringency receptor that mediates bacterial infection. We localized green fluorescent protein (GFP)-tagged LYK3 in M. truncatula and found that it has a punctate distribution at the cell periphery consistent with a plasma membrane or membrane-tethered vesicle localization. In buffer-treated control roots, LYK3:GFP puncta are dynamic. After inoculation with compatible S. meliloti, LYK3:GFP puncta are relatively stable. We show that increased LYK3:GFP stability depends on bacterial NF and NF structure but that NF is not sufficient for the change in LYK3:GFP dynamics. In uninoculated root hairs, LYK3:GFP has little codistribution with mCherry-tagged FLOTILLIN4 (FLOT4), another punctate plasma membrane-associated protein required for infection. In inoculated root hairs, we observed an increase in FLOT4:mCherry and LYK3:GFP colocalization; both proteins localize to positionally stable puncta. We also demonstrate that the localization of tagged FLOT4 is altered in plants carrying a mutation that inactivates the kinase domain of LYK3. Our work indicates that LYK3 protein localization and dynamics are altered in response to symbiotic bacteria.

  11. The NDR/LATS kinase Cbk1 controls the activity of the transcriptional regulator Bcr1 during biofilm formation in Candida albicans.

    Directory of Open Access Journals (Sweden)

    Pilar Gutiérrez-Escribano

    Full Text Available In nature, many microorganisms form specialized complex, multicellular, surface-attached communities called biofilms. These communities play critical roles in microbial pathogenesis. The fungal pathogen Candida albicans is associated with catheter-based infections due to its ability to establish biofilms. The transcription factor Bcr1 is a master regulator of C. albicans biofilm development, although the full extent of its regulation remains unknown. Here, we report that Bcr1 is a phosphoprotein that physically interacts with the NDR kinase Cbk1 and undergoes Cbk1-dependent phosphorylation. Mutating the two putative Cbk1 phosphoacceptor residues in Bcr1 to alanine markedly impaired Bcr1 function during biofilm formation and virulence in a mouse model of disseminated candidiasis. Cells lacking Cbk1, or any of its upstream activators, also had reduced biofilm development. Notably, mutating the two putative Cbk1 phosphoacceptor residues in Bcr1 to glutamate in cbk1Δ cells upregulated the transcription of Bcr1-dependent genes and partially rescued the biofilm defects of a cbk1Δ strain. Therefore, our data uncovered a novel role of the NDR/LATS kinase Cbk1 in the regulation of biofilm development through the control of Bcr1.

  12. Drosophila melanogaster deoxyribonucleoside kinase activates gemcitabine

    Energy Technology Data Exchange (ETDEWEB)

    Knecht, Wolfgang [BioCentrum-DTU, Technical University of Denmark, DK-2800 Lyngby (Denmark); Mikkelsen, Nils Egil [Department of Molecular Biology, Swedish University of Agricultural Sciences, Biomedical Centre, SE-751 24 Uppsala (Sweden); Clausen, Anders Ranegaard [Cell and Organism Biology, Lund University, Soelvegatan 35, SE-22362 Lund (Sweden); Willer, Mette [ZGene A/S, Agern Alle 7, DK-2970 Horsholm (Denmark); Eklund, Hans [Department of Molecular Biology, Swedish University of Agricultural Sciences, Biomedical Centre, SE-751 24 Uppsala (Sweden); Gojkovic, Zoran [ZGene A/S, Agern Alle 7, DK-2970 Horsholm (Denmark); Piskur, Jure, E-mail: Jure.Piskur@cob.lu.se [BioCentrum-DTU, Technical University of Denmark, DK-2800 Lyngby (Denmark); Cell and Organism Biology, Lund University, Soelvegatan 35, SE-22362 Lund (Sweden)

    2009-05-01

    Drosophila melanogaster multisubstrate deoxyribonucleoside kinase (Dm-dNK) can additionally sensitize human cancer cell lines towards the anti-cancer drug gemcitabine. We show that this property is based on the Dm-dNK ability to efficiently phosphorylate gemcitabine. The 2.2 A resolution structure of Dm-dNK in complex with gemcitabine shows that the residues Tyr70 and Arg105 play a crucial role in the firm positioning of gemcitabine by extra interactions made by the fluoride atoms. This explains why gemcitabine is a good substrate for Dm-dNK.

  13. Rho kinases in cardiovascular physiology and pathophysiology.

    Science.gov (United States)

    Loirand, Gervaise; Guérin, Patrice; Pacaud, Pierre

    2006-02-17

    Rho kinases (ROCKs) are the first and the best-characterized effectors of the small G-protein RhoA. In addition to their effect on actin organization, or through this effect, ROCKs have been found to regulate a wide range of fundamental cell functions such as contraction, motility, proliferation, and apoptosis. Abnormal activation of the RhoA/ROCK pathway has been observed in major cardiovascular disorders such as atherosclerosis, restenosis, hypertension, pulmonary hypertension, and cardiac hypertrophy. This review, based on recent molecular, cellular, and animal studies, focuses on the current understanding of ROCK signaling and its roles in cardiovascular physiology and pathophysiology.

  14. Protein kinase CK2 in human diseases

    DEFF Research Database (Denmark)

    Guerra, Barbara; Issinger, Olaf-Georg

    2008-01-01

    in various disease processes including cancer has been gained in recent years, and the present review may help to further elucidate its aberrant role in many disease states. Its peculiar structural features [3-9] may be advantageous in designing tailor-made compounds with the possibility to specifically...... target this protein kinase [10]. Since not all the aspects of what has been published on CK2 can be covered in this review, we would like to recommend the following reviews; (i) for general information on CK2 [11-18] and (ii) with a focus on aberrant CK2 [19-22]....

  15. Conformation-specific inhibitors of Raf kinases.

    Science.gov (United States)

    Wang, Xiaolun; Schleicher, Kristin

    2013-01-01

    Since the discovery linking B-Raf mutations to human tumors in 2002, significant advances in the development of Raf inhibitors have been made, leading to the recent approval of two Raf inhibitor drugs. This chapter includes a brief introduction to B-Raf as a validated target and focuses on the three different binding modes observed with Raf small-molecule inhibitors. These various binding modes lock the Raf kinase in different conformations that impact the toxicity profiles of the inhibitors. Possible solutions to mitigate the side effects caused by inhibitor-induced dimerization are also discussed.

  16. Determination of aspartate kinase activity in maize tissues

    OpenAIRE

    Ferreira,Renato Rodrigues; Vendemiatti,Ariane; Gratão, Priscila Lupino; Lea, Peter John; Azevedo, Ricardo Antunes

    2005-01-01

    Lysine, threonine, methionine and isoleucine are synthesized from aspartate in a branched pathway in higher plants. Aspartate kinase plays a key role in the control of the aspartate pathway. The enzyme is very sensitive to manipulation and storage and the hydroxamate assay normally used to determine aspartate kinase activity has to be altered according to the plant species and tissue to be analyzed. We have optimized the assay for the determination of aspartate kinase in maize plants callus c...

  17. Protein kinases are potential targets to treat inflammatory bowel disease

    Institute of Scientific and Technical Information of China (English)

    Lei; Yang; Yutao; Yan

    2014-01-01

    Protein kinases play a crucial role in the pathogenesis of inflammatory bowel disease(IBD), the two main forms of which are ulcerative colitis and Crohn’s dis-ease. In this article, we will review the mechanisms of involvement of protein kinases in the pathogenesis of and intervention against IBD, in terms of their effects on genetics, microbiota, mucous layer and tight junc-tion, and the potential of protein kinases as therapeutic targets against IBD.

  18. Involvement of the MAP kinase cascade in Xenopus mesoderm induction.

    OpenAIRE

    Gotoh, Y.; Masuyama, N; Suzuki, A.; Ueno, N; Nishida, E

    1995-01-01

    Mitogen-activated protein kinase (MAPK) is activated by MAPK kinase (MAPKK) in a variety of signaling pathways. This kinase cascade has been shown to function in cell proliferation and differentiation, but its role in early vertebrate development remains to be investigated. During early vertebrate embryogenesis, the induction and patterning of mesoderm are thought to be determined by signals from intercellular factors such as members of the fibroblast growth factor (FGF) family and members of...

  19. Clinical stage EGFR inhibitors irreversibly alkylate Bmx kinase.

    Science.gov (United States)

    Hur, Wooyoung; Velentza, Anastasia; Kim, Sungjoon; Flatauer, Laura; Jiang, Xinnong; Valente, David; Mason, Daniel E; Suzuki, Melissa; Larson, Brad; Zhang, Jianming; Zagorska, Anna; Didonato, Michael; Nagle, Advait; Warmuth, Markus; Balk, Steven P; Peters, Eric C; Gray, Nathanael S

    2008-11-15

    Irreversible HER/erbB inhibitors selectively inhibit HER-family kinases by targeting a unique cysteine residue located within the ATP-binding pocket. Sequence alignment reveals that this rare cysteine is also present in ten other protein kinases including all five Tec-family members. We demonstrate that the Tec-family kinase Bmx is potently inhibited by irreversible modification at Cys496 by clinical stage EGFR inhibitors such as CI-1033. This cross-reactivity may have significant clinical implications.

  20. Peptide substrates for Rho-associated kinase 2 (Rho-kinase 2/ROCK2.

    Directory of Open Access Journals (Sweden)

    Jeong-Hun Kang

    Full Text Available Peptide substrates sensitive for a certain protein kinase could be important for new-drug development and to understand the mechanism of diseases. Rho-associated kinase (Rho-kinase/ROCK is a serine/threonine kinase, and plays an important part in cardiovascular disease, migration and invasion of tumor cells, and in neurological disorders. The purpose of this study was to find substrates with high affinity and sensitivity for ROCK2. We synthesized 136 peptide substrates from protein substrates for ROCK2 with different lengths and charged peptides. Incorporation of (32P [counts per minute (CPM] for each peptide substrate was determined by the radiolabel assay using [γ-(32P]ATP. When the top five peptide substrates showing high CPMs (R4, R22, R133, R134, and R135 were phosphorylated by other enzymes (PKA, PKCα, and ERK1, R22, R133, and R135 displayed the highest CPM level for ROCK2 compared with other enzymes, whereas R4 and R134 showed similar CPM levels for ROCK2 and PKCα. We hypothesize that R22, R133, and R135 can be useful peptide substrates for ROCK2.

  1. p21-activated Kinase1(PAK1) can promote ERK activation in a kinase independent manner

    DEFF Research Database (Denmark)

    Wang, Zhipeng; Fu, Meng; Wang, Lifeng

    2013-01-01

    204) although phosphorylation of b-Raf (Ser445) and c-Raf (Ser 338) remained unchanged. Furthermore, increased activation of the PAK1 activator Rac1 induced the formation of a triple complex of Rac1, PAK1 and Mek1, independent of the kinase activity of PAK1. These data suggest that PAK1 can stimulate...

  2. Nonmuscle Myosin IIA Regulates Platelet Contractile Forces Through Rho Kinase and Myosin Light-Chain Kinase.

    Science.gov (United States)

    Feghhi, Shirin; Tooley, Wes W; Sniadecki, Nathan J

    2016-10-01

    Platelet contractile forces play a major role in clot retraction and help to hold hemostatic clots against the vessel wall. Platelet forces are produced by its cytoskeleton, which is composed of actin and nonmuscle myosin filaments. In this work, we studied the role of Rho kinase, myosin light-chain kinase, and myosin in the generation of contractile forces by using pharmacological inhibitors and arrays of flexible microposts to measure platelet forces. When platelets were seeded onto microposts, they formed aggregates on the tips of the microposts. Forces produced by the platelets in the aggregates were measured by quantifying the deflection of the microposts, which bent in proportion to the force of the platelets. Platelets were treated with small molecule inhibitors of myosin activity: Y-27632 to inhibit the Rho kinase (ROCK), ML-7 to inhibit myosin light-chain kinase (MLCK), and blebbistatin to inhibit myosin ATPase activity. ROCK inhibition reduced platelet forces, demonstrating the importance of the assembly of actin and myosin phosphorylation in generating contractile forces. Similarly, MLCK inhibition caused weaker platelet forces, which verifies that myosin phosphorylation is needed for force generation in platelets. Platelets treated with blebbistatin also had weaker forces, which indicates that myosin's ATPase activity is necessary for platelet forces. Our studies demonstrate that myosin ATPase activity and the regulation of actin-myosin assembly by ROCK and MLCK are needed for the generation of platelet forces. Our findings illustrate and explain the importance of myosin for clot compaction in hemostasis and thrombosis.

  3. Structures of down syndrome kinases, DYRKs, reveal mechanisms of kinase activation and substrate recognition

    DEFF Research Database (Denmark)

    Soundararajan, M.; Roos, A.K.; Savitsky, P.

    2013-01-01

    Dual-specificity tyrosine-(Y)-phosphorylation-regulated kinases (DYRKs) play key roles in brain development, regulation of splicing, and apoptosis, and are potential drug targets for neurodegenerative diseases and cancer. We present crystal structures of one representative member of each DYRK sub...

  4. Targeting mitogen-activated protein kinase kinase (MEK) in solid tumors.

    Science.gov (United States)

    Duffy, Austin; Kummar, Shivaani

    2009-12-01

    The Raf-mitogen activated protein kinase kinase (MEK)-extracellular signal-regulated kinase (ERK) protein kinase signaling cascade is an important intracellular pathway whose activation influences many fundamental cellular processes and whose aberrancy is associated with cancer cell growth. In addition to activation from within by, for example, Raf mutations, this pathway is frequently activated from above by mutated Ras or epidermal growth factor receptor (EGFR). Given the near ubiquity of derangements affecting at least part of this network in cancer, there is a strong and clear rationale for interrupting it. In recent times, in colorectal and lung cancer, Ras and EGFR mutant status have been shown to be critically important and mutually exclusive predictors of response to anti-EGFR therapies. These developments underline the importance of targeting downstream effectors, and MEK inhibition has been the subject of intense scientific and clinical research for some time now. This article reviews the current status of MEK inhibitors with regard to their clinical development.

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

    Science.gov (United States)

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

    2014-07-01

    Receptor tyrosine kinases (RTKs) are essential components of signal transduction pathways that mediate cell-to-cell communication and their function as relay points for signaling pathways. They have a key role in numerous processes that control cellular proliferation and differentiation, regulate cell growth and cellular metabolism, and promote cell survival and apoptosis. Recently, the role of RTKs including TCR, FLT-3, c-Kit, c-Fms, PDGFR, ephrin, neurotrophin receptor, and TAM receptor in autoimmune disorder, especially rheumatoid arthritis and multiple sclerosis has been suggested. In multiple sclerosis pathogenesis, RTKs and their tyrosine kinase enzymes are selective important targets for tyrosine kinase inhibitor (TKI) agents. TKIs, compete with the ATP binding site of the catalytic domain of several tyrosine kinases, and act as small molecules that have a favorable safety profile in disease treatment. Up to now, the efficacy of TKIs in numerous animal models of MS has been demonstrated, but application of these drugs in human diseases should be tested in future clinical trials.

  6. Peptide substrates for Rho-associated kinase 2 (Rho-kinase 2/ROCK2).

    Science.gov (United States)

    Kang, Jeong-Hun; Asai, Daisuke; Tsuchiya, Akira; Mori, Takeshi; Niidome, Takuro; Katayama, Yoshiki

    2011-01-01

    Peptide substrates sensitive for a certain protein kinase could be important for new-drug development and to understand the mechanism of diseases. Rho-associated kinase (Rho-kinase/ROCK) is a serine/threonine kinase, and plays an important part in cardiovascular disease, migration and invasion of tumor cells, and in neurological disorders. The purpose of this study was to find substrates with high affinity and sensitivity for ROCK2. We synthesized 136 peptide substrates from protein substrates for ROCK2 with different lengths and charged peptides. Incorporation of (32)P [counts per minute (CPM)] for each peptide substrate was determined by the radiolabel assay using [γ-(32)P]ATP. When the top five peptide substrates showing high CPMs (R4, R22, R133, R134, and R135) were phosphorylated by other enzymes (PKA, PKCα, and ERK1), R22, R133, and R135 displayed the highest CPM level for ROCK2 compared with other enzymes, whereas R4 and R134 showed similar CPM levels for ROCK2 and PKCα. We hypothesize that R22, R133, and R135 can be useful peptide substrates for ROCK2.

  7. Distribution of protein kinase Mzeta and the complete protein kinase C isoform family in rat brain

    DEFF Research Database (Denmark)

    Naik, M U; Benedikz, Eirikur; Hernandez, I

    2000-01-01

    Protein kinase C (PKC) is a multigene family of at least ten isoforms, nine of which are expressed in brain (alpha, betaI, betaII, gamma, delta, straightepsilon, eta, zeta, iota/lambda). Our previous studies have shown that many of these PKCs participate in synaptic plasticity in the CA1 region o...

  8. Protein kinase C gamma mutations in spinocerebellar ataxia 14 increase kinase activity and alter membrane targeting

    NARCIS (Netherlands)

    Verbeek, D. S.; Knight, M. A.; Harmison, G. G.; Fischbeck, K. H.; Howell, B. W.

    2005-01-01

    The protein kinase C gamma (PKCgamma) gene is mutated in spinocerebellar ataxia type 14 (SCA14). In this study, we investigated the effects of two SCA14 missense mutations, G118D and C150F, on PKCgamma function. We found that these mutations increase the intrinsic activity of PKCgamma. Direct visual

  9. Diacylglycerol kinase counteracts protein kinase C-mediated inactivation of the EGF receptor

    NARCIS (Netherlands)

    Baal, van J.; Widt, de J.; Divecha, N.; Blitterswijk, van W.J.

    2012-01-01

    Epidermal growth factor receptor (EGFR) activation is negatively regulated by protein kinase C (PKC)signaling. Stimulation of A431 cells with EGF, bradykinin or UTP increased EGFR phosphorylation at Thr654 in a PKC-dependent manner. Inhibition of PKC signaling enhanced EGFR activation, as assessed b

  10. A-Raf kinase is a new interacting partner of protein kinase CK2 beta subunit

    DEFF Research Database (Denmark)

    Boldyreff, B; Issinger, O G

    1997-01-01

    In a search for protein kinase CK2 beta subunit binding proteins using the two-hybrid system, more than 1000 positive clones were isolated. Beside clones for the alpha' and beta subunit of CK2, there were clones coding for a so far unknown protein, whose partial cDNA sequence was already deposite...

  11. The acetate kinase of Clostridum acetobutylicum strain P262.

    Science.gov (United States)

    Diez-Gonzalez, F; Russell, J B; Hunter, J B

    1996-12-01

    Clostridum acetobutylicum strain P262 fermented glucose, pyruvate, or lactate, and the butyrate production was substrate-dependent. Differences in butyrate yield could not be explained by changes in butyrate kinase activities, but the butyrate production was inversely related to acetate kinase activity. The acetate kinase had a pH optimum of 8.0, a Km for acetate of 160 mM, and a kcat of 16, 800 min-1. The enyzme had a native molecular mass of 78 kDa; the size of 42 kDa on SDS-PAGE indicated that the acetate kinase of strain P262 was a homodimer.

  12. Diversity, classification and function of the plant protein kinase superfamily.

    Science.gov (United States)

    Lehti-Shiu, Melissa D; Shiu, Shin-Han

    2012-09-19

    Eukaryotic protein kinases belong to a large superfamily with hundreds to thousands of copies and are components of essentially all cellular functions. The goals of this study are to classify protein kinases from 25 plant species and to assess their evolutionary history in conjunction with consideration of their molecular functions. The protein kinase superfamily has expanded in the flowering plant lineage, in part through recent duplications. As a result, the flowering plant protein kinase repertoire, or kinome, is in general significantly larger than other eukaryotes, ranging in size from 600 to 2500 members. This large variation in kinome size is mainly due to the expansion and contraction of a few families, particularly the receptor-like kinase/Pelle family. A number of protein kinases reside in highly conserved, low copy number families and often play broadly conserved regulatory roles in metabolism and cell division, although functions of plant homologues have often diverged from their metazoan counterparts. Members of expanded plant kinase families often have roles in plant-specific processes and some may have contributed to adaptive evolution. Nonetheless, non-adaptive explanations, such as kinase duplicate subfunctionalization and insufficient time for pseudogenization, may also contribute to the large number of seemingly functional protein kinases in plants.

  13. Recent advances in designing substrate-competitive protein kinase inhibitors.

    Science.gov (United States)

    Han, Ki-Cheol; Kim, So Yeon; Yang, Eun Gyeong

    2012-01-01

    Protein kinases play central roles in cellular signaling pathways and their abnormal phosphorylation activity is inseparably linked with various human diseases. Therefore, modulation of kinase activity using potent inhibitors is an attractive strategy for the treatment of human disease. While most protein kinase inhibitors in clinical development are mainly targeted to the highly conserved ATP-binding sites and thus likely promiscuously inhibit multiple kinases including kinases unrelated to diseases, protein substrate-competitive inhibitors are more selective and expected to be promising therapeutic agents. Most substrate-competitive inhibitors mimic peptides derived from substrate proteins, or from inhibitory domains within kinases or inhibitor proteins. In addition, bisubstrate inhibitors are generated by conjugating substrate-competitive peptide inhibitors to ATP-competitive inhibitors to improve affinity and selectivity. Although structural information on protein kinases provides invaluable guidance in designing substrate-competitive inhibitors, other strategies including bioinformatics, computational modeling, and high-throughput screening are often employed for developing specific substrate-competitive kinase inhibitors. This review focuses on recent advances in the design and discovery of substrate-competitive inhibitors of protein kinases.

  14. A Broad Specificity Nucleoside Kinase from Thermoplasma acidophilum

    OpenAIRE

    Elkin, Sarah R.; Kumar, Abhinav; Price, Carol W.; Columbus, Linda

    2013-01-01

    The crystal structure of Ta0880, determined at 1.91 A resolution, from Thermoplasma acidophilum revealed a dimer with each monomer composed of an α/β /α sandwich domain and a smaller lid domain. The overall fold belongs to the PfkB family of carbohydrate kinases (a family member of the Ribokinase clan) which include ribokinases, 1-phosphofructokinases, 6-phosphofructo-2-kinase, inosine/guanosine kinases, frutokinases, adenosine kinases, and many more. Based on its general fold, Ta0880 had bee...

  15. Deoxyribonucleoside kinases: two enzyme families catalyze the same reaction

    DEFF Research Database (Denmark)

    Sandrini, Michael; Piskur, Jure

    2005-01-01

    Mammals have four deoxyribonucleoside kinases, the cytoplasmic (TK1) and mitochondrial (TK2) thymidine kinases, and the deoxycytidine (dCK) and deoxyguanosine (dGK) kinases, which salvage the precursors for nucleic acids synthesis. In addition to the native deoxyribonucleoside substrates, the kin......, the kinases can phosphorylate and thereby activate a variety of anti-cancer and antiviral prodrugs. Recently, the crystal structure of human TK1 has been solved and has revealed that enzymes with fundamentally different origins and folds catalyze similar, crucial cellular reactions....

  16. Tyrosine Kinase Inhibition: An Approach to Drug Development

    Science.gov (United States)

    Levitzki, Alexander; Gazit, Aviv

    1995-03-01

    Protein tyrosine kinases (PTKs) regulate cell proliferation, cell differentiation, and signaling processes in the cells of the immune system. Uncontrolled signaling from receptor tyrosine kinases and intracellular tyrosine kinases can lead to inflammatory responses and to diseases such as cancer, atherosclerosis, and psoriasis. Thus, inhibitors that block the activity of tyrosine kinases and the signaling pathways they activate may provide a useful basis for drug development. This article summarizes recent progress in the development of PTK inhibitors and demonstrates their potential use in the treatment of disease.

  17. Constitutive Activity in an Ancestral Form of Abl Tyrosine Kinase.

    Science.gov (United States)

    Aleem, Saadat U; Craddock, Barbara P; Miller, W Todd

    2015-01-01

    The c-abl proto-oncogene encodes a nonreceptor tyrosine kinase that is found in all metazoans, and is ubiquitously expressed in mammalian tissues. The Abl tyrosine kinase plays important roles in the regulation of mammalian cell physiology. Abl-like kinases have been identified in the genomes of unicellular choanoflagellates, the closest relatives to the Metazoa, and in related unicellular organisms. Here, we have carried out the first characterization of a premetazoan Abl kinase, MbAbl2, from the choanoflagellate Monosiga brevicollis. The enzyme possesses SH3, SH2, and kinase domains in a similar arrangement to its mammalian counterparts, and is an active tyrosine kinase. MbAbl2 lacks the N-terminal myristoylation and cap sequences that are critical regulators of mammalian Abl kinase activity, and we show that MbAbl2 is constitutively active. When expressed in mammalian cells, MbAbl2 strongly phosphorylates cellular proteins on tyrosine, and transforms cells much more potently than mammalian Abl kinase. Thus, MbAbl2 appears to lack the autoinhibitory mechanism that tightly constrains the activity of mammalian Abl kinases, suggesting that this regulatory apparatus arose more recently in metazoan evolution.

  18. Constitutive Activity in an Ancestral Form of Abl Tyrosine Kinase.

    Directory of Open Access Journals (Sweden)

    Saadat U Aleem

    Full Text Available The c-abl proto-oncogene encodes a nonreceptor tyrosine kinase that is found in all metazoans, and is ubiquitously expressed in mammalian tissues. The Abl tyrosine kinase plays important roles in the regulation of mammalian cell physiology. Abl-like kinases have been identified in the genomes of unicellular choanoflagellates, the closest relatives to the Metazoa, and in related unicellular organisms. Here, we have carried out the first characterization of a premetazoan Abl kinase, MbAbl2, from the choanoflagellate Monosiga brevicollis. The enzyme possesses SH3, SH2, and kinase domains in a similar arrangement to its mammalian counterparts, and is an active tyrosine kinase. MbAbl2 lacks the N-terminal myristoylation and cap sequences that are critical regulators of mammalian Abl kinase activity, and we show that MbAbl2 is constitutively active. When expressed in mammalian cells, MbAbl2 strongly phosphorylates cellular proteins on tyrosine, and transforms cells much more potently than mammalian Abl kinase. Thus, MbAbl2 appears to lack the autoinhibitory mechanism that tightly constrains the activity of mammalian Abl kinases, suggesting that this regulatory apparatus arose more recently in metazoan evolution.

  19. Deoxyribonucleoside kinases: two enzyme families catalyze the same reaction

    DEFF Research Database (Denmark)

    Sandrini, Michael; Piskur, Jure

    2005-01-01

    Mammals have four deoxyribonucleoside kinases, the cytoplasmic (TK1) and mitochondrial (TK2) thymidine kinases, and the deoxycytidine (dCK) and deoxyguanosine (dGK) kinases, which salvage the precursors for nucleic acids synthesis. In addition to the native deoxyribonucleoside substrates, the kin......, the kinases can phosphorylate and thereby activate a variety of anti-cancer and antiviral prodrugs. Recently, the crystal structure of human TK1 has been solved and has revealed that enzymes with fundamentally different origins and folds catalyze similar, crucial cellular reactions....

  20. Mapping C-terminal transactivation domains of the nuclear HER family receptor tyrosine kinase HER3.

    Science.gov (United States)

    Brand, Toni M; Iida, Mari; Luthar, Neha; Wleklinski, Matthew J; Starr, Megan M; Wheeler, Deric L

    2013-01-01

    Nuclear localized HER family receptor tyrosine kinases (RTKs) have been observed in primary tumor specimens and cancer cell lines for nearly two decades. Inside the nucleus, HER family members (EGFR, HER2, and HER3) have been shown to function as co-transcriptional activators for various cancer-promoting genes. However, the regions of each receptor that confer transcriptional potential remain poorly defined. The current study aimed to map the putative transactivation domains (TADs) of the HER3 receptor. To accomplish this goal, various intracellular regions of HER3 were fused to the DNA binding domain of the yeast transcription factor Gal4 (Gal4DBD) and tested for their ability to transactivate Gal4 UAS-luciferase. Results from these analyses demonstrated that the C-terminal domain of HER3 (CTD, amino acids distal to the tyrosine kinase domain) contained potent transactivation potential. Next, nine HER3-CTD truncation mutants were constructed to map minimal regions of transactivation potential using the Gal4 UAS-luciferase based system. These analyses identified a bipartite region of 34 (B₁) and 27 (B₂) amino acids in length that conferred the majority of HER3's transactivation potential. Next, we identified full-length nuclear HER3 association and regulation of a 122 bp region of the cyclin D1 promoter. To understand how the B₁ and B₂ regions influenced the transcriptional functions of nuclear HER3, we performed cyclin D1 promoter-luciferase assays in which HER3 deleted of the B₁ and B₂ regions was severely hindered in regulating this promoter. Further, the overexpression of HER3 enhanced cyclin D1 mRNA expression, while HER3 deleted of its identified TADs was hindered at doing so. Thus, the ability for HER3 to function as a transcriptional co-activator may be dependent on specific C-terminal TADs.