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Sample records for acyl-coenzyme a-dependent lysophosphatidic

  1. SLC1 and SLC4 encode partially redundant acyl-coenzyme A 1-acylglycerol-3-phosphate O-acyltransferases of budding yeast

    DEFF Research Database (Denmark)

    Benghezal, Mohammed; Roubaty, Carole; Veepuri, Vijayanath

    2007-01-01

    Phosphatidic acid is the intermediate, from which all glycerophospholipids are synthesized. In yeast, it is generated from lysophosphatidic acid, which is acylated by Slc1p, an sn-2-specific, acyl-coenzyme A-dependent 1-acylglycerol-3-phosphate O-acyltransferase. Deletion of SLC1 is not lethal an......-phosphate O-acyltransferases but also be involved in fatty acid exchange at the sn-2-position of mature glycerophospholipids....

  2. Acyl-coenzyme A binding protein (ACBP)

    DEFF Research Database (Denmark)

    Kragelund, B B; Knudsen, J; Poulsen, F M

    1999-01-01

    Acyl-coenzyme A binding proteins are known from a large group of eukaryote species and to bind a long chain length acyl-CoA ester with very high affinity. Detailed biochemical mapping of ligand binding properties has been obtained as well as in-depth structural studies on the bovine apo-protein...... and of the complex with palmitoyl-CoA using NMR spectroscopy. In the four alpha-helix bundle structure, a set of 21 highly conserved residues present in more that 90% of all known sequences of acyl-coenzyme A binding proteins constitutes three separate mini-cores. These residues are predominantly located...... at the helix-helix interfaces. From studies of a large set of mutant proteins the role of the conserved residues has been related to structure, function, folding and stability....

  3. Acyl-coenzyme A binding protein, ACBP

    DEFF Research Database (Denmark)

    Kragelund, Birthe Brandt; Knudsen, J.; Poulsen, Flemming

    1999-01-01

    Acyl-coenzyme A binding proteins are known from a large group of eukaryote species and to bind a long chain length acyl-CoA ester with very high affinity. Detailed biochemical mapping of ligand binding properties has been obtained as well as in-depth structural studies on the bovine apo-protein...... and of the complex with palmitoyl-CoA using NMR spectroscopy. In the four a-helix bundle structure, a set of 21 highly conserved residues present in more that 90% of all known sequences of acyl-coenzyme A binding proteins constitutes three separate mini-cores. These residues are predominantly located at the helix......-helix interfaces. From studies of a large set of mutant proteins the role of the conserved residues has been related to structure, function, folding and stability....

  4. Acyl-coenzyme A:cholesterol acyltransferases

    OpenAIRE

    Chang, Ta-Yuan; Li, Bo-Liang; Chang, Catherine C.Y.; Urano, Yasuomi

    2009-01-01

    The enzymes acyl-coenzyme A (CoA):cholesterol acyltransferases (ACATs) are membrane-bound proteins that utilize long-chain fatty acyl-CoA and cholesterol as substrates to form cholesteryl esters. In mammals, two isoenzymes, ACAT1 and ACAT2, encoded by two different genes, exist. ACATs play important roles in cellular cholesterol homeostasis in various tissues. This chapter summarizes the current knowledge on ACAT-related research in two areas: 1) ACAT genes and proteins and 2) ACAT enzymes as...

  5. Acyl-coenzyme A organizes laterally in membranes and is recognized specifically by acyl-coenzyme A binding protein

    DEFF Research Database (Denmark)

    Cohen Simonsen, A; Bernchou Jensen, U; Færgeman, Nils J.;

    2003-01-01

    Long chain acyl-coenzyme A (acyl-CoA) is a biochemically important amphiphilic molecule that is known to partition strongly into membranes by insertion of the acyl chain. At present, microscopically resolved evidence is lacking on how acyl-CoA influences and organizes laterally in membranes...

  6. Continuous recording of long-chain acyl-coenzyme A synthetase activity using fluorescently labeled bovine serum albumin

    DEFF Research Database (Denmark)

    Demant, Erland J.F.; Nystrøm, Birthe T.

    2001-01-01

    acyl-Coenzyme A, synthetase, activity assay, fluorescence recording, fatty acid probe, serum albumin, hydroxycoumarin, detergent, micelles, Pseudomonas fragi, rat liver microsomes......acyl-Coenzyme A, synthetase, activity assay, fluorescence recording, fatty acid probe, serum albumin, hydroxycoumarin, detergent, micelles, Pseudomonas fragi, rat liver microsomes...

  7. Roles of Long-chain Acyl Coenzyme A Synthetase in Absorption and Transport of Fatty Acid

    Institute of Scientific and Technical Information of China (English)

    Fan Gao; Xue-feng Yang; Nian Fu; Yang Hu; Yan Ouyang; Kai Qing

    2016-01-01

    Abstract Long-chain acyl coenzyme A synthetase (ACSL) is a member of the synthetase family encoded by a multigene family; it plays an important role in the absorption and transport of fatty acid. Here we review the roles of ACSL in the regulating absorption and transport of fatty acid, as well as the connection between ACSL and some metabolic diseases.

  8. Adult peroxisomal acyl-coenzyme A oxidase deficiency with cerebellar and brainstem atrophy

    NARCIS (Netherlands)

    S. Ferdinandusse; S. Barker; K. Lachlan; M. Duran; H.R. Waterham; R.J.A. Wanders; S. Hammans

    2010-01-01

    Peroxisomal acyl-coenzyme A oxidase deficiency ( formerly also called pseudoneonatal adrenoleucodystrophy) is a disorder of peroxisomal fatty acid oxidation with a severe presentation. Most patients present at birth or in early infancy, and the mean age of death was 5 years in a recently published c

  9. Very long chain acyl-coenzyme A dehydrogenase deficiency with adult onset

    DEFF Research Database (Denmark)

    Smelt, A H; Poorthuis, B J; Onkenhout, W;

    1998-01-01

    Very long chain acyl-coenzyme A (acyl-CoA) dehydrogenase (VLCAD) deficiency is a severe disorder of mitochondrial beta-oxidation in infants. We report adult onset of attacks of painful rhabdomyolysis. Gas chromatography identified strongly elevated levels of tetradecenoic acid, 14:1(n-9), tetrade......Very long chain acyl-coenzyme A (acyl-CoA) dehydrogenase (VLCAD) deficiency is a severe disorder of mitochondrial beta-oxidation in infants. We report adult onset of attacks of painful rhabdomyolysis. Gas chromatography identified strongly elevated levels of tetradecenoic acid, 14:1(n-9......), tetradecadienoic acid, 14:2(n-6), and hexadecadienoic acid, 16:2(n-6). Palmitoyl-CoA and behenoyl-CoA dehydrogenase in fibroblasts were deficient. Muscle VLCAD activity was very low. DNA analysis revealed compound heterozygosity for two missense mutations in the VLCAD gene. The relatively mild clinical course may...

  10. Use of Limited Proteolysis and Mutagenesis To Identify Folding Domains and Sequence Motifs Critical for Wax Ester Synthase/Acyl Coenzyme A:Diacylglycerol Acyltransferase Activity

    Science.gov (United States)

    Villa, Juan A.; Cabezas, Matilde; de la Cruz, Fernando

    2014-01-01

    Triacylglycerols and wax esters are synthesized as energy storage molecules by some proteobacteria and actinobacteria under stress. The enzyme responsible for neutral lipid accumulation is the bifunctional wax ester synthase/acyl-coenzyme A (CoA):diacylglycerol acyltransferase (WS/DGAT). Structural modeling of WS/DGAT suggests that it can adopt an acyl-CoA-dependent acyltransferase fold with the N-terminal and C-terminal domains connected by a helical linker, an architecture demonstrated experimentally by limited proteolysis. Moreover, we found that both domains form an active complex when coexpressed as independent polypeptides. The structural prediction and sequence alignment of different WS/DGAT proteins indicated catalytically important motifs in the enzyme. Their role was probed by measuring the activities of a series of alanine scanning mutants. Our study underscores the structural understanding of this protein family and paves the way for their modification to improve the production of neutral lipids. PMID:24296496

  11. Immunolocalization of acyl-coenzyme A:cholesterol O-acyltransferase in macrophages.

    Science.gov (United States)

    Khelef, N; Buton, X; Beatini, N; Wang, H; Meiner, V; Chang, T Y; Farese, R V; Maxfield, F R; Tabas, I

    1998-05-01

    Macrophages in atherosclerotic lesions accumulate large amounts of cholesteryl-fatty acyl esters ("foam cell" formation) through the intracellular esterification of cholesterol by acyl-coenzyme A:cholesterol O-acyltransferase (ACAT). In this study, we sought to determine the subcellular localization of ACAT in macrophages. Using mouse peritoneal macrophages and immunofluorescence microscopy, we found that a major portion of ACAT was in a dense reticular cytoplasmic network and in the nuclear membrane that colocalized with the luminal endoplasmic reticulum marker protein-disulfide isomerase (PDI) and that was in a similar distribution as the membrane-bound endoplasmic reticulum marker ribophorin. Remarkably, another portion of the macrophage ACAT pattern did not overlap with PDI or ribophorin, but was found in as yet unidentified cytoplasmic structures that were juxtaposed to the nucleus. Compartments containing labeled beta-very low density lipoprotein, an atherogenic lipoprotein, did not overlap with the ACAT label, but rather were embedded in the dense reticular network of ACAT. Furthermore, cell-surface biotinylation experiments revealed that freshly harvested, non-attached macrophages, but not those attached to tissue culture dishes, contained approximately 10-15% of ACAT on the cell surface. In summary, ACAT was found in several sites in macrophages: a cytoplasmic reticular/nuclear membrane site that overlaps with PDI and ribophorin and has the characteristics of the endoplasmic reticulum, a perinuclear cytoplasmic site that does not overlap with PDI or ribophorin and may be another cytoplasmic structure or possibly a unique subcompartment of the endoplasmic reticulum, and a cell-surface site in non-attached macrophages. Understanding possible physiological differences of ACAT in these locations may reveal an important component of ACAT regulation and macrophage foam cell formation.

  12. Patients with medium-chain acyl-coenzyme a dehydrogenase deficiency have impaired oxidation of fat during exercise but no effect of L-carnitine supplementation

    DEFF Research Database (Denmark)

    Madsen, K L; Preisler, N; Orngreen, M C

    2013-01-01

    It is not clear to what extent skeletal muscle is affected in patients with medium-chain acyl-coenzyme A dehydrogenase deficiency (MCADD). l-Carnitine is commonly used as a supplement in patients with MCADD, although its beneficial effect has not been verified.......It is not clear to what extent skeletal muscle is affected in patients with medium-chain acyl-coenzyme A dehydrogenase deficiency (MCADD). l-Carnitine is commonly used as a supplement in patients with MCADD, although its beneficial effect has not been verified....

  13. Actinobacterial acyl coenzyme A synthetases involved in steroid side-chain catabolism.

    Science.gov (United States)

    Casabon, Israël; Swain, Kendra; Crowe, Adam M; Eltis, Lindsay D; Mohn, William W

    2014-02-01

    Bacterial steroid catabolism is an important component of the global carbon cycle and has applications in drug synthesis. Pathways for this catabolism involve multiple acyl coenzyme A (CoA) synthetases, which activate alkanoate substituents for β-oxidation. The functions of these synthetases are poorly understood. We enzymatically characterized four distinct acyl-CoA synthetases from the cholate catabolic pathway of Rhodococcus jostii RHA1 and the cholesterol catabolic pathway of Mycobacterium tuberculosis. Phylogenetic analysis of 70 acyl-CoA synthetases predicted to be involved in steroid metabolism revealed that the characterized synthetases each represent an orthologous class with a distinct function in steroid side-chain degradation. The synthetases were specific for the length of alkanoate substituent. FadD19 from M. tuberculosis H37Rv (FadD19Mtb) transformed 3-oxo-4-cholesten-26-oate (kcat/Km = 0.33 × 10(5) ± 0.03 × 10(5) M(-1) s(-1)) and represents orthologs that activate the C8 side chain of cholesterol. Both CasGRHA1 and FadD17Mtb are steroid-24-oyl-CoA synthetases. CasG and its orthologs activate the C5 side chain of cholate, while FadD17 and its orthologs appear to activate the C5 side chain of one or more cholesterol metabolites. CasIRHA1 is a steroid-22-oyl-CoA synthetase, representing orthologs that activate metabolites with a C3 side chain, which accumulate during cholate catabolism. CasI had similar apparent specificities for substrates with intact or extensively degraded steroid nuclei, exemplified by 3-oxo-23,24-bisnorchol-4-en-22-oate and 1β(2'-propanoate)-3aα-H-4α(3″-propanoate)-7aβ-methylhexahydro-5-indanone (kcat/Km = 2.4 × 10(5) ± 0.1 × 10(5) M(-1) s(-1) and 3.2 × 10(5) ± 0.3 × 10(5) M(-1) s(-1), respectively). Acyl-CoA synthetase classes involved in cholate catabolism were found in both Actinobacteria and Proteobacteria. Overall, this study provides insight into the physiological roles of acyl-CoA synthetases in steroid

  14. Genetic Basis for Correction of Very‐Long‐Chain Acyl-Coenzyme A Dehydrogenase Deficiency by Bezafibrate in Patient Fibroblasts: Toward a Genotype‐Based Therapy

    DEFF Research Database (Denmark)

    Gobin‐Limballe, S.; Djouadi, F.; Aubey, F.

    2007-01-01

    Very‐long‐chain acyl-coenzyme A dehydrogenase (VLCAD) deficiency is an inborn mitochondrial fatty‐acid β‐oxidation (FAO) defect associated with a broad mutational spectrum, with phenotypes ranging from fatal cardiopathy in infancy to adolescent‐onset myopathy, and for which there is no establishe...

  15. Involvement of Acyl Coenzyme A Oxidase Isozymes in Biotransformation of Methyl Ricinoleate into γ-Decalactone by Yarrowia lipolytica

    Science.gov (United States)

    Waché, Yves; Laroche, Céline; Bergmark, Karin; Møller-Andersen, Charlotte; Aguedo, Mario; Le Dall, Marie-Thérèse; Wang, Huijie; Nicaud, Jean-Marc; Belin, Jean-Marc

    2000-01-01

    We reported previously on the function of acyl coenzyme A (acyl-CoA) oxidase isozymes in the yeast Yarrowia lipolytica by investigating strains disrupted in one or several acyl-CoA oxidase-encoding genes (POX1 through POX5) (H. Wang et al., J. Bacteriol. 181:5140–5148, 1999). Here, these mutants were studied for lactone production. Monodisrupted strains produced similar levels of lactone as the wild-type strain (50 mg/liter) except for Δpox3, which produced 220 mg of γ-decalactone per liter after 24 h. The Δpox2 Δpox3 double-disrupted strain, although slightly affected in growth, produced about 150 mg of lactone per liter, indicating that Aox2p was not essential for the biotransformation. The Δpox2 Δpox3 Δpox5 triple-disrupted strain produced and consumed lactone very slowly. On the contrary, the Δpox2 Δpox3 Δpox4 Δpox5 multidisrupted strain did not grow or biotransform methyl ricinoleate into γ-decalactone, demonstrating that Aox4p is essential for the biotransformation. PMID:10698800

  16. Involvement of acyl coenzyme A oxidase isozymes in biotransformation of methyl ricinoleate into gamma-decalactone by Yarrowia lipolytica.

    Science.gov (United States)

    Waché, Y; Laroche, C; Bergmark, K; Møller-Andersen, C; Aguedo, M; Le Dall, M T; Wang, H; Nicaud, J M; Belin, J M

    2000-03-01

    We reported previously on the function of acyl coenzyme A (acyl-CoA) oxidase isozymes in the yeast Yarrowia lipolytica by investigating strains disrupted in one or several acyl-CoA oxidase-encoding genes (POX1 through POX5) (H. Wang et al., J. Bacteriol. 181:5140-5148, 1999). Here, these mutants were studied for lactone production. Monodisrupted strains produced similar levels of lactone as the wild-type strain (50 mg/liter) except for Deltapox3, which produced 220 mg of gamma-decalactone per liter after 24 h. The Deltapox2 Deltapox3 double-disrupted strain, although slightly affected in growth, produced about 150 mg of lactone per liter, indicating that Aox2p was not essential for the biotransformation. The Deltapox2 Deltapox3 Deltapox5 triple-disrupted strain produced and consumed lactone very slowly. On the contrary, the Deltapox2 Deltapox3 Deltapox4 Deltapox5 multidisrupted strain did not grow or biotransform methyl ricinoleate into gamma-decalactone, demonstrating that Aox4p is essential for the biotransformation.

  17. Structure of YciA from Haemophilus influenzae (HI0827), a Hexameric Broad Specificity Acyl-Coenzyme A Thioesterase

    Energy Technology Data Exchange (ETDEWEB)

    Willis, Mark A.; Zhuang, Zhihao; Song, Feng; Howard, Andrew; Dunaway-Mariano, Debra; Herzberg, Osnat (UNM); (IIT); (UMBI)

    2008-04-02

    The crystal structure of HI0827 from Haemophilus influenzae Rd KW20, initially annotated 'hypothetical protein' in sequence databases, exhibits an acyl-coenzyme A (acyl-CoA) thioesterase 'hot dog' fold with a trimer of dimers oligomeric association, a novel assembly for this enzyme family. In studies described in the preceding paper [Zhuang, Z., Song, F., Zhao, H., Li, L., Cao, J., Eisenstein, E., Herzberg, O., and Dunaway-Mariano, D. (2008) Biochemistry 47, 2789-2796], HI0827 is shown to be an acyl-CoA thioesterase that acts on a wide range of acyl-CoA compounds. Two substrate binding sites are located across the dimer interface. The binding sites are occupied by two CoA molecules, one with full occupancy and the second only partially occupied. The CoA molecules, acquired from HI0827-expressing Escherichia coli cells, remained tightly bound to the enzyme through the protein purification steps. The difference in CoA occupancies indicates a different substrate affinity for each of the binding sites, which in turn implies that the enzyme might be subject to allosteric regulation. Mutagenesis studies have shown that the replacement of the putative catalytic carboxylate Asp44 with an alanine residue abolishes activity. The impact of this mutation is seen in the crystal structure of D44A HI0827. Whereas the overall fold and assembly of the mutant protein are the same as those of the wild-type enzyme, the CoA ligands are absent. The dimer interface is perturbed, and the channel that accommodates the thioester acyl chain is more open and wider than that observed in the wild-type enzyme. A model of intact substrate bound to wild-type HI0827 provides a structural rationale for the broad substrate range.

  18. Purification of a jojoba embryo fatty acyl-coenzyme A reductase and expression of its cDNA in high erucic acid rapeseed.

    Science.gov (United States)

    Metz, J G; Pollard, M R; Anderson, L; Hayes, T R; Lassner, M W

    2000-03-01

    The jojoba (Simmondsia chinensis) plant produces esters of long-chain alcohols and fatty acids (waxes) as a seed lipid energy reserve. This is in contrast to the triglycerides found in seeds of other plants. We purified an alcohol-forming fatty acyl-coenzyme A reductase (FAR) from developing embryos and cloned the cDNA encoding the enzyme. Expression of a cDNA in Escherichia coli confers FAR activity upon those cells and results in the accumulation of fatty alcohols. The FAR sequence shows significant homology to an Arabidopsis protein of unknown function that is essential for pollen development. When the jojoba FAR cDNA is expressed in embryos of Brassica napus, long-chain alcohols can be detected in transmethylated seed oils. Resynthesis of the gene to reduce its A plus T content resulted in increased levels of alcohol production. In addition to free alcohols, novel wax esters were detected in the transgenic seed oils. In vitro assays revealed that B. napus embryos have an endogenous fatty acyl-coenzyme A: fatty alcohol acyl-transferase activity that could account for this wax synthesis. Thus, introduction of a single cDNA into B. napus results in a redirection of a portion of seed oil synthesis from triglycerides to waxes.

  19. Acyl-coenzyme A:cholesterol acyltransferase inhibitor, avasimibe, stimulates bile acid synthesis and cholesterol 7α-hydroxylase in cultured rat hepatocytes and in vivo in the rat

    NARCIS (Netherlands)

    Post, S.M.; Paul Zoeteweij, J.; Bos, M.H.A.; Wit, E.C.M. de; Havinga, R.; Kuipers, F.; Princen, H.M.G.

    1999-01-01

    Acyl-coenzyme A:cholesterol acyltransferase (ACAT) inhibitors are currently in clinical development as potential lipid-lowering and antiatherosclerotic agents. We investigated the effect of avasimibe (C1- 1011), a novel ACAT inhibitor, on bile acid synthesis and cholesterol 7α- hydroxylase in cultur

  20. Acyl coenzyme A synthetase long-chain 1 (ACSL1 gene polymorphism (rs6552828 and elite endurance athletic status: a replication study.

    Directory of Open Access Journals (Sweden)

    Thomas Yvert

    Full Text Available The aim of this study was to determine the association between the rs6552828 polymorphism in acyl coenzyme A synthetase (ACSL1 and elite endurance athletic status. We studied 82 Caucasian (Spanish World/Olympic-class endurance male athletes, and a group of sex and ethnically matched healthy young adults (controls, n=197. The analyses were replicated in a cohort of a different ethnic origin (Chinese of the Han ethnic group, composed of elite endurance athletes (runners [cases, n=241 (128 male] and healthy sedentary adults [controls, n=504 (267 male]. In the Spanish cohort, genotype (P=0.591 and minor allele (A frequencies were similar in cases and controls (P=0.978. In the Chinese cohort, genotype (P=0.973 and minor allele (G frequencies were comparable in female endurance athletes and sedentary controls (P=0.881, whereas in males the frequency of the G allele was higher in endurance athletes (0.40 compared with their controls (0.32, P=0.040. The odds ratio (95%CI for an elite endurance Chinese athlete to carry the G allele compared with ethnically matched controls was 1.381 (1.015-1.880 (P-value=0.04. Our findings suggest that the ACSL1 gene polymorphism rs6552828 is not associated with elite endurance athletic status in Caucasians, yet a marginal association seems to exist for the Chinese (Han male population.

  1. Shrinking the FadE proteome of Mycobacterium tuberculosis: insights into cholesterol metabolism through identification of an α2β2 heterotetrameric acyl coenzyme A dehydrogenase family.

    Science.gov (United States)

    Wipperman, Matthew F; Yang, Meng; Thomas, Suzanne T; Sampson, Nicole S

    2013-10-01

    The ability of the pathogen Mycobacterium tuberculosis to metabolize steroids like cholesterol and the roles that these compounds play in the virulence and pathogenesis of this organism are increasingly evident. Here, we demonstrate through experiments and bioinformatic analysis the existence of an architecturally distinct subfamily of acyl coenzyme A (acyl-CoA) dehydrogenase (ACAD) enzymes that are α2β2 heterotetramers with two active sites. These enzymes are encoded by two adjacent ACAD (fadE) genes that are regulated by cholesterol. FadE26-FadE27 catalyzes the dehydrogenation of 3β-hydroxy-chol-5-en-24-oyl-CoA, an analog of the 5-carbon side chain cholesterol degradation intermediate. Genes encoding the α2β2 heterotetrameric ACAD structures are present in multiple regions of the M. tuberculosis genome, and subsets of these genes are regulated by four different transcriptional repressors or activators: KstR1 (also known as KstR), KstR2, Mce3R, and SigE. Homologous ACAD gene pairs are found in other Actinobacteria, as well as Proteobacteria. Their structures and genomic locations suggest that the α2β2 heterotetrameric structural motif has evolved to enable catalysis of dehydrogenation of steroid- or polycyclic-CoA substrates and that they function in four subpathways of cholesterol metabolism.

  2. Rosiglitazone inhibits expression of acyl-coenzyme A:cholesterol acyltransferase-1 in THP-1 macrophages induced by advanced glycation end-products

    Institute of Scientific and Technical Information of China (English)

    Yang Qihong; Xu Qiang; Zhang Hong; Si Liangyi

    2008-01-01

    Objective: To investigate the effects of rosiglitazone, a synthetic ligand of peroxisome proliferators-activated receptor gamma (PPARγ), on the expression of acyl-coenzyme A: cholesterol acyltransferase-1 (ACAT-1) in phorbol myristate acetate (PMA)-pretreated THP-1 cells after the inducement of advanced glycation end products (AGEs). Methods: After THP-1 cells were cultured in the presence of 0.1 umol/L PMA for 72 h to induce phagocytic differentiation, the obtained THP-1 macrophages were treated with rosiglitazone for 4 h at different concentrations (1,5 or 10 μmol/L) and then exposed to AGEs-modified bovine serum albumin (AGEs-BSA) for 24 h at a concentration of 200 mg/L. Reverse transcription polymerase chain reaction (RT-PCR) and Western blot analysis were performed to detect the mRNA and protein expressions of ACAT-1 respectively. Results: Administration of AGEs-BSA (200 mg/L) into the THP-1 macrophages resulted in up-regulation of ACAT-1 at mRNA and protein levels when compared with the expressions in macrophages incubated with serum-free RPM11640. Pretreatment of rosiglitazone inhibited significantly the increased expression of ACAT-1 induced by AGEs-BSA in a concentration-dependent manner. Conclusion: PPARγ activation by rosiglitazone down-regulates ACAT-1 expression induced by AGEs in THP-1 macrophages, which might provide a new way for treating atherogenesis in diabetic patients.

  3. Identification of a single nucleotide polymorphism at intron 16 of the caprine acyl-coenzyme A: diacylglycerol acyltransferase 1 (DGAT1) gene.

    Science.gov (United States)

    Angiolillo, Antonella; Amills, Marcel; Urrutia, Baltasar; Doménech, Anna; Sastre, Yolanda; Badaoui, Bouabid; Jordana, Jordi

    2007-02-01

    The DGAT1 gene encodes a microsomal enzyme that catalyses the only committed step in triacylglycerol synthesis by joining diacylglycerol and fatty acyl coenzyme A. In cattle, a K232A substitution in the DGAT1 molecule has a significant effect on enzyme activity and milk fat content. The prominent role of this gene in lipid metabolism led us to undertake the structural characterization of DGAT1 in goats. In this way, we have sequenced a 1552 bp fragment of the goat DGAT1 cDNA, which encompasses most of the coding sequence (from exon 1 to 17), and a genomic fragment covering exons 12 to 17. Multiple alignment of the goat DGAT1 sequences revealed the existence of a single nucleotide polymorphism (SNP) involving a T to C substitution at intron 16. We optimized a primer extension based genotyping method that allowed us to determine that the C variant is a minority allele with frequencies ranging from 0.062 (Murciano-Granadina) to 0.109 (Malagueña). This SNP, although not expected to have any functional effect, might be useful as a genetic marker in association studies to detect additional DGAT1 polymorphisms which might influence fat milk content and other traits of economic interest.

  4. FAR5, a fatty acyl-coenzyme A reductase, is involved in primary alcohol biosynthesis of the leaf blade cuticular wax in wheat (Triticum aestivum L.).

    Science.gov (United States)

    Wang, Yong; Wang, Meiling; Sun, Yulin; Wang, Yanting; Li, Tingting; Chai, Guaiqiang; Jiang, Wenhui; Shan, Liwei; Li, Chunlian; Xiao, Enshi; Wang, Zhonghua

    2015-03-01

    A waxy cuticle that serves as a protective barrier against non-stomatal water loss and environmental damage coats the aerial surfaces of land plants. It comprises a cutin polymer matrix and waxes. Cuticular waxes are complex mixtures of very long chain fatty acids (VLCFAs) and their derivatives. Results show that primary alcohols are the major components of bread wheat (Triticum aestivum L.) leaf blade cuticular waxes. Here, the characterization of TaFAR5 from wheat cv Xinong 2718, which is allelic to TAA1b, an anther-specific gene, is reported. Evidence is presented for a new function for TaFAR5 in the biosynthesis of primary alcohols of leaf blade cuticular wax in wheat. Expression of TaFAR5 cDNA in yeast (Saccharomyces cerevisiae) led to production of C22:0 primary alcohol. The transgenic expression of TaFAR5 in tomato (Solanum lycopersicum) cv MicroTom leaves resulted in the accumulation of C26:0, C28:0, and C30:0 primary alcohols. TaFAR5 encodes an alcohol-forming fatty acyl-coenzyme A reductase (FAR). Expression analysis revealed that TaFAR5 was expressed at high levels in the leaf blades, anthers, pistils, and seeds. Fully functional green fluorescent protein-tagged TaFAR5 protein was localized to the endoplasmic reticulum (ER), the site of primary alcohol biosynthesis. SDS-PAGE analysis indicated that the TaFAR5 protein possessed a molecular mass of 58.4kDa, and it was also shown that TaFAR5 transcript levels were regulated in response to drought, cold, and abscisic acid (ABA). Overall, these data suggest that TaFAR5 plays an important role in the synthesis of primary alcohols in wheat leaf blade.

  5. Acyl-coenzyme A-binding protein regulates Beta-oxidation required for growth and survival of non-small cell lung cancer.

    Science.gov (United States)

    Harris, Fredrick T; Rahman, S M Jamshedur; Hassanein, Mohamed; Qian, Jun; Hoeksema, Megan D; Chen, Heidi; Eisenberg, Rosana; Chaurand, Pierre; Caprioli, Richard M; Shiota, Masakazu; Massion, Pierre P

    2014-07-01

    We identified acyl-coenzyme A-binding protein (ACBP) as part of a proteomic signature predicting the risk of having lung cancer. Because ACBP is known to regulate β-oxidation, which in turn controls cellular proliferation, we hypothesized that ACBP contributes to regulation of cellular proliferation and survival of non-small cell lung cancer (NSCLC) by modulating β-oxidation. We used matrix-assisted laser desorption/ionization-imaging mass spectrometry (MALDI-IMS) and immunohistochemistry (IHC) to confirm the tissue localization of ABCP in pre-invasive and invasive NSCLCs. We correlated ACBP gene expression levels in NSCLCs with clinical outcomes. In loss-of-function studies, we tested the effect of the downregulation of ACBP on cellular proliferation and apoptosis in normal bronchial and NSCLC cell lines. Using tritiated-palmitate ((3)H-palmitate), we measured β-oxidation levels and tested the effect of etomoxir, a β-oxidation inhibitor, on proliferation and apoptosis. MALDI-IMS and IHC analysis confirmed that ACBP is overexpressed in pre-invasive and invasive lung cancers. High ACBP gene expression levels in NSCLCs correlated with worse survival (HR = 1.73). We observed a 40% decrease in β-oxidation and concordant decreases in proliferation and increases in apoptosis in ACBP-depleted NSCLC cells as compared with bronchial airway epithelial cells. Inhibition of β-oxidation by etomoxir in ACBP-overexpressing cells produced dose-dependent decrease in proliferation and increase in apoptosis (P = 0.01 and P oxidation.

  6. Mangiferin treatment inhibits hepatic expression of acyl-coenzyme A:diacylglycerol acyltransferase-2 in fructose-fed spontaneously hypertensive rats: a link to amelioration of fatty liver.

    Science.gov (United States)

    Xing, Xiaomang; Li, Danyang; Chen, Dilong; Zhou, Liang; Chonan, Ritsu; Yamahara, Johji; Wang, Jianwei; Li, Yuhao

    2014-10-15

    Mangiferin, a xanthone glucoside, and its associated traditional herbs have been demonstrated to improve abnormalities of lipid metabolism. However, its underlying mechanisms remain largely unclear. This study investigated the anti-steatotic effect of mangiferin in fructose-fed spontaneously hypertensive rat (SHR)s that have a mutation in sterol regulatory element binding protein (SREBP)-1. The results showed that co-administration of mangiferin (15 mg/kg, once daily, by oral gavage) over 7 weeks dramatically diminished fructose-induced increases in hepatic triglyceride content and Oil Red O-stained area in SHRs. However, blood pressure, fructose and chow intakes, white adipose tissue weight and metabolic parameters (plasma concentrations of glucose, insulin, triglyceride, total cholesterol and non-esterified fatty acids) were unaffected by mangiferin treatment. Mechanistically, mangiferin treatment suppressed acyl-coenzyme A:diacylglycerol acyltransferase (DGAT)-2 expression at the mRNA and protein levels in the liver. In contrast, mangiferin treatment was without effect on hepatic mRNA and/or protein expression of SREBP-1/1c, carbohydrate response element binding protein, liver pyruvate kinase, fatty acid synthase, acetyl-CoA carboxylase-1, stearoyl-CoA desaturase-1, DGAT-1, monoacyglycerol acyltransferase-2, microsomal triglyceride transfer protein, peroxisome proliferator-activated receptor-alpha, carnitine palmitoyltransferase-1 and acyl-CoA oxidase. Collectively, our results suggest that mangiferin treatment ameliorates fatty liver in fructose-fed SHRs by inhibiting hepatic DGAT-2 that catalyzes the final step in triglyceride biosynthesis. The anti-steatotic effect of mangiferin may occur independently of the hepatic signals associated with de novo fatty acid synthesis and oxidation.

  7. The wax ester synthase/acyl coenzyme A:diacylglycerol acyltransferase from Acinetobacter sp. strain ADP1: characterization of a novel type of acyltransferase.

    Science.gov (United States)

    Stöveken, Tim; Kalscheuer, Rainer; Malkus, Ursula; Reichelt, Rudolf; Steinbüchel, Alexander

    2005-02-01

    The wax ester synthase/acyl coenzyme A (acyl-CoA):diacylglycerol acyltransferase (WS/DGAT) catalyzes the final steps in triacylglycerol (TAG) and wax ester (WE) biosynthesis in the gram-negative bacterium Acinetobacter sp. strain ADP1. It constitutes a novel class of acyltransferases which is fundamentally different from acyltransferases involved in TAG and WE synthesis in eukaryotes. The enzyme was purified by a three-step purification protocol to apparent homogeneity from the soluble fraction of recombinant Escherichia coli Rosetta (DE3)pLysS (pET23a::atfA). Purified WS/DGAT revealed a remarkably low substrate specificity, accepting a broad range of various substances as alternative acceptor molecules. Besides having DGAT and WS activity, the enzyme possesses acyl-CoA:monoacylglycerol acyltransferase (MGAT) activity. The sn-1 and sn-3 positions of acylglycerols are accepted with higher specificity than the sn-2 position. Linear alcohols ranging from ethanol to triacontanol are efficiently acylated by the enzyme, which exhibits highest specificities towards medium-chain-length alcohols. The acylation of cyclic and aromatic alcohols, such as cyclohexanol or phenylethanol, further underlines the unspecific character of this enzyme. The broad range of possible substrates may lead to biotechnological production of interesting wax ester derivatives. Determination of the native molecular weight revealed organization as a homodimer. The large number of WS/DGAT-homologous genes identified in pathogenic mycobacteria and their possible importance for the pathogenesis and latency of these bacteria makes the purified WS/DGAT from Acinetobacter sp. strain ADP1 a valuable model for studying this group of proteins in pathogenic mycobacteria.

  8. Acyl-coenzyme A oxidases 1 and 3 in brown trout (Salmo trutta f. fario): Can peroxisomal fatty acid β-oxidation be regulated by estrogen signaling?

    Science.gov (United States)

    Madureira, Tânia Vieira; Castro, L Filipe C; Rocha, Eduardo

    2016-02-01

    Acyl-coenzyme A oxidases 1 (Acox1) and 3 (Acox3) are key enzymes in the regulation of lipid homeostasis. Endogenous and exogenous factors can disrupt their normal expression/activity. This study presents for the first time the isolation and characterization of Acox1 and Acox3 in brown trout (Salmo trutta f. fario). Additionally, as previous data point to the existence of a cross-talk between two nuclear receptors, namely peroxisome proliferator-activated receptors and estrogen receptors, it was here evaluated after in vitro exposures of trout hepatocytes the interference caused by ethynylestradiol in the mRNA levels of an inducible (by peroxisome proliferators) and a non-inducible oxidase. The isolated Acox1 and Acox3 show high levels of sequence conservation compared to those of other teleosts. Additionally, it was found that Acox1 has two alternative splicing isoforms, corresponding to 3I and 3II isoforms of exon 3 splicing variants. Both isoforms display tissue specificity, with Acox1-3II presenting a more ubiquitous expression in comparison with Acox1-3I. Acox3 was expressed in almost all brown trout tissues. According to real-time PCR data, the highest estrogenic stimulus was able to cause a down-regulation of Acox1 and an up-regulation of Acox3. So, for Acox1 we found a negative association between an estrogenic input and a directly activated PPARα target gene. In conclusion, changes in hormonal estrogenic stimulus may impact the mobilization of hepatic lipids to the gonads, with ultimate consequences in reproduction. Further studies using in vivo assays will be fundamental to clarify these issues.

  9. Mangiferin treatment inhibits hepatic expression of acyl-coenzyme A:diacylglycerol acyltransferase-2 in fructose-fed spontaneously hypertensive rats: a link to amelioration of fatty liver

    Energy Technology Data Exchange (ETDEWEB)

    Xing, Xiaomang; Li, Danyang; Chen, Dilong; Zhou, Liang [Faculty of Basic Medical Sciences, Chongqing Medical University, Chongqing 400016 China (China); Chonan, Ritsu [Koei Kogyo Co., Ltd., Tokyo, 101-0063 Japan (Japan); Yamahara, Johji [Pharmafood Institute, Kyoto, 602-8136 Japan (Japan); Wang, Jianwei, E-mail: wangjianwei1968@gmail.com [Department of Traditional Chinese Medicine, Chongqing Medical University, Chongqing 400016 China (China); Li, Yuhao, E-mail: yuhao@sitcm.edu.au [Endocrinology and Metabolism Group, Sydney Institute of Health Sciences/Sydney Institute of Traditional Chinese Medicine, NSW 2000 Australia (Australia)

    2014-10-15

    Mangiferin, a xanthone glucoside, and its associated traditional herbs have been demonstrated to improve abnormalities of lipid metabolism. However, its underlying mechanisms remain largely unclear. This study investigated the anti-steatotic effect of mangiferin in fructose-fed spontaneously hypertensive rat (SHR)s that have a mutation in sterol regulatory element binding protein (SREBP)-1. The results showed that co-administration of mangiferin (15 mg/kg, once daily, by oral gavage) over 7 weeks dramatically diminished fructose-induced increases in hepatic triglyceride content and Oil Red O-stained area in SHRs. However, blood pressure, fructose and chow intakes, white adipose tissue weight and metabolic parameters (plasma concentrations of glucose, insulin, triglyceride, total cholesterol and non-esterified fatty acids) were unaffected by mangiferin treatment. Mechanistically, mangiferin treatment suppressed acyl-coenzyme A:diacylglycerol acyltransferase (DGAT)-2 expression at the mRNA and protein levels in the liver. In contrast, mangiferin treatment was without effect on hepatic mRNA and/or protein expression of SREBP-1/1c, carbohydrate response element binding protein, liver pyruvate kinase, fatty acid synthase, acetyl-CoA carboxylase-1, stearoyl-CoA desaturase-1, DGAT-1, monoacyglycerol acyltransferase-2, microsomal triglyceride transfer protein, peroxisome proliferator-activated receptor-alpha, carnitine palmitoyltransferase-1 and acyl-CoA oxidase. Collectively, our results suggest that mangiferin treatment ameliorates fatty liver in fructose-fed SHRs by inhibiting hepatic DGAT-2 that catalyzes the final step in triglyceride biosynthesis. The anti-steatotic effect of mangiferin may occur independently of the hepatic signals associated with de novo fatty acid synthesis and oxidation. - Highlights: • We investigated the anti-steatotic effect of mangiferin (MA) in fructose-fed SHR. • MA (15 mg/kg/day for 7 weeks) ameliorated fructose-induced fatty liver in

  10. Photoaffinity Labeling of Developing Jojoba Seed Microsomal Membranes with a Photoreactive Analog of Acyl-Coenzyme A (Acyl-CoA) (Identification of a Putative Acyl-CoA:Fatty Alcohol Acyltransferase.

    Science.gov (United States)

    Shockey, J. M.; Rajasekharan, R.; Kemp, J. D.

    1995-01-01

    Jojoba (Simmondsia chinensis, Link) is the only plant known that synthesizes liquid wax. The final step in liquid wax biosynthesis is catalyzed by an integral membrane enzyme, fatty acyl-coenzyme A (CoA):fatty alcohol acyltransferase, which transfers an acyl chain from acyl-CoA to a fatty alcohol to form the wax ester. To purify the acyltransferase, we have labeled the enzyme with a radioiodinated, photoreactive analog of acyl-CoA, 12-[N-(4-azidosalicyl)amino] dodecanoyl-CoA (ASD-CoA). This molecule acts as an inhibitor of acyltransferase activity in the dark and as an irreversible inhibitor upon exposure to ultraviolet light. Oleoyl-CoA protects enzymatic activity in a concentration-dependent manner. Photolysis of microsomal membranes with labeled ASD-CoA resulted in strong labeling of two polypeptides of 57 and 52 kD. Increasing concentrations of oleoyl-CoA reduced the labeling of the 57-kD polypeptide dramatically, whereas the labeling of the 52-kD polypeptide was much less responsive to oleoyl-CoA. Also, unlike the other polypeptide, the labeling of the 57-kD polypeptide was enhanced considerably when photolyzed in the presence of dodecanol. These results suggest that a 57-kD polypeptide from jojoba microsomes may be the acyl-CoA:fatty alcohol acyltransferase.

  11. Bioconversion of α-linolenic acid to n-3 LCPUFA and expression of PPAR-alpha, acyl Coenzyme A oxidase 1 and carnitine acyl transferase I are incremented after feeding rats with α-linolenic acid-rich oils.

    Science.gov (United States)

    González-Mañán, Daniel; Tapia, Gladys; Gormaz, Juan Guillermo; D'Espessailles, Amanda; Espinosa, Alejandra; Masson, Lilia; Varela, Patricia; Valenzuela, Alfonso; Valenzuela, Rodrigo

    2012-07-01

    High dietary intake of n-6 fatty acids in relation to n-3 fatty acids may generate health disorders, such as cardiovascular and other chronic diseases. Fish consumption rich in n-3 fatty acids is low in Latin America, it being necessary to seek other alternatives to provide α-linolenic acid (ALA), precursor of n-3 LCPUFA (EPA and DHA). Two innovative oils were assayed, chia (Salvia hispanica) and rosa mosqueta (Rosa rubiginosa). This study evaluated hepatic bioconversion of ALA to EPA and DHA, expression of PPAR-α, acyl-Coenzyme A oxidase 1 (ACOX1) and carnitine acyltransferase I (CAT-I), and accumulation of EPA and DHA in plasma and adipose tissue in Sprague-Dawley rats. Three experimental groups were fed 21 days: sunflower oil (SFO, control); chia oil (CO); rosa mosqueta oil (RMO). Fatty acid composition of total lipids and phospholipids from plasma, hepatic and adipose tissue was assessed by gas-liquid chromatography and TLC. Expression of PPAR-α (RT-PCR) and ACOX1 and CAT-I (Western blot). CO and RMO increased plasma, hepatic and adipose tissue levels of ALA, EPA and DHA and decreased n-6:n-3 ratio compared to SFO (p < 0.05, One-way ANOVA and Newman-Keuls test). CO increased levels of ALA and EPA compared to RMO (p < 0.05). No significant differences were observed for DHA levels. CO also increased the expression of PPAR-α, ACOX1 and CAT-I. Only CAT-I levels were increased by RO. CO and RMO may be a nutritional alternative to provide ALA for its bioconversion to EPA and DHA, and to increase the expression of PPAR-α, ACOX1 and CAT-I, especially CO-oil.

  12. Signalling properties of lysophosphatidic acid.

    Science.gov (United States)

    Durieux, M E; Lynch, K R

    1993-06-01

    Lysophosphatidic acid (LPA) is the simplest natural phospholipid, primarily known as a membrane component and metabolic intermediate. However, a remarkable variety of biological effects of this compound have come to light, seemingly pointing to an additional role for LPA as a signalling molecule. In this review, Marcel Durieux and Kevin Lynch integrate the recent information that indicates that LPA could be an intercellular messenger, possibly acting through a G protein-coupled receptor, and with a role in cell growth and motility.

  13. [Lysophosphatidic acid and human erythrocyte aggregation].

    Science.gov (United States)

    Sheremet'ev, Iu A; Popovicheva, A N; Levin, G Ia

    2014-01-01

    The effects of lysophosphatidic acid on the morphology and aggregation of human erythrocytes has been studied. Morphology of erythrocytes and their aggregates were studied by light microscopy. It has been shown that lysophosphatidic acid changes the shape of red blood cells: diskocyte become echinocytes. Aggregation of red blood cells (rouleaux) was significantly reduced in autoplasma. At the same time there is a strong aggregation of echinocytes. This was accompanied by the formation of microvesicles. Adding normal plasma to echinocytes restores shape and aggregation of red blood cells consisting of "rouleaux". A possible mechanism of action of lysophosphatidic acid on erythrocytes is discussed.

  14. Lysophosphatidic acid as a phospholipid mediator: pathways of synthesis.

    Science.gov (United States)

    Gaits, F; Fourcade, O; Le Balle, F; Gueguen, G; Gaigé, B; Gassama-Diagne, A; Fauvel, J; Salles, J P; Mauco, G; Simon, M F; Chap, H

    1997-06-23

    From very recent studies, including molecular cloning of cDNA coding for membrane receptors, lysophosphatidic acid (LPA) reached the status of a novel phospholipid mediator with various biological activities. Another strong argument supporting this view was the discovery that LPA is secreted from activated platelets, resulting in its appearance in serum upon blood coagulation. The metabolic pathways as well as the enzymes responsible for LPA production are poorly characterized. However, a survey of literature data indicates some interesting issues which might be used as the basis for further molecular characterization of phospholipases A able to degrade phosphatidic acid.

  15. Tetracyclines increase lipid phosphate phosphatase expression on plasma membranes and turnover of plasma lysophosphatidate.

    Science.gov (United States)

    Tang, Xiaoyun; Zhao, Yuan Y; Dewald, Jay; Curtis, Jonathan M; Brindley, David N

    2016-04-01

    Extracellular lysophosphatidate and sphingosine 1-phosphate (S1P) are important bioactive lipids, which signal through G-protein-coupled receptors to stimulate cell growth and survival. The lysophosphatidate and S1P signals are terminated partly by degradation through three broad-specificity lipid phosphate phosphatases (LPPs) on the cell surface. Significantly, the expression of LPP1 and LPP3 is decreased in many cancers, and this increases the impact of lysophosphatidate and S1P signaling. However, relatively little is known about the physiological or pharmacological regulation of the expression of the different LPPs. We now show that treating several malignant and nonmalignant cell lines with 1 μg/ml tetracycline, doxycycline, or minocycline significantly increased the extracellular degradation of lysophosphatidate. S1P degradation was also increased in cells that expressed high LPP3 activity. These results depended on an increase in the stabilities of the three LPPs and increased expression on the plasma membrane. We tested the physiological significance of these results and showed that treating rats with doxycycline accelerated the clearance of lysophosphatidate, but not S1P, from the circulation. However, administering 100 mg/kg/day doxycycline to mice decreased plasma concentrations of lysophosphatidate and S1P. This study demonstrates a completely new property of tetracyclines in increasing the plasma membrane expression of the LPPs.

  16. Biological effects of lysophosphatidic acid in the nervous system.

    Science.gov (United States)

    Frisca, Frisca; Sabbadini, Roger A; Goldshmit, Yona; Pébay, Alice

    2012-01-01

    Lysophosphatidic acid (LPA) is a bioactive lipid that regulates a broad range of cellular effects in various cell types, leading to a variety of responses in tissues, including in the nervous system. LPA and its receptors are found in the nervous system, with different cellular and temporal profiles. Through its ability to target most cells of the nervous system and its induction of pleiotropic effects, LPA mediates events during neural development and adulthood. In this review, we summarize the current knowledge on the effects of LPA in the nervous system, during development and adulthood, and in various pathologies of the nervous system. We also explore potential LPA intervention strategies for anti-LPA therapeutics.

  17. Lysophosphatidic Acid (LPA Signaling in Human and Ruminant Reproductive Tract

    Directory of Open Access Journals (Sweden)

    Izabela Wocławek-Potocka

    2014-01-01

    Full Text Available Lysophosphatidic acid (LPA through activating its G protein-coupled receptors (LPAR 1–6 exerts diverse cellular effects that in turn influence several physiological processes including reproductive function of the female. Studies in various species of animals and also in humans have identified important roles for the receptor-mediated LPA signaling in multiple aspects of human and animal reproductive tract function. These aspects range from ovarian and uterine function, estrous cycle regulation, early embryo development, embryo implantation, decidualization to pregnancy maintenance and parturition. LPA signaling can also have pathological consequences, influencing aspects of endometriosis and reproductive tissue associated tumors. The review describes recent progress in LPA signaling research relevant to human and ruminant reproduction, pointing at the cow as a relevant model to study LPA influence on the human reproductive performance.

  18. Lysophosphatidic acid metabolism and elimination in cardiovascular disease

    Science.gov (United States)

    Salous, Abdelghaffar Kamal

    The bioactive lipids lysophosphatidic acid (LPA) and sphingosine 1-phosphate (S1P) are present in human and mouse plasma at a concentration of ~0.1-1 microM and regulate physiological and pathophysiological processes in the cardiovascular system including atherothrombosis, intimal hyperplasia, and immune function, edema formation, and permeability. PPAP2B, the gene encoding LPP3, a broad activity integral membrane enzyme that terminates LPA actions in the vasculature, has a single nucleotide polymorphism that been recently associated with coronary artery disease risk. The synthesis and signaling of LPA and S1P in the cardiovascular system have been extensively studied but the mechanisms responsible for their elimination are less well understood. The broad goal of this research was to examine the role of LPP3 in the termination of LPA signaling in models of cardiovascular disease involving vascular wall cells, investigate the role of LPP3 in the elimination of plasma LPA, and further characterize the elimination of plasma LPA. The central hypothesis is that LPP3 plays an important role in attenuating the pathological responses to LPA signaling and that it mediates the elimination of exogenously applied bioactive lipids from the plasma. These hypotheses were tested using molecular biological approaches, in vitro studies, synthetic lysophospholipid mimetics, modified surgical procedures, and mass spectrometry assays. My results indicated that LPP3 played a critical role in attenuating LPA signaling mediating the pathological processes of intimal hyperplasia and vascular leak in mouse models of disease. Additionally, enzymatic inactivation of lysophospholipids by LPP and PLA enzymes in the plasma was not a primary mechanism for the rapid elimination of plasma LPA and S1P. Instead, evidence strongly suggested a transcellular uptake mechanism by hepatic non-parenchymal cells as the predominant mechanism for elimination of these molecules. These results support a model in

  19. Altered food consumption in mice lacking lysophosphatidic acid receptor-1.

    Science.gov (United States)

    Dusaulcy, R; Daviaud, D; Pradère, J P; Grès, S; Valet, Ph; Saulnier-Blache, J S

    2009-12-01

    The release of lysophosphatidic acid (LPA) by adipocytes has previously been proposed to play a role in obesity and associated pathologies such as insulin resistance and diabetes. In the present work, the sensitivity to diet-induced obesity was studied in mice lacking one of the LPA receptor subtype (LPA1R). Conversely to what was observed in wild type (WT) mice, LPA1R-KO-mice fed a high fat diet (HFD) showed no significant increase in body weight or fat mass when compared to low fat diet (LFD). In addition, in contrast to what was observed in WT mice, LPA1R-KO mice did not exhibit over-consumption of food associated with HFD. Surprisingly, when fed a LFD, LPA1R-KO mice exhibited significant higher plasma leptin concentration and higher level of adipocyte leptin mRNA than WT mice. In conclusion, LPA1R-KO mice were found to be resistant to diet-induced obesity consecutive to a resistance to fat-induced over-consumption of food that may result at least in part from alterations in leptin expression and production.

  20. Roles of lysophosphatidic acid in cardiovascular physiology and disease.

    Science.gov (United States)

    Smyth, Susan S; Cheng, Hsin-Yuan; Miriyala, Sumitra; Panchatcharam, Manikandan; Morris, Andrew J

    2008-09-01

    The bioactive lipid mediator lysophosphatidic acid (LPA) exerts a range of effects on the cardiovasculature that suggest a role in a variety of critical cardiovascular functions and clinically important cardiovascular diseases. LPA is an activator of platelets from a majority of human donors identifying a possible role as a regulator of acute thrombosis and platelet function in atherogenesis and vascular injury responses. Of particular interest in this context, LPA is an effective phenotypic modulator of vascular smooth muscle cells promoting the de-differentiation, proliferation and migration of these cells that are required for the development of intimal hyperplasia. Exogenous administration of LPA results in acute and systemic changes in blood pressure in different animal species, suggesting a role for LPA in both normal blood pressure regulation and hypertension. Advances in our understanding of the molecular machinery responsible for the synthesis, actions and inactivation of LPA now promise to provide the tools required to define the role of LPA in cardiovascular physiology and disease. In this review we discuss aspects of LPA signaling in the cardiovasculature focusing on recent advances and attempting to highlight presently unresolved issues and promising avenues for further investigation.

  1. Lysophosphatidate induces chemo-resistance by releasing breast cancer cells from taxol-induced mitotic arrest.

    Directory of Open Access Journals (Sweden)

    Nasser Samadi

    Full Text Available BACKGROUND: Taxol is a microtubule stabilizing agent that arrests cells in mitosis leading to cell death. Taxol is widely used to treat breast cancer, but resistance occurs in 25-69% of patients and it is vital to understand how Taxol resistance develops to improve chemotherapy. The effects of chemotherapeutic agents are overcome by survival signals that cancer cells receive. We focused our studies on autotaxin, which is a secreted protein that increases tumor growth, aggressiveness, angiogenesis and metastasis. We discovered that autotaxin strongly antagonizes the Taxol-induced killing of breast cancer and melanoma cells by converting the abundant extra-cellular lipid, lysophosphatidylcholine, into lysophosphatidate. This lipid stimulates specific G-protein coupled receptors that activate survival signals. METHODOLOGY/PRINCIPAL FINDINGS: In this study we determined the basis of these antagonistic actions of lysophosphatidate towards Taxol-induced G2/M arrest and cell death using cultured breast cancer cells. Lysophosphatidate does not antagonize Taxol action in MCF-7 cells by increasing Taxol metabolism or its expulsion through multi-drug resistance transporters. Lysophosphatidate does not lower the percentage of cells accumulating in G2/M by decreasing exit from S-phase or selective stimulation of cell death in G2/M. Instead, LPA had an unexpected and remarkable action in enabling MCF-7 and MDA-MB-468 cells, which had been arrested in G2/M by Taxol, to normalize spindle structure and divide, thus avoiding cell death. This action involves displacement of Taxol from the tubulin polymer fraction, which based on inhibitor studies, depends on activation of LPA receptors and phosphatidylinositol 3-kinase. CONCLUSIONS/SIGNIFICANCE: This work demonstrates a previously unknown consequence of lysophosphatidate action that explains why autotaxin and lysophosphatidate protect against Taxol-induced cell death and promote resistance to the action of this

  2. 溶血磷脂酸受体2%Research progress on lysophosphatidic acid receptor 2

    Institute of Scientific and Technical Information of China (English)

    龚晓华; 饶勇; 马卫列; 张志珍

    2013-01-01

    Lysophosphatidic acid receptor 2 (LPA2), also known as endothelial differentiation gene receptor 4 (EDG4), is a G-protein coupled receptor (GPCR) activated by lysophosphatidic acid. It has high affinity to lysophosphatidic acid, and can mediate many kinds of cellular activities. Recent researches have shown that LPA2/EDG4 is overexpressed in ovarian cancer cells. In addition, it is closely connected with breast carcinoma, colon cancer, atherosclerosis, respiratory disease and hypertensivedisorder complicating pregnancy.%溶血磷脂酸受体2 (lysophosphatidic acid receptor 2,LPA2),也称内皮分化基因受体4(endothelial differentiation gene receptor4,EDG4),是溶血磷脂酸G蛋白偶联受体类的一种,对溶血磷脂酸有较高亲和力,可介导多种细胞活动.近年研究发现,LPA2/EDG4在卵巢癌细胞中过表达,同时与乳腺癌、结直肠癌、动脉粥样硬化、呼吸道疾病、妊娠性高血压等有着密切关系.

  3. Rac activation by lysophosphatidic acid LPA1 receptors through the guanine nucleotide exchange factor Tiam1

    NARCIS (Netherlands)

    Van Leeuwen, Frank N; Olivo, Cristina; Grivell, Shula; Giepmans, Ben N G; Collard, John G; Moolenaar, Wouter H

    2003-01-01

    Lysophosphatidic acid (LPA) is a serum-borne phospholipid that activates its own G protein-coupled receptors present in numerous cell types. In addition to stimulating cell proliferation, LPA also induces cytoskeletal changes and promotes cell migration in a RhoA- and Rac-dependent manner. Whereas R

  4. Regulation of lipolytic activity by long-chain acyl-coenzyme A in islets and adipocytes

    DEFF Research Database (Denmark)

    Hu, Liping; Deeney, Jude T; Nolan, Christopher J;

    2005-01-01

    normal and hormone-sensitive lipase (HSL)-null mice and in phosphatase-treated islets, indicating that the stimulatory effect was neither on HSL nor phosphorylation dependent. In contrast, we reproduced the previously published observations showing inhibition of HSL activity by LC-CoA in adipocytes....... The inhibitory effect of LC-CoA on adipocyte HSL was dependent on phosphorylation and enhanced by acyl-CoA-binding protein (ACBP). In contrast, the stimulatory effect on islet lipase activity was blocked by ACBP, presumably due to binding and sequestration of LC-CoA. These data suggest the following intertissue...

  5. Polyunsaturated fatty acyl-coenzyme As are inhibitors of cholesterol biosynthesis in zebrafish and mice

    Directory of Open Access Journals (Sweden)

    Santhosh Karanth

    2013-11-01

    Lipid disorders pose therapeutic challenges. Previously we discovered that mutation of the hepatocyte β-hydroxybutyrate transporter Slc16a6a in zebrafish causes hepatic steatosis during fasting, marked by increased hepatic triacylglycerol, but not cholesterol. This selective diversion of trapped ketogenic carbon atoms is surprising because acetate and acetoacetate can exit mitochondria and can be incorporated into both fatty acids and cholesterol in normal hepatocytes. To elucidate the mechanism of this selective diversion of carbon atoms to fatty acids, we fed wild-type and slc16a6a mutant animals high-protein ketogenic diets. We find that slc16a6a mutants have decreased activity of the rate-limiting enzyme of cholesterol biosynthesis, 3-hydroxy-3-methylglutaryl-coenzyme A reductase (Hmgcr, despite increased Hmgcr protein abundance and relative incorporation of mevalonate into cholesterol. These observations suggest the presence of an endogenous Hmgcr inhibitor. We took a candidate approach to identify such inhibitors. First, we found that mutant livers accumulate multiple polyunsaturated fatty acids (PUFAs and PUFA-CoAs, and we showed that human HMGCR is inhibited by PUFA-CoAs in vitro. Second, we injected mice with an ethyl ester of the PUFA eicosapentaenoic acid and observed an acute decrease in hepatic Hmgcr activity, without alteration in Hmgcr protein abundance. These results elucidate a mechanism for PUFA-mediated cholesterol lowering through direct inhibition of Hmgcr.

  6. Thermodynamics of ligand binding to acyl-coenzyme A binding protein studied by titration calorimetry

    DEFF Research Database (Denmark)

    Færgeman, Nils J.; Sigurskjold, B W; Kragelund, B B

    1996-01-01

    Ligand binding to recombinant bovine acyl-CoA binding protein (ACBP) was examined using isothermal microcalorimetry. Microcalorimetric measurements confirm that the binding affinity of acyl-CoA esters for ACBP is strongly dependent on the length of the acyl chain with a clear preference for acyl......-CoA esters containing more than eight carbon atoms and that the 3'-phosphate of the ribose accounts for almost half of the binding energy. Binding of acyl-CoA esters, with increasing chain length, to ACBP was clearly enthalpically driven with a slightly unfavorable entropic contribution. Accessible surface...... areas derived from the measured enthalpies were compared to those calculated from sets of three-dimensional solution structures and showed reasonable correlation, confirming the enthalphically driven binding. Binding of dodecanoyl-CoA to ACBP was studied at various temperatures and was characterized...

  7. Cell Shrinkage is Essential in Lysophosphatidic Acid Signaling in Ehrlich Ascites

    DEFF Research Database (Denmark)

    Pedersen, Susanne; Hoffmann, Else Kay; Hougaard, Charlotte;

    2000-01-01

    -induced Ca(2+) mobilization were estimated at 0.03 nm and 0.4 nm LPA in the presence and absence of extracellular Ca(2+), respectively. The LPA-induced increase in [Ca(2+)](i) resulted in (i) co-activation of Ca(2+)-activated, charybdotoxin (ChTX)-sensitive K(+) and niflumic acid-sensitive Cl(-) currents......The present study aimed at elucidating the initial intracellular lysophosphatidic acid (LPA)-induced signaling events, in order to investigate the sequence in which LPA affects the intracellular concentration of free, cytosolic Ca(2+), [Ca(2+)](i), ion channels, the F-actin cytoskeleton, cell...

  8. The autotaxin-lysophosphatidic acid pathway in pathogenesis of rheumatoid arthritis.

    Science.gov (United States)

    Orosa, Beatriz; García, Samuel; Conde, Carmen

    2015-10-15

    Lysophosphatidic acid (LPA) is a phospholipid that is mainly produced by the hydrolysis of lysophosphatidylcholine (LPC) by lysophospholipase D, which is also called autotaxin (ATX). LPA interacts with specific G-protein coupled receptors and is involved in the regulation of cellular survival, proliferation, differentiation and motility. LPA also has roles in several pathological disorders, such as cancer and pulmonary, dermal and renal fibrosis. The involvement of the ATX-LPA pathway has recently been demonstrated in inflammatory responses and apoptosis of fibroblast-like synoviocytes (FLS) from patients with rheumatoid arthritis and during the development of experimental arthritis. This review summarises the current literature of the ATX-LPA pathway in rheumatoid arthritis.

  9. Lysophosphatidic Acid Receptor Is a Functional Marker of Adult Hippocampal Precursor Cells

    Directory of Open Access Journals (Sweden)

    Tara L. Walker

    2016-04-01

    Full Text Available Here, we show that the lysophosphatidic acid receptor 1 (LPA1 is expressed by a defined population of type 1 stem cells and type 2a precursor cells in the adult mouse dentate gyrus. LPA1, in contrast to Nestin, also marks the quiescent stem cell population. Combining LPA1-GFP with EGFR and prominin-1 expression, we have enabled the prospective separation of both proliferative and non-proliferative precursor cell populations. Transcriptional profiling of the isolated proliferative precursor cells suggested immune mechanisms and cytokine signaling as molecular regulators of adult hippocampal precursor cell proliferation. In addition to LPA1 being a marker of this important stem cell population, we also show that the corresponding ligand LPA is directly involved in the regulation of adult hippocampal precursor cell proliferation and neurogenesis, an effect that can be attributed to LPA signaling via the AKT and MAPK pathways.

  10. A novel highly potent autotaxin/ENPP2 inhibitor produces prolonged decreases in plasma lysophosphatidic acid formation in vivo and regulates urethral tension.

    Directory of Open Access Journals (Sweden)

    Hiroshi Saga

    Full Text Available Autotaxin, also known as ectonucleotide pyrophosphatase/phosphodiesterase 2 (ENPP2, is a secreted enzyme that has lysophospholipase D activity, which converts lysophosphatidylcholine to bioactive lysophosphatidic acid. Lysophosphatidic acid activates at least six G-protein coupled recpetors, which promote cell proliferation, survival, migration and muscle contraction. These physiological effects become dysfunctional in the pathology of cancer, fibrosis, and pain. To date, several autotaxin/ENPP2 inhibitors have been reported; however, none were able to completely and continuously inhibit autotaxin/ENPP2 in vivo. In this study, we report the discovery of a highly potent autotaxin/ENPP2 inhibitor, ONO-8430506, which decreased plasma lysophosphatidic acid formation. The IC50 values of ONO-8540506 for lysophospholipase D activity were 6.4-19 nM for recombinant autotaxin/ENPP2 proteins and 4.7-11.6 nM for plasma from various animal species. Plasma lysophosphatidic acid formation during 1-h incubation was almost completely inhibited by the addition of >300 nM of the compound to human plasma. In addition, when administered orally to rats at a dose of 30 mg/kg, the compound demonstrated good pharmacokinetics in rats and persistently inhibited plasma lysophosphatidic acid formation even at 24 h after administration. Smooth muscle contraction is a known to be promoted by lysophosphatidic acid. In this study, we showed that dosing rats with ONO-8430506 decreased intraurethral pressure accompanied by urethral relaxation. These findings demonstrate the potential of this autotaxin/ENPP2 inhibitor for the treatment of various diseases caused by lysophosphatidic acid, including urethral obstructive disease such as benign prostatic hyperplasia.

  11. Toluene diisocyanate: Induction of the autotaxin-lysophosphatidic acid axis and its association with airways symptoms

    Energy Technology Data Exchange (ETDEWEB)

    Broström, Julia M. [Division of Occupational and Environmental Medicine, Lund University, SE 221 85 Lund (Sweden); Ye, Zhi-wei [Institute of Environmental Medicine, Karolinska Institutet, Box 210, SE 171 77 Stockholm (Sweden); Axmon, Anna; Littorin, Margareta; Tinnerberg, Håkan; Lindh, Christian H. [Division of Occupational and Environmental Medicine, Lund University, SE 221 85 Lund (Sweden); Zheng, Huiyuan; Ghalali, Aram; Stenius, Ulla [Institute of Environmental Medicine, Karolinska Institutet, Box 210, SE 171 77 Stockholm (Sweden); Jönsson, Bo A.G. [Division of Occupational and Environmental Medicine, Lund University, SE 221 85 Lund (Sweden); Högberg, Johan, E-mail: johan.hogberg@ki.se [Institute of Environmental Medicine, Karolinska Institutet, Box 210, SE 171 77 Stockholm (Sweden)

    2015-09-15

    Diisocyanates are industrial chemicals which have a wide range of applications in developed and developing countries. They are notorious lung toxicants and respiratory sensitizers. However, the mechanisms behind their adverse effects are not adequately characterized. Autotaxin (ATX) is an enzyme producing lysophosphatidic acid (LPA), and the ATX-LPA axis has been implicated in lung related inflammatory conditions and diseases, including allergic asthma, but not to toxicity of environmental low-molecular-weight chemicals. We investigated effects of toluene diisocyanate (TDI) on ATX induction in human lung epithelial cell models, and we correlated LPA-levels in plasma to biomarkers of TDI exposure in urine collected from workers exposed to < 5 ppb (parts per billion). Information on workers' symptoms was collected through interviews. One nanomolar TDI robustly induced ATX release within 10 min in vitro. A P2X7- and P2X4-dependent microvesicle formation was implicated in a rapid ATX release and a subsequent protein synthesis. Co-localization between purinergic receptors and ATX was documented by immunofluorescence and confocal microscopy. The release was modulated by monocyte chemoattractant protein-1 (MCP-1) and by extracellular ATP. In workers, we found a dose–response relationship between TDI exposure biomarkers in urine and LPA levels in plasma. Among symptomatic workers reporting “sneezing”, the LPA levels were higher than among non-symptomatic workers. This is the first report indicating induction of the ATX-LPA axis by an environmental low-molecular-weight chemical, and our data suggest a role for the ATX-LPA axis in TDI toxicity. - Highlights: • Human epithelial cells release autotaxin in response to 1 nM toluene diisocyanate (TDI). • The release involves P2X4 and P2X7 receptors and is modulated by ATP and MCP-1. • Lysophosphatidic acid (LPA) was measured in workers exposed to < 5 ppb TDI. • LPA in plasma correlated to TDI exposure

  12. Frequent mutations of lysophosphatidic acid receptor-1 gene in rat liver tumors

    Energy Technology Data Exchange (ETDEWEB)

    Obo, Yumi; Yamada, Takanori; Furukawa, Mami; Hotta, Mayuko [Laboratory of Cancer Biology and Bioinformatics, Department of Life Science, Faculty of Science and Engineering, Kinki University, 3-4-1, Kowakae, Higashiosaka, Osaka 577-8502 (Japan); Honoki, Kanya [Department of Orthopedic Surgery, Nara Medical University, 840 Shijo-cho, Kashihara, Nara 634-8521 (Japan); Fukushima, Nobuyuki [Laboratory of Molecular Neurobiology, Department of Life Science, Faculty of Science and Technology, Kinki University, 3-4-1, Kowakae, Higashiosaka, Osaka 577-8502 (Japan); Tsujiuchi, Toshifumi [Laboratory of Cancer Biology and Bioinformatics, Department of Life Science, Faculty of Science and Engineering, Kinki University, 3-4-1, Kowakae, Higashiosaka, Osaka 577-8502 (Japan)], E-mail: ttujiuch@life.kindai.ac.jp

    2009-01-15

    Lysophosphatidic acid (LPA) is a bioactive phospholipid that stimulates cell proliferation, migration, and protects cells from apoptosis. It interacts with specific G protein-coupled transmembrane receptors, including LPA1 to LPA5. In the present study, to clarify an involvement of LPA1 gene alterations in the development of hepatocellular carcinomas (HCCs) we investigated the LPA1 mutations in rat HCCs induced by exogenous and endogenous liver carcinogenesis models. We induced HCCs in rats with N-nitrosodiethylamine (DEN) and a choline-deficient L-amino acid-defined (CDAA) diet. RNAs were extracted from 15 HCCs induced by DEN and 12 HCCs induced by the CDAA diet. To identify LPA1 mutations, reverse transcription (RT) - polymerase chain reaction (PCR) - single strand conformation polymorphism (SSCP) analysis, followed by nucleotide sequencing, was performed. Missense mutations were detected in 7 out of 15 HCCs (46.7%) induced by DEN. Five out of 12 HCCs (41.7%) induced by the CDAA diet also showed missense mutations. These results demonstrated that mutations in LPA1 gene occur in rat HCCs induced by DEN and the CDAA diet, suggesting that LPA1 mutations may be essentially involved in rat liver carcinogenesis.

  13. Elevated Serum Levels of Arachidonoyl-lysophosphatidic Acid and Sphingosine 1-Phosphate in Systemic Sclerosis

    Directory of Open Access Journals (Sweden)

    Akira Tokumura, Laura D. Carbone, Yasuko Yoshioka, Junichi Morishige, Masaki Kikuchi, Arnold Postlethwaite, Mitchell A. Watsky

    2009-01-01

    Full Text Available Systemic sclerosis (SSc is an often fatal disease characterized by autoimmunity and inflammation, leading to widespread vasculopathy and fibrosis. Lysophosphatidic acid (LPA, a bioactive phospholipid in serum, is generated from lysophospholipids secreted from activated platelets in part by the action of lysophospholipase D (lysoPLD. Sphingosine 1-phosphate (S1P, a member of the bioactive lysophospholipid family, is also released from activated platelets. Because activated platelets are a hallmark of SSc, we wanted to determine whether subjects with SSc have altered serum lysophospholipid levels or lysoPLD activity. Lysophospholipid levels were measured using mass spectrometric analysis. LysoPLD activity was determined by quantifying choline released from exogenous lysophosphatidylcholine (LPC. The major results were that serum levels of arachidonoyl (20:4-LPA and S1P were significantly higher in SSc subjects versus controls. Furthermore, serum LPA:LPC ratios of two different polyunsaturated phospholipid molecular species, and also the ratio of all species combined, were significantly higher in SSc subjects versus controls. No significant differences were found between other lysophospholipid levels or lysoPLD activities. Elevated 20:4 LPA, S1P levels and polyunsaturated LPA:LPC ratios may be markers for and/or play a significant role in the etiology of SSc and may be future pharmacological targets for SSc treatment.

  14. Increased urinary lysophosphatidic acid in mouse with subtotal nephrectomy: potential involvement in chronic kidney disease.

    Science.gov (United States)

    Mirzoyan, Koryun; Baïotto, Anna; Dupuy, Aude; Marsal, Dimitri; Denis, Colette; Vinel, Claire; Sicard, Pierre; Bertrand-Michel, Justine; Bascands, Jean-Loup; Schanstra, Joost P; Klein, Julie; Saulnier-Blache, Jean-Sébastien

    2016-12-01

    Increased incidence of chronic kidney disease (CKD) with consecutive progression to end-stage renal disease represents a significant burden to healthcare systems. Renal tubulointerstitial fibrosis (TIF) is a classical hallmark of CKD and is well correlated with the loss of renal function. The bioactive lysophospholipid lysophosphatidic acid (LPA), acting through specific G-protein-coupled receptors, was previously shown to be involved in TIF development in a mouse model of unilateral ureteral obstruction. Here, we study the role of LPA in a mouse subjected to subtotal nephrectomy (SNx), a more chronic and progressive model of CKD. Five months after surgical nephron reduction, SNx mice showed massive albuminuria, extensive TIF, and glomerular hypertrophy when compared to sham-operated animals. Urinary and plasma levels of LPA were analyzed using liquid chromatography tandem mass spectrometry. LPA was significantly increased in SNx urine, not in plasma, and was significantly correlated with albuminuria and TIF. Moreover, SNx mice showed significant downregulation in the renal expression of lipid phosphate phosphohydrolases (LPP1, 2, and 3) that might be involved in reduced LPA bioavailability through dephosphorylation. We concluded that SNx increases urinary LPA through a mechanism that could involve co-excretion of plasma LPA with albumin associated with a reduction of its catabolism in the kidney. Because of the previously demonstrated profibrotic activity of LPA, the association of urinary LPA with TIF suggests the potential involvement of LPA in the development of advanced CKD in the SNx mouse model. Targeting LPA metabolism might represent an interesting approach in CKD treatment.

  15. Expression of rapeseed microsomal lysophosphatidic acid acyltransferase isozymes enhances seed oil content in Arabidopsis.

    Science.gov (United States)

    Maisonneuve, Sylvie; Bessoule, Jean-Jacques; Lessire, René; Delseny, Michel; Roscoe, Thomas J

    2010-02-01

    In higher plants, lysophosphatidic acid acyltransferase (LPAAT), located in the cytoplasmic endomembrane compartment, plays an essential role in the synthesis of phosphatidic acid, a key intermediate in the biosynthesis of membrane phospholipids in all tissues and storage lipids in developing seeds. In order to assess the contribution of LPAATs to the synthesis of storage lipids, we have characterized two microsomal LPAAT isozymes, the products of homoeologous genes that are expressed in rapeseed (Brassica napus). DNA sequence homologies, complementation of a bacterial LPAAT-deficient mutant, and enzymatic properties confirmed that each of two cDNAs isolated from a Brassica napus immature embryo library encoded a functional LPAAT possessing the properties of a eukaryotic pathway enzyme. Analyses in planta revealed differences in the expression of the two genes, one of which was detected in all rapeseed tissues and during silique and seed development, whereas the expression of the second gene was restricted predominantly to siliques and developing seeds. Expression of each rapeseed LPAAT isozyme in Arabidopsis (Arabidopsis thaliana) resulted in the production of seeds characterized by a greater lipid content and seed mass. These results support the hypothesis that increasing the expression of glycerolipid acyltransferases in seeds leads to a greater flux of intermediates through the Kennedy pathway and results in enhanced triacylglycerol accumulation.

  16. 1-Oleoyl lysophosphatidic acid: a new mediator of emotional behavior in rats.

    Directory of Open Access Journals (Sweden)

    Estela Castilla-Ortega

    Full Text Available The role of lysophosphatidic acid (LPA in the control of emotional behavior remains to be determined. We analyzed the effects of the central administration of 1-oleoyl-LPA (LPA 18∶1 in rats tested for food consumption and anxiety-like and depression-like behaviors. For this purpose, the elevated plus-maze, open field, Y maze, forced swimming and food intake tests were performed. In addition, c-Fos expression in the dorsal periaqueductal gray matter (DPAG was also determined. The results revealed that the administration of LPA 18∶1 reduced the time in the open arms of the elevated plus-maze and induced hypolocomotion in the open field, suggesting an anxiogenic-like phenotype. Interestingly, these effects were present following LPA 18∶1 infusion under conditions of novelty but not under habituation conditions. In the forced swimming test, the administration of LPA 18∶1 dose-dependently increased depression-like behavior, as evaluated according to immobility time. LPA treatment induced no effects on feeding. However, the immunohistochemical analysis revealed that LPA 18∶1 increased c-Fos expression in the DPAG. The abundant expression of the LPA1 receptor, one of the main targets for LPA 18∶1, was detected in this brain area, which participates in the control of emotional behavior, using immunocytochemistry. These findings indicate that LPA is a relevant transmitter potentially involved in normal and pathological emotional responses, including anxiety and depression.

  17. Effects of lysophosphatidic acid on human colon cancer cells and its mechanisms of action

    Institute of Scientific and Technical Information of China (English)

    Hong Sun; Juan Ren; Qing Zhu; Fan-Zhong Kong; Lei Wu; Bo-Rong Pan

    2009-01-01

    AIM: To study the effects of lysophosphatidic acid (LPA) on proliferation, adhesion, migration, and apoptosisin the human colon cancer cell line, SW480, and its mechanisms of action. METHODS: Methyl tetrazolium assay was used to assess cell proliferation. Flow cytometry was employed to detect cell apoptosis. Cell migration was measured by using a Boyden transwell migration chamber. Cell adhesion assay was performed in 96-well plates according to protocol.RESULTS: LPA significantly stimulated SW480 cell proliferation in a dose-dependent and time-dependent manner compared with the control group (P < 0.05) while the mitogen-activated protein kinase (MAPK) inhibitor,PD98059, significantly blocked the LPA stimulation effect on proliferation. LPA also significantly stimulated adhesion and migration of SW480 cells in a dosedependent manner (P < 0.05). Rho kinase inhibitor,Y-27632, significantly inhibited the up-regulatory effect of LPA on adhesion and migration (P < 0.05). LPA significantly protected cells from apoptosis induced by the chemotherapeutic drugs, cisplatin and 5-FU (P < 0.05),but the phosphoinositide 3-kinase (PI3K) inhibitor,LY294002, significantly blocked the protective effect of LPA on apoptosis.CONCLUSION: LPA stimulated proliferation, adhesion,migration of SW480 cells, and protected from apoptosis.The Ras/Raf-MAPK, G12/13-Rho-RhoA and PI3KAKT/ PKB signal pathways may be involved.

  18. Multiple actions of lysophosphatidic acid on fibroblasts revealed by transcriptional profiling

    Directory of Open Access Journals (Sweden)

    Moolenaar Wouter H

    2008-08-01

    Full Text Available Abstract Background Lysophosphatidic acid (LPA is a lipid mediator that acts through specific G protein-coupled receptors to stimulate the proliferation, migration and survival of many cell types. LPA signaling has been implicated in development, wound healing and cancer. While LPA signaling pathways have been studied extensively, it remains unknown how LPA affects global gene expression in its target cells. Results We have examined the temporal program of global gene expression in quiescent mouse embryonic fibroblasts stimulated with LPA using 32 k oligonucleotide microarrays. In addition to genes involved in growth stimulation and cytoskeletal reorganization, LPA induced many genes that encode secreted factors, including chemokines, growth factors, cytokines, pro-angiogenic and pro-fibrotic factors, components of the plasminogen activator system and metalloproteases. Strikingly, epidermal growth factor induced a broadly overlapping expression pattern, but some 7% of the genes (105 out of 1508 transcripts showed differential regulation by LPA. The subset of LPA-specific genes was enriched for those associated with cytoskeletal remodeling, in keeping with LPA's ability to regulate cell shape and motility. Conclusion This study highlights the importance of LPA in programming fibroblasts not only to proliferate and migrate but also to produce many paracrine mediators of tissue remodeling, angiogenesis, inflammation and tumor progression. Furthermore, our results show that G protein-coupled receptors and receptor tyrosine kinases can signal independently to regulate broadly overlapping sets of genes in the same cell type.

  19. Studies on lysophosphatidic acid action during in vitro preimplantation embryo development.

    Science.gov (United States)

    Boruszewska, D; Sinderewicz, E; Kowalczyk-Zieba, I; Grycmacher, K; Woclawek-Potocka, I

    2016-01-01

    Assisted reproductive technologies, including in vitro embryo production (IVP), have been successfully used in animal reproduction to optimize breeding strategies for improved production and health in animal husbandry. Despite the progress in IVP techniques over the years, further improvements in in vitro embryo culture systems are required for the enhancement of oocyte and embryo developmental competence. One of the most important issues associated with IVP procedures is the optimization of the in vitro culture of oocytes and embryos. Studies in different species of animals and in humans have identified important roles for receptor-mediated lysophosphatidic acid (LPA) signaling in multiple aspects of human and animal reproductive tract function. The data on LPA signaling in the ovary and uterus suggest that LPA can directly contribute to embryo-maternal interactions via its influence on early embryo development beginning from the influence of the ovarian environment on the oocyte to the influence of the uterine environment on the preimplantation embryo. This review discusses the current status of LPA as a potential supplement in oocyte maturation, fertilization, and embryo culture media and current views on the potential involvement of the LPA signaling pathway in early embryo development.

  20. Ethylmalonic aciduria is associated with an amino acid variant of short chain acyl-coenzyme A dehydrogenase

    DEFF Research Database (Denmark)

    Corydon, M J; Gregersen, N; Lehnert, W

    1996-01-01

    metabolized by propionyl-CoA carboxylase to EMA. We have recently detected a guanine to adenine polymorphism in the SCAD gene at position 625 in the SCAD cDNA, which changes glycine 209 to serine (G209S). The variant allele (A625) is present in homozygous and in heterozygous form in 7 and 34.8% of the general...

  1. Elucidation of the mechanism of inhibition of cyclooxygenases by acyl-coenzyme A and acylglucuronic conjugates of ketoprofen.

    Science.gov (United States)

    Levoin, Nicolas; Blondeau, Céline; Guillaume, Cécile; Grandcolas, Line; Chretien, Françoise; Jouzeau, Jean-Yves; Benoit, Etienne; Chapleur, Yves; Netter, Patrick; Lapicque, Françoise

    2004-11-15

    Nonsteroidal anti-inflammatory drugs (NSAIDs) inhibit the cyclooxygenase (COX) isoforms which accounts for their clinical effects. The differential inhibition of COX-1 and COX-2 is not sufficient to explain the absence of a correlation between in vitro and in vivo effects, especially for 2-aryl-propionates, thus indicating the participation of metabolites. Conjugates to glucuronic acid and to coenzyme-A are mainly produced, and have been shown to be chemically reactive. Therefore, we studied the interaction of the ketoprofen metabolites with the COX enzymes. After incubation with bovine pulmonary artery endothelial cells (BPAEC), COX-1 was inhibited stereoselectively by S-ketoprofen acylglucuronide, and more significantly by CoA-thioester. After washing-out the medium, COX-1 activity was essentially recovered, indicating a reversible inhibition. In LPS-stimulated J774.2 cells, COX activity (mainly inducible COX-2) was inhibited reversibly and stereospecifically by S-ketoprofen glucuronide, whereas it disappeared totally and was not recovered after incubation with CoA-thioester. Correspondingly, inhibition of purified COX-2 with this compound was observed to be rapid and irreversible. Using an anti-ketoprofen antibody, COX immunoprecipitated from cells exhibited adduct formation for COX-2 but not for COX-1. This was observed after incubation with CoA-thioester, and, surprisingly, also with glucuronide. Molecular docking gave support to explain this discrepancy: the glucuronide was found to establish a strong interaction with Y115 located in the membrane binding domain, whereas the thioester was preferentially bound to the active site of the enzyme. Overall, our results suggest a contribution of CoA-thioester metabolites of carboxylic NSAIDs to their pharmacological action by irreversibly and selectively inhibiting COX-2.

  2. Conserved residues and their role in the structure, function, and stability of acyl-coenzyme A binding protein

    DEFF Research Database (Denmark)

    Kragelund, B B; Poulsen, K; Andersen, K V;

    1999-01-01

    measured by the extent of binding of the ligand dodecanoyl-CoA using isothermal titration calorimetry, and effects on protein stability were measured with chemical denaturation followed by intrinsic tryptophan and tyrosine fluorescence. The sequence sites that have been conserved for direct functional...

  3. Determination of an ensemble of structures representing the denatured state of the bovine acyl-coenzyme a binding protein

    DEFF Research Database (Denmark)

    Lindorff-Larsen, Kresten; Kristjansdottir, Sigridur; Teilum, Kaare;

    2004-01-01

    is highly heterogeneous. The high sensitivity of the computational method that we present, however, enabled us to identify long-range interactions between two regions, located near the N- and C-termini, that include both native and non-native elements. The preferential formation of these contacts suggests...

  4. Transient structure formation in unfolded acyl-coenzyme A-binding protein observed by site-directed spin labelling

    DEFF Research Database (Denmark)

    Teilum, Kaare; Kragelund, Birthe B; Poulsen, Flemming M

    2002-01-01

    are not affected in the native folded structure. It is suggested that the experiment is recording the formation of many discrete and transient structures in the polypeptide chain in the preface of protein folding. Analysis of secondary chemical shifts shows a high propensity for alpha-helix formation in the C......-terminal part of the polypeptide chain, which forms an alpha-helix in the native structure and a high propensity for turn formation in two regions of the polypeptide that form turns in the native structure. The results contribute to the idea that native-like structural elements form transiently in the unfolded...

  5. Lysophosphatidic acid rescues bone mesenchymal stem cells from hydrogen peroxide-induced apoptosis.

    Science.gov (United States)

    Wang, Xian-Yun; Fan, Xue-Song; Cai, Lin; Liu, Si; Cong, Xiang-Feng; Chen, Xi

    2015-03-01

    The increase of reactive oxygen species in infracted heart significantly reduces the survival of donor mesenchymal stem cells, thereby attenuating the therapeutic efficacy for myocardial infarction. In our previous study, we demonstrated that lysophosphatidic acid (LPA) protects bone marrow-derived mesenchymal stem cells (BMSCs) against hypoxia and serum deprivation-induced apoptosis. However, whether LPA protects BMSCs from H2O2-induced apoptosis was not examined. In this study, we report that H2O2 induces rat BMSC apoptosis whereas LPA pre-treatment effectively protects BMSCs from H2O2-induced apoptosis. LPA protection of BMSC from the induced apoptosis is mediated mostly through LPA3 receptor. Furthermore, we found that membrane G protein Gi2 and Gi3 are involved in LPA-elicited anti-apoptotic effects through activation of ERK1/2- and PI3 K-pathways. Additionally, H2O2 increases levels of type II of light chain 3B (LC3B II), an autophagy marker, and H2O2-induced autophagy thus protected BMSCs from apoptosis. LPA further increases the expression of LC3B II in the presence of H2O2. In contrast, autophagy flux inhibitor bafilomycin A1 has no effect on LPA's protection of BMSC from H2O2-induced apoptosis. Taken together, our data suggest that LPA rescues H2O2-induced apoptosis mainly by interacting with Gi-coupled LPA3, resulting activation of the ERK1/2- and PI3 K/AKT-pathways and inhibition caspase-3 cleavage, and LPA protection of BMSCs against the apoptosis is independent of it induced autophagy.

  6. Yangxueqingnao particles inhibit rat vascular smooth muscle cell proliferation induced by lysophosphatidic acid

    Institute of Scientific and Technical Information of China (English)

    CAI Wei; XU Yi; CHEN Jun-zhu; HUANG Shu-ru; LU Zhen-ya; WANG Zhan-kun

    2005-01-01

    Objective: To observe the effect of Yangxueqingnao particles on rat vascular smooth muscle cell (VSMC) proliferation induced by lysophosphatidic acid (LPA). Methods: The amount of3H-TdR (3H-thymidine) admixed in cultured rat VSMC was measured and mitogen-activated protein kinase (MAPK) activity and lipid peroxidation end product malondialdehyde (MDA)content of the VSMC were assayed. Results: 1×10-9, 1×10-8, 1×10-7 mol/L LPA in a concentration dependent manner, induced the amount of 3H-TdR admixed, MAP kinase activity, and MDA content of the cultured rat VSMC to increase. However, 5%, 10%,and 15% Yangxueqingnao serum preincubation resulted in a decrease of 23.0%, 42.0%, and 52.0% (P<0.01) respectively in the amount of 3H-TdR admixed, a decline in VSMC MAP kinase activity of 13.9% (P<0.05), 29.6% (P<0.01), and 48.9% (P<0.01)respectively, and also, a decrease in MDA content of VSMC of 19.4%, 24.7%, and 43.2% (P<0.01) respectively, in the 1×10-7mol/L LPA-treated VSMC. Conclusions: LPA activates the proliferation and lipid peroxidation of VSMC in a concentration dependent manner. The LPA-induced VSMC proliferation is related to the activity of MAP kinases, enzymes involved in an intracellular signalling pathway. The results of the present study showed that Yangxueqingnao particles can effectively inhibit LPA-induced VSMC proliferation, MAP kinase activation, and reduce lipid peroxidative lesion.

  7. Gintonin enhances performance of mice in rotarod test: Involvement of lysophosphatidic acid receptors and catecholamine release.

    Science.gov (United States)

    Lee, Byung-Hwan; Kim, Jisu; Lee, Ra Mi; Choi, Sun-Hye; Kim, Hyeon-Joong; Hwang, Sung-Hee; Lee, Myung Koo; Bae, Chun-Sik; Kim, Hyoung-Chun; Rhim, Hyewon; Lim, Kiwon; Nah, Seung-Yeol

    2016-01-26

    Ginseng has a long history of use as a tonic for restoration of vigor. One example of ginseng-derived tonic effect is that it can improve physical stamina under conditions of stress. However, the active ingredient and the underlying molecular mechanism responsible for the ergogenic effect are unknown. Recent studies show that ginseng contains a novel ingredient, gintonin, which consists of a unique class of herbal-medicine lysophosphatidic acids (LPAs). Gintonin activates G protein-coupled LPA receptors to produce a transient [Ca(2+)]i signal, which is coupled to diverse intra- and inter-cellular signal transduction pathways that stimulate hormone or neurotransmitter release. However, relatively little is known about how gintonin-mediated cellular modulation is linked to physical endurance. In the present study, systemic administration of gintonin, but not ginsenosides, in fasted mice increased blood glucose concentrations in a dose-dependent manner. Gintonin treatment elevated blood glucose to a maximum level after 30min. This elevation in blood glucose level could be abrogated by the LPA1/3 receptor antagonist, Ki16425, or the β-adrenergic receptor antagonist, propranolol. Furthermore, gintonin-dependent enhanced performance of fasted mice in rotarod test was likewise abrogated by Ki16425. Gintonin also elevated plasma epinephrine and norepinephrine concentrations. The present study shows that gintonin mediates catecholamine release through activation of the LPA receptor and that activation of the β-adrenergic receptor is coupled to liver glycogenolysis, thereby increasing the supply of glucose and enhancing performance in the rotarod test. Thus, gintonin acts via the LPA-catecholamine-glycogenolysis axis, representing a candidate mechanism that can explain how ginseng treatment enhances physical stamina.

  8. Lysophosphatidic acid acyltransferase β (LPAATβ promotes the tumor growth of human osteosarcoma.

    Directory of Open Access Journals (Sweden)

    Farbod Rastegar

    Full Text Available BACKGROUND: Osteosarcoma is the most common primary malignancy of bone with poorly characterized molecular pathways important in its pathogenesis. Increasing evidence indicates that elevated lipid biosynthesis is a characteristic feature of cancer. We sought to investigate the role of lysophosphatidic acid acyltransferase β (LPAATβ, aka, AGPAT2 in regulating the proliferation and growth of human osteosarcoma cells. LPAATβ can generate phosphatidic acid, which plays a key role in lipid biosynthesis as well as in cell proliferation and survival. Although elevated expression of LPAATβ has been reported in several types of human tumors, the role of LPAATβ in osteosarcoma progression has yet to be elucidated. METHODOLOGY/PRINCIPAL FINDINGS: Endogenous expression of LPAATβ in osteosarcoma cell lines is analyzed by using semi-quantitative PCR and immunohistochemical staining. Adenovirus-mediated overexpression of LPAATβ and silencing LPAATβ expression is employed to determine the effect of LPAATβ on osteosarcoma cell proliferation and migration in vitro and osteosarcoma tumor growth in vivo. We have found that expression of LPAATβ is readily detected in 8 of the 10 analyzed human osteosarcoma lines. Exogenous expression of LPAATβ promotes osteosarcoma cell proliferation and migration, while silencing LPAATβ expression inhibits these cellular characteristics. We further demonstrate that exogenous expression of LPAATβ effectively promotes tumor growth, while knockdown of LPAATβ expression inhibits tumor growth in an orthotopic xenograft model of human osteosarcoma. CONCLUSIONS/SIGNIFICANCE: Our results strongly suggest that LPAATβ expression may be associated with the aggressive phenotypes of human osteosarcoma and that LPAATβ may play an important role in regulating osteosarcoma cell proliferation and tumor growth. Thus, targeting LPAATβ may be exploited as a novel therapeutic strategy for the clinical management of osteosarcoma. This

  9. Lysophosphatidic Acid Up-Regulates Hexokinase II and Glycolysis to Promote Proliferation of Ovarian Cancer Cells

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    Abir Mukherjee

    2015-09-01

    Full Text Available Lysophosphatidic acid (LPA, a blood-borne lipid mediator, is present in elevated concentrations in ascites of ovarian cancer patients and other malignant effusions. LPA is a potent mitogen in cancer cells. The mechanism linking LPA signal to cancer cell proliferation is not well understood. Little is known about whether LPA affects glucose metabolism to accommodate rapid proliferation of cancer cells. Here we describe that in ovarian cancer cells, LPA enhances glycolytic rate and lactate efflux. A real time PCR-based miniarray showed that hexokinase II (HK2 was the most dramatically induced glycolytic gene to promote glycolysis in LPA-treated cells. Analysis of the human HK2 gene promoter identified the sterol regulatory element-binding protein as the primary mediator of LPA-induced HK2 transcription. The effects of LPA on HK2 and glycolysis rely on LPA2, an LPA receptor subtype overexpressed in ovarian cancer and many other malignancies. We further examined the general role of growth factor-induced glycolysis in cell proliferation. Like LPA, epidermal growth factor (EGF elicited robust glycolytic and proliferative responses in ovarian cancer cells. Insulin-like growth factor 1 (IGF-1 and insulin, however, potently stimulated cell proliferation but only modestly induced glycolysis. Consistent with their differential effects on glycolysis, LPA and EGF-dependent cell proliferation was highly sensitive to glycolytic inhibition while the growth-promoting effect of IGF-1 or insulin was more resistant. These results indicate that LPA- and EGF-induced cell proliferation selectively involves up-regulation of HK2 and glycolytic metabolism. The work is the first to implicate LPA signaling in promotion of glucose metabolism in cancer cells.

  10. Lysophosphatidic acid induces chemotaxis in MC3T3-E1 osteoblastic cells

    Energy Technology Data Exchange (ETDEWEB)

    Masiello, Lisa M.; Fotos, Joseph S.; Galileo, Deni S.; Karin, Norm J.

    2006-07-01

    Lysophosphatidic acid (LPA) is a bioactive lipid that has pleiotropic effects on a variety of cell types and enhances the migration of endothelial and cancer cells, but it is not known if this lipid can alter osteoblast motility. We performed transwell migration assays using MC3T3-E1 osteoblastic cells and found LPA to be a potent chemotactic agent. Quantitative time-lapse video analysis of osteoblast migration after wounds were introduced into cell monolayers indicated that LPA stimulated both migration velocity and the average migration distance per cell. LPA also elicited substantial changes in cell shape and actin cytoskeletal structure; lipid-treated cells contained fewer stress fibers and displayed long membrane processes that were enriched in F-actin. Quantitative RT-PCR analysis showed that MC3T3-E1 cells express all four known LPA-specific G protein-coupled receptors (LPA1-LPA4) with a relative mRNA abundance of LPA1 > LPA4 > LPA2 >> LPA3. LPA-induced changes in osteoblast motility and morphology were antagonized by both pertussis toxin and Ki16425, a subtype-specific blocker of LPA1 and LPA3 receptor function. Cell migration in many cell types is linked to changes in intracellular Ca2+. Ki16425 also inhibited LPA-induced Ca2+ signaling in a dose-dependent manner, suggesting a link between LPA-induced Ca2+ transients and osteoblast chemotaxis. Our data show that LPA stimulates MC3T3-E1 osteoblast motility via a mechanism that is linked primarily to the G protein-coupled receptor LPA1.

  11. TAZ Mediates Lysophosphatidic Acid-Induced Migration and Proliferation of Epithelial Ovarian Cancer Cells

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    Geun Ok Jeong

    2013-07-01

    Full Text Available Background: Transcriptional co-activator with PDZ-binding motif (TAZ, a downstream effector of the Hippo pathway, has been reported to regulate organ size, tissue homeostasis, and tumorigenesis by acting as a transcriptional co-activator. Lysophosphatidic acid (LPA is a bioactive lipid implicated in tumorigenesis and metastasis of ovarian cancer through activation of G protein-coupled receptors. However, the involvement of TAZ in LPA-induced tumorigenesis of ovarian cancer has not been elucidated. Methods: In order to demonstrate the role of TAZ in LPA-stimulated tumorigenesis, the effects of LPA on TAZ expression and cell migration were determined by Western blotting and chemotaxis analyses in R182 human epithelial ovarian cancer cells. Results and Conclusion: Treatment of R182 cells with the LPA receptor inhibitor Ki16425 blocked LPA-induced cell migration. In addition, transfection of R182 cells with small interfering RNA specific for LPA receptor 1 resulted in abrogation of LPA-stimulated cell migration. LPA induced phosphorylation of ERK and p38 MAP kinase in R182 cells and pretreatment of cells with the MEK-ERK pathway inhibitor U0126, but not the p38 MAPK inhibitor SB202190, resulted in abrogation of LPA-induced cell migration. Pretreatment of R182 cells with U0126 attenuated LPA-induced mRNA levels of TAZ and its transcriptional target genes, such as CTGF and CYR61, without affecting phosphorylation level of YAP. These results suggest that MEK-ERK pathway plays a key role in LPA-induced cell migration and mRNA expression of TAZ in R182 cells, without affecting stability of TAZ protein. In addition, small interfering RNA-mediated silencing of TAZ expression attenuated LPA-stimulated migration of R182 cells. These results suggest that TAZ plays a key role in LPA-stimulated migration of epithelial ovarian cancer cells.

  12. Ginseng pharmacology: a new paradigm based on gintonin-lysophosphatidic acid receptor interactions

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    Seung-Yeol eNah

    2015-10-01

    Full Text Available Ginseng, the root of Panax ginseng, is used as a traditional medicine. Despite the long history of the use of ginseng, there is no specific scientific or clinical rationale for ginseng pharmacology besides its application as a general tonic. The ambiguous description of ginseng pharmacology might be due to the absence of a predominant active ingredient that represents ginseng pharmacology. Recent studies show that ginseng abundantly contains lysophosphatidic acids (LPAs, which are phospholipid-derived growth factor with diverse biological functions including those claimed to be exhibited by ginseng. LPAs in ginseng form a complex with ginseng proteins, which can bind and deliver LPA to its cognate receptors with a high affinity. As a first messenger, gintonin produces second messenger Ca2+ via G protein-coupled LPA receptors. Ca2+ is an intracellular mediator of gintonin and initiates a cascade of amplifications for further intercellular communications by activation of Ca2+-dependent kinases, receptors, gliotransmitter and neurotransmitter release. Ginsenosides, which have been regarded as primary ingredients of ginseng, cannot elicit intracellular [Ca2+]i transients, since they lack specific cell surface receptor. However, ginsenosides exhibit non-specific ion channel and receptor regulations. This is the key characteristic that distinguishes gintonin from ginsenosides. Although the current discourse on ginseng pharmacology is focused on ginsenosides, gintonin can definitely provide a mode of action for ginseng pharmacology that ginsenosides cannot. This review article introduces a novel concept of ginseng ligand-LPA receptor interaction and proposes to establish a paradigm that shifts the focus from ginsenosides to gintonin as a major ingredient representing ginseng pharmacology.

  13. Calcium-dependent generation of N-acylethanolamines and lysophosphatidic acids by glycerophosphodiesterase GDE7.

    Science.gov (United States)

    Rahman, Iffat Ara Sonia; Tsuboi, Kazuhito; Hussain, Zahir; Yamashita, Ryouhei; Okamoto, Yoko; Uyama, Toru; Yamazaki, Naoshi; Tanaka, Tamotsu; Tokumura, Akira; Ueda, Natsuo

    2016-12-01

    N-Acylethanolamines form a class of lipid mediators and include an endocannabinoid arachidonoylethanolamide (anandamide), analgesic and anti-inflammatory palmitoylethanolamide, and appetite-suppressing oleoylethanolamide. In animal tissues, N-acylethanolamines are synthesized from N-acylated ethanolamine phospholipids directly by N-acylphosphatidylethanolamine-hydrolyzing phospholipase D or through multi-step pathways via N-acylethanolamine lysophospholipids. We previously reported that glycerophosphodiesterase (GDE) 4, a member of the GDE family, has lysophospholipase D (lysoPLD) activity hydrolyzing N-acylethanolamine lysophospholipids to N-acylethanolamines. Recently, GDE7 was shown to have lysoPLD activity toward lysophosphatidylcholine to produce lysophosphatidic acid (LPA). Here, we examined the reactivity of GDE7 with N-acylethanolamine lysophospholipids as well as the requirement of divalent cations for its catalytic activity. When overexpressed in HEK293 cells, recombinant GDE7 proteins of human and mouse showed lysoPLD activity toward N-palmitoyl, N-oleoyl, and N-arachidonoyl-lysophosphatidylethanolamines and N-palmitoyl-lysoplasmenylethanolamine to generate their corresponding N-acylethanolamines and LPAs. However, GDE7 hardly hydrolyzed glycerophospho-N-palmitoylethanolamine. Overexpression of GDE7 in HEK293 cells increased endogenous levels of N-acylethanolamines and LPAs. Interestingly, GDE7 was stimulated by micromolar concentrations of Ca(2+) but not by millimolar concentrations of Mg(2+), while GDE4 was stimulated by Mg(2+) but was insensitive to Ca(2+). GDE7 was widely distributed in various tissues of humans and mice with the highest levels in their kidney tissues. These results suggested that GDE7 is a novel Ca(2+)-dependent lysoPLD, which is involved in the generation of both N-acylethanolamines and LPAs.

  14. Lysophosphatidic acid (LPA 18:1 transcriptional regulation of primary human gingival fibroblasts

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    D. Roselyn Cerutis

    2014-12-01

    Full Text Available The pleiotropic, bioactive lipid lysophosphatidic acid [(LPA, 1-acyl-sn-glycerol-3-phosphate] exerts critical regulatory actions in physiology and pathophysiology in many systems. It is present in normal bodily fluids, and is elevated in pathology (1. In vivo, “LPA” exists as distinct molecular species, each having a single fatty acid of varying chain length and degree of unsaturation covalently attached to the glycerol backbone via an acyl, alkyl, or alkenyl link. These species differ in affinities for the individual LPA receptors [(LPARs, LPA1-6] and coupling to G proteins (2. However, LPA 18:1 has been and continues to be the most commonly utilized species in reported studies. The actions of “LPA” remain poorly defined in oral biology and pathophysiology. Our laboratory has addressed this knowledge gap by studying in vitro the actions of the major human salivary LPA species [18:1, 18:0, and 16:0 (3] in human oral cells (4–7. This includes gingival fibroblasts (GF, which our flow cytometry data from multiple donors found that they express LPA1-5 (6. We have also reported that these species are ten-fold elevated to pharmacologic levels in the saliva and gingival crevicular fluid obtained from patients with moderate–severe periodontitis (8. As the potential of LPA to regulate transcriptional activity had not been examined in the oral system, this study used whole human genome microarray analysis to test the hypothesis that LPA 18:1-treated human GF would show significant changes in gene transcripts relevant to their biology, wound-healing, and inflammatory responses. LPA 18:1 was found to significantly regulate a large, complex set of genes critical to GF biology in these categories and to periodontal disease. The raw data has been deposited at NCBI's GEO database as record GSE57496.

  15. Expression of lysophosphatidic acid and its receptor in human pancreatic cancer and its clinical evaluation of diagnosis and therapy

    Institute of Scientific and Technical Information of China (English)

    WANG Shao-kai; TAO Chen-jie; WANG Wei-dong; L(U)Guang-mei; GONG Yong-ling

    2011-01-01

    Lysophosphatidic acid(LPA) is a naturally occurring phospholipid with diverse effects in various cells, ranging from immediate morphological alteration to long lasting cellular function changes, such as induction of stimulation of cell proliferation, survival, drug resistance and motility. Like many other biomediators, LPA interacts with cells through specific cell surface receptors(G protein-coupled receptors). LPA1/Edg-2,LPA2/Edg-4 and LPA3/Edg-7, named as Edg/LP subfamily, are the three most common lysophosphatidic acid receptors. LPA plays a critical role as a general growth, survival and pro-angiogenic factor in the regulation of pathophysiological processes in vivo and in vitro. Recent literatures suggest that abnormalities in LPA metabolism and function in pancreatic cancer patients may contribute to the initiation and progression of the disease. Thus, LPA might be a potential target for clinical pancreatic cancer diagnosis and therapy. Herein we review the expression of LPA and its receptors in the carcinogenesis of human malignancies, with focus on human pancreatic cancer, and also clinical diagnosis and treatment has been evaluated.

  16. Clinical significance of plasma lysophosphatidic acid levels in the differential diagnosis of ovarian cancer

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    Yun-Jie Zhang

    2015-01-01

    Full Text Available Objective: To investigate the value of lysophosphatidic acid (LPA in the diagnosis of ovarian cancer. Materials and Methods: We first performed a hospital-based, case-control study involving 123 ovarian cancer patients and 101 benign ovarian tumor patients, and then conducted a meta-analysis with 19 case-control studies to assess the correlation between ovarian cancer and plasma LPA levels. Results: The case-control study results demonstrated that ovarian cancer patients have increased LPA and cancer antigen (CA-125 levels compared to patients with benign ovarian tumor (LPA: Ovarian cancer vs benign ovarian tumor: 5.28 ± 1.52 vs 1.82 ± 0.77 μmol/L; CA-125: Ovarian cancer vs benign ovarian tumor: 87.17 ± 45.81 vs. 14.03 ± 10.14 U/mL, which showed statistically significant differences (both P < 0.05. LPA with advanced sensitivity, specificity, positive predictive value, negative predictive value, and accuracy rate of diagnosis excelled CA-125 in the diagnosis of ovarian cancer (both P < 0.05. The areas under the receiver operating characteristic (ROC curve in the diagnosis of ovarian cancer (LPA: 0.983; CA-125: 0.910 were statistically significant compared with the reference (both P < 0.001 and the difference of the areas of ROC curve between LPA and CA-125 in the diagnosis of ovarian cancer showed statistically significant difference (P < 0.05. The meta-analysis results suggested that plasma LPA levels were higher in ovarian cancer tissues than in benign tissues (standardized mean difference (SMD =2.36, 95% confidence interval (CI: 1.61-3.11, P < 0.001 and normal tissues (SMD = 2.32, 95% CI: 1.77-2.87, P < 0.001. Conclusion: LPA shows greater value in the diagnosis of ovarian cancer compared to CA-125 and may be employed as a biological index to diagnose ovarian cancer.

  17. Lysophosphatidic acid receptor-5 negatively regulates cellular responses in mouse fibroblast 3T3 cells

    Energy Technology Data Exchange (ETDEWEB)

    Dong, Yan; Hirane, Miku; Araki, Mutsumi [Division of Cancer Biology and Bioinformatics, Department of Life Science, Faculty of Science and Engineering, Kinki University, 3-4-1, Kowakae, Higashiosaka, Osaka 577-8502 (Japan); Fukushima, Nobuyuki [Division of Molecular Neurobiology, Department of Life Science, Faculty of Science and Engineering, Kinki University, 3-4-1, Kowakae, Higashiosaka, Osaka 577-8502 (Japan); Tsujiuchi, Toshifumi, E-mail: ttujiuch@life.kindai.ac.jp [Division of Cancer Biology and Bioinformatics, Department of Life Science, Faculty of Science and Engineering, Kinki University, 3-4-1, Kowakae, Higashiosaka, Osaka 577-8502 (Japan)

    2014-04-04

    Highlights: • LPA{sub 5} inhibits the cell growth and motile activities of 3T3 cells. • LPA{sub 5} suppresses the cell motile activities stimulated by hydrogen peroxide in 3T3 cells. • Enhancement of LPA{sub 5} on the cell motile activities inhibited by LPA{sub 1} in 3T3 cells. • The expression and activation of Mmp-9 were inhibited by LPA{sub 5} in 3T3 cells. • LPA signaling via LPA{sub 5} acts as a negative regulator of cellular responses in 3T3 cells. - Abstract: Lysophosphatidic acid (LPA) signaling via G protein-coupled LPA receptors (LPA{sub 1}–LPA{sub 6}) mediates a variety of biological functions, including cell migration. Recently, we have reported that LPA{sub 1} inhibited the cell motile activities of mouse fibroblast 3T3 cells. In the present study, to evaluate a role of LPA{sub 5} in cellular responses, Lpar5 knockdown (3T3-L5) cells were generated from 3T3 cells. In cell proliferation assays, LPA markedly stimulated the cell proliferation activities of 3T3-L5 cells, compared with control cells. In cell motility assays with Cell Culture Inserts, the cell motile activities of 3T3-L5 cells were significantly higher than those of control cells. The activity levels of matrix metalloproteinases (MMPs) were measured by gelatin zymography. 3T3-L5 cells stimulated the activation of Mmp-2, correlating with the expression levels of Mmp-2 gene. Moreover, to assess the co-effects of LPA{sub 1} and LPA{sub 5} on cell motile activities, Lpar5 knockdown (3T3a1-L5) cells were also established from Lpar1 over-expressing (3T3a1) cells. 3T3a1-L5 cells increased the cell motile activities of 3T3a1 cells, while the cell motile activities of 3T3a1 cells were significantly lower than those of control cells. These results suggest that LPA{sub 5} may act as a negative regulator of cellular responses in mouse fibroblast 3T3 cells, similar to the case for LPA{sub 1}.

  18. LPA5 is abundantly expressed by human mast cells and important for lysophosphatidic acid induced MIP-1β release.

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    Anders Lundequist

    Full Text Available BACKGROUND: Lysophosphatidic acid (LPA is a bioactive lipid inducing proliferation, differentiation as well as cytokine release by mast cells through G-protein coupled receptors. Recently GPR92/LPA5 was identified as an LPA receptor highly expressed by cells of the immune system, which prompted us to investigate its presence and influence on mast cells. PRINCIPAL FINDINGS: Transcript analysis using quantitative real-time PCR revealed that LPA5 is the most prevalent LPA-receptor in human mast cells. Reduction of LPA5 levels using shRNA reduced calcium flux and abolished MIP-1β release in response to LPA. CONCLUSIONS: LPA5 is a bona fide LPA receptor on human mast cells responsible for the majority of LPA induced MIP-1β release.

  19. Golgi membrane fission requires the CtBP1-S/BARS-induced activation of lysophosphatidic acid acyltransferase δ.

    Science.gov (United States)

    Pagliuso, Alessandro; Valente, Carmen; Giordano, Lucia Laura; Filograna, Angela; Li, Guiling; Circolo, Diego; Turacchio, Gabriele; Marzullo, Vincenzo Manuel; Mandrich, Luigi; Zhukovsky, Mikhail A; Formiggini, Fabio; Polishchuk, Roman S; Corda, Daniela; Luini, Alberto

    2016-07-12

    Membrane fission is an essential cellular process by which continuous membranes split into separate parts. We have previously identified CtBP1-S/BARS (BARS) as a key component of a protein complex that is required for fission of several endomembranes, including basolateral post-Golgi transport carriers. Assembly of this complex occurs at the Golgi apparatus, where BARS binds to the phosphoinositide kinase PI4KIIIβ through a 14-3-3γ dimer, as well as to ARF and the PKD and PAK kinases. We now report that, when incorporated into this complex, BARS binds to and activates a trans-Golgi lysophosphatidic acid (LPA) acyltransferase type δ (LPAATδ) that converts LPA into phosphatidic acid (PA); and that this reaction is essential for fission of the carriers. LPA and PA have unique biophysical properties, and their interconversion might facilitate the fission process either directly or indirectly (via recruitment of proteins that bind to PA, including BARS itself).

  20. The Src homology 3 binding domain is required for lysophosphatidic acid 3 receptor-mediated cellular viability in melanoma cells.

    Science.gov (United States)

    Jia, Wei; Tran, Sterling K; Ruddick, Caitlin A; Murph, Mandi M

    2015-01-28

    The LPA3 receptor is a G protein-coupled receptor that binds extracellular lysophosphatidic acid and mediates intracellular signaling cascades. Although we previously reported that receptor inhibition using siRNA or chemical inhibition obliterates the viability of melanoma cells, the mechanism was unclear. Herein we hypothesized that amino acids comprising the Src homology 3 (SH3) ligand binding motif, R/K-X-X-V/P-X-X-P or (216)-KTNVLSP-(222), within the third intracellular loop of LPA3 were critical in mediating this outcome. Therefore, we performed site-directed mutagenesis of the lysine, valine and proline, replacing these amino acids with alanines, and evaluated the changes in viability, proliferation, ERK1/2 signaling and calcium in response to lysophosphatidic acid. Our results show that enforced LPA3 expression in SK-MEL-2 cells enhanced their resiliency by allowing these cells to oppose any loss of viability during growth in serum-free medium for up to 96 h, in contrast to parental SK-MEL-2 cells, which show a significant decline in viability. Similarly, site-directed alanine substitutions of valine and proline, V219A/P222A or 2aa-SK-MEL-2 cells, did not significantly alter viability, but adding a further alanine to replace the lysine, K216A/V219A/P222A or 3aa-SK-MEL-2 cells, obliterated this function. In addition, an inhibitor of the LPA3 receptor had no impact on the parental SK-MEL-2, 2aa-SK-MEL-2 or 3aa-SK-MEL-2 cells, but significantly reduced viability among wt-LPA3-SK-MEL-2 cells. Taken together, the data suggest that the SH3 ligand binding domain of LPA3 is required to mediate viability in melanoma cells.

  1. Structurally divergent lysophosphatidic acid acyltransferases with high selectivity for saturated medium chain fatty acids from Cuphea seeds.

    Science.gov (United States)

    Kim, Hae Jin; Silva, Jillian E; Iskandarov, Umidjon; Andersson, Mariette; Cahoon, Rebecca E; Mockaitis, Keithanne; Cahoon, Edgar B

    2015-12-01

    Lysophosphatidic acid acyltransferase (LPAT) catalyzes acylation of the sn-2 position on lysophosphatidic acid by an acyl CoA substrate to produce the phosphatidic acid precursor of polar glycerolipids and triacylglycerols (TAGs). In the case of TAGs, this reaction is typically catalyzed by an LPAT2 from microsomal LPAT class A that has high specificity for C18 fatty acids containing Δ9 unsaturation. Because of this specificity, the occurrence of saturated fatty acids in the TAG sn-2 position is infrequent in seed oils. To identify LPATs with variant substrate specificities, deep transcriptomic mining was performed on seeds of two Cuphea species producing TAGs that are highly enriched in saturated C8 and C10 fatty acids. From these analyses, cDNAs for seven previously unreported LPATs were identified, including cDNAs from Cuphea viscosissima (CvLPAT2) and Cuphea avigera var. pulcherrima (CpuLPAT2a) encoding microsomal, seed-specific class A LPAT2s and a cDNA from C. avigera var. pulcherrima (CpuLPATB) encoding a microsomal, seed-specific LPAT from the bacterial-type class B. The activities of these enzymes were characterized in Camelina sativa by seed-specific co-expression with cDNAs for various Cuphea FatB acyl-acyl carrier protein thioesterases (FatB) that produce a variety of saturated medium-chain fatty acids. CvLPAT2 and CpuLPAT2a expression resulted in accumulation of 10:0 fatty acids in the Camelina sativa TAG sn-2 position, indicating a 10:0 CoA specificity that has not been previously described for plant LPATs. CpuLPATB expression generated TAGs with 14:0 at the sn-2 position, but not 10:0. Identification of these LPATs provides tools for understanding the structural basis of LPAT substrate specificity and for generating altered oil functionalities.

  2. Autotaxin activity increases locally following lung injury, but is not required for pulmonary lysophosphatidic acid production or fibrosis.

    Science.gov (United States)

    Black, Katharine E; Berdyshev, Evgeny; Bain, Gretchen; Castelino, Flavia V; Shea, Barry S; Probst, Clemens K; Fontaine, Benjamin A; Bronova, Irina; Goulet, Lance; Lagares, David; Ahluwalia, Neil; Knipe, Rachel S; Natarajan, Viswanathan; Tager, Andrew M

    2016-06-01

    Lysophosphatidic acid (LPA) is an important mediator of pulmonary fibrosis. In blood and multiple tumor types, autotaxin produces LPA from lysophosphatidylcholine (LPC) via lysophospholipase D activity, but alternative enzymatic pathways also exist for LPA production. We examined the role of autotaxin (ATX) in pulmonary LPA production during fibrogenesis in a bleomycin mouse model. We found that bleomycin injury increases the bronchoalveolar lavage (BAL) fluid levels of ATX protein 17-fold. However, the LPA and LPC species that increase in BAL of bleomycin-injured mice were discordant, inconsistent with a substrate-product relationship between LPC and LPA in pulmonary fibrosis. LPA species with longer chain polyunsaturated acyl groups predominated in BAL fluid after bleomycin injury, with 22:5 and 22:6 species accounting for 55 and 16% of the total, whereas the predominant BAL LPC species contained shorter chain, saturated acyl groups, with 16:0 and 18:0 species accounting for 56 and 14% of the total. Further, administration of the potent ATX inhibitor PAT-048 to bleomycin-challenged mice markedly decreased ATX activity systemically and in the lung, without effect on pulmonary LPA or fibrosis. Therefore, alternative ATX-independent pathways are likely responsible for local generation of LPA in the injured lung. These pathways will require identification to therapeutically target LPA production in pulmonary fibrosis.-Black, K. E., Berdyshev, E., Bain, G., Castelino, F. V., Shea, B. S., Probst, C. K., Fontaine, B. A., Bronova, I., Goulet, L., Lagares, D., Ahluwalia, N., Knipe, R. S., Natarajan, V., Tager, A. M. Autotaxin activity increases locally following lung injury, but is not required for pulmonary lysophosphatidic acid production or fibrosis.

  3. Lysophosphatidic acid is a major serum noncytokine survival factor for murine macrophages which acts via the phosphatidylinositol 3-kinase signaling pathway.

    OpenAIRE

    Koh, J. S.; Lieberthal, W; Heydrick, S; Levine, J. S.

    1998-01-01

    Lysophosphatidic acid (LPA) is the smallest and structurally simplest of all the glycerophospholipids. It occurs normally in serum and binds with high affinity to albumin, while retaining its biological activity. The effects of LPA are pleiotropic and range from mitogenesis to stress fiber formation. We show a novel role for LPA: as a macrophage survival factor with potency equivalent to serum. Administration of LPA protects macrophages from apoptosis induced by serum deprivation, and protect...

  4. Effects of lysophosphatidic acid on the in vitro proliferation and differentiation of a novel porcine preadipocyte cell line.

    Science.gov (United States)

    Nobusue, Hiroyuki; Kondo, Daisuke; Yamamoto, Makiko; Kano, Koichiro

    2010-12-01

    We examined the effects of lysophosphatidic acid (LPA) on in vitro proliferation and differentiation of a porcine preadipocyte cell line, DFAT-P, and a mouse preadipocyte cell line, 3T3-L1. During the proliferation and differentiation phases, DFAT-P and 3T3-L1 cells expressed only the endothelial differentiation gene (EDG)-2 receptor and not EDG-4 and EDG-7 receptors. LPA promoted the proliferation of DFAT-P cells more extensively than that of 3T3-L1 cells. After adipogenic induction, LPA inhibited glycerol-3-phosphate dehydrogenase activity and lipid droplet accumulation, and suppressed peroxisome proliferator-activated receptor γ (PPARγ) protein expression, this inhibitory effect in DFAT-P cells was twice as high as that in 3T3-L1 cells. Furthermore, treatments with low LPA concentrations significantly inhibited adipocyte differentiation in DFAT-P cells but not in 3T3-L1 cells. We conclude that LPA promotes the proliferation of porcine preadipocytes through the EDG-2 receptor but inhibits their differentiation, and these effects depend on the down-regulation of PPARγ expression via the EDG-2 receptor. Furthermore, DFAT-P cells are more sensitive to LPA than 3T3-L1 cells. These findings in a porcine model will contribute to the understanding of LPA action mechanisms on in vitro proliferation and differentiation of preadipocytes in domestic animals and/or humans.

  5. Inhibition of lysophosphatidic acid receptors 1 and 3 attenuates atherosclerosis development in LDL-receptor deficient mice.

    Science.gov (United States)

    Kritikou, Eva; van Puijvelde, Gijs H M; van der Heijden, Thomas; van Santbrink, Peter J; Swart, Maarten; Schaftenaar, Frank H; Kröner, Mara J; Kuiper, Johan; Bot, Ilze

    2016-11-24

    Lysophosphatidic acid (LPA) is a natural lysophospholipid present at high concentrations within lipid-rich atherosclerotic plaques. Upon local accumulation in the damaged vessels, LPA can act as a potent activator for various types of immune cells through its specific membrane receptors LPA1/3. LPA elicits chemotactic, pro-inflammatory and apoptotic effects that lead to atherosclerotic plaque progression. In this study we aimed to inhibit LPA signaling by means of LPA1/3 antagonism using the small molecule Ki16425. We show that LPA1/3 inhibition significantly impaired atherosclerosis progression. Treatment with Ki16425 also resulted in reduced CCL2 production and secretion, which led to less monocyte and neutrophil infiltration. Furthermore, we provide evidence that LPA1/3 blockade enhanced the percentage of non-inflammatory, Ly6C(low) monocytes and CD4(+) CD25(+) FoxP3(+) T-regulatory cells. Finally, we demonstrate that LPA1/3 antagonism mildly reduced plasma LDL cholesterol levels. Therefore, pharmacological inhibition of LPA1/3 receptors may prove a promising approach to diminish atherosclerosis development.

  6. Dual Action of Lysophosphatidate-Functionalised Titanium: Interactions with Human (MG63) Osteoblasts and Methicillin Resistant Staphylococcus aureus.

    Science.gov (United States)

    Skindersoe, Mette Elena; Krogfelt, Karen A; Blom, Ashley; Zhang, Jianxing; Jiang, Guowei; Prestwich, Glenn D; Mansell, Jason Peter

    2015-01-01

    Titanium (Ti) is a widely used material for surgical implants; total joint replacements (TJRs), screws and plates for fixing bones and dental implants are forged from Ti. Whilst Ti integrates well into host tissue approximately 10% of TJRs will fail in the lifetime of the patient through a process known as aseptic loosening. These failures necessitate revision arthroplasties which are more complicated and costly than the initial procedure. Finding ways of enhancing early (osseo)integration of TJRs is therefore highly desirable and continues to represent a research priority in current biomaterial design. One way of realising improvements in implant quality is to coat the Ti surface with small biological agents known to support human osteoblast formation and maturation at Ti surfaces. Lysophosphatidic acid (LPA) and certain LPA analogues offer potential solutions as Ti coatings in reducing aseptic loosening. Herein we present evidence for the successful bio-functionalisation of Ti using LPA. This modified Ti surface heightened the maturation of human osteoblasts, as supported by increased expression of alkaline phosphatase. These functionalised surfaces also deterred the attachment and growth of Staphylococcus aureus, a bacterium often associated with implant failures through sepsis. Collectively we provide evidence for the fabrication of a dual-action Ti surface finish, a highly desirable feature towards the development of next-generation implantable devices.

  7. Dual Action of Lysophosphatidate-Functionalised Titanium: Interactions with Human (MG63 Osteoblasts and Methicillin Resistant Staphylococcus aureus.

    Directory of Open Access Journals (Sweden)

    Mette Elena Skindersoe

    Full Text Available Titanium (Ti is a widely used material for surgical implants; total joint replacements (TJRs, screws and plates for fixing bones and dental implants are forged from Ti. Whilst Ti integrates well into host tissue approximately 10% of TJRs will fail in the lifetime of the patient through a process known as aseptic loosening. These failures necessitate revision arthroplasties which are more complicated and costly than the initial procedure. Finding ways of enhancing early (osseointegration of TJRs is therefore highly desirable and continues to represent a research priority in current biomaterial design. One way of realising improvements in implant quality is to coat the Ti surface with small biological agents known to support human osteoblast formation and maturation at Ti surfaces. Lysophosphatidic acid (LPA and certain LPA analogues offer potential solutions as Ti coatings in reducing aseptic loosening. Herein we present evidence for the successful bio-functionalisation of Ti using LPA. This modified Ti surface heightened the maturation of human osteoblasts, as supported by increased expression of alkaline phosphatase. These functionalised surfaces also deterred the attachment and growth of Staphylococcus aureus, a bacterium often associated with implant failures through sepsis. Collectively we provide evidence for the fabrication of a dual-action Ti surface finish, a highly desirable feature towards the development of next-generation implantable devices.

  8. P2X7 receptors on osteoblasts couple to production of lysophosphatidic acid: a signaling axis promoting osteogenesis.

    Science.gov (United States)

    Panupinthu, Nattapon; Rogers, Joseph T; Zhao, Lin; Solano-Flores, Luis Pastor; Possmayer, Fred; Sims, Stephen M; Dixon, S Jeffrey

    2008-06-02

    Nucleotides are released from cells in response to mechanical stimuli and signal in an autocrine/paracrine manner through cell surface P2 receptors. P2rx7-/- mice exhibit diminished appositional growth of long bones and impaired responses to mechanical loading. We find that calvarial sutures are wider in P2rx7-/- mice. Functional P2X7 receptors are expressed on osteoblasts in situ and in vitro. Activation of P2X7 receptors by exogenous nucleotides stimulates expression of osteoblast markers and enhances mineralization in cultures of rat calvarial cells. Moreover, osteogenesis is suppressed in calvarial cell cultures from P2rx7-/- mice compared with the wild type. P2X7 receptors couple to production of the potent lipid mediators lysophosphatidic acid (LPA) and prostaglandin E2. Either an LPA receptor antagonist or cyclooxygenase (COX) inhibitors abolish the stimulatory effects of P2X7 receptor activation on osteogenesis. We conclude that P2X7 receptors enhance osteoblast function through a cell-autonomous mechanism. Furthermore, a novel signaling axis links P2X7 receptors to production of LPA and COX metabolites, which in turn stimulate osteogenesis.

  9. Embryo spacing and implantation timing are differentially regulated by LPA3-mediated lysophosphatidic acid signaling in mice.

    Science.gov (United States)

    Hama, Kotaro; Aoki, Junken; Inoue, Asuka; Endo, Tomoko; Amano, Tomokazu; Motoki, Rie; Kanai, Motomu; Ye, Xiaoqin; Chun, Jerold; Matsuki, Norio; Suzuki, Hiroshi; Shibasaki, Masakatsu; Arai, Hiroyuki

    2007-12-01

    In polytocous animals, blastocysts are evenly distributed along each uterine horn and implant. The molecular mechanisms underlying these precise events remain elusive. We recently showed that lysophosphatidic acid (LPA) has critical roles in the establishment of early pregnancy by affecting embryo spacing and subsequent implantation through its receptor, LPA3. Targeted deletion of Lpa3 in mice resulted in delayed implantation and embryo crowding, which is associated with a dramatic decrease in the prostaglandins and prostaglandin-endoperoxide synthase 2 expression levels. Exogenous administration of prostaglandins rescued the delayed implantation but did not rescue the defects in embryo spacing, suggesting the role of prostaglandins in implantation downstream of LPA3 signaling. In the present study, to know how LPA3 signaling regulates the embryo spacing, we determined the time course distribution of blastocysts during the preimplantation period. In wild-type (WT) uteri, blastocysts were distributed evenly along the uterine horns at Embryonic Day 3.8 (E3.8), whereas in the Lpa3-deficient uteri, they were clustered in the vicinity of the cervix, suggesting that the mislocalization and resulting crowding of the embryos are the cause of the delayed implantation. However, embryos transferred singly into E2.5 pseudopregnant Lpa3-deficient uterine horns still showed delayed implantation but on-time implantation in WT uteri, indicating that embryo spacing and implantation timing are two segregated events. We also found that an LPA3-specific agonist induced rapid uterine contraction in WT mice but not in Lpa3-deficient mice. Because the uterine contraction is critical for embryo spacing, our results suggest that LPA3 signaling controls embryo spacing via uterine contraction around E3.5.

  10. Cyclic phosphatidic acid and lysophosphatidic acid induce hyaluronic acid synthesis via CREB transcription factor regulation in human skin fibroblasts.

    Science.gov (United States)

    Maeda-Sano, Katsura; Gotoh, Mari; Morohoshi, Toshiro; Someya, Takao; Murofushi, Hiromu; Murakami-Murofushi, Kimiko

    2014-09-01

    Cyclic phosphatidic acid (cPA) is a naturally occurring phospholipid mediator and an analog of the growth factor-like phospholipid lysophosphatidic acid (LPA). cPA has a unique cyclic phosphate ring at the sn-2 and sn-3 positions of its glycerol backbone. We showed before that a metabolically stabilized cPA derivative, 2-carba-cPA, relieved osteoarthritis pathogenesis in vivo and induced hyaluronic acid synthesis in human osteoarthritis synoviocytes in vitro. This study focused on hyaluronic acid synthesis in human fibroblasts, which retain moisture and maintain health in the dermis. We investigated the effects of cPA and LPA on hyaluronic acid synthesis in human fibroblasts (NB1RGB cells). Using particle exclusion and enzyme-linked immunosorbent assays, we found that both cPA and LPA dose-dependently induced hyaluronic acid synthesis. We revealed that the expression of hyaluronan synthase 2 messenger RNA and protein is up-regulated by cPA and LPA treatment time dependently. We then characterized the signaling pathways up-regulating hyaluronic acid synthesis mediated by cPA and LPA in NB1RGB cells. Pharmacological inhibition and reporter gene assays revealed that the activation of the LPA receptor LPAR1, Gi/o protein, phosphatidylinositol-3 kinase (PI3K), extracellular-signal-regulated kinase (ERK), and cyclic adenosine monophosphate response element-binding protein (CREB) but not nuclear factor κB induced hyaluronic acid synthesis by the treatment with cPA and LPA in NB1RGB cells. These results demonstrate for the first time that cPA and LPA induce hyaluronic acid synthesis in human skin fibroblasts mainly through the activation of LPAR1-Gi/o followed by the PI3K, ERK, and CREB signaling pathway.

  11. Lysophosphatidic acid receptor 1 antagonist ki16425 blunts abdominal and systemic inflammation in a mouse model of peritoneal sepsis.

    Science.gov (United States)

    Zhao, Jing; Wei, Jianxin; Weathington, Nathaniel; Jacko, Anastasia M; Huang, Hai; Tsung, Allan; Zhao, Yutong

    2015-07-01

    Lysophosphatidic acid (LPA) is a bioactive lipid mediator of inflammation via the LPA receptors 1-6. We and others have previously described proinflammatory and profibrotic activities of LPA signaling in bleomycin- or lipopolysaccharide (LPS)-induced pulmonary fibrosis or lung injury models. In this study, we investigated if LPA signaling plays a role in the pathogenesis of systemic sepsis from an abdominal source. We report here that antagonism of the LPA receptor LPA1 with the small molecule ki16425 reduces the severity of abdominal inflammation and organ damage in the setting of peritoneal endotoxin exposure. Pretreatment of mice with intraperitoneal ki16425 eliminates LPS-induced peritoneal neutrophil chemokine and cytokine production, liver oxidative stress, liver injury, and cellular apoptosis in visceral organs. Mice pretreated with ki16425 are also protected from LPS-induced mortality. Tissue myeloperoxidase activity is not affected by LPA1 antagonism. We have shown that LPA1 is associated with LPS coreceptor CD14 and the association is suppressed by ki16425. LPS-induced phosphorylation of protein kinase C δ (PKCδ) and p38 mitogen-activated protein kinase (p38 MAPK) in liver cells and interleukin 6 production in Raw264 cells are likewise blunted by LPA1 antagonism. These studies indicate that the small molecule inhibitor of LPA1, ki16425, suppresses cytokine responses and inflammation in a peritoneal sepsis model by blunting downstream signaling through the LPA1-CD14-toll-like receptor 4 receptor complex. This anti-inflammatory effect may represent a therapeutic strategy for the treatment of systemic inflammatory responses to infection of the abdominal cavity.

  12. Lysophosphatidic Acid and Sphingosine-1-Phosphate: A Concise Review of Biological Function and Applications for Tissue Engineering.

    Science.gov (United States)

    Binder, Bernard Y K; Williams, Priscilla A; Silva, Eduardo A; Leach, J Kent

    2015-12-01

    The presentation and controlled release of bioactive signals to direct cellular growth and differentiation represents a widely used strategy in tissue engineering. Historically, work in this field has primarily focused on the delivery of large cytokines and growth factors, which can be costly to manufacture and difficult to deliver in a sustained manner. There has been a marked increase over the past decade in the pursuit of lipid mediators due to their wide range of effects over multiple cell types, low cost, and ease of scale-up. Lysophosphatidic acid (LPA) and sphingosine-1-phosphate (S1P) are two bioactive lysophospholipids (LPLs) that have gained attention for use as pharmacological agents in tissue engineering applications. While these lipids can have similar effects on cellular response, they possess distinct chemical backbones, mechanisms of synthesis and degradation, and signaling pathways using a discrete set of G-protein-coupled receptors (GPCRs). LPA and S1P predominantly act extracellularly on their GPCRs and can directly regulate cell survival, differentiation, cytokine secretion, proliferation, and migration--each of the important functions that must be considered in regenerative medicine. In addition to these potent physiological functions, these LPLs play pivotal roles in a number of pathophysiological processes. To capitalize on the promise of these molecules in tissue engineering, these lipids have been incorporated into biomaterials for in vivo delivery. Here, we survey the effects of LPA and S1P on both cellular- and tissue-level phenotypes, with an eye toward regulating stem/progenitor cell growth and differentiation. In particular, we examine work that has translational applications for cell-based tissue engineering strategies in promoting cell survival, bone and cartilage engineering, and therapeutic angiogenesis.

  13. Differential requirement of the epidermal growth factor receptor for G protein-mediated activation of transcription factors by lysophosphatidic acid

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    Dent Paul

    2010-01-01

    Full Text Available Abstract Background The role of the epidermal growth factor receptor (EGFR and other receptor tyrosine kinases (RTKs in provoking biological actions of G protein-coupled receptors (GPCRs has been one of the most disputed subjects in the field of GPCR signal transduction. The purpose of the current study is to identify EGFR-mediated mechanisms involved in activation of G protein cascades and the downstream transcription factors by lysophosphatidic acid (LPA. Results In ovarian cancer cells highly responsive to LPA, activation of AP-1 by LPA was suppressed by inhibition of EGFR, an effect that could be reversed by co-stimulation of another receptor tyrosine kinase c-Met with hepatocyte growth factor, indicating that LPA-mediated activation of AP-1 requires activity of a RTK, not necessarily EGFR. Induction of AP-1 components by LPA lied downstream of Gi, G12/13, and Gq. Activation of the effectors of Gi, but not Gq or G12/13 was sensitive to inhibition of EGFR. In contrast, LPA stimulated another prominent transcription factor NF-κB via the Gq-PKC pathway in an EGFR-independent manner. Consistent with the importance of Gi-elicited signals in a plethora of biological processes, LPA-induced cytokine production, cell proliferation, migration and invasion require intact EGFR. Conclusions An RTK activity is required for activation of the AP-1 transcription factor and other Gi-dependent cellular responses to LPA. In contrast, activation of G12/13, Gq and Gq-elicited NF-κB by LPA is independent of such an input. These results provide a novel insight into the role of RTK in GPCR signal transduction and biological functions.

  14. Role of lysophosphatidic acid receptor LPA2 in the development of allergic airway inflammation in a murine model of asthma

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    Chun Jerold

    2009-11-01

    Full Text Available Abstract Background Lysophosphatidic acid (LPA plays a critical role in airway inflammation through G protein-coupled LPA receptors (LPA1-3. We have demonstrated that LPA induced cytokine and lipid mediator release in human bronchial epithelial cells. Here we provide evidence for the role of LPA and LPA receptors in Th2-dominant airway inflammation. Methods Wild type, LPA1 heterozygous knockout mice (LPA1+/-, and LPA2 heterozygous knockout mice (LPA2+/- were sensitized with inactivated Schistosoma mansoni eggs and local antigenic challenge with Schistosoma mansoni soluble egg Ag (SEA in the lungs. Bronchoalveolar larvage (BAL fluids and lung tissues were collected for analysis of inflammatory responses. Further, tracheal epithelial cells were isolated and challenged with LPA. Results BAL fluids from Schistosoma mansoni egg-sensitized and challenged wild type mice (4 days of challenge showed increase of LPA level (~2.8 fold, compared to control mice. LPA2+/- mice, but not LPA1+/- mice, exposed to Schistosoma mansoni egg revealed significantly reduced cell numbers and eosinophils in BAL fluids, compared to challenged wild type mice. Both LPA2+/- and LPA1+/- mice showed decreases in bronchial goblet cells. LPA2+/- mice, but not LPA1+/- mice showed the decreases in prostaglandin E2 (PGE2 and LPA levels in BAL fluids after SEA challenge. The PGE2 production by LPA was reduced in isolated tracheal epithelial cells from LPA2+/- mice. These results suggest that LPA and LPA receptors are involved in Schistosoma mansoni egg-mediated inflammation and further studies are proposed to understand the role of LPA and LPA receptors in the inflammatory process.

  15. Identification of a Chlamydomonas plastidial 2-lysophosphatidic acid acyltransferase and its use to engineer microalgae with increased oil content.

    Science.gov (United States)

    Yamaoka, Yasuyo; Achard, Dorine; Jang, Sunghoon; Legéret, Bertrand; Kamisuki, Shogo; Ko, Donghwi; Schulz-Raffelt, Miriam; Kim, Yeongho; Song, Won-Yong; Nishida, Ikuo; Li-Beisson, Yonghua; Lee, Youngsook

    2016-11-01

    Despite a strong interest in microalgal oil production, our understanding of the biosynthetic pathways that produce algal lipids and the genes involved in the biosynthetic processes remains incomplete. Here, we report that Chlamydomonas reinhardtii Cre09.g398289 encodes a plastid-targeted 2-lysophosphatidic acid acyltransferase (CrLPAAT1) that acylates the sn-2 position of a 2-lysophosphatidic acid to form phosphatidic acid, the first common precursor of membrane and storage lipids. In vitro enzyme assays showed that CrLPAAT1 prefers 16:0-CoA to 18:1-CoA as an acyl donor. Fluorescent protein-tagged CrLPAAT1 was localized to the plastid membrane in C. reinhardtii cells. Furthermore, expression of CrLPAAT1 in plastids led to a > 20% increase in oil content under nitrogen-deficient conditions. Taken together, these results demonstrate that CrLPAAT1 is an authentic plastid-targeted LPAAT in C. reinhardtii, and that it may be used as a molecular tool to genetically increase oil content in microalgae.

  16. Derivatives of Dictyostelium differentiation-inducing factors inhibit lysophosphatidic acid–stimulated migration of murine osteosarcoma LM8 cells

    Energy Technology Data Exchange (ETDEWEB)

    Kubohara, Yuzuru, E-mail: ykuboha@juntendo.ac.jp [Department of Molecular and Cellular Biology, Institute for Molecular and Cellular Regulation (IMCR), Gunma University, Maebashi 371-8512 (Japan); Department of Health Science, Juntendo University Graduate School of Health and Sports Science, Inzai 270-1695 (Japan); Komachi, Mayumi [Department of Molecular and Cellular Biology, Institute for Molecular and Cellular Regulation (IMCR), Gunma University, Maebashi 371-8512 (Japan); Department of Radiation Oncology, Gunma University Graduate School of Medicine, Maebashi 371-8511 (Japan); Homma, Yoshimi [Department of Biomolecular Science, Institute of Biomedical Sciences, Fukushima Medical University School of Medicine, Fukushima 960-1295 (Japan); Kikuchi, Haruhisa; Oshima, Yoshiteru [Laboratory of Natural Product Chemistry, Tohoku University Graduate School of Pharmaceutical Sciences, Aoba-yama, Aoba-ku, Sendai 980-8578 (Japan)

    2015-08-07

    Osteosarcoma is a common metastatic bone cancer that predominantly develops in children and adolescents. Metastatic osteosarcoma remains associated with a poor prognosis; therefore, more effective anti-metastatic drugs are needed. Differentiation-inducing factor-1 (DIF-1), −2, and −3 are novel lead anti-tumor agents that were originally isolated from the cellular slime mold Dictyostelium discoideum. Here we investigated the effects of a panel of DIF derivatives on lysophosphatidic acid (LPA)-induced migration of mouse osteosarcoma LM8 cells by using a Boyden chamber assay. Some DIF derivatives such as Br-DIF-1, DIF-3(+2), and Bu-DIF-3 (5–20 μM) dose-dependently suppressed LPA-induced cell migration with associated IC{sub 50} values of 5.5, 4.6, and 4.2 μM, respectively. On the other hand, the IC{sub 50} values of Br-DIF-1, DIF-3(+2), and Bu-DIF-3 versus cell proliferation were 18.5, 7.2, and 2.0 μM, respectively, in LM8 cells, and >20, 14.8, and 4.3 μM, respectively, in mouse 3T3-L1 fibroblasts (non-transformed). Together, our results demonstrate that Br-DIF-1 in particular may be a valuable tool for the analysis of cancer cell migration, and that DIF derivatives such as DIF-3(+2) and Bu-DIF-3 are promising lead anti-tumor agents for the development of therapies that suppress osteosarcoma cell proliferation, migration, and metastasis. - Highlights: • LPA induces cell migration (invasion) in murine osteosarcoma LM8 cells. • DIFs are novel lead anti-tumor agents found in Dictyostelium discoideum. • We examined the effects of DIF derivatives on LPA-induced LM8 cell migration in vitro. • Some of the DIF derivatives inhibited LPA-induced LM8 cell migration.

  17. Autotaxin, a synthetic enzyme of lysophosphatidic acid (LPA, mediates the induction of nerve-injured neuropathic pain

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    Chun Jerold

    2008-02-01

    Full Text Available Abstract Recently, we reported that lysophosphatidic acid (LPA induces long-lasting mechanical allodynia and thermal hyperalgesia as well as demyelination and upregulation of pain-related proteins through one of its cognate receptors, LPA1. In addition, mice lacking the LPA1 receptor gene (lpa1-/- mice lost these nerve injury-induced neuropathic pain behaviors and phenomena. However, since lpa1-/- mice did not exhibit any effects on the basal nociceptive threshold, it is possible that nerve injury-induced neuropathic pain and its machineries are initiated by LPA via defined biosynthetic pathways that involve multiple enzymes. Here, we attempted to clarify the involvement of a single synthetic enzyme of LPA known as autotaxin (ATX in nerve injury-induced neuropathic pain. Wild-type mice with partial sciatic nerve injury showed robust mechanical allodynia starting from day 3 after the nerve injury and persisting for at least 14 days, along with thermal hyperalgesia. On the other hand, heterozygous mutant mice for the autotaxin gene (atx+/-, which have 50% ATX protein and 50% lysophospholipase D activity compared with wild-type mice, showed approximately 50% recovery of nerve injury-induced neuropathic pain. In addition, hypersensitization of myelinated Aβ˜ MathType@MTEF@5@5@+=feaafiart1ev1aaatCvAUfKttLearuWrP9MDH5MBPbIqV92AaeXatLxBI9gBaebbnrfifHhDYfgasaacPC6xNi=xH8viVGI8Gi=hEeeu0xXdbba9frFj0xb9qqpG0dXdb9aspeI8k8fiI+fsY=rqGqVepae9pg0db9vqaiVgFr0xfr=xfr=xc9adbaqaaeGacaGaaiaabeqaaeqabiWaaaGcbaGafqOSdiMbaGaaaaa@2D83@- or Aδ-fiber function following nerve injury was observed in electrical stimuli-induced paw withdrawal tests using a Neurometer®. The hyperalgesia was completely abolished in lpa1-/- mice, and reduced by 50% in atx+/- mice. Taken together, these findings suggest that LPA biosynthesis through ATX is the source of LPA for LPA1 receptor-mediated neuropathic pain. Therefore, targeted inhibition of ATX-mediated LPA biosynthesis as well as

  18. Systematic Analysis of Gene Expression Alterations and Clinical Outcomes for Long-Chain Acyl-Coenzyme A Synthetase Family in Cancer.

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    Wei-Ching Chen

    Full Text Available Dysregulated lipid metabolism contributes to cancer progression. Our previous study indicates that long-chain fatty acyl-Co A synthetase (ACSL 3 is essential for lipid upregulation induced by endoplasmic reticulum stress. In this report, we aimed to identify the role of ACSL family in cancer with systematic analysis and in vitro experiment. We explored the ACSL expression using Oncomine database to determine the gene alteration during carcinogenesis and identified the association between ACSL expression and the survival of cancer patient using PrognoScan database. ACSL1 may play a potential oncogenic role in colorectal and breast cancer and play a potential tumor suppressor role in lung cancer. Co-expression analysis revealed that ACSL1 was coexpressed with MYBPH, PTPRE, PFKFB3, SOCS3 in colon cancer and with LRRFIP1, TSC22D1 in lung cancer. In accordance with PrognoScan analysis, downregulation of ACSL1 in colon and breast cancer cell line inhibited proliferation, migration, and anchorage-independent growth. In contrast, increase of oncogenic property was observed in lung cancer cell line by attenuating ACSL1. High ACSL3 expression predicted a better prognosis in ovarian cancer; in contrast, high ACSL3 predicted a worse prognosis in melanoma. ACSL3 was coexpressed with SNUPN, TRIP13, and SEMA5A in melanoma. High expression of ACSL4 predicted a worse prognosis in colorectal cancer, but predicted better prognosis in breast, brain and lung cancer. ACSL4 was coexpressed with SERPIN2, HNRNPCL1, ITIH2, PROCR, LRRFIP1. High expression of ACSL5 predicted good prognosis in breast, ovarian, and lung cancers. ACSL5 was coexpressed with TMEM140, TAPBPL, BIRC3, PTPRE, and SERPINB1. Low ACSL6 predicted a worse prognosis in acute myeloid leukemia. ACSL6 was coexpressed with SOX6 and DARC. Altogether, different members of ACSLs are implicated in diverse types of cancer development. ACSL-coexpressed molecules may be used to further investigate the role of ACSL family in individual type of cancers.

  19. Potentials of the circulating pruritogenic mediator lysophosphatidic acid in development of allergic skin inflammation in mice: role of blood cell-associated lysophospholipase D activity of autotaxin.

    Science.gov (United States)

    Shimizu, Yoshibumi; Morikawa, Yoshiyuki; Okudaira, Shinichi; Kimoto, Shigenobu; Tanaka, Tamotsu; Aoki, Junken; Tokumura, Akira

    2014-05-01

    Itching and infiltration of immune cells are important hallmarks of atopic dermatitis (AD). Although various studies have focused on peripheral mediator-mediated mechanisms, systemic mediator-mediated mechanisms are also important in the pathogenesis and development of AD. Herein, we found that intradermal injection of lysophosphatidic acid (LPA), a bioactive phospholipid, induces scratching responses by Institute of Cancer Research mice through LPA1 receptor- and opioid μ receptor-mediating mechanisms, indicating its potential as a pruritogen. The circulating level of LPA in Naruto Research Institute Otsuka Atrichia mice, a systemic AD model, with severe scratching was found to be higher than that of control BALB/c mice, probably because of the increased lysophospholipase D activity of autotaxin (ATX) in the blood (mainly membrane associated) rather than in plasma (soluble). Heparan sulfate proteoglycan was shown to be involved in the association of ATX with blood cells. The sequestration of ATX protein on the blood cells by heparan sulfate proteoglycan may accelerate the transport of LPA to the local apical surface of vascular endothelium with LPA receptors, promoting the hyperpermeability of venules and the pathological uptake of immune cells, aggravating lesion progression and itching in Naruto Research Institute Otsuka Atrichia mice.

  20. Mechanism of sphingosine 1-phosphate- and lysophosphatidic Acid-induced up-regulation of adhesion molecules and eosinophil chemoattractant in nerve cells.

    LENUS (Irish Health Repository)

    Costello, Richard W

    2012-02-01

    The lysophospholipids sphingosine 1-phosphate (S1P) and lysophosphatidic acid (LPA) act via G-protein coupled receptors S1P(1-5) and LPA(1-3) respectively, and are implicated in allergy. Eosinophils accumulate at innervating cholinergic nerves in asthma and adhere to nerve cells via intercellular adhesion molecule-1 (ICAM-1). IMR-32 neuroblastoma cells were used as an in vitro cholinergic nerve cell model. The G(i) coupled receptors S1P(1), S1P(3), LPA(1), LPA(2) and LPA(3) were expressed on IMR-32 cells. Both S1P and LPA induced ERK phosphorylation and ERK- and G(i)-dependent up-regulation of ICAM-1 expression, with differing time courses. LPA also induced ERK- and G(i)-dependent up-regulation of the eosinophil chemoattractant, CCL-26. The eosinophil granule protein eosinophil peroxidase (EPO) induced ERK-dependent up-regulation of transcription of S1P(1), LPA(1), LPA(2) and LPA(3), providing the situation whereby eosinophil granule proteins may enhance S1P- and\\/or LPA- induced eosinophil accumulation at nerve cells in allergic conditions.

  1. Mechanism of sphingosine 1-phosphate- and lysophosphatidic Acid-induced up-regulation of adhesion molecules and eosinophil chemoattractant in nerve cells.

    LENUS (Irish Health Repository)

    Costello, Richard W

    2011-05-01

    The lysophospholipids sphingosine 1-phosphate (S1P) and lysophosphatidic acid (LPA) act via G-protein coupled receptors S1P(1-5) and LPA(1-3) respectively, and are implicated in allergy. Eosinophils accumulate at innervating cholinergic nerves in asthma and adhere to nerve cells via intercellular adhesion molecule-1 (ICAM-1). IMR-32 neuroblastoma cells were used as an in vitro cholinergic nerve cell model. The G(i) coupled receptors S1P(1), S1P(3), LPA(1), LPA(2) and LPA(3) were expressed on IMR-32 cells. Both S1P and LPA induced ERK phosphorylation and ERK- and G(i)-dependent up-regulation of ICAM-1 expression, with differing time courses. LPA also induced ERK- and G(i)-dependent up-regulation of the eosinophil chemoattractant, CCL-26. The eosinophil granule protein eosinophil peroxidase (EPO) induced ERK-dependent up-regulation of transcription of S1P(1), LPA(1), LPA(2) and LPA(3), providing the situation whereby eosinophil granule proteins may enhance S1P- and\\/or LPA- induced eosinophil accumulation at nerve cells in allergic conditions.

  2. Lysophosphatidic acid transactivates both c-Met and epidermal growth factor receptor, and induces cyclooxygenase-2 expression in human colon cancer LoVo cells

    Institute of Scientific and Technical Information of China (English)

    Dai Shida; Joji Kitayama; Hironori Yamaguchi; Hiroharu Yamashita; Ken Mori; Toshiaki Watanabe; Hirokazu Nagawa

    2005-01-01

    AIM: To examine whether lysophosphatidic acid (LPA)induces phosphorylation of c-Met and epidermal growth factor receptor (EGFR), both of which have been proposed as prognostic markers of colorectal cancer, and whether LPA induces cyclooxygenase-2 (COX-2) expression in human colon cancer cells.METHODS: Using a human colon cancer cell line, LoVo cells, we performed immunoprecipitation analysis,followed by Western blot analysis. We also examined whether LPA induced COX-2 expression, by Western blot analysis.RESULTS: Immunoprecipitation analysis revealed that 10 μmol/L LPA induced tyrosine phosphorylation of c-Met and EGFR in LoVo cells within a few minutes. We found that c-Met tyrosine phosphorylation induced by LPA was not attenuated by pertussis toxin or a matrix metalloproteinase inhibitor, in marked contrast to the results for EGFR. In addition, 0.2-40 μmol/L LPA induced COX-2 expression in a dose-dependent manner.CONCLUSION: Our results suggest that LPA acts upstream of various receptor tyrosine kinases (RTKs) and COX-2,and thus may act as a potent stimulator of colorectal cancer.

  3. Lysophosphatidic acid activates peroxisome proliferator activated receptor-γ in CHO cells that over-express glycerol 3-phosphate acyltransferase-1.

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    Cliona M Stapleton

    Full Text Available Lysophosphatidic acid (LPA is an agonist for peroxisome proliferator activated receptor-γ (PPARγ. Although glycerol-3-phosphate acyltransferase-1 (GPAT1 esterifies glycerol-3-phosphate to form LPA, an intermediate in the de novo synthesis of glycerolipids, it has been assumed that LPA synthesized by this route does not have a signaling role. The availability of Chinese Hamster Ovary (CHO cells that stably overexpress GPAT1, allowed us to analyze PPARγ activation in the presence of LPA produced as an intracellular intermediate. LPA levels in CHO-GPAT1 cells were 6-fold higher than in wild-type CHO cells, and the mRNA abundance of CD36, a PPARγ target, was 2-fold higher. Transactivation assays showed that PPARγ activity was higher in the cells that overexpressed GPAT1. PPARγ activity was enhanced further in CHO-GPAT1 cells treated with the PPARγ ligand troglitazone. Extracellular LPA, phosphatidic acid (PA or a membrane-permeable diacylglycerol had no effect, showing that PPARγ had been activated by LPA generated intracellularly. Transient transfection of a vector expressing 1-acylglycerol-3-phosphate acyltransferase-2, which converts endogenous LPA to PA, markedly reduced PPARγ activity, as did over-expressing diacylglycerol kinase, which converts DAG to PA, indicating that PA could be a potent inhibitor of PPARγ. These data suggest that LPA synthesized via the glycerol-3-phosphate pathway can activate PPARγ and that intermediates of de novo glycerolipid synthesis regulate gene expression.

  4. Heart-type fatty-acid-binding protein (FABP3 is a lysophosphatidic acid-binding protein in human coronary artery endothelial cells

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    Ryoko Tsukahara

    2014-01-01

    Full Text Available Fatty-acid-binding protein 3, muscle and heart (FABP3, also known as heart-type FABP, is a member of the family of intracellular lipid-binding proteins. It is a small cytoplasmic protein with a molecular mass of about 15 kDa. FABPs are known to be carrier proteins for transporting fatty acids and other lipophilic substances from the cytoplasm to the nucleus, where these lipids are released to a group of nuclear receptors such as peroxisome proliferator-activated receptors (PPARs. In this study, using lysophosphatidic acid (LPA-coated agarose beads, we have identified FABP3 as an LPA carrier protein in human coronary artery endothelial cells (HCAECs. Administration of LPA to HCAECs resulted in a dose-dependent increase in PPARγ activation. Furthermore, the LPA-induced PPARγ activation was abolished when the FABP3 expression was reduced using small interfering RNA (siRNA. We further show that the nuclear fraction of control HCAECs contained a significant amount of exogenously added LPA, whereas FABP3 siRNA-transfected HCAECs had a decreased level of LPA in the nucleus. Taken together, these results suggest that FABP3 governs the transcriptional activities of LPA by targeting them to cognate PPARγ in the nucleus.

  5. Eicosopentaneoic Acid and Other Free Fatty Acid Receptor Agonists Inhibit Lysophosphatidic Acid- and Epidermal Growth Factor-Induced Proliferation of Human Breast Cancer Cells

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    Mandi M. Hopkins

    2016-01-01

    Full Text Available Many key actions of ω-3 (n-3 fatty acids have recently been shown to be mediated by two G protein-coupled receptors (GPCRs in the free fatty acid receptor (FFAR family, FFA1 (GPR40 and FFA4 (GPR120. n-3 Fatty acids inhibit proliferation of human breast cancer cells in culture and in animals. In the current study, the roles of FFA1 and FFA4 were investigated. In addition, the role of cross-talk between GPCRs activated by lysophosphatidic acid (LPA, and the tyrosine kinase receptor activated by epidermal growth factor (EGF, was examined. In MCF-7 and MDA-MB-231 human breast cancer cell lines, both LPA and EGF stimulated proliferation, Erk activation, Akt activation, and CCN1 induction. LPA antagonists blocked effects of LPA and EGF on proliferation in MCF-7 and MDA-MB-231, and on cell migration in MCF-7. The n-3 fatty acid eicosopentaneoic acid inhibited LPA- and EGF-induced proliferation in both cell lines. Two synthetic FFAR agonists, GW9508 and TUG-891, likewise inhibited LPA- and EGF-induced proliferation. The data suggest a major role for FFA1, which was expressed by both cell lines. The results indicate that n-3 fatty acids inhibit breast cancer cell proliferation via FFARs, and suggest a mechanism involving negative cross-talk between FFARS, LPA receptors, and EGF receptor.

  6. Expression of lysophosphatidic acid receptor 1 and relation with cell proliferation, apoptosis, and angiogenesis on preneoplastic changes induced by cadmium chloride in the rat ventral prostate.

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    Riánsares Arriazu

    Full Text Available BACKGROUND: Lysophosphatidic acid (LPA is a phospholipid growth factor involved in cell proliferation, differentiation, migration, inflammation, angiogenesis, wound healing, cancer invasion, and survival. This study was directed to evaluate the immunoexpression of LPA-1, cell proliferation, apoptosis, and angiogenesis markers in preneoplastic lesions induced with cadmium chloride in rat prostate. METHODS: The following parameters were calculated in ventral prostate of normal rats and rats that received Cd in drinking water during 24 months: percentages of cells immunoreactive to LPA-1 (LILPA1, PCNA (LIPCNA, MCM7 (LIMCM7, ubiquitin (LIUBI, apoptotic cells (LIAPO, and p53 (LIp53; volume fraction of Bcl-2 (VFBcl-2; and length of microvessels per unit of volume (LVMV/mm3. Data were analyzed using Student's t-test and Pearson correlation test. RESULTS: The LILPA1 in dysplastic lesions and normal epithelium of Cd-treated rats was significantly higher than those in the control group. Markers of proliferation were significantly increased in dysplastic lesions, whereas some apoptotic markers were significantly decreased. No significant differences between groups were found in VFBcl-2. Dysplastic lesions showed a significant increase of LIp53. The length of microvessels per unit of volume was elevated in dysplastic acini. Statistically significant correlations were found only between LILPA1 and LIUBI. CONCLUSIONS: Our results suggest that LPA-1 might be implicated in dysplastic lesions induced by cadmium chloride development. More studies are needed to confirm its potential contribution to the disease.

  7. Adult Lysophosphatidic Acid Receptor 1-Deficient Rats with Hyperoxia-Induced Neonatal Chronic Lung Disease Are Protected against Lipopolysaccharide-Induced Acute Lung Injury

    Science.gov (United States)

    Chen, Xueyu; Walther, Frans J.; Laghmani, El H.; Hoogeboom, Annemarie M.; Hogen-Esch, Anne C. B.; van Ark, Ingrid; Folkerts, Gert; Wagenaar, Gerry T. M.

    2017-01-01

    Aim: Survivors of neonatal chronic lung disease or bronchopulmonary dysplasia (BPD) suffer from compromised lung function and are at high risk for developing lung injury by multiple insults later in life. Because neonatal lysophosphatidic acid receptor-1 (LPAR1)-deficient rats are protected against hyperoxia-induced lung injury, we hypothesize that LPAR1-deficiency may protect adult survivors of BPD from a second hit response against lipopolysaccharides (LPS)-induced lung injury. Methods: Directly after birth, Wistar control and LPAR1-deficient rat pups were exposed to hyperoxia (90%) for 8 days followed by recovery in room air. After 7 weeks, male rats received either LPS (2 mg kg−1) or 0.9% NaCl by intraperitoneal injection. Alveolar development and lung inflammation were investigated by morphometric analysis, IL-6 production, and mRNA expression of cytokines, chemokines, coagulation factors, and an indicator of oxidative stress. Results: LPAR1-deficient and control rats developed hyperoxia-induced neonatal emphysema, which persisted into adulthood, as demonstrated by alveolar enlargement and decreased vessel density. LPAR1-deficiency protected against LPS-induced lung injury. Adult controls with BPD exhibited an exacerbated response toward LPS with an increased expression of pro-inflammatory mRNAs, whereas LPAR1-deficient rats with BPD were less sensitive to this “second hit” with a decreased pulmonary influx of macrophages and neutrophils, interleukin-6 (IL-6) production, and mRNA expression of IL-6, monocyte chemoattractant protein-1, cytokine-induced neutrophil chemoattractant 1, plasminogen activator inhibitor-1, and tissue factor. Conclusion: LPAR1-deficient rats have increased hyperoxia-induced BPD survival rates and, despite the presence of neonatal emphysema, are less sensitive to an aggravated “second hit” than Wistar controls with BPD. Intervening in LPA-LPAR1-dependent signaling may not only have therapeutic potential for neonatal chronic

  8. Función del ácido lisofosfatídico como regulador lipídico modulador del comportamiento / Role of lysophosphatidic acid as lipid mediator in behavior

    Directory of Open Access Journals (Sweden)

    Fernando Rodríguez de Fonseca

    2011-12-01

    Full Text Available Lysophosphatidic acid (LPA is an endogenous phospholipid which is involved in many different cellular processes through specific G-protein coupled receptors (LPA1-6. The finding of a lysophosphatidic acid (LPA signaling pathway in the developing and adult brain led to the characterization of the functional roles of LPA in normal and diseased brain. Previous studies using pharmacological or genetic approaches such as receptor null mice have been demonstrated as indispensable to determine the requirement of, at least, LPA1 receptor for normal brain function and its influence in many different processes including neural cell proliferation and differentiation, cell survival, synapsis, neural transmission, or neurochemical balance in a variety of cerebral areas although, remarkably, the hippocampus. To date numerous contributions have showed behavioral alterations affecting cognition and emotional behavior in correlation with structural and neurochemical observations. Here we review the functions of LPA in behavior, principally particularized to those mediated by LPA1 receptor, and also discuss their relevance to psychiatric disorders.

  9. Regulation of gene expression and subcellular protein distribution in MLO-Y4 osteocytic cells by lysophosphatidic acid: Relevance to dendrite outgrowth.

    Energy Technology Data Exchange (ETDEWEB)

    Waters, Katrina M.; Jacobs, Jon M.; Gritsenko, Marina A.; Karin, Norman J.

    2011-02-26

    Osteoblastic and osteocytic cells are highly responsive to the lipid growth factor lysophosphatidic acid (LPA) but the mechanisms by which LPA alters bone cell functions are largely unknown. A major effect of LPA on osteocytic cells is the stimulation of dendrite membrane outgrowth, a process that we predicted to require changes in gene expression and protein distribution. We employed DNA microarrays for global transcriptional profiling of MLO-Y4 osteocytic cells grown for 6 and 24h in the presence or absence of LPA. We identified 932 transcripts that displayed statistically significant changes in abundance of at least 1.25-fold in response to LPA treatment. Gene ontology (GO) analysis revealed that the regulated gene products were linked to diverse cellular processes, including DNA repair, response to unfolded protein, ossification, protein-RNA complex assembly, and amine biosynthesis. Gene products associated with the regulation of actin microfilament dynamics displayed the most robust expression changes, and LPA-induced dendritogenesis in vitro was blocked by the stress fiber inhibitor cytochalasin D. Mass spectrometry-based proteomic analysis of MLO-Y4 cells revealed significant LPA-induced changes in the abundance of 284 proteins at 6h and 844 proteins at 24h. GO analysis of the proteomic data linked the effects of LPA to cell processes that control of protein distribution and membrane outgrowth, including protein localization, protein complex assembly, Golgi vesicle transport, cytoskeleton-dependent transport, and membrane invagination/endocytosis. Dendrites were isolated from LPA-treated MLO-Y4 cells and subjected to proteomic analysis to quantitatively assess the subcellular distribution of proteins. Sets of 129 and 36 proteins were enriched in the dendrite fraction as compared to whole cells after 6h and 24h of LPA exposure, respectively. Protein markers indicated that membranous organelles were largely excluded from the dendrites. Highly represented among

  10. Regulation of gene expression and subcellular protein distribution in MLO-Y4 osteocytic cells by lysophosphatidic acid: Relevance to dendrite outgrowth.

    Science.gov (United States)

    Waters, Katrina M; Jacobs, Jon M; Gritsenko, Marina A; Karin, Norman J

    2011-06-01

    Osteoblastic and osteocytic cells are highly responsive to the lipid growth factor lysophosphatidic acid (LPA) but the mechanisms by which LPA alters bone cell functions are largely unknown. A major effect of LPA on osteocytic cells is the stimulation of dendrite membrane outgrowth, a process that we predicted to require changes in gene expression and protein distribution. We employed DNA microarrays for global transcriptional profiling of MLO-Y4 osteocytic cells grown for 6 and 24h in the presence or absence of LPA. We identified 932 transcripts that displayed statistically significant changes in abundance of at least 1.25-fold in response to LPA treatment. Gene ontology (GO) analysis revealed that the regulated gene products were linked to diverse cellular processes, including DNA repair, response to unfolded protein, ossification, protein-RNA complex assembly, and amine biosynthesis. Gene products associated with the regulation of actin microfilament dynamics displayed the most robust expression changes, and LPA-induced dendritogenesis in vitro was blocked by the stress fiber inhibitor cytochalasin D. Mass spectrometry-based proteomic analysis of MLO-Y4 cells revealed significant LPA-induced changes in the abundance of 284 proteins at 6h and 844 proteins at 24h. GO analysis of the proteomic data linked the effects of LPA to cell processes that control of protein distribution and membrane outgrowth, including protein localization, protein complex assembly, Golgi vesicle transport, cytoskeleton-dependent transport, and membrane invagination/endocytosis. Dendrites were isolated from LPA-treated MLO-Y4 cells and subjected to proteomic analysis to quantitatively assess the subcellular distribution of proteins. Sets of 129 and 36 proteins were enriched in the dendrite fraction as compared to whole cells after 6h and 24h of LPA exposure, respectively. Protein markers indicated that membranous organelles were largely excluded from the dendrites. Highly represented among

  11. Lysophosphatidic acid increases SLC26A3 expression in inflamed intestine and reduces diarrheal severity in C57BL/6 mice with dextran-sodium-sulfate-induced colitis

    Institute of Scientific and Technical Information of China (English)

    Xu Lihong; Xiao Fang; He Jiayi; Lan Xiaoqin; Ding Qiang; Li Junhua; Ursula Seidler

    2014-01-01

    Background Diarrhea is a common clinical feature of ulcerative colitis resulting from unbalanced intestinal fluid and salt absorption and secretion.The Cl-/HCO3-exchanger SLC26A3 is strongly expressed in the mid-distal colon and plays an essential role in colonic Cl-absorption and HCO3-secretion.Sic26a3 expression is up-regulated by lysophosphatidic acid (LPA) in vitro.Our study was designed to investigate the effects of LPA on SLC26A3 expression and the diarrheal phenotype in a mouse colitis model.Methods Colitis was induced in C57BL/6 mice by adding 4% of dextran sodium sulfate (DSS) to the drinking water.The mice were assigned to LPA treatment DSS group,phosphate-buffered saline (PBS) treatment DSS group,DSS only group and untreated mice with a completely randomized design.Diarrhea severity was evaluated by measuring mice weight,disease activity index (DAI),stool water content and macroscopic evaluation of colonic damage.The effect of LPA treatment on Sic26a3 mRNA level and protein expression in the different groups of mice was investigated by quantitative PCR and Western blotting.Results All mice treated with DSS lost weight,but the onset and severity of weight loss was attenuated in the LPA treatment DSS group.The increases in stool water content and the macroscopic inflammation score in LPA treatment DSS group were significantly lower compared to DSS control group or PBS treatment DSS group ((18.89±8.67)% vs.(28.97±6.95)% or (29.48±6.71)%,P=0.049,P=0.041,respectively and 2.67±0.81 vs.4.5±0.83 or 4.5±0.54,P=0.020,P=0.006,respectively),as well as the increase in DAI (P=0.004,P=0.008,respectively).LPA enema resulted in higher Slc26a3 mRNA and protein expression levels compared to PBS-treated and untreated DSS colitis mice.Conclusion LPA increases Slc26a3 expression in the inflamed intestine and reduces diarrhea severity in DSS-induced colitis,suggesting LPA might be a therapeutic strategy in the treatment of colitis associated diarrhea.

  12. β-Arrestin2 regulates lysophosphatidic acid-induced human breast tumor cell migration and invasion via Rap1 and IQGAP1.

    Directory of Open Access Journals (Sweden)

    Mistre Alemayehu

    Full Text Available β-Arrestins play critical roles in chemotaxis and cytoskeletal reorganization downstream of several receptor types, including G protein-coupled receptors (GPCRs, which are targets for greater than 50% of all pharmaceuticals. Among them, receptors for lysophosphatidic acid (LPA, namely LPA(1 are overexpressed in breast cancer and promote metastatic spread. We have recently reported that β-arrestin2 regulates LPA(1-mediated breast cancer cell migration and invasion, although the underlying molecular mechanisms are not clearly understood. We show here that LPA induces activity of the small G protein, Rap1 in breast cancer cells in a β-arrestin2-dependent manner, but fails to activate Rap1 in non-malignant mammary epithelial cells. We found that Rap1A mRNA levels are higher in human breast tumors compared to healthy patient samples and Rap1A is robustly expressed in human ductal carcinoma in situ and invasive tumors, in contrast to the normal mammary ducts. Rap1A protein expression is also higher in aggressive breast cancer cells (MDA-MB-231 and Hs578t relative to the weakly invasive MCF-7 cells or non-malignant MCF10A mammary cells. Depletion of Rap1A expression significantly impaired LPA-stimulated migration of breast cancer cells and invasiveness in three-dimensional Matrigel cultures. Furthermore, we found that β-arrestin2 associates with the actin binding protein IQGAP1 in breast cancer cells, and is necessary for the recruitment of IQGAP1 to the leading edge of migratory cells. Depletion of IQGAP1 blocked LPA-stimulated breast cancer cell invasion. Finally, we have identified that LPA enhances the binding of endogenous Rap1A to β-arrestin2, and also stimulates Rap1A and IQGAP1 to associate with LPA(1. Thus our data establish novel roles for Rap1A and IQGAP1 as critical regulators of LPA-induced breast cancer cell migration and invasion.

  13. 偏头痛急性期患者溶血磷脂酸与TCD的变化%Changes of lysophosphatidic acid and TCD in patients with migraine during acute stage

    Institute of Scientific and Technical Information of China (English)

    宋叶华; 牛建平; 汤婷; 叶良灶; 何倪靖

    2013-01-01

    Objective Observation the changesof plasma lysophosphatidic acid (LPA) 、lysophosphatidic acid similar levels of phospholipids(AP) and Transcranial Doppler ultrasound (TCD) in acute migraine patients. Methodes Determination plasma LPA and AP in migraine patients with acute period (migraine without aura and migraine with aura group) ,at the same time,were examined by TCD, compared with normal patients. Results Migraine patients with LPA 、AP increased significantly, especial y for migraine with aura group increased more significantly, suggesting that platelet activation in patients with acute migraine attacks, and blood flow velocity in patients with migraine acute stage significantly increased in patients with migraine.The results support functions - vascular nerve disorder in patients with migraine.%目的观察偏头痛急性发作期患者血浆溶血磷脂酸(LPA)及溶血磷脂酸相似磷脂水平(AP)、经颅多普勒超声(TCD)的变化。方法测定偏头痛急性期患者(无先兆偏头痛组及有先兆偏头痛组)血浆LPA及AP水平,同时对患者行TCD检查,与正常患者进行对照。结果偏头痛组血浆LPA水平高于对照组,尤其是有先兆偏头痛组,且偏头痛急性期TCD以血流速度增快为主,尤其是患侧大脑中动脉血流速度。结论偏头痛发作期患者LPA、AP明显升高,尤其是有先兆偏头痛组升高更明显,提示偏头痛急性发作期患者存在血小板活化过程,同时偏头痛急性期患者血流速度常明显增快,支持偏头痛患者存在神经-血管功能紊乱。

  14. RhoA Kinase (Rock) and p90 Ribosomal S6 Kinase (p90Rsk) phosphorylation of the sodium hydrogen exchanger (NHE1) is required for lysophosphatidic acid-induced transport, cytoskeletal organization and migration.

    Science.gov (United States)

    Wallert, Mark A; Hammes, Daniel; Nguyen, Tony; Kiefer, Lea; Berthelsen, Nick; Kern, Andrew; Anderson-Tiege, Kristina; Shabb, John B; Muhonen, Wallace W; Grove, Bryon D; Provost, Joseph J

    2015-03-01

    The sodium hydrogen exchanger isoform one (NHE1) plays a critical role coordinating asymmetric events at the leading edge of migrating cells and is regulated by a number of phosphorylation events influencing both the ion transport and cytoskeletal anchoring required for directed migration. Lysophosphatidic acid (LPA) activation of RhoA kinase (Rock) and the Ras-ERK growth factor pathway induces cytoskeletal reorganization, activates NHE1 and induces an increase in cell motility. We report that both Rock I and II stoichiometrically phosphorylate NHE1 at threonine 653 in vitro using mass spectrometry and reconstituted kinase assays. In fibroblasts expressing NHE1 alanine mutants for either Rock (T653A) or ribosomal S6 kinase (Rsk; S703A) we show that each site is partially responsible for the LPA-induced increase in transport activity while NHE1 phosphorylation by either Rock or Rsk at their respective site is sufficient for LPA stimulated stress fiber formation and migration. Furthermore, mutation of either T653 or S703 leads to a higher basal pH level and a significantly higher proliferation rate. Our results identify the direct phosphorylation of NHE1 by Rock and suggest that both RhoA and Ras pathways mediate NHE1-dependent ion transport and migration in fibroblasts.

  15. Protein kinase C alpha-CARMA3 signaling axis links Ras to NF-kappa B for lysophosphatidic acid-induced urokinase plasminogen activator expression in ovarian cancer cells.

    Science.gov (United States)

    Mahanivong, C; Chen, H M; Yee, S W; Pan, Z K; Dong, Z; Huang, S

    2008-02-21

    We reported previously that a signaling pathway consisting of G(i)-Ras-NF-kappaB mediates lysophosphatidic acid (LPA)-induced urokinase plasminogen activator (uPA) upregulation in ovarian cancer cells. However, it is not clear what signaling components link Ras to nuclear factor (NF)-kappaB for this LPA-induced event. In the present study, we found that treatment of protein kinase C (PKC) inhibitors including conventional PKC (cPKC) inhibitor Gö6976 abolished LPA-induced uPA upregulation in ovarian cancer cell lines tested, indicating the importance of cPKC activity in this LPA-induced event. Indeed, LPA stimulation led to the activation of PKCalpha and Ras-PKCalpha interaction. Although constitutively active mutants of PKCalpha (a cPKC), PKCtheta (a novel PKC (nPKC)) and PKCzeta (an atypical PKC (aPKC)) were all able to activate NF-kappaB and upregulate uPA expression, only dominant-negative PKCalpha mutant attenuated LPA-induced NF-kappaB activation and uPA upregulation. These results suggest that PKCalpha, rather than PKC isoforms in other PKC classes, participates in LPA-induced NF-kappaB activation and uPA upregulation in ovarian cancer cells. To determine the signaling components downstream of PKCalpha mediating LPA-induced uPA upregulation, we showed that forced expression of dominant-negative CARMA3 or silencing CARMA3, Bcl10 and MALT1 with specific siRNAs diminished these LPA-induced events. Furthermore, we demonstrated that PKCalpha/CARMA3 signaling axis is important in LPA-induced ovarian cancer cell in vitro invasion.

  16. Possible Role of Different Yeast and Plant Lysophospholipid:Acyl-CoA Acyltransferases (LPLATs) in Acyl Remodelling of Phospholipids.

    Science.gov (United States)

    Jasieniecka-Gazarkiewicz, Katarzyna; Demski, Kamil; Lager, Ida; Stymne, Sten; Banaś, Antoni

    2016-01-01

    Recent results have suggested that plant lysophosphatidylcholine:acyl-coenzyme A acyltransferases (LPCATs) can operate in reverse in vivo and thereby catalyse an acyl exchange between the acyl-coenzyme A (CoA) pool and the phosphatidylcholine. We have investigated the abilities of Arabidopsis AtLPCAT2, Arabidopsis lysophosphatidylethanolamine acyltransferase (LPEAT2), S. cerevisiae lysophospholipid acyltransferase (Ale1) and S. cerevisiae lysophosphatidic acid acyltransferase (SLC1) to acylate lysoPtdCho, lysoPtdEtn and lysoPtdOH and act reversibly on the products of the acylation; the PtdCho, PtdEtn and PtdOH. The tested LPLATs were expressed in an S. cervisiae ale1 strain and enzyme activities were assessed in assays using microsomal preparations of the different transformants. The results show that, despite high activity towards lysoPtdCho, lysoPtdEtn and lysoPtdOH by the ALE1, its capacities to operate reversibly on the products of the acylation were very low. Slc1 readily acylated lysoPtdOH, lysoPtdCho and lysoPtdEtn but showed no reversibility towards PtdCho, very little reversibility towards PtdEtn and very high reversibility towards PtdOH. LPEAT2 showed the highest levels of reversibility towards PtdCho and PtdEtn of all LPLATs tested but low ability to operate reversibly on PtdOH. AtLPCAT2 showed good reversible activity towards PtdCho and PtdEtn and very low reversibility towards PtdOH. Thus, it appears that some of the LPLATs have developed properties that, to a much higher degree than other LPLATs, promote the reverse reaction during the same assay conditions and with the same phospholipid. The results also show that the capacity of reversibility can be specific for a particular phospholipid, albeit the lysophospholipid derivatives of other phospholipids serve as good acyl acceptors for the forward reaction of the enzyme.

  17. Identification of heparin-binding EGF-like growth factor (HB-EGF as a biomarker for lysophosphatidic acid receptor type 1 (LPA1 activation in human breast and prostate cancers.

    Directory of Open Access Journals (Sweden)

    Marion David

    Full Text Available Lysophosphatidic acid (LPA is a natural bioactive lipid with growth factor-like functions due to activation of a series of six G protein-coupled receptors (LPA₁₋₆. LPA receptor type 1 (LPA₁ signaling influences the pathophysiology of many diseases including cancer, obesity, rheumatoid arthritis, as well as lung, liver and kidney fibrosis. Therefore, LPA₁ is an attractive therapeutic target. However, most mammalian cells co-express multiple LPA receptors whose co-activation impairs the validation of target inhibition in patients because of missing LPA receptor-specific biomarkers. LPA₁ is known to induce IL-6 and IL-8 secretion, as also do LPA₂ and LPA₃. In this work, we first determined the LPA induced early-gene expression profile in three unrelated human cancer cell lines expressing different patterns of LPA receptors (PC3: LPA₁,₂,₆; MDA-MB-231: LPA1,2; MCF-7: LPA₂,₆. Among the set of genes upregulated by LPA only in LPA₁-expressing cells, we validated by QPCR and ELISA that upregulation of heparin-binding EGF-like growth factor (HB-EGF was inhibited by LPA₁-₃ antagonists (Ki16425, Debio0719. Upregulation and downregulation of HB-EGF mRNA was confirmed in vitro in human MDA-B02 breast cancer cells stably overexpressing LPA₁ (MDA-B02/LPA₁ and downregulated for LPA₁ (MDA-B02/shLPA1, respectively. At a clinical level, we quantified the expression of LPA₁ and HB-EGF by QPCR in primary tumors of a cohort of 234 breast cancer patients and found a significantly higher expression of HB-EGF in breast tumors expressing high levels of LPA₁. We also generated human xenograph prostate tumors in mice injected with PC3 cells and found that a five-day treatment with Ki16425 significantly decreased both HB-EGF mRNA expression at the primary tumor site and circulating human HB-EGF concentrations in serum. All together our results demonstrate that HB-EGF is a new and relevant biomarker with potentially high value in

  18. The relationship between lysophosphatidic molecules and their receptors and multiple sclerosis%溶血磷脂类分子及其受体与多发性硬化的关系

    Institute of Scientific and Technical Information of China (English)

    蒋东晓; 连艳芬; 詹霞; 林勇; 吴喜娟

    2015-01-01

    Multiple sclerosis is an inflammatory demyelinating diseases of central nervous system, with multiple lesions and clinical course of repeated for characteristics,the pathology shows the damage of lymphocytes and macrophages to myelin and secondary axonal damage.Lysophosphatidic molecules is an important signal molecule in regulation of cell survival and apoptosis, with relationship to the pathological processes of tumor,atherosclerosis and ischemic cardio cerebral vascular diseases.Recent studies show that lysophospholipids molecules not only participate in the production of a variety of inflammatory cytokines,but also have closely relation to the infiltration of inflammatory cells,and have an important impact on the central nervous system demyelination, axonal damage.This article reviewed the research progress of relationship of lysophospholipid molecule and multiple sclerosis.%多发性硬化是一种以中枢神经系统炎性脱髓鞘疾病,临床以多发病灶及病程反复为特点,其病理表现主要是淋巴细胞和巨噬细胞对髓鞘的破坏及继发性轴索损害. 溶血磷脂酸分子是调节细胞生存与凋亡的重要信号分子,与肿瘤、动脉粥样硬化及缺血性心脑血管病等病理过程有关. 近年来研究表明,溶血磷脂类分子不仅参与各种炎性细胞因子的产生,而且同炎性细胞的浸润密切相关,对中枢神经系统的髓鞘脱失、轴索损害产生重要影响,本文就溶血磷脂类分子与多发性硬化关系的研究进展作一综述.

  19. 花生溶血磷脂酸酰基转移酶基因的克隆与表达分析%Cloning and Expression Analysis of Lysophosphatidic Acid Acyltransferase (LPAT) Encoding Gene in Peanut

    Institute of Scientific and Technical Information of China (English)

    陈四龙; 黄家权; 雷永; 任小平; 文奇根; 陈玉宁; 姜慧芳; 晏立英; 廖伯寿

    2012-01-01

    Lysophosphatidic acid acyltransferase (LPAT) is a key enzyme in biosynthesis pathway of vegetable oil in plant. It is important for oil crops to improve oil quality and increase seed oil content through genetic engineering. We constructed a full-length cDNA library of peanut (Arachis hypogaea) seed via a large number of sequences of expressed sequence tag (EST) and gene functional annotation, a lysophosphatidic acid acyltransferase gene, designated AhLPAT, and its genomic DNA sequence were isolated from peanut. The sequence of AhLPAT cDN A was 1 629 bp, and its genomic sequence was 5 331 bp. Bioinformatic analysis showed that AhLPAT was composed of 11 exons and 10 introns with typical GT-AG characteristic in comparison of its sequences of genomic DNA and cDNA by Splign in NCBI. A peptide of 387 amino acid residues with protein molecular weight of 43.2 kD and isoelectric point (p7) of 9.42 were deduced from AhLPAT. Conserved domains prediction indicated that AhLPAT comprised a typical conserved acyltransferase domain and a conserved lysophospholipid acyltransferases domain. The deduced amino acid had a high identity with the LPAT proteins reported from other species. Amino acid similarities of LPAT protein be tween peanut and Tropaeolum majus, Brassica napus, Crambe hispanica subsp. Abyssinica, Ricinus communis, and Arabidopsis thaliana were 90%, 89%, 89%, 88%, and 87%, respectively. A phylogenetic tree was constructed by the Neighbor-Joining method using MEGA5.0. The phylogenetic tree suggested that AhLPAT and AtLPAT2 derived from Arabidopsis thaliana were grouped into the same class. Both AhLPAT and AtLPAT2 were endoplasmic reticulum type LPATs. The tissue specific expression analysis by using quantitative RT-PCR assays indicated that AhLPAT was ubiquitously expressed in root, stem, leaf, flower, gynophore, seed of peanut with the highest level in gynophore and seed. The expression level reached a peak in the stage from 50 to 60 days after flowering. The

  20. Yeast Interacting Proteins Database: YHL003C, YKL008C [Yeast Interacting Proteins Database

    Lifescience Database Archive (English)

    Full Text Available 6(acyl)-coenzyme A and dihydrosphingosine or phytosphingosine, functionally equivalent to Lac1p Rows with th...nthesis of ceramide from C26(acyl)-coenzyme A and dihydrosphingosine or phytosphingosine, functionally...e A and dihydrosphingosine or phytosphingosine, functionally equivalent to Lac1p Rows with this bait as bait...ynthesis of ceramide from C26(acyl)-coenzyme A and dihydrosphingosine or phytosphingosine, functionally equi

  1. Nucleon effective mass and the A dependence of structure functions

    Energy Technology Data Exchange (ETDEWEB)

    Garcia Canal, C.A.; Santangelo, E.M.; Vucetich, H.

    1984-10-08

    The nucleon effective mass was successfully used, as the only free parameter, to adjust the ratio R(A) of structure functions measured in a nucleus of mass number A and in the deuteron, for each A value in the SLAC set of experimental data. The resulting A dependence of the effective mass, being linear in A/sup -1/3/, is consistent with the behavior expected from nuclear structure considerations. The extrapolated value of the effective mass for nuclear matter agrees with previous estimations.

  2. A Dependent Hidden Markov Model of Credit Quality

    Directory of Open Access Journals (Sweden)

    Małgorzata Wiktoria Korolkiewicz

    2012-01-01

    Full Text Available We propose a dependent hidden Markov model of credit quality. We suppose that the "true" credit quality is not observed directly but only through noisy observations given by posted credit ratings. The model is formulated in discrete time with a Markov chain observed in martingale noise, where "noise" terms of the state and observation processes are possibly dependent. The model provides estimates for the state of the Markov chain governing the evolution of the credit rating process and the parameters of the model, where the latter are estimated using the EM algorithm. The dependent dynamics allow for the so-called "rating momentum" discussed in the credit literature and also provide a convenient test of independence between the state and observation dynamics.

  3. Plasma Atherogenic Index and Acyl-coenzyme A Cholesterol Acyltransferase 2 of Obese Adolescents after Weight Reduction%4周运动减肥对肥胖青少年血浆致动脉粥样硬化指数和脂酰辅酶A胆固醇酰基转移酶2的影响

    Institute of Scientific and Technical Information of China (English)

    林云; 陈文鹤

    2012-01-01

    Objective To observe the effect of weight reduction on plasma atherogenic index and cyl-coenzyme A cholesterol acyltransferase 2 (ACAT2) of obese adolescents. Methods 30 obese adolescents took part in 4-week aerobic exercise and diet program for weight reduction. Results After 4 weeks of exercise and diet,the body weight,fat percentage,BMI and serum insulin of the subjects reduced significantly(P < 0.01);the level of LPL and the ratio of HDL-C/LDL-C increased and the level of TG, TC,HDL-C,LDL-C,atherogenic index and ACAT2 decreased significantly (P < 0.01). Conclusion Weight reduction through exercise and proper diet effectively improves somatometric measurements,lipid metabolism and insulin resistance to a certain extent in obese adolescents.%目的:观察运动减肥对肥胖青少年血浆致动脉粥样硬化指数和脂酰辅酶A胆固醇酰基转移酶等动脉粥样硬化致病相关因子的影响.方法:以参加2011年上海巅峰运动减肥夏令营的30名肥胖青少年为对象,进行4周中小强度有氧运动为主结合适当饮食控制的减肥运动训练.分别于入营第一天和出营前一天进行身体形态及血液指标测试.结果:经过夏令营4周有氧运动训练,肥胖青少年体重、体脂率、BMI与运动前相比显著下降,空腹血清胰岛素、血脂水平与运动前相比均明显改善,血浆致动脉粥样硬化指数、脂酰辅酶A胆固醇酰基转移酶2水平显著降低.结论:4周有氧运动明显降低了肥胖青少年的肥胖程度和胰岛素水平,改善血脂代谢,在一定程度上降低了动脉粥样硬化发病的风险.

  4. Effect of lysophosphatidic acid on differentiation of embryonic neural stem cells into neuroglial cells in rats in vitro%溶血磷脂酸对离体培养的大鼠胚胎神经干细胞向神经胶质细胞分化的影响

    Institute of Scientific and Technical Information of China (English)

    崔慧林; 乔健天

    2007-01-01

    To study the effect of lysophosphatidic acid(LPA) on the differentiation of embryonic neural stem cells(NSCs)into neuroglial cells in rats in vitro,both oligodendrocytes and astrocytes were detected by their marker proteins galactocerebroside(GalC)and glial fibrillary acidic protein(GFAP),respectively,using double-labeling immunocytochemistry.RT-PCR assay was also used for analyzing the expression of LPA receptors in NSCs.Our results showed that:(1)LPA at different concentrations(0.01-3.0 μmol/L)was added to culture medium and cell counting was carried out on the 7th day in all groups.Exposure to LPA led to a dose-dependent increase of oligodendrocytes with the response peaked at 1.0 μmol/L,with an increased percentage of 32.6%(P<0.01)of total cells as compared to that of 8.5% in the vehicle group.(2)LPA showed no effect on the differentiation of NSCs into astrocytes.(3)RT-PCR assay showed that LPA1 and LPA3 receptors were strongly expressed while LPA2 receptor expressed weakly in NSCs.These results suggest that LPA at low concentration might act as an extracellular signal through the receptors in NSCs,mainly LPA1 and LPA3 receptors,to promote the differentiation of NSCs into oligodendrocytes,while it exhibits little,if any,conceivable effect on the differentiation of NSCs into astrocytes.%本研究用免疫细胞化学荧光双标技术观察了溶血磷脂酸(lysophosphatidic acid,LPA)对大鼠胚胎神经干细胞(neural stem cells,NSCs)分化为少突胶质细胞(galactocerebroside-positive,Gal-C阳性)和星形胶质细胞(glial fibrillary acidic protein-positive,GFAP阳性)的影响,并且用RT-PCR技术对NSCs可能表达的LPA受体进行分析.结果显示:(1)加入不同浓度(0.01~3.0μmol/L)LPA,第7天进行检测时,少突胶质细胞数量呈明显的剂量依赖性增加,峰值出现在1.0μmol/L LPA组,少突胶质细胞所占百分比从对照组的8.5%增加到32.6%:(2)星形胶质细胞的分化几乎不受LPA的影响,第7天时各LPA处理

  5. The expression of lysophosphatidic acid, its receptors, and IL-6 and IL-8 in breast cancer%溶血磷脂酸及其受体和IL-6 IL-8在乳腺癌进展中的表达变化与意义

    Institute of Scientific and Technical Information of China (English)

    涂福平; 黄莉; 王祥财; 许明君; 王钇力; 衷敬华

    2013-01-01

    Objective:This work aimed to investigate the expression level of lysophosphatidic acid (LPA) and its receptors. The paper also discussed the interrelationship among the LPA, the receptors, and IL-6 and IL-8 in breast cancer tissues. Methods:The ex-pressions of the 3 hypo-types of LPA receptor in the breast cancer and paraneoplastic tissues were detected using semi-quantitative re-verse transcription polymerase chain reaction. The plasma levels of LPA, IL-6 and IL-8 were respectively detected in healthy subjects and in patients with benign breast tumor using the LPA biochemistry and enzyme linked immunosorbent assay kits. Results:The plas-ma LPA level was significantly higher in patients with breast cancer metastasis than in those with local breast cancer (P<0.01), benign breast tumor (P<0.01), and healthy volunteers (P<0.01). In addition, the IL-6 and IL-8 plasma levels were higher in the group with me-tastasis compared with the other three groups, too (P<0.01). LPA1 expression level was higher in breast cancer tissue than in benign breast tumor (P<0.05) and in normal breast tissue (P<0.05). There was a significantly positive correlation between the plasma LPA and the plasma IL-6 in patients with breast cancer (P<0.01), and between the plasma LPA and IL-8 (P<0.01). Conclusion:LPA expressions on the endogenous IL-6 and IL-8 in patients with breast cancer may have an up-regulation. Moreover, the detection of the LPA, IL-6, and IL-8 expression levels may have some predictable effects on metastatic breast cancer, especially bone metastases.%  目的:探讨溶血磷脂酸(lysophosphatidic acid,LPA)及其受体和IL-6与IL-8在乳腺癌进展中的表达及临床意义。方法:采用半定量RT-PCR方法检测乳腺肿瘤组织和瘤旁组织中LPA受体的表达水平。采用LPA生化测定法和酶联免疫吸附(ELISA)法分别检测乳腺肿瘤患者和健康妇女的血浆LPA、IL-6和IL-8水平。结果:术后复发转

  6. Relationship between levels of plasma lysophosphatidic acid, matrix metalloproteinase-9 and coronary stenosis%血浆溶血磷脂酸、基质金属蛋白酶-9水平与冠状动脉狭窄程度的相关性研究

    Institute of Scientific and Technical Information of China (English)

    杨波; 林琍; 宗文霞

    2011-01-01

    目的:观察冠心病患者血浆溶血磷脂酸(lysophosphatidic acid,LPA)及基质金属蛋白酶-9(matrix metalloproteinase-9,MMP-9)水平与冠状动脉病变的关系,探讨其在冠心病中的临床意义.方法:140例冠脉造影者根据病情及冠脉造影结果分为急性心肌梗死(AMI)组(n=40)、不稳定性心绞痛(UAP)组(n=35)、稳定性心绞痛(SAP)组(n=35)、对照组(n=30).用Gensini积分评定冠状动脉狭窄程度,根据评分四分位间距分组将患者分为4组:Ⅰ组(0~7分)35例,Ⅱ组(8~25分)36例,Ⅲ组(26~46分)26例及Ⅳ组(>46分)43例.分别用无机磷定量法和酶联免疫吸附法测定血浆LPA、MMP-9水平.结果:冠心病各组血浆LPA、 MMP-9水平及Genisi评分均显著高于对照组(P<0.01),AMI组高于UAP组及SAP组(P<0.01),UAP组高于SAP组(P<0.01).不同Genisi评分各组之间LPA、 MMP-9水平均差异有统计学意义(P<0.01).LPA与MMP-9水平呈正相关(r=0.22,P<0.05).结论:冠心病患者血浆LPA与MMP-9水平显著增高,且与冠心病严重程度及冠脉狭窄程度密切相关.%Objective: To investigate the relationship between levels of plasma lysophosphatidic acid ( LPA), matrix metalloproteinase-9(MMP-9) and the severity of coronary artery disease in patients with acute coronary heart disease (CHD) and to explore the potential clinical significance. Methods: One hundred and forty cases undergone coronary arteriography were divided into 4 groups according to the state of illness and results of coronary angiography: acute myocardial infarction (AMI) group( n= 40 ), unstable angina pectoris (UAP) group ( n=35 ), stable angina pectoris (SAP) group (n= 35) and control group(n=30 ). The degree of coronary artery stenosis was determined by Gensini's scores system, and the patients were redivided into 4 groups based on the interquartile of the Gensini's scores: group Ⅰ (0-7 scores,n= 35), group Ⅱ (8-25 scores,n= 36), group Ⅲ (26-46 scores,n= 26 ) and group Ⅳ (>46 scores

  7. A system level approach for a dependable heterogeneous MPSoC

    NARCIS (Netherlands)

    Ibrahim, Ahmed; Kerkhoff, Hans G.

    2015-01-01

    Mission-critical systems require a dependable operation during their lifetime. However, the current ongoing aggressive scaling of technology has resulted in increasing reliability issues. Dependability of such systems has become a major concern in the design process. In this work we aim to enhance t

  8. 脑脊液溶血磷脂酸含量与动脉瘤性蛛网膜下腔出血后脑积水的关系%Relationship between lysophosphatidic acid levels in cerebrospinal fluid and hydrocephalus after aneurysmal subarachnoid hemorrhage

    Institute of Scientific and Technical Information of China (English)

    步星耀; 郭晓鹤; 闫兆月; 魏振宇; 周伟; 马春晓; 郭锁成; 张建国; 邢亚洲

    2012-01-01

    Objective To explore the changes of lysophosphatidic acid (LPA) levels in cerebrospinal fluid (CSF) in patients with aneurismal subarachnoid hemorrhage (aSAH) and study its relationship with hydrocephalus. Methods Levels of LPA in CSF of 72 patients with aSAH diagnozed by clinical and accessory examinations were measured on day 1,7, 14 and 28 after the symptoms onset. Time - phase correlation between the LPA levels and onset of hydrocephalus was observed. Results Of the 72 SAH patients, 23 patients (31.9% ) developed hydrocephalus, and the average onset time was (12. 6 ± 9.7) days. The LPA level on day 7 of the aSAH attack was obviously higher than that on day 1 (P = 0.002). LPA level began to decline on day 14. On day 28, the LPA level was not significantly different from that of day 1 level (P = 0. 931). On day 7 and 14, the levels of LPA in the patients with hydrocephalus were significantly higher than those without hydrocephalus (t1 = 11.15,P1 =0.000; t2 =4.90,P2 = 0.001) , on day 1 and 28, the differences being not significant (t1 = 1. 126, P1 = 0.286; t2 = 0. 726, P2 = 0.484). Conclusions The LPA levels in CSF increase markedly from day 7 to day 14 after the attack of aSAH, which is obviously associated with the occurrence of hydrocephalus on time course. LPA in CSF of patients with aSAH may be involved in the formation of hydrocephalus. To estimate the content of LPA in CSF may be of importance in predicting the occurrence of hydrocephalus after aSAH.%目的 探讨脑脊液溶血磷脂酸(LPA)含量与动脉瘤性蛛网膜下腔出血(aSAH)后脑积水的关系.方法 选取经临床和辅助检查确诊的aSAH患者72例,于发病1、7、14、28d测定脑脊液中LPA含量,观察LPA含量与脑积水发生在时程上的相关性.结果 72例SAH患者中有23例(31.9%)发生脑积水,平均发生时间为(12.6±9.7)d.发病7d时aSAH患者脑脊液中LPA含量与发病1d时相比明显升高(P=0.002),14 d时开始下降,28d时与发病1d

  9. 溶血磷脂酸调控RhoA/ROCK2信号通路对乳腺癌细胞增殖的影响%Influence of lysophosphatidic acid on proliferation of breast cancer cell by adjusted RhoA/ROCK2 signal pathway

    Institute of Scientific and Technical Information of China (English)

    许海; 段刚峰

    2013-01-01

    目的 探讨溶血磷脂酸(LPA)与RhoA/ROCK2信号通路对乳腺癌细胞增殖的影响及其作用机制.方法 以不同浓度LPA干预乳腺癌MDA-MB-231细胞,每隔24 h以细胞计数法观察和记录细胞的增殖.以最佳LPA促增殖浓度作用于MDA-MB-231细胞,观察Rho激酶抑制剂(Y-27632)对癌细胞的影响;以Pull-down及Western blot法检测各组细胞内RhoA活性及RhoA、ROCK2蛋白表达.结果 LPA以时间及剂量依赖性关系显著促进MDA-MB-231细胞的增殖(P<0.05);Y-27632可以显著抑制LPA的促增殖作用;LPA干预后RhoA活性及RhoA、ROCK2蛋白表达显著升高(P<0.05),Y-27632干预后RhoA活性及RhoA、ROCK2蛋白表达显著下降(P<0.05).结论 LPA可能通过调控RhoA/ROCK2信号通路促进乳腺癌细胞的增殖,为乳腺癌的临床治疗提供了新思路.%Objective To investigate the influence and mechanism of lysophosphatidic acid and RhoA/ROCK2 signal pathway on proliferation of breast cancer cell. Methods After treatment with different concentration of LPA, the proliferation of breast cancer cell MDA-MB-231 was observed and recorded by cell count method every of 24 h. MDA-MB-231 treated with optimal concentration of LPA and observed the effect of Rho kinase inhibitor( Y-27632) on LPA-induced proliferation. The activity of RhoA was tested by a pull-down way. The protein expression of RhoA and ROCK2 were determined by Western blot. Results LPA could promote MDA-MB-231 proliferation in a time and dose-dependent manner (P 〈 0. 05). ROCK inhibitor significantly inhibited LPA-induced cell proliferation (P 〈 0. 05 ). The activity of RhoA and expressionof RhoA, ROCK2 were enhanced significantly after LPA intervention (P 〈0. 05). However Y-27632 markedly decreased LPA-induced the increase of RhoA activity and protein expression of RhoA and ROCK2 ( P 〈 0. 05). Conclusions LPA may promote breast cancer cell proliferation through regulating RhoA/ROCK2 signal pathway. It provides a new idea

  10. DksA-dependent transcriptional regulation in Salmonella experiencing nitrosative stress

    Directory of Open Access Journals (Sweden)

    Matthew A Crawford

    2016-03-01

    Full Text Available Redox-based signaling is fundamental to the capacity of bacteria to sense, and respond to, nitrosative and oxidative stress encountered in natural and host environments. The conserved RNA polymerase regulatory protein DksA is a thiol-based sensor of reactive nitrogen and oxygen species. DksA-dependent transcriptional control promotes antinitrosative and antioxidative defenses that contribute to Salmonella pathogenesis. The specific adaptive changes mediated by DksA in response to reactive species, however, have not been elucidated. Herein, we characterize DksA-dependent changes in gene expression in Salmonella enterica experiencing nitrosative stress. Genome-wide expression analysis of wild-type and delta-dksA Salmonella exposed to the nitric oxide (•NO donor DETA NONOate demonstrated •NO- and DksA-dependent regulatory control of 427 target genes. Transcriptional changes centered primarily on genes encoding aspects of cellular metabolism. Several antioxidants and oxidoreductases important in redox buffering, •NO detoxification, and damage repair were also observed to be up-regulated in an •NO- and DksA-dependent manner. Compared to wild-type bacteria, •NO-treated delta-dksA Salmonella exhibited a de-repression of genes encoding components of iron homeostasis and failed to activate sulfur assimilation and cysteine biosynthetic operons. As cysteine is integral to efficient antinitrosative and antioxidative defense and repair programs, we further examined the redox-responsive transcriptional control of cysteine biosynthesis by DksA. These investigations revealed that the activation of genes comprising cysteine biosynthesis also occurs in response to hydrogen peroxide, is dependent upon the redox-sensing zinc finger domain of DksA, and requires the transcriptional regulator CysB. Our observations demonstrate that DksA mediates global adaptation to nitrosative stress in Salmonella and provide unique insight into a novel regulatory mechanism

  11. Pro-lipogenic Action of Lysophosphatidic Acid in Ovarian Cancer

    Science.gov (United States)

    2014-04-01

    their nonselective suppression of fatty acid synthesis in both normal andmalignant tissues, which could deteriorate weight loss , anorexia, fatigue, and... supplemented LPA did not fully reverse the effect of the iPLA2β inhibitor BEL on cell cycling (8), suggesting involvement of additional bioactive...dominant-negative form was found to inhibit growth of ovarian cancer cell lines (Fig. 5). Furthermore, inhibition of carnitine palmitoyl transferase 1A

  12. Lysophosphatidic Acid Regulation and Roles in Human Prostate Cancer

    Science.gov (United States)

    2006-08-01

    Received 23 June 2005 C©2005 Biochemical Society Glycoconjugate Journal 20, 39–47, 2004 C© 2004 Kluwer Academic Publishers. Manufactured in The Netherlands...neuroblastoma, lymphoma, and ovarian cancer cells [105,106]. Thus, antibod- ies against specific gangliosides have received consideration as...is nearly as effective a chemoattractant for DC as are C5a and CCL19 [34]. Like T cells, DC express CCR7 and migrate in response to its ligands CCL19

  13. Requirements for E1A dependent transcription in the yeast Saccharomyces cerevisiae

    Directory of Open Access Journals (Sweden)

    Mymryk Joe S

    2009-04-01

    Full Text Available Abstract Background The human adenovirus type 5 early region 1A (E1A gene encodes proteins that are potent regulators of transcription. E1A does not bind DNA directly, but is recruited to target promoters by the interaction with sequence specific DNA binding proteins. In mammalian systems, E1A has been shown to contain two regions that can independently induce transcription when fused to a heterologous DNA binding domain. When expressed in Saccharomyces cerevisiae, each of these regions of E1A also acts as a strong transcriptional activator. This allows yeast to be used as a model system to study mechanisms by which E1A stimulates transcription. Results Using 81 mutant yeast strains, we have evaluated the effect of deleting components of the ADA, COMPASS, CSR, INO80, ISW1, NuA3, NuA4, Mediator, PAF, RSC, SAGA, SAS, SLIK, SWI/SNF and SWR1 transcriptional regulatory complexes on E1A dependent transcription. In addition, we examined the role of histone H2B ubiquitylation by Rad6/Bre1 on transcriptional activation. Conclusion Our analysis indicates that the two activation domains of E1A function via distinct mechanisms, identify new factors regulating E1A dependent transcription and suggest that yeast can serve as a valid model system for at least some aspects of E1A function.

  14. PUMA promotes Bax translocation in FOXO3a-dependent pathway during STS-induced apoptosis

    Science.gov (United States)

    Zhang, Yingjie; Chen, Qun

    2009-08-01

    PUMA (p53 up-regulated modulator of apoptosis, also called Bbc3) was first identified as a BH3-only Bcl-2 family protein that is transcriptionally up-regulated by p53 and activated upon p53-dependent apoptotic stimuli, such as treatment with DNA-damaging drugs or UV irradiation. Recently studies have been shown that Puma is also up-regulated in response to certain p53-independent apoptotic stimuli, such as growth factor deprivation or treatment with glucocorticoids or STS (staurosporine). However, the molecular mechanisms of PUMA up-regulation and how PUMA functions in response to p53-independent apoptotic stimuli remain poorly understood. In this study, based on real-time single cell analysis, flow cytometry and western blotting technique, we investigated the function of PUMA in living human lung adenocarcinoma cells (ASTC-a-1) after STS treatment. Our results show that FOXO3a was activated by STS stimulation and then translocated from cytosol to nucleus. The expression of PUMA was up-regulated via a FOXO3a-dependent manner after STS treatment, while p53 had little function in this process. Moreover, cell apoptosis and Bax translocation induced by STS were not blocked by Pifithrin-α (p53 inhibitor), which suggested that p53 was not involved in this signaling pathway. Taken together, these results indicate that PUMA promoted Bax translocation in a FOXO3a-dependment pathway during STS-induced apoptosis, while p53 was dispensable in this process.

  15. Function of the N-terminal segment of the RecA-dependent nuclease Ref.

    Science.gov (United States)

    Gruber, Angela J; Olsen, Tayla M; Dvorak, Rachel H; Cox, Michael M

    2015-02-18

    The bacteriophage P1 Ref (recombination enhancement function) protein is a RecA-dependent, HNH endonuclease. It can be directed to create targeted double-strand breaks within a displacement loop formed by RecA. The 76 amino acid N-terminal region of Ref is positively charged (25/76 amino acid residues) and inherently unstructured in solution. Our investigation of N-terminal truncation variants shows this region is required for DNA binding, contains a Cys involved in incidental dimerization and is necessary for efficient Ref-mediated DNA cleavage. Specifically, Ref N-terminal truncation variants lacking between 21 and 47 amino acids are more effective RecA-mediated targeting nucleases. We propose a more refined set of options for the Ref-mediated cleavage mechanism, featuring the N-terminal region as an anchor for at least one of the DNA strand cleavage events.

  16. A-dependence of the Spectra of the F Isotopes from ab initio Calculations

    Science.gov (United States)

    Barrett, Bruce R.; Dikmen, Erdal; Maris, Pieter; Vary, James P.; Shirokov, Andrey M.

    2016-03-01

    Using a succession of Okubo-Lee-Suzuki transformations within the No Core Shell Model (NCSM) formalism, we derive an ab initio, non-perturbative procedure for calculating the input for standard shell-model (SSM) calculations within one major shell. We have used this approach for calculating the spectra of the F isotopes from A=18 to A=25, so as to study the A-dependence of the results. In particular, we are interested in seeing if the theoretical input is weak enough, so that a single set of two-body effective interactions can be used for all of the F isotopes investigated. We will present results from SSM calculations based on input obtained with the JISP16 nucleon-nucleon interaction in an initial 4 ℏΩ NCSM basis space. This work supported in part by TUBITAK-BIDEB, the US DOE, the US NSF, NERSC, and the Russian Ministry of Education and Science.

  17. A Dependence of the Electronuclear System Parameters on the Component Concentration in Fuel MOX

    CERN Document Server

    Barashenkov, V S

    2001-01-01

    A dependence of the parameters of a electronuclear system with U-Pu fuel MOX on the relative share of plutonium and its isotope ^{240}Pu in fuel is investigated by means of mathematical modelling. As an example, we consider an experimental system with a heat power 10-20 kW designed in Dubna on the basis of the 660 MeV proton phasotron. The 2 % admixture of ^{240}Pu decreases the value of the neutron multiplication coefficient from 0.95 down to 0.90, neutron yield and heat power are diminished almost twice. Such a decrease can be compensated by the increase of Plutonium share in MOX from 25 up to 27 %.

  18. Metabolism of β-valine via a CoA-dependent ammonia lyase pathway.

    Science.gov (United States)

    Otzen, Marleen; Crismaru, Ciprian G; Postema, Christiaan P; Wijma, Hein J; Heberling, Matthew M; Szymanski, Wiktor; de Wildeman, Stefaan; Janssen, Dick B

    2015-11-01

    Pseudomonas species strain SBV1 can rapidly grow on medium containing β-valine as a sole nitrogen source. The tertiary amine feature of β-valine prevents direct deamination reactions catalyzed by aminotransferases, amino acid dehydrogenases, and amino acid oxidases. However, lyase- or aminomutase-mediated conversions would be possible. To identify enzymes involved in the degradation of β-valine, a PsSBV1 gene library was prepared and used to complement the β-valine growth deficiency of a closely related Pseudomonas strain. This resulted in the identification of a gene encoding β-valinyl-coenzyme A ligase (BvaA) and two genes encoding β-valinyl-CoA ammonia lyases (BvaB1 and BvaB2). The BvaA protein demonstrated high sequence identity to several known phenylacetate CoA ligases. Purified BvaA enzyme did not convert phenyl acetic acid but was able to activate β-valine in an adenosine triphosphate (ATP)- and CoA-dependent manner. The substrate range of the enzyme appears to be narrow, converting only β-valine and to a lesser extent, 3-aminobutyrate and β-alanine. Characterization of BvaB1 and BvaB2 revealed that both enzymes were able to deaminate β-valinyl-CoA to produce 3-methylcrotonyl-CoA, a common intermediate in the leucine degradation pathway. Interestingly, BvaB1 and BvaB2 demonstrated no significant sequence identity to known CoA-dependent ammonia lyases, suggesting they belong to a new family of enzymes. BLAST searches revealed that BvaB1 and BvaB2 show high sequence identity to each other and to several enoyl-CoA hydratases, a class of enzymes that catalyze a similar reaction with water instead of amine as the leaving group.

  19. RecA-dependent programmable endonuclease Ref cleaves DNA in two distinct steps.

    Science.gov (United States)

    Ronayne, Erin A; Cox, Michael M

    2014-04-01

    The bacteriophage P1 recombination enhancement function (Ref) protein is a RecA-dependent programmable endonuclease. Ref targets displacement loops formed when an oligonucleotide is bound by a RecA filament and invades homologous double-stranded DNA sequences. Mechanistic details of this reaction have been explored, revealing that (i) Ref is nickase, cleaving the two target strands of a displacement loop sequentially, (ii) the two strands are cleaved in a prescribed order, with the paired strand cut first and (iii) the two cleavage events have different requirements. Cutting the paired strand is rapid, does not require RecA-mediated ATP hydrolysis and is promoted even by Ref active site variant H153A. The displaced strand is cleaved much more slowly, requires RecA-mediated ATP hydrolysis and does not occur with Ref H153A. The two cleavage events are also affected differently by solution conditions. We postulate that the second cleavage (displaced strand) is limited by some activity of RecA protein.

  20. Nuclear Envelope Protein SUN2 Promotes Cyclophilin-A-Dependent Steps of HIV Replication

    Directory of Open Access Journals (Sweden)

    Xavier Lahaye

    2016-04-01

    Full Text Available During the early phase of replication, HIV reverse transcribes its RNA and crosses the nuclear envelope while escaping host antiviral defenses. The host factor Cyclophilin A (CypA is essential for these steps and binds the HIV capsid; however, the mechanism underlying this effect remains elusive. Here, we identify related capsid mutants in HIV-1, HIV-2, and SIVmac that are restricted by CypA. This antiviral restriction of mutated viruses is conserved across species and prevents nuclear import of the viral cDNA. Importantly, the inner nuclear envelope protein SUN2 is required for the antiviral activity of CypA. We show that wild-type HIV exploits SUN2 in primary CD4+ T cells as an essential host factor that is required for the positive effects of CypA on reverse transcription and infection. Altogether, these results establish essential CypA-dependent functions of SUN2 in HIV infection at the nuclear envelope.

  1. Arousal effect of orexin A depends on activation of the histaminergic system.

    Science.gov (United States)

    Huang, Z L; Qu, W M; Li, W D; Mochizuki, T; Eguchi, N; Watanabe, T; Urade, Y; Hayaishi, O

    2001-08-14

    Orexin neurons are exclusively localized in the lateral hypothalamic area and project their fibers to the entire central nervous system, including the histaminergic tuberomammillary nucleus (TMN). Dysfunction of the orexin system results in the sleep disorder narcolepsy, but the role of orexin in physiological sleep-wake regulation and the mechanisms involved remain to be elucidated. Here we provide several lines of evidence that orexin A induces wakefulness by means of the TMN and histamine H(1) receptor (H1R). Perfusion of orexin A (5 and 25 pmol/min) for 1 hr into the TMN of rats through a microdialysis probe promptly increased wakefulness for 2 hr after starting the perfusion by 2.5- and 4-fold, respectively, concomitant with a reduction in rapid eye movement (REM) and non-REM sleep. Microdialysis studies showed that application of orexin A to the TMN increased histamine release from both the medial preoptic area and the frontal cortex by approximately 2-fold over the baseline for 80 to 160 min in a dose-dependent manner. Furthermore, infusion of orexin A (1.5 pmol/min) for 6 hr into the lateral ventricle of mice produced a significant increase in wakefulness during the 8 hr after starting infusion to the same level as the wakefulness observed during the active period in wild-type mice, but not at all in H1R gene knockout mice. These findings strongly indicate that the arousal effect of orexin A depends on the activation of histaminergic neurotransmission mediated by H1R.

  2. A-dependence of nuclear transparency in quasielastic A(e,e{prime}p) at high Q{sup 2}

    Energy Technology Data Exchange (ETDEWEB)

    O`Neill, T.G.; Lorenzon, W.; Arrington, J. [California Institute of Technology, Pasadena, CA (United States). W.K. Kellogg Radiation Lab.] [and others

    1994-05-01

    The A-dependence of the quasielastic A(e,e{prime}p) reaction has been studied with {sup 3}H, C, Fe, and Au nuclei at momentum transfers Q{sup 2} = 1, 3, 5, and 6.8(GeV/c){sup 2}. The authors extract the nuclear transparency T(A,Q{sup 2}), a measure of the average probability of escape of a proton from a nucleus A. Several calculations predict a significant increase in T with momentum transfer, a phenomenon known as color transparency. No statistically significant rise is seen for any of the nuclei studied.

  3. Arabidopsis Deficient in Cutin Ferulate encodes a transferase required for feruloylation of ω-hydroxy fatty acids in cutin polyester.

    Science.gov (United States)

    Rautengarten, Carsten; Ebert, Berit; Ouellet, Mario; Nafisi, Majse; Baidoo, Edward E K; Benke, Peter; Stranne, Maria; Mukhopadhyay, Aindrila; Keasling, Jay D; Sakuragi, Yumiko; Scheller, Henrik Vibe

    2012-02-01

    The cuticle is a complex aliphatic polymeric layer connected to the cell wall and covers surfaces of all aerial plant organs. The cuticle prevents nonstomatal water loss, regulates gas exchange, and acts as a barrier against pathogen infection. The cuticle is synthesized by epidermal cells and predominantly consists of an aliphatic polymer matrix (cutin) and intracuticular and epicuticular waxes. Cutin monomers are primarily C(16) and C(18) unsubstituted, ω-hydroxy, and α,ω-dicarboxylic fatty acids. Phenolics such as ferulate and p-coumarate esters also contribute to a minor extent to the cutin polymer. Here, we present the characterization of a novel acyl-coenzyme A (CoA)-dependent acyl-transferase that is encoded by a gene designated Deficient in Cutin Ferulate (DCF). The DCF protein is responsible for the feruloylation of ω-hydroxy fatty acids incorporated into the cutin polymer of aerial Arabidopsis (Arabidopsis thaliana) organs. The enzyme specifically transfers hydroxycinnamic acids using ω-hydroxy fatty acids as acyl acceptors and hydroxycinnamoyl-CoAs, preferentially feruloyl-CoA and sinapoyl-CoA, as acyl donors in vitro. Arabidopsis mutant lines carrying DCF loss-of-function alleles are devoid of rosette leaf cutin ferulate and exhibit a 50% reduction in ferulic acid content in stem insoluble residues. DCF is specifically expressed in the epidermis throughout all green Arabidopsis organs. The DCF protein localizes to the cytosol, suggesting that the feruloylation of cutin monomers takes place in the cytoplasm.

  4. Internalization of a thiazole-modified peptide in Sinorhizobium meliloti occurs by BacA-dependent and -independent mechanisms.

    Science.gov (United States)

    Wehmeier, Silvia; Arnold, Markus F F; Marlow, Victoria L; Aouida, Mustapha; Myka, Kamila K; Fletcher, Vivien; Benincasa, Monica; Scocchi, Marco; Ramotar, Dindial; Ferguson, Gail P

    2010-09-01

    BacA proteins play key roles in the chronic intracellular infections of Sinorhizobium meliloti, Brucella abortus and Mycobacterium tuberculosis within their respective hosts. S. meliloti, B. abortus and M. tuberculosis BacA-deficient mutants have increased resistance to the thiazole-modified peptide bleomycin. BacA has been previously hypothesized, but not experimentally verified, to be involved in bleomycin uptake. In this paper, we show that a BacA-dependent mechanism is the major route of bleomycin internalization in S. meliloti. We also determined that the B. abortus and S. meliloti BacA proteins are functional homologues and that the B. abortus BacA protein is involved in the uptake of both bleomycin and proline-rich peptides. Our findings also provide evidence that there is a second, BacA-independent minor mechanism for bleomycin internalization in S. meliloti. We determined that the BacA-dependent and -independent mechanisms of bleomycin uptake are energy-dependent, consistent with both mechanisms of bleomycin uptake involving transport systems.

  5. The minor C-allele of rs2014355 in ACADS is associated with reduced insulin release following an oral glucose load

    OpenAIRE

    Pisinger Charlotta; Lauritzen Torsten; Sandbæk Annelli; Andersson Åsa; Sandholt Camilla H; Krarup Nikolaj T; Justesen Johanne M; Banasik Karina; Hornbak Malene; Witte Daniel R; Sørensen Thorkild IA; Pedersen Oluf; Hansen Torben

    2011-01-01

    Abstract Background A genome-wide association study (GWAS) using metabolite concentrations as proxies for enzymatic activity, suggested that two variants: rs2014355 in the gene encoding short-chain acyl-coenzyme A dehydrogenase (ACADS) and rs11161510 in the gene encoding medium-chain acyl-coenzyme A dehydrogenase (ACADM) impair fatty acid β-oxidation. Chronic exposure to fatty acids due to an impaired β-oxidation may down-regulate the glucose-stimulated insulin release and result in an increa...

  6. Group boundary permeability moderates the effect of a dependency meta-stereotype on help-seeking behaviour.

    Science.gov (United States)

    Zhang, Lange; Kou, Yu; Zhao, Yunlong; Fu, Xinyuan

    2016-08-01

    Previous studies have found that when low-status group members are aware that their in-group is stereotyped as dependent by a specific out-group (i.e. a dependency meta-stereotype is salient), they are reluctant to seek help from the high-status out-group to avoid confirming the negative meta-stereotype. However, it is unclear whether low-status group members would seek more help in the context of a salient dependency meta-stereotype when there is low (vs. high) group boundary permeability. Therefore, we conducted two experiments to examine the moderating effect of permeability on meta-stereotype confirmation with a real group. In study 1, we manipulated the salience of the dependency meta-stereotype, measured participants' perceived permeability and examined their help-seeking behaviour in a real-world task. Participants who perceived low permeability sought more help when the meta-stereotype was salient (vs. not salient), whereas participants who perceived high permeability sought the same amount of help across conditions. In study 2, we manipulated the permeability levels and measured the dependency meta-stereotype. Participants who endorsed a high-dependency meta-stereotype sought more help than participants who endorsed a low-dependency meta-stereotype; this effect was particularly strong in the low-permeability condition. The implications of these results for social mobility and intergroup helping are discussed.

  7. The difference between observed and expected prevalence of MCAD deficiency in The Netherlands : a genetic epidemiological study

    NARCIS (Netherlands)

    Derks, Terry G J; Duran, Marinus; Waterham, Hans R; Reijngoud, Dirk-Jan; Ten Kate, Leo P; Smit, G Peter A

    2005-01-01

    Medium chain acyl coenzyme A dehydrogenase ( MCAD) deficiency is assumed to be the most common inherited disorder of mitochondrial fatty acid oxidation. Few reports mention the difference between the expected and observed prevalence of MCAD deficiency on the basis of the carrier frequency in the pop

  8. The difference between observed and expected prevalence of MCAD deficiency in The Netherlands: a genetic epidemiological study

    NARCIS (Netherlands)

    Derks, T.G.J.; Duran, M.; Waterham, H.R.; Reijngoud, D.J.; Kate, L.P. ten; Smit, G.P.A.

    2005-01-01

    Medium chain acyl coenzyme A dehydrogenase (MCAD) deficiency is assumed to be the most common inherited disorder of mitochondrial fatty acid oxidation. Few reports mention the difference between the expected and observed prevalence of MCAD deficiency on the basis of the carrier frequency in the popu

  9. The acylation of 1-acylglycero-3-phosphorylcholines by rat-liver microsomes

    NARCIS (Netherlands)

    Bosch, H. van den; Golde, L.M.G. van; Eibl, H.; Deenen, L.L.M. van

    1967-01-01

    1. 1. The transfer of acyl groups from acyl-coenzyme A derivatives to phosphatidylcholine by rat-liver microsomes was found to be significantly stimulated by the addition of synthetic 1-acylglycero-3-phosphorylcholines. Unsaturated acyl chains were transferred in preference to saturated ones, partic

  10. Neuropsychological Outcomes in Fatty Acid Oxidation Disorders: 85 Cases Detected by Newborn Screening

    Science.gov (United States)

    Waisbren, Susan E.; Landau, Yuval; Wilson, Jenna; Vockley, Jerry

    2013-01-01

    Mitochondrial fatty acid oxidation disorders include conditions in which the transport of activated acyl-Coenzyme A (CoA) into the mitochondria or utilization of these substrates is disrupted or blocked. This results in a deficit in the conversion of fat into energy. Most patients with fatty acid oxidation defects are now identified through…

  11. Disturbed hepatic carbohydrate management during high metabolic demand in medium-chain acyl-CoA dehydrogenase (MCAD)-deficient mice

    NARCIS (Netherlands)

    Herrema, H.J.; Derks, T.G.; Dijk, van T.H.; Bloks, V.W.; Gerding, A.; Havinga, R.; Tietge, U.J.; Müller, M.R.; Smit, G.P.; Kuipers, F.; Reijngoud, D.J.

    2008-01-01

    Medium-chain acyl-coenzyme A (CoA) dehydrogenase (MCAD) catalyzes crucial steps in mitochondrial fatty acid oxidation, a process that is of key relevance for maintenance of energy homeostasis, especially during high metabolic demand. To gain insight into the metabolic consequences of MCAD deficiency

  12. Safe and unsafe duration of fasting for children with MCAD deficiency

    NARCIS (Netherlands)

    Derks, Terry G J; van Spronsen, Francjan J; Rake, Jan Peter; van der Hilst, Christian S; Span, Mark M; Smit, G Peter A

    2007-01-01

    OBJECTIVE: To study the safe and unsafe duration of fasting in children with medium chain acyl-Coenzyme A dehydrogenase (MCAD) deficiency, the literature and the database on Dutch MCAD-deficient patients were searched for data on fasting studies in patients with MCAD deficiency. MATERIALS AND METHOD

  13. Effect of the DGAT1 K232A genotype of dairy cows on the milk metabolome and proteome

    NARCIS (Netherlands)

    Lu, J.; Boeren, S.; Hooijdonk, van A.C.M.; Vervoort, J.J.M.; Hettinga, K.A.

    2015-01-01

    Diglyceride O-acyltransferase 1 (DGAT1) is the enzyme that catalyzes the synthesis of triglycerides from diglycerides and acyl-coenzyme A. The DGAT1 K232A polymorphism was previously shown to have a significant influence on bovine milk production characteristics (milk yield, protein content, fat con

  14. Risk stratification by residual enzyme activity after newborn screening for medium-chain acyl-CoA dehyrogenase deficiency : data from a cohort study

    NARCIS (Netherlands)

    Touw, Catharina M. L.; Smit, G. Peter A.; de Vries, Maaike; de Klerk, Johannis B. C.; Bosch, Annet M.; Visser, Gepke; Mulder, Margot F.; Rubio-Gozalbo, M. Estela; Elvers, Bert; Niezen-Koning, Klary E.; Wanders, Ronald J. A.; Waterham, Hans R.; Reijngoud, Dirk-Jan; Derks, Terry G. J.

    2012-01-01

    Background: Since the introduction of medium-chain acyl coenzyme A dehydrogenase (MCAD) deficiency in population newborn bloodspot screening (NBS) programs, subjects have been identified with variant ACADM (gene encoding MCAD enzyme) genotypes that have never been identified in clinically ascertaine

  15. Risk stratification by residual enzyme activity after newborn screening for medium-chain acyl-CoA dehyrogenase deficiency: Data from a cohort study

    NARCIS (Netherlands)

    C.M.L. Touw (Catharina M L); G.P. Smit; M. de Vries (Maaike); J.B.C. de Klerk (Johannes); A.M. Bosch (Annet); G. Visser (G.); M.F. Mulder; M.E. Rubio-Gozalbo (Estela); L.H. Elvers; K.E. Niezen-Koning; R.J.A. Wanders (Ronald); H.R. Waterham; D.J. Reijngoud; T.G.J. Derks (Terry G J)

    2012-01-01

    textabstractBackground. Since the introduction of medium-chain acyl coenzyme A dehydrogenase (MCAD) deficiency in population newborn bloodspot screening (NBS) programs, subjects have been identified with variant ACADM (gene encoding MCAD enzyme) genotypes that have never been identified in clinicall

  16. Experimental evidence for protein oxidative damage and altered antioxidant defense in patients with medium-chain acyl-CoA dehydrogenase deficiency

    NARCIS (Netherlands)

    Derks, Terry G J; Touw, Catharina M L; Ribas, Graziela S; Biancini, Giovana B; Vanzin, Camila S; Negretto, Giovanna; Mescka, Caroline P; Reijngoud, Dirk Jan; Smit, G Peter A; Wajner, Moacir; Vargas, Carmen R

    2014-01-01

    The objective of this study was to test whether macromolecule oxidative damage and altered enzymatic antioxidative defenses occur in patients with medium-chain acyl coenzyme A dehydrogenase (MCAD) deficiency. We performed a cross-sectional observational study of in vivo parameters of lipid and prote

  17. Spinal cord injury: Role of endothelial differentiation gene family lysophosphatidic acid receptors

    OpenAIRE

    Santos Nogueira, Eva

    2015-01-01

    El daño tisular secundario que se produce tras una lesión de la médula espinal contribuye de manera significativa a las pérdidas funcionales que se observan pacientes que padecen este tipo de afectación. Aunque la regeneración axonal y la sustitución de las neuronas dañadas tras el traumatismo medular son objetivos importantes para reparar estas lesiones, el desarrollo de estrategias experimentales que tengan como meta evitar el daño secundario sobre axones, neuronas, mielina y las células gl...

  18. Definition of a Dependent Child

    CERN Multimedia

    Human Resources Department

    2005-01-01

    The Department of Human Resources wishes to remind members of the personnel that, under the provisions of § 6 of Administrative Circular No. 5 “Dependent child”, in the case of a child over 18 years of age the status of dependent child comes to an end once a course of studies is completed. Consequently, the payment of the dependent child allowance and the child's membership of the CERN Health Insurance Scheme terminate with effect from the last day of the month in which the course of study concerned ends. In this connection, members of the personnel are reminded that children who are no longer dependent according to the Staff Rules and Regulations and who are less than 26 years of age can nevertheless opt for membership of the normal health insurance under the terms and conditions laid down in the CERN Health Insurance Rules. The Department of Human Resources also wishes to remind members of the personnel that, pursuant to Article R IV 1.17 of the Staff Regulations, a member of the personnel is requ...

  19. A liver-specific defect of Acyl-CoA degradation produces hyperammonemia, hypoglycemia and a distinct hepatic Acyl-CoA pattern.

    Directory of Open Access Journals (Sweden)

    Nicolas Gauthier

    Full Text Available Most conditions detected by expanded newborn screening result from deficiency of one of the enzymes that degrade acyl-coenzyme A (CoA esters in mitochondria. The role of acyl-CoAs in the pathophysiology of these disorders is poorly understood, in part because CoA esters are intracellular and samples are not generally available from human patients. We created a mouse model of one such condition, deficiency of 3-hydroxy-3-methylglutaryl-CoA lyase (HL, in liver (HLLKO mice. HL catalyses a reaction of ketone body synthesis and of leucine degradation. Chronic HL deficiency and acute crises each produced distinct abnormal liver acyl-CoA patterns, which would not be predictable from levels of urine organic acids and plasma acylcarnitines. In HLLKO hepatocytes, ketogenesis was undetectable. Carboxylation of [2-(14C] pyruvate diminished following incubation of HLLKO hepatocytes with the leucine metabolite 2-ketoisocaproate (KIC. HLLKO mice also had suppression of the normal hyperglycemic response to a systemic pyruvate load, a measure of gluconeogenesis. Hyperammonemia and hypoglycemia, cardinal features of many inborn errors of acyl-CoA metabolism, occurred spontaneously in some HLLKO mice and were inducible by administering KIC. KIC loading also increased levels of several leucine-related acyl-CoAs and reduced acetyl-CoA levels. Ultrastructurally, hepatocyte mitochondria of KIC-treated HLLKO mice show marked swelling. KIC-induced hyperammonemia improved following administration of carglumate (N-carbamyl-L-glutamic acid, which substitutes for the product of an acetyl-CoA-dependent reaction essential for urea cycle function, demonstrating an acyl-CoA-related mechanism for this complication.

  20. Creating Directed Double-strand Breaks with the Ref Protein: A Novel Rec A-Dependent Nuclease from Bacteriophage P1

    Energy Technology Data Exchange (ETDEWEB)

    Gruenig, Marielle C.; Lu, Duo; Won, Sang Joon; Dulberger, Charles L.; Manlick, Angela J.; Keck, James L.; Cox, Michael M. (UW)

    2012-03-16

    The bacteriophage P1-encoded Ref protein enhances RecA-dependent recombination in vivo by an unknown mechanism. We demonstrate that Ref is a new type of enzyme; that is, a RecA-dependent nuclease. Ref binds to ss- and dsDNA but does not cleave any DNA substrate until RecA protein and ATP are added to form RecA nucleoprotein filaments. Ref cleaves only where RecA protein is bound. RecA functions as a co-nuclease in the Ref/RecA system. Ref nuclease activity can be limited to the targeted strands of short RecA-containing D-loops. The result is a uniquely programmable endonuclease activity, producing targeted double-strand breaks at any chosen DNA sequence in an oligonucleotide-directed fashion. We present evidence indicating that cleavage occurs in the RecA filament groove. The structure of the Ref protein has been determined to 1.4 {angstrom} resolution. The core structure, consisting of residues 77-186, consists of a central 2-stranded {beta}-hairpin that is sandwiched between several {alpha}-helical and extended loop elements. The N-terminal 76 amino acid residues are disordered; this flexible region is required for optimal activity. The overall structure of Ref, including several putative active site histidine residues, defines a new subclass of HNH-family nucleases. We propose that enhancement of recombination by Ref reflects the introduction of directed, recombinogenic double-strand breaks.

  1. Creating directed double-strand breaks with the Ref protein: a novel RecA-dependent nuclease from bacteriophage P1.

    Science.gov (United States)

    Gruenig, Marielle C; Lu, Duo; Won, Sang Joon; Dulberger, Charles L; Manlick, Angela J; Keck, James L; Cox, Michael M

    2011-03-11

    The bacteriophage P1-encoded Ref protein enhances RecA-dependent recombination in vivo by an unknown mechanism. We demonstrate that Ref is a new type of enzyme; that is, a RecA-dependent nuclease. Ref binds to ss- and dsDNA but does not cleave any DNA substrate until RecA protein and ATP are added to form RecA nucleoprotein filaments. Ref cleaves only where RecA protein is bound. RecA functions as a co-nuclease in the Ref/RecA system. Ref nuclease activity can be limited to the targeted strands of short RecA-containing D-loops. The result is a uniquely programmable endonuclease activity, producing targeted double-strand breaks at any chosen DNA sequence in an oligonucleotide-directed fashion. We present evidence indicating that cleavage occurs in the RecA filament groove. The structure of the Ref protein has been determined to 1.4 Å resolution. The core structure, consisting of residues 77-186, consists of a central 2-stranded β-hairpin that is sandwiched between several α-helical and extended loop elements. The N-terminal 76 amino acid residues are disordered; this flexible region is required for optimal activity. The overall structure of Ref, including several putative active site histidine residues, defines a new subclass of HNH-family nucleases. We propose that enhancement of recombination by Ref reflects the introduction of directed, recombinogenic double-strand breaks.

  2. A Dependence Study of $\\Xi^{*0}$ and $\\bar{\\Xi}^{*0}$ in 250 GeV/c $\\pi^-$. $K^-$ -nucleon Interactions

    Energy Technology Data Exchange (ETDEWEB)

    Seixas de Rezende, Fabio Antonio; /Rio de Janeiro, CBPF

    2005-04-01

    A direct measurement of the mass number (A) dependence of the production of the hyperon {Xi}*{sup 0} and its opposite {bar {Xi}}*{sup 0} in {pi}{sup -}, K{sup -} beam-nucleon interactions at 250 GeV/c is reported. The data derive from the experiment E769 at Fermilab. The results were obtained for different targets: Be, Al, Cu and W. It was observed the data are found to be well described by the parametrization {sigma}{sub A} = {sigma}{sub 0}A{sup {alpha}}, {alpha} being calculated for different beams. The results obtained are compared with those results of E769 experiment. The results shown here are preliminary.

  3. A Dependência do Calibre nos Cálculos Modelo do Fator de Forma Transverso Elétrico em Núcleos Complexos

    OpenAIRE

    1996-01-01

    Neste trabalho estudamos a dependência do calibre nos cálculos modelo do fator de forma transverso elétrico para núcleos complexos. Foram consideradas as diferentes prescrições que procuram levar parcialmente em consideração os efeitos da corrente de dois corpos, no cálculo do fator de forma transverso elétrico, através dos vínculos impostos pela equação da continuidade. Quando os estados nucleares satisfazem a equação de Schrödinger e a corrente a equação da continuidade as prescrições coinc...

  4. Public perception of alcohol dependence A percepção popular sobre a dependência alcoólica

    Directory of Open Access Journals (Sweden)

    Érica de Toledo Piza Peluso

    2008-03-01

    Full Text Available OBJECTIVE: To describe how the population of the city of São Paulo identifies alcohol dependence, which causes they attribute to this disorder and what is reported regarding stigma perception, risk of violence and emotional reactions. METHOD: Cross sectional study involving a probabilistic sample of 500 individuals in São Paulo aged 18 to 65 years. A structured questionnaire was used and started with a vignette describing an individual with alcohol dependence according to the DSM-IV and ICD-10, followed by a questionnaire divided into various sections examining the causes, stigma, risk of violence and emotional reactions to the case presented in the vignette. RESULTS: Less than 20% of the subjects reported to believe this was a mental illness. The causes considered most relevant were psychosocial ones, followed by moral causes. Alcohol dependence was associated with a high risk of violence and stigma by the part of others. In contrast, reactions declared by the subjects about their probable attitude were mostly positive. CONCLUSION: Alcohol dependence is seen as a psychosocial and moral problem. Negative images predominate regarding individuals with this disorder.OBJETIVO: Descrever como a população da cidade de São Paulo identifica a dependência alcoólica, quais causas atribui para explicar esse transtorno, e avaliar o que é percebido em relação ao estigma, risco de violência e as reações emocionais. MÉTODO: Foi realizado estudo de corte transversal com uma amostra probabilística de 500 indivíduos residentes em São Paulo, com idade entre 18 e 65 anos. Utilizou-se um questionário estruturado que se iniciava com a apresentação de uma vinheta descrevendo um indivíduo com dependência alcoólica (segundo o DSM-IV e a CID 10, seguido de um questionário dividido em várias seções examinando as causas, estigma, risco de violência e as reações emocionais ao caso apresentado na vinheta. RESULTADOS: Menos de 20% dos

  5. SIRT3 interacts with the daf-16 homolog FOXO3a in the Mitochondria, as well as increases FOXO3a Dependent Gene expression

    Science.gov (United States)

    Jacobs, Kristi Muldoon; Pennington, J. Daniel; Bisht, Kheem S.; Aykin-Burns, Nukhet; Kim, Hyun-Seok; Mishra, Mark; Sun, Lunching; Nguyen, Phuongmai; Ahn, Bong-Hyun; Leclerc, Jaime; Deng, Chu-Xia; Spitz, Douglas R.; Gius, David

    2008-01-01

    Cellular longevity is a complex process relevant to age-related diseases including but not limited to chronic illness such as diabetes and metabolic syndromes. Two gene families have been shown to play a role in the genetic regulation of longevity; the Sirtuin and FOXO families. It is also established that nuclear Sirtuins interact with and under specific cellular conditions regulate the activity of FOXO gene family proteins. Thus, we hypothesize that a mitochondrial Sirtuin (SIRT3) might also interact with and regulate the activity of the FOXO proteins. To address this we used HCT116 cells overexpressing either wild-type or a catalytically inactive dominant negative SIRT3. For the first time we establish that FOXO3a is also a mitochondrial protein and forms a physical interaction with SIRT3 in mitochondria. Overexpression of a wild-type SIRT3 gene increase FOXO3a DNA-binding activity as well as FOXO3a dependent gene expression. Biochemical analysis of HCT116 cells over expressing the deacetylation mutant, as compared to wild-type SIRT3 gene, demonstrated an overall oxidized intracellular environment, as monitored by increase in intracellular superoxide and oxidized glutathione levels. As such, we propose that SIRT3 and FOXO3a comprise a potential mitochondrial signaling cascade response pathway. PMID:18781224

  6. The superoxide dismutase SodA is targeted to the periplasm in a SecA-dependent manner by a novel mechanism.

    Science.gov (United States)

    Krehenbrink, Martin; Edwards, Anne; Downie, J Allan

    2011-10-01

    The manganese/iron-type superoxide dismutase (SodA) of Rhizobium leguminosarum bv. viciae 3841 is exported to the periplasm of R. l. bv. viciae and Escherichia coli. However, it does not possess a hydrophobic cleaved N-terminal signal peptide typically present in soluble proteins exported by the Sec-dependent (Sec) pathway or the twin-arginine translocation (TAT) pathway. A tatC mutant of R. l. bv. viciae exported SodA to the periplasm, ruling out export of SodA as a complex with a TAT substrate as a chaperone. The export of SodA was unaffected in a secB mutant of E. coli, but its export from R. l. bv. viciae was inhibited by azide, an inhibitor of SecA ATPase activity. A temperature-sensitive secA mutant of E. coli was strongly reduced for SodA export. The 10 N-terminal amino acid residues of SodA were sufficient to target the reporter protein alkaline phosphatase to the periplasm. Our results demonstrate the export of a protein lacking a classical signal peptide to the periplasm by a SecA-dependent, but SecB-independent targeting mechanism. Export of the R. l. bv. viciae SodA to the periplasm was not limited to the genus Rhizobium, but was also observed in other proteobacteria.

  7. BabA dependent binding of Helicobacter pylori to human gastric mucins cause aggregation that inhibits proliferation and is regulated via ArsS

    Science.gov (United States)

    Skoog, Emma C.; Padra, Médea; Åberg, Anna; Gideonsson, Pär; Obi, Ikenna; Quintana-Hayashi, Macarena P.; Arnqvist, Anna; Lindén, Sara K.

    2017-01-01

    Mucins in the gastric mucus layer carry a range of glycan structures, which vary between individuals, can have antimicrobial effect or act as ligands for Helicobacter pylori. Mucins from various individuals and disease states modulate H. pylori proliferation and adhesin gene expression differently. Here we investigate the relationship between adhesin mediated binding, aggregation, proliferation and adhesin gene expression using human gastric mucins and synthetic adhesin ligand conjugates. By combining measurements of optical density, bacterial metabolic activity and live/dead stains, we could distinguish bacterial aggregation from viability changes, enabling elucidation of mechanisms behind the anti-prolific effects that mucins can have. Binding of H. pylori to Leb-glycoconjugates inhibited the proliferation of the bacteria in a BabA dependent manner, similarly to the effect of mucins carrying Leb. Furthermore, deletion of arsS lead to a decrease in binding to Leb-glycoconjugates and Leb-decorated mucins, accompanied by decreased aggregation and absence of anti-prolific effect of mucins and Leb-glycoconjugates. Inhibition of proliferation caused by adhesin dependent binding to mucins, and the subsequent aggregation suggests a new role of mucins in the host defense against H. pylori. This aggregating trait of mucins may be useful to incorporate into the design of adhesin inhibitors and other disease intervention molecules. PMID:28106125

  8. BabA dependent binding of Helicobacter pylori to human gastric mucins cause aggregation that inhibits proliferation and is regulated via ArsS.

    Science.gov (United States)

    Skoog, Emma C; Padra, Médea; Åberg, Anna; Gideonsson, Pär; Obi, Ikenna; Quintana-Hayashi, Macarena P; Arnqvist, Anna; Lindén, Sara K

    2017-01-20

    Mucins in the gastric mucus layer carry a range of glycan structures, which vary between individuals, can have antimicrobial effect or act as ligands for Helicobacter pylori. Mucins from various individuals and disease states modulate H. pylori proliferation and adhesin gene expression differently. Here we investigate the relationship between adhesin mediated binding, aggregation, proliferation and adhesin gene expression using human gastric mucins and synthetic adhesin ligand conjugates. By combining measurements of optical density, bacterial metabolic activity and live/dead stains, we could distinguish bacterial aggregation from viability changes, enabling elucidation of mechanisms behind the anti-prolific effects that mucins can have. Binding of H. pylori to Le(b)-glycoconjugates inhibited the proliferation of the bacteria in a BabA dependent manner, similarly to the effect of mucins carrying Le(b). Furthermore, deletion of arsS lead to a decrease in binding to Le(b)-glycoconjugates and Le(b)-decorated mucins, accompanied by decreased aggregation and absence of anti-prolific effect of mucins and Le(b)-glycoconjugates. Inhibition of proliferation caused by adhesin dependent binding to mucins, and the subsequent aggregation suggests a new role of mucins in the host defense against H. pylori. This aggregating trait of mucins may be useful to incorporate into the design of adhesin inhibitors and other disease intervention molecules.

  9. Negociando a dependência: relações militares Brasil-Estados Unidos no início da Guerra Fria

    Directory of Open Access Journals (Sweden)

    Eduardo Svartman

    2014-12-01

    Full Text Available Durante a Guerra Fria, os laços estabelecidos entre as forças armadas dos Estados Unidos e os militares de sues aliados em diferentes regiões do mundo foi elemento importante tanto da estratégia anticomunista de Washington quanto da projeção deste país em áreas até então influenciadas pelas potências europeias. O artigo argumenta que o Brasil foi um caso ilustrativo deste processo, no qual apesar da profunda dependência e alinhamento, essas relações militares não foram lineares nem desprovidas de conflitos. O caráter instrumental conferido pelo Brasil a essas relações e a coincidência apenas parcial de objetivos entre os dois países fez com que, em determinadas ocasiões, os militares brasileiros procurassem renegociar a dependência. O impacto político dessa interação proporcionou a criação de espaços de formulação ideológica e de articulação política, polarizou a oficialidade em torno de temas como exploração de petróleo e participação na Guerra da Coreia e reforçou disposições para o protagonismo na política interna.

  10. O processo saúde-doença e a dependência química: interfaces e evolução

    Directory of Open Access Journals (Sweden)

    Elisângela Maria Machado Pratta

    Full Text Available O uso de drogas atualmente é considerado um grave e complexo problema de saúde pública. Falar sobre drogadição é discutir o processo saúde/doença, considerando-se os modelos que contribuem para a compreensão do fenômeno no momento atual e das estratégias de intervenção estabelecidas. Discutir a dependência química hoje exige uma reflexão sobre como a droga foi encarada ao longo da história, tendo em vista as questões de saúde/doença e os paradigmas hegemônicos em cada momento. Este estudo visa: a mostrar as bases teórico-conceituais de três eixos (saúde, doença e dependência química e suas interseções; b propiciar uma reflexão crítica sobre a questão da promoção da saúde frente à dependência de drogas, de acordo com o modelo biopsicossocial presente na atualidade. Esse modelo considera o ser humano integral, dotado de subjetividade, de saberes e fazeres próprios, ativo no processo saúde/doença, ressaltando a necessidade de rompimento com o modelo cartesiano ainda predominante na saúde.

  11. Cannabidiol stimulates Aml-1a-dependent glial differentiation and inhibits glioma stem-like cells proliferation by inducing autophagy in a TRPV2-dependent manner.

    Science.gov (United States)

    Nabissi, Massimo; Morelli, Maria Beatrice; Amantini, Consuelo; Liberati, Sonia; Santoni, Matteo; Ricci-Vitiani, Lucia; Pallini, Roberto; Santoni, Giorgio

    2015-10-15

    Glioma stem-like cells (GSCs) correspond to a tumor cell subpopulation, involved in glioblastoma multiforme (GBM) tumor initiation and acquired chemoresistance. Currently, drug-induced differentiation is considered as a promising approach to eradicate this tumor-driving cell population. Recently, the effect of cannabinoids (CBs) in promoting glial differentiation and inhibiting gliomagenesis has been evidenced. Herein, we demonstrated that cannabidiol (CBD) by activating transient receptor potential vanilloid-2 (TRPV2) triggers GSCs differentiation activating the autophagic process and inhibits GSCs proliferation and clonogenic capability. Above all, CBD and carmustine (BCNU) in combination overcome the high resistance of GSCs to BCNU treatment, by inducing apoptotic cell death. Acute myeloid leukemia (Aml-1) transcription factors play a pivotal role in GBM proliferation and differentiation and it is known that Aml-1 control the expression of several nociceptive receptors. So, we evaluated the expression levels of Aml-1 spliced variants (Aml-1a, b and c) in GSCs and during their differentiation. We found that Aml-1a is upregulated during GSCs differentiation, and its downregulation restores a stem cell phenotype in differentiated GSCs. Since it was demonstrated that CBD induces also TRPV2 expression and that TRPV2 is involved in GSCs differentiation, we evaluated if Aml-1a interacted directly with TRPV2 promoters. Herein, we found that Aml-1a binds TRPV2 promoters and that Aml-1a expression is upregulated by CBD treatment, in a TRPV2 and PI3K/AKT dependent manner. Altogether, these results support a novel mechanism by which CBD inducing TRPV2-dependent autophagic process stimulates Aml-1a-dependent GSCs differentiation, abrogating the BCNU chemoresistance in GSCs.

  12. Notch-1 signaling activates NF-κB in human breast carcinoma MDA-MB-231 cells via PP2A-dependent AKT pathway.

    Science.gov (United States)

    Li, Li; Zhang, Jing; Xiong, Niya; Li, Shun; Chen, Yu; Yang, Hong; Wu, Chunhui; Zeng, Hongjuan; Liu, Yiyao

    2016-04-01

    Breast cancer has a high incidence in the world and is becoming a leading cause of death in female patients due to its high metastatic ability. High expression of Notch-1 and its ligand Jagged-1 correlates with poor prognosis in breast cancer. Our previous work has shown that Notch-1 signaling pathway upregulates NF-κB transcriptional activity and induces the adhesion, migration and invasion of human breast cancer cell line MDA-MB-231. However, the role of Notch-1 in NF-κB activation is still poorly understood. Here, we aim to understand the exact mechanism that Notch-1 regulates NF-κB activity. In MDA-MB-231 cells where Notch-1 is constitutively activated, the phosphorylation of p85 and AKT (Tyr308/Ser473) is upregulated, indicating PI3K/AKT pathway is activated. Notch-1 activation caused the increase of PP2A phosphorylation at Tyr307, indicating Notch-1 inhibits PP2A activity. NF-κB transcriptional activity was evaluated by dual-luciferase reporter assay, and the results showed that, while silencing of Notch-1, PP2A activity was upregulated and NF-κB activity was downregulated, whereas PP2A inhibitor okadaic acid (OA) restored NF-κB activity. Immunofluorescence and Western blots showed that OA treatment antagonized the decrease of p65 nuclear translocation caused by Notch-1 silencing. Moreover, OA treatment also upregulated MMP-2, MMP-9 and VEGF mRNA expression levels, indicating OA rescues Notch-1 silencing that caused low cell invasion. Taken together, our results suggest that Notch-1-activating PI3K/AKT/NF-κB pathway is PP2A dependent; PP2A may be a potential therapeutic target in breast cancer.

  13. Lysophosphatidic acid inhibition of the accumulation of Pseudomonas aeruginosa PAO1 alginate, pyoverdin, elastase and LasA

    DEFF Research Database (Denmark)

    Laux, D.C.; Corson, J.M.; Givskov, Michael Christian;

    2002-01-01

    The pathogenesis of Pseudomonas aeruginosa is at least partially attributable to its ability to synthesize and secrete the siderophore pyoverdin and the two zinc metal loproteases elastase and LasA, and its ability to form biofilms in which bacterial cells are embedded in an alginate matrix...... pyoverdin. MPPA also inhibited biofilm formation. The inhibitory effects of MPPA occur independently of rpoS expression and without affecting the accumulation of the autoinducers N-(3-oxododecanoyl) homoserine lactone and N-butyryl-(L)-homoserine lactone, and may be due, at least in part, to the ability...

  14. Severe infantile hypotonia with ethylmalonic aciduria: case report.

    Science.gov (United States)

    Okuyaz, Cetin; Ezgü, Fatih Süheyl; Biberoglu, Gürsel; Zeviani, Massimo; Tiranti, Valeria; Yilgör, Esat

    2008-06-01

    An 8-month-old girl was admitted to an outpatient clinic with significant hypotonia and weakness. Organic acid analysis in urine revealed a significant increase in ethylmalonic acid. A deoxyribonucleic analysis revealed the presence of a 625G>A (G-to-A substitution at nucleotide 625) variant short-chain acyl-coenzyme A dehydrogenase gene polymorphism. With the clinical, biochemical and molecular findings, short-chain acyl-coenzyme A dehydrogenase deficiency was suspected. Because 625G>A and 511C>T (C-to-T substitution at nucleotide 511) genetic variations are also present in 14% of the general population, these are considered to be genetic sensitivity variations rather than causing a disease themselves and to result in possible short-chain acyl-coenzyme A dehydrogenase deficiency in the presence of environmental factors such as fever and hunger as well as cellular, biochemical, and other genetic factors. It was stressed that severe infantile hypotonia could also be the only manifestation of ethylmalonic aciduria spectrum disorders.

  15. 3-Methylcholanthrene, an AhR agonist, caused cell-cycle arrest by histone deacetylation through a RhoA-dependent recruitment of HDAC1 and pRb2 to E2F1 complex.

    Directory of Open Access Journals (Sweden)

    Chih-Cheng Chang

    Full Text Available We previously showed that treating vascular endothelial cells with 3-methylcholanthrene (3MC caused cell-cycle arrest in the Go/G1 phase; this resulted from the induction of p21 and p27 and a decreased level and activity of the cyclin-dependent kinase, Cdk2. We further investigated the molecular mechanisms that modulate cell-cycle regulatory proteins through the aryl-hydrocarbon receptor (AhR/Ras homolog gene family, member A (RhoA dependent epigenetic modification of histone. AhR/RhoA activation mediated by 3MC was essential for the upregulation of retinoblastoma 2 (pRb2 and histone deacetylase 1 (HDAC1, whereas their nuclear translocation was primarily modulated by RhoA activation. The combination of increased phosphatase and tensin homolog (PTEN activity and decreased phosphatidylinositide 3-kinase (PI3K activation by 3MC led to the inactivation of the Ras-cRaf pathway, which contributed to pRb2 hypophosphorylation. Increased HDAC1/pRb2 recruitment to the E2F1 complex decreased E2F1-transactivational activity and H3/H4 deacetylation, resulting in the downregulation of cell-cycle regulatory proteins (Cdk2/4 and Cyclin D3/E. Co-immunoprecipitation and electrophoretic mobility shift assay (EMSA results showed that simvastatin prevented the 3MC-increased binding activities of E2F1 proteins in their promoter regions. Additionally, RhoA inhibitors (statins reversed the effect of 3MC in inhibiting DNA synthesis by decreasing the nuclear translocation of pRb2/HDAC1, leading to a recovery of the levels of cell-cycle regulatory proteins. In summary, 3MC decreased cell proliferation by the epigenetic modification of histone through an AhR/RhoA-dependent mechanism that can be rescued by statins.

  16. A Influência da Confiança, Dependência e Comprometimento na Orientação de Longo Prazo de Varejistas para com os Fabricantes Líderes do Mercado de Bebidas

    Directory of Open Access Journals (Sweden)

    Ivan Lapuente Garrido

    2013-09-01

    Full Text Available O objetivo principal deste estudo é analisar como a confiança, o comprometimento e a dependência influenciam a orientação de longo prazo de varejistas para com o relacionamento com as empresas fabricantes de cervejas e refrigerantes líderes no mercado nacional. Para atingir o objetivo proposto, fizeram-se necessárias duas etapas de pesquisa. Na primeira etapa de cunho exploratório, foram realizadas entrevistas em profundidade com varejistas que possuíam relacionamento comercial com as empresas líderes do mercado nacional de cervejas e refrigerantes; esta etapa possibilitou aprofundamento sobre o campo de estudo e adequação de escalas aplicadas a segunda etapa de pesquisa. Na segunda etapa de pesquisa, por meio de questionário composto por questões fechadas, 299 varejistas avaliaram seu relacionamento com os fabricantes líderes de mercado. Utilizando-se de regressão múltipla, os resultados indicam que: (a a dependência dos varejistas influencia positivamente o comprometimento para com os fabricantes, (b a confiança influencia positivamente a orientação de longo prazo dos varejistas para com os fabricantes e, (c o comprometimento influencia positivamente a orientação de longo prazo dos varejistas para com os fabricantes. As hipóteses que consideravam a relação entre confiança e comprometimento e entre dependência e orientação de longo prazo não obtiveram significância estatística. DOI: 10.5585/remark.v12i3.2328

  17. An ultra-high performance liquid chromatography-tandem mass spectrometric assay for quantifying 3-ketocholanoic acid: Application to the human liver microsomal CYP3A-dependent lithocholic acid 3-oxidation assay.

    Science.gov (United States)

    Bansal, Sumit; Chai, Swee Fen; Lau, Aik Jiang

    2016-06-15

    Lithocholic acid (LCA), a hepatotoxic and carcinogenic bile acid, is metabolized to 3-ketocholanoic acid (3-KCA) by cytochrome P450 3A (CYP3A). In the present study, the objectives were to develop and validate an ultra-high performance liquid chromatography-tandem mass spectrometric (UPLC-MS/MS) method to quantify 3-KCA and apply it to the human liver microsomal CYP3A-dependent LCA 3-oxidation assay. Chromatographic separation was achieved on a Waters ACQUITY™ UPLC C18 column (50×2.1mm, 1.7μm) with a gradient system consisting of 0.1% v/v formic acid in water (solvent A) and 0.1% v/v formic acid in acetonitrile (solvent B). The retention time was 3.73min for 3-KCA and 2.73min for cortisol (internal standard). Positive electrospray ionization with multiple reaction monitoring (MRM) mode was used to quantify 3-KCA (m/z 375.4→135.2) and cortisol (m/z 363.5→121.0). The limit of detection of 3-KCA was 10μM, the lower limit of quantification was 33.3μM, and the calibration curve was linear from 0.05-10μM with r(2)>0.99. Intra-day and inter-day accuracy and precision were Michaelis-Menten model with an apparent Km of 26±7μM and Vmax of 303±50pmol/min/mg protein. This novel UPLC-MS/MS method for quantifying 3-KCA offers a specific, sensitive, and fast approach to determine liver microsomal LCA 3-oxidation.

  18. The Nonenzymatic Reactivity of the Acyl-Linked Metabolites of Mefenamic Acid toward Amino and Thiol Functional Group Bionucleophiles

    OpenAIRE

    2013-01-01

    Mefenamic acid (MFA), a carboxylic acid–containing nonsteroidal anti-inflammatory drug, is metabolized into the chemically-reactive MFA-1-O-acyl-glucuronide (MFA-1-O-G), MFA-acyl-adenylate (MFA-AMP), and the MFA-S-acyl-coenzyme A (MFA-CoA), all of which are electrophilic and capable of acylating nucleophilic sites on biomolecules. In this study, we investigate the nonenzymatic ability of each MFA acyl-linked metabolite to transacylate amino and thiol functional groups on the acceptor biomolec...

  19. Formation of Native and Non-native Interactions in Ensembles of Denatured ACBP Molecules from Paramagnetic Relaxation Enhancement Studies

    DEFF Research Database (Denmark)

    Kristjansdottir, S.; Lindorff-Larsen, Kresten; Fieber, W.;

    2005-01-01

    in the denatured states with those in the transition state for folding we also provided new insights into the mechanism of formation of the native state of this protein. Keywords: protein folding; denatured state; NMR; molecular dynamics; structural studies Abbreviations: ACBP, acyl coenzyme A binding protein; Gu...... of the residual structure in the denatured state of ACBP under these different conditions has enabled us to infer that regions in the N and C-terminal parts of the protein sequence have a high tendency to interact in the unfolded state under physiological conditions. By comparing the structural features...

  20. Reference: 782 [Arabidopsis Phenome Database[Archive

    Lifescience Database Archive (English)

    Full Text Available 782 http://metadb.riken.jp/db/SciNetS_ria224i/cria224u4ria224u18621978i Li Fenglin...ulate to high concentrations in the seed oils of a few plant species, including j...e report the identification and characterization of WSD1, a member of the bifunctional wax ester synthase/diacylglycerol acyltransfe... of diacylglycerol acyltransferase activity. Expression of the WSD1 gene in Sacch...ntification of the wax ester synthase/acyl-coenzyme A: diacylglycerol acyltransfe

  1. Model-independent interpretation of NMR relaxation data for unfolded proteins: the acid-denatured state of ACBP

    DEFF Research Database (Denmark)

    Modig, Kristofer; Poulsen, Flemming

    2008-01-01

    We have investigated the acid-unfolded state of acyl-coenzyme A binding protein (ACBP) using (15)N laboratory frame nuclear magnetic resonance (NMR) relaxation experiments at three magnetic field strengths. The data have been analyzed using standard model-free fitting and models involving....... The analysis also shows that the relaxation data are consistent with and complementary to information obtained from other parameters, especially secondary chemical shifts and residual dipolar couplings, and strengthens the conclusions of previous observations that three out of the four regions that form...

  2. Cloning and Expression of a Ralstonia eutropha HF39 Gene Mediating Indigo Formation in Escherichia coli

    Science.gov (United States)

    Drewlo, Sascha; Brämer, Christian O.; Madkour, Mohamed; Mayer, Frank; Steinbüchel, Alexander

    2001-01-01

    On complex medium Escherichia coli strains carrying hybrid plasmid pBEC/EE:11.0, pSKBEC/BE:9.0, pSKBEC/PP:3.3, or pSKBEC/PP:2.4 harboring genomic DNA of Ralstonia eutropha HF39 produced a blue pigment characterized as indigo by several chemical and spectroscopic methods. A 1,251-bp open reading frame (bec) was cloned and sequenced. The deduced amino acid sequence of bec showed only weak similarities to short-chain acyl-coenzyme A dehydrogenases, and the gene product catalyzed formation of indoxyl, a reactive preliminary stage for production of indigo. PMID:11282658

  3. Very long-chain acyl CoA dehydrogenase deficiency which was accepted as infanticide.

    Science.gov (United States)

    Eminoglu, Tuba F; Tumer, Leyla; Okur, Ilyas; Ezgu, Fatih S; Biberoglu, Gursel; Hasanoglu, Alev

    2011-07-15

    Very-long-chain acyl-coenzyme A (CoA) dehydrogenase deficiency (VLCADD) (OMIM #201475) is an autosomal recessive disorder of fatty acid oxidation. Major phenotypic expressions are hypoketotic hypoglycemia, hepatomegaly, cardiomyopathy, myopathy, rhabdomyolysis, elevated creatinine kinase, and lipid infiltration of liver and muscle. At the same time, it is a rare cause of Sudden Infant Death Syndrome (SIDS) or unexplained death in the neonatal period [1-4]. We report a patient with VLCADD whose parents were investigated for infanticide because her three previous siblings had suddenly died after normal deliveries.

  4. Clinical and biochemical monitoring of patients with fatty acid oxidation disorders

    DEFF Research Database (Denmark)

    Lund, Allan Meldgaard; Skovby, Flemming; Vestergaard, Helle

    2010-01-01

    carnitine is measured to monitor carnitine supplementation in patients with multiple acyl-coenzyme A dehydrogenase deficiency (MADD) and carnitine transporter deficiency (CTD) and to follow metabolic control and disclose deficiency states in other FAO disorders. We are evaluating long-chain acylcarnitines......-chain and carnitine transporter deficiencies. Eye examination is done in all, and liver ultrasonography in some patients with long-chain 3-hydroxyacyl-coenzyme A dehydrogenase/tri-functional protein (LCHAD/TFP) deficiencies. Biochemical follow-up includes determination of free carnitine and acylcarnitines. Free...

  5. The MLK-related kinase (MRK) is a novel RhoC effector that mediates lysophosphatidic acid (LPA)-stimulated tumor cell invasion.

    Science.gov (United States)

    Korkina, Olga; Dong, Zhiwan; Marullo, Allison; Warshaw, Gregg; Symons, Marc; Ruggieri, Rosamaria

    2013-02-22

    The small GTPase RhoC is overexpressed in many invasive tumors and is essential for metastasis. Despite its high structural homology to RhoA, RhoC appears to perform functions that are different from those controlled by RhoA. The identity of the signaling components that are differentially regulated by these two GTPases is only beginning to emerge. Here, we show that the MAP3K protein MRK directly binds to the GTP-bound forms of both RhoA and RhoC in vitro. However, siRNA-mediated depletion of MRK in cells phenocopies depletion of RhoC, rather than that of RhoA. MRK depletion, like that of RhoC, inhibits LPA-stimulated cell invasion, while depletion of RhoA increases invasion. We also show that active MRK enhances LPA-stimulated invasion, further supporting a role for MRK in the regulation of invasion. Depletion of either RhoC or MRK causes sustained myosin light chain phosphorylation after LPA stimulation. In addition, activation of MRK causes a reduction in myosin light chain phosphorylation. In contrast, as expected, depletion of RhoA inhibits myosin light chain phosphorylation. We also present evidence that both RhoC and MRK are required for LPA-induced stimulation of the p38 and ERK MAP kinases. In conclusion, we have identified MRK as a novel RhoC effector that controls LPA-stimulated cell invasion at least in part by regulating myosin dynamics, ERK and p38.

  6. Lysophosphatidic Acid Acyltransferase from Coconut Endosperm Mediates the Insertion of Laurate at the sn-2 Position of Triacylglycerols in Lauric Rapeseed Oil and Can Increase Total Laurate Levels

    Science.gov (United States)

    Knutzon, Deborah S.; Hayes, Thomas R.; Wyrick, Annette; Xiong, Hui; Maelor Davies, H.; Voelker, Toni A.

    1999-01-01

    Expression of a California bay laurel (Umbellularia californica) 12:0-acyl-carrier protein thioesterase, bay thioesterase (BTE), in developing seeds of oilseed rape (Brassica napus) led to the production of oils containing up to 50% laurate. In these BTE oils, laurate is found almost exclusively at the sn-1 and sn-3 positions of the triacylglycerols (T.A. Voelker, T.R. Hayes, A.C. Cranmer, H.M. Davies [1996] Plant J 9: 229–241). Coexpression of a coconut (Cocos nucifera) 12:0-coenzyme A-preferring lysophosphatitic acid acyltransferase (D.S. Knutzon, K.D. Lardizabal, J.S. Nelsen, J.L. Bleibaum, H.M. Davies, J.G. Metz [1995] Plant Physiol 109: 999–1006) in BTE oilseed rape seeds facilitates efficient laurate deposition at the sn-2 position, resulting in the acccumulation of trilaurin. The introduction of the coconut protein into BTE oilseed rape lines with laurate above 50 mol % further increases total laurate levels. PMID:10398708

  7. Detection of Serum Lysophosphatidic Acids Using Affinity Binding and Surface Enhanced Laser Desorption/Ionization (SELDI) Time of Flight Mass Spectrometry

    Science.gov (United States)

    2005-04-01

    VIB), Ghent University, Faculty of Medicine and Health Sciences, Albert Baertsoenkaai 3, B-9000 Ghent, Belgium, 2Department of Medicinal Chemistry, the...Chemistry 280, 4415-4421 20 Camus , D., Lyon, J. A., Reaudjareed, T., Haynes, J. D., and Diggs, C. L. (1987) Molecular and Biochemical Parasitology 26, 21-27

  8. The multigene family of lysophosphatidate acyltransferase (LPAT)-related enzymes in Ricinus communis: cloning and molecular characterization of two LPAT genes that are expressed in castor seeds.

    Science.gov (United States)

    Arroyo-Caro, José María; Chileh, Tarik; Kazachkov, Michael; Zou, Jitao; Alonso, Diego López; García-Maroto, Federico

    2013-02-01

    The multigene family encoding proteins related to lysophosphatidyl-acyltransferases (LPATs) has been analyzed in the castor plant Ricinus communis. Among them, two genes designated RcLPAT2 and RcLPATB, encoding proteins with LPAT activity and expressed in the developing seed, have been cloned and characterized in some detail. RcLPAT2 groups with well characterized members of the so-called A-class LPATs and it shows a generalized expression pattern in the plant and along seed development. Enzymatic assays of RcLPAT2 indicate a preference for ricinoleoyl-CoA over other fatty acid thioesters when ricinoleoyl-LPA is used as the acyl acceptor, while oleoyl-CoA is the preferred substrate when oleoyl-LPA is employed. RcLPATB groups with B-class LPAT enzymes described as seed specific and selective for unusual fatty acids. However, RcLPATB exhibit a broad specificity on the acyl-CoAs, with saturated fatty acids (12:0-16:0) being the preferred substrates. RcLPATB is upregulated coinciding with seed triacylglycerol accumulation, but its expression is not restricted to the seed. These results are discussed in the light of a possible role for LPAT isoenzymes in the channelling of ricinoleic acid into castor bean triacylglycerol.

  9. Detection of Serum Lysophosphatidic Acids Using Affinity Binding and Surface Enhanced Laser Deorption/Ionization (SELDI) Time of Flight Mass Spectrometry

    Science.gov (United States)

    2006-04-01

    tested these LPA analogues on insect Sf9 cells induced to express human LPA1, LPA2, and LPA3 receptors. While none of the analogues were found to be more...at all three receptors. The R-fluoromethylene phosphonate analogue 15 activated calcium release in LPA3-transfected insect Sf9 cells at a...of increases in intracellular calcium ([Ca2+]i) by 1-oleoyl LPA and 15 in LPA1, LPA2, or LPA3 expressing Sf9 cells. Sf9 cells expressing LPA1, LPA2

  10. Dual Action of Lysophosphatidate-Functionalised Titanium: Interactions with Human (MG63) Osteoblasts and Methicillin Resistant Staphylococcus aureus

    DEFF Research Database (Denmark)

    Skindersø, Mette Elena; Krogfelt, Karen Angeliki; Blom, Ashley;

    2015-01-01

    Titanium (Ti) is a widely used material for surgical implants; total joint replacements (TJRs), screws and plates for fixing bones and dental implants are forged from Ti. Whilst Ti integrates well into host tissue approximately 10% of TJRs will fail in the lifetime of the patient through a process...... known as aseptic loosening. These failures necessitate revision arthroplasties which are more complicated and costly than the initial procedure. Finding ways of enhancing early (osseo)integration of TJRs is therefore highly desirable and continues to represent a research priority in current biomaterial...

  11. The nicotine dependence associated with alcohol use and other psychoactive substance A dependência da nicotina associada ao uso de álcool e outras substâncias psicoativas

    Directory of Open Access Journals (Sweden)

    Rafaela Serra Bacchi

    2008-10-01

    Full Text Available To examine an association between nicotine dependence with alcohol, other psychoactive use, and depressive disorder. Smokers were recruited from Centro de Referência de Abordagem e Tratamento do Tabagismo at the Hospital das Clínicas da Universidade Estadual de Londrina (AHC/ UEL. All subjects were informed and gave then written consent for the research as approved by the Ethics Research Committee of Universidade Estadual de Londrina. The measures used were: structured questionnaire, alcohol, smoking, and psychoactive substance involvement screening test (ASSIST v 3.0, the Fagerström test for Nicotine Dependence (FTND, and the Diagnostic Interview for Research on Depressive disorder of the World Health Organization. Smokers presented the following socio-demographic characteristics: prevalence of the female sex and mean age of 47 years old with capacity for domestic activities and work. The mean age of onset of cigarette use for smokers was 16 years old. Fagerström’s test presented a medium punctuation of 6, so much for users of substances psicoativas, as for the ones that they don’t use them. Relationship between serious depression and the of psychoactive substances use was relevant for the research. This study evidenced an association among the use of the tobacco and other psychoactive substances, and depressive disorder. The health professional in smoking cessation intervention would be to identify subgroups of adult smokers, associated with depression, psychoactive substance use, and promote an intervention in both comorbidities and larger effectiveness of the smoking cessation. Analisar a associação entre a dependência de nicotina com o uso de álcool, outras substâncias psicoativas e transtorno depressivo. Os tabagistas foram recrutados a partir do Centro de Referência de Abordagem e Tratamento do Tabagismo no Hospital de Clínicas da Universidade Estadual de Londrina (AHC/UEL. Todos os participantes foram informados e

  12. Patatin-related phospholipase pPLAIIIδ increases seed oil content with long-chain fatty acids in Arabidopsis.

    Science.gov (United States)

    Li, Maoyin; Bahn, Sung Chul; Fan, Chuchuan; Li, Jia; Phan, Tien; Ortiz, Michael; Roth, Mary R; Welti, Ruth; Jaworski, Jan; Wang, Xuemin

    2013-05-01

    The release of fatty acids from membrane lipids has been implicated in various metabolic and physiological processes, but in many cases, the enzymes involved and their functions in plants remain unclear. Patatin-related phospholipase As (pPLAs) constitute a major family of acyl-hydrolyzing enzymes in plants. Here, we show that pPLAIIIδ promotes the production of triacylglycerols with 20- and 22-carbon fatty acids in Arabidopsis (Arabidopsis thaliana). Of the four pPLAIIIs (α, β, γ, δ), only pPLAIIIδ gene knockout results in a decrease in seed oil content, and pPLAIIIδ is most highly expressed in developing embryos. The overexpression of pPLAIIIδ increases the content of triacylglycerol and 20- and 22-carbon fatty acids in seeds with a corresponding decrease in 18-carbon fatty acids. Several genes in the glycerolipid biosynthetic pathways are up-regulated in pPLAIIIδ-overexpressing siliques. pPLAIIIδ hydrolyzes phosphatidylcholine and also acyl-coenzyme A to release fatty acids. pPLAIIIδ-overexpressing plants have a lower level, whereas pPLAIIIδ knockout plants have a higher level, of acyl-coenzyme A than the wild type. Whereas seed yield decreases in transgenic plants that ubiquitously overexpress pPLAIIIδ, seed-specific overexpression of pPLAIIIδ increases seed oil content without any detrimental effect on overall seed yield. These results indicate that pPLAIIIδ-mediated phospholipid turnover plays a role in fatty acid remodeling and glycerolipid production.

  13. Intracellular mediators of JAM-A-dependent epithelial barrier function.

    Science.gov (United States)

    Monteiro, Ana C; Parkos, Charles A

    2012-06-01

    Junctional adhesion molecule-A (JAM-A) is a critical signaling component of the apical junctional complex, a structure composed of several transmembrane and scaffold molecules that controls the passage of nutrients and solutes across epithelial surfaces. Observations from JAM-A-deficient epithelial cells and JAM-A knockout animals indicate that JAM-A is an important regulator of epithelial paracellular permeability; however, the mechanism(s) linking JAM-A to barrier function are not understood. This review highlights recent findings relevant to JAM-A-mediated regulation of epithelial permeability, focusing on the role of upstream and downstream signaling candidates. We draw on what is known about proteins reported to associate with JAM-A in other pathways and on known modulators of barrier function to propose candidate effectors that may mediate JAM-A regulation of epithelial paracellular permeability. Further investigation of pathways highlighted in this review may provide ideas for novel therapeutics that target debilitating conditions associated with barrier dysfunction, such as inflammatory bowel disease.

  14. A-dependence of weak nuclear structure functions

    Energy Technology Data Exchange (ETDEWEB)

    Haider, H.; Athar, M. Sajjad [Department of Physics, Aligarh Muslim University, Aligarh-202 002 (India); Simo, I. Ruiz [Dipartimento di Fisica, Universitá degli studi di Trento Via Sommarive 14, Povo (Trento) I-38123 (Italy)

    2015-05-15

    Effect of nuclear medium on the weak structure functions F{sub 2}{sup A}(x, Q{sup 2}) and F{sub 3}{sup A}(x, Q{sup 2}) have been studied using charged current (anti)neutrino deep inelastic scattering on various nuclear targets. Relativistic nuclear spectral function which incorporate Fermi motion, binding and nucleon correlations are used for the calculations. We also consider the pion and rho meson cloud contributions calculated from a microscopic model for meson-nucleus self-energies. Using these structure functions, F{sub i}{sup A}/F{sub i}{sup proton} and F{sub i}{sup A}/F{sub i}{sup deuteron}(i=2,3, A={sup 12}C, {sup 16}O, CH and H{sub 2}O) are obtained.

  15. Development of a Dependency Theory Toolbox for Database Design.

    Science.gov (United States)

    1987-12-01

    SDS) .......................... 15 2.3.3 Ceri and Gottlob .................................. 16 2.3.4 Relational Database Design Aid Version 1 (REDi...Ceri and Gottlob [8] - closure of a set of attributes - find minimal cover - determine keys - test for lossless joins - 3NF design - BCNF design 4...3.3 Ceri and Gottilob. In their normalization tool, Ceri and Gottlob implement several database design algorithms in the Prolog programming language

  16. Care plan for the patient with a dependent personality disorder

    Directory of Open Access Journals (Sweden)

    Ana María Ruiz Galán

    2010-11-01

    Full Text Available Personality is unique for each individual and can be defined as the dynamic collection of characteristics relative to emotions, thought and behaviour.Personality trout’s only mean a Personality Disorder (PD when they are inflexible and maladjusted and cause notable functional deterioration or uneasiness.According to Bermudez personality is “the enduring organization of structural and functional features, innate and acquired under the special conditions of each one’s development that shape the particular and specific collection of behaviour to face different situations”.According to the Diagnostic a Statistical Manual of Mental Disorders (DSM-IV, a Personality Disorder is “an enduring pattern of inner experience and behavior that deviates markedly from the expectations of the person’s culture is pervasive and an inflexible, is stable over time and leads to distress or impairment. The onset of these patterns of behaviour is the beginning of the adulthood and, in rare instances, early adolescence”.There are several types of Personality Disorders (paranoid, schizoid, borderline, antisocial, dependent…. Dependent Personality Disorder is one of the most frequent in the Mental Health Services.People who suffer from this disorder are unable to take a decision by themselves because they don’t have confidence in themselves. They need a lot of social support and affection until the point of deny their individuality by subordinating their desires to other person’s desires and permitting these persons to manage their lives. Maybe they feel desolated by separation and loss and can support any situation, even maltreatment to keep a relationship.As we a deduce this diagnosis is sensible to cultural influences. This work aims to elaborate an standarized plan of cares for the patient with Dependent Personality Disorder by using nursing Diagnosis of NANDA II, Outcomes Criteria (NOC and Interventions Criteria (NIC.

  17. The Avalon Beowulf Cluster: A Dependable Tool for Scientific Simulation

    Science.gov (United States)

    Warren, Michael

    2000-03-01

    Avalon is a 140 processor Alpha/Linux Beowulf cluster constructed entirely from commodity personal computer technology and freely available software. Computational Physics simulations performed on Avalon resulted in the award of a 1998 Gordon Bell price/performance prize for significant achievement in parallel processing. Avalon ranked as the 113th fastest computer in the world on the November 1998 TOP500 list, obtaining a result of 48.6 Gigaflops on the parallel Linpack benchmark. The price of hardware and final assembly labor for Avalon totalled 313,000 dollars in the fall of 1998. Avalon currently provides over 15,000 node-hours of production computing time per week, split among about 10 production users. Obtaining an equivalent amount of computing through Los Alamos institutional sources would cost a minimicrons of 30,000 per week. The machine also supports code development for another 60 users. Significant simulations have been performed on Avalon in fields of astrophysics, molecular dynamics, nonlinear dynamics as well as other areas. The largest single simulation performed on Avalon computed a total of over 10^16 floating point operations. We will describe some of the applications which have obtained good performance on Avalon, and their characteristics. Our goal has been to provide dependable cycles for computational physics, and not to perform research into clustered computing systems. One of the main lessons learned from the Avalon project is that the details of the hardware are not nearly as important as the attitudes and expectations of the users and managers of the hardware.

  18. Design of a dependable Interlock System for linear colliders

    CERN Document Server

    Nouvel, Patrice

    For high energy accelerators, the interlock system is a key part of the machine protection. The interlock principle is to inhibit the beam either on failure of critical equipment and/or on low beam quality evaluation. The dependability of such a system is the most critical parameter. This thesis presents the design of an dependable interlock system for linear collider with an application to the CLIC (Compact Linear Collider) project. This design process is based on the IEEE 1220 standard and is is divided in four steps. First, the specifications are established, with a focus on the dependability, more precisely the reliability and the availability of the system. The second step is the design proposal based on a functional analysis, the CLIC and interfaced systems architecture. Third, the feasibility study is performed, applying the concepts in an accelerator facility. Finally, the last step is the hardware verification. Its aim is to prove that the proposed design is able to reach the requirements.

  19. The Integrated periodogram of a dependent extremal event sequence

    DEFF Research Database (Denmark)

    Mikosch, Thomas Valentin; Zhao, Yuwei

    2015-01-01

    We investigate the asymptotic properties of the integrated periodogram calculated from a sequence of indicator functions of dependent extremal events. An event in Euclidean space is extreme if it occurs far away from the origin. We use a regular variation condition on the underlying stationary.......i.d. case a Brownian bridge appears. In the general case, we propose a stationary bootstrap procedure for approximating the distribution of the limiting process. The developed theory can be used to construct classical goodness-of-fit tests such as the Grenander–Rosenblatt and Cramér–von Mises tests which...

  20. Measuring the impact of a dependence among insured life lengths

    NARCIS (Netherlands)

    M. Denuit; J. Dhaene; C. Le Bailly De Tilleghem; S. Teghem

    2001-01-01

    Actuaries usually compute multiple life premiums based on the unrealistic assumption of independence of the lifelengths of insured persons. Many clinical studies, however, have demonstrated dependence of the lifetimes of paired lives such as husband and wife. In this respect, the present article tri

  1. Dicty_cDB: Contig-U10831-1 [Dicty_cDB

    Lifescience Database Archive (English)

    Full Text Available mplet... 43 0.033 CP000615_24( CP000615 |pid:none) Burkholderia vietnamiensis G4 chr... 43 0.0... ) RecName: Full=Peroxisomal acyl-coenzyme A oxidase 1; ... 87 2e-15 T20569( T20569 ) hypothetical protein F08A8.3 - Caenorhabdi...icus su... 51 1e-04 AF142581_1( AF142581 |pid:none) Streptomyces coelicolor acyl-CoA d... 51 1e-04 CP000903_2301( CP000903 |pid...i rw*isf*s**ikriiskfkyi**kifngtiin**ts*tnatsmfrdysitigpie*ygke skeisimv*idinv*sisiieicssi*f...re E Sequences producing significant alignments: (bits) Value N ( U87813 ) Dictyostelium discoideum AcoA (aco

  2. 常用英文缩略语名词解释

    Institute of Scientific and Technical Information of China (English)

    2015-01-01

    AMD:年龄相关性黄斑变性(age—related macular degeneration)DR:糖尿病视网膜病变(diabetic retinopathy)OIR:氧诱导视网膜病变(oxygen—induced retinopathy)Nrf2:核因子E2相关因子2(nuclear factor—E2-related factor 2)ARE:抗氧化反应元件(antioxidant response element)Hrd1:羟甲基戊二酰辅酶A还原酶降解蛋白1(hydroxymethyl glutaric acyl coenzyme A reductase degradation protein 1)。

  3. Micro method for determination of nonesterified fatty acid in whole blood obtained by fingertip puncture

    DEFF Research Database (Denmark)

    Hansen, Jesper Søndergaard; Munk, Jens; Gaster, Michael;

    2006-01-01

    background fluorescence reading, NEFAs were converted to acyl-CoA by the acyl-CoA synthetase and the NEFA content was calculated from fluorescence emission changes using palmitic acid as external standard. The FACI-50 NEFA method was compared with two commercially available methods for quantification of NEFA.......Diagnostic tools for early identification of subjects at high risk for type 2 diabetes and other obesity-related disorders are important in prevention of these diseases. Nonesterified fatty acids (NEFAs) have been suggested to serve as a prediagnostic marker of diabetes and obesity......-related disorders. In the current study, we developed a sensitive and reproducible micro method for quantification of NEFA in less than 10 microl whole blood. The method involves only two steps: (i) conversion of NEFA to fatty acid acyl-coenzyme A (acyl-CoA) esters using an acyl-CoA synthetase and (ii...

  4. Analysis on differentially expressed genes in watermelon rind color based on RNA-Seq

    Institute of Scientific and Technical Information of China (English)

    杨侃侃; 梁志怀; 吴才君

    2016-01-01

    In order to screen the genes controlling watermelon rind color and luster, the experiment was carried out with yellow watermelon skin mutants as tester and green wild type watermelon as control, and transcriptome sequencing and bioinformatics analysis were done. The results show that 34.27Gb clean data were got by transcriptome sequencing. There are 261 differentially expressed genes among Y1_vs_G1, Y2_vs_G2 and Y3_vs_G3.The pathways contenting most differentially expressed genes are plant hormone signal transduction pathway, phenylpropanoid biosynthesis pathway, photosynthesis pathway, starch and sucrose metabolism pathway. 9-cis-epoxycarotenoid dioxygenase (Cla002942), alcohol dehydrogenase (Cla004992), photosystem I reaction center subunit III, chloroplastic (precursor) (Cla009181), long-chain acyl coenzyme A synthetase (Cla017341), threonine dehydratase biosynthetic (Cla018352) candidates genes were screened out.

  5. Formation of hydrogen bonds precedes the rate-limiting formation of persistent structure in the folding of ACBP

    DEFF Research Database (Denmark)

    Teilum, K; Kragelund, B B; Knudsen, J;

    2000-01-01

    A burst phase in the early folding of the four-helix two-state folder protein acyl-coenzyme A binding protein (ACBP) has been detected using quenched-flow in combination with site-specific NMR-detected hydrogen exchange. Several of the burst phase structures coincide with a structure consisting...... of eight conserved hydrophobic residues at the interface between the two N and C-terminal helices. Previous mutation studies have shown that the formation of this structure is rate limiting for the final folding of ACBP. The burst phase structures observed in ACBP are different from the previously reported...... collapsed types of burst phase intermediates observed in the folding of other proteins....

  6. Thioredoxin-interacting protein regulates lipid metabolism via Akt/mTOR pathway in diabetic kidney disease.

    Science.gov (United States)

    Du, Chunyang; Wu, Ming; Liu, Huan; Ren, Yunzhuo; Du, Yunxia; Wu, Haijiang; Wei, Jinying; Liu, Chuxin; Yao, Fang; Wang, Hui; Zhu, Yan; Duan, Huijun; Shi, Yonghong

    2016-10-01

    Abnormal lipid metabolism contributes to the renal lipid accumulation, which is associated with diabetic kidney disease, but its precise mechanism remains unclear. The growing evidence demonstrates that thioredoxin-interacting protein is involved in regulating cellular glucose and lipid metabolism. Here, we investigated the effects of thioredoxin-interacting protein on lipid accumulation in diabetic kidney disease. In contrast to the diabetic wild-type mice, the physical and biochemical parameters were improved in the diabetic thioredoxin-interacting protein knockout mice. The increased renal lipid accumulation, expression of acetyl-CoA carboxylase, fatty acid synthase and sterol regulatory element binding protein-1, and phosphorylated Akt and mTOR associated with diabetes in wild-type mice was attenuated in diabetic thioredoxin-interacting protein knockout mice. Furthermore, thioredoxin-interacting protein knockout significantly increased the expression of peroxisome proliferator-activated receptor-α, acyl-coenzyme A oxidase 1 and carnitine palmitoyltransferaser 1 in diabetic kidneys. In vitro experiments, using HK-2 cells, revealed that knockdown of thioredoxin-interacting protein inhibited high glucose-mediated lipid accumulation, expression of acetyl-CoA carboxylase, fatty acid synthase and sterol regulatory element binding protein-1, as well as activation of Akt and mTOR. Moreover, knockdown of thioredoxin-interacting protein reversed high glucose-induced reduction of peroxisome proliferator-activated receptor-α, acyl-coenzyme A oxidase 1 and carnitine palmitoyltransferaser 1 expression in HK-2 cells. Importantly, blockade of Akt/mTOR signaling pathway with LY294002, a specific PI3K inhibitor, replicated these effects of thioredoxin-interacting protein silencing. Taken together, these data suggest that thioredoxin-interacting protein deficiency alleviates diabetic renal lipid accumulation through regulation of Akt/mTOR pathway, thioredoxin

  7. Green tea extract suppresses adiposity and affects the expression of lipid metabolism genes in diet-induced obese zebrafish

    Directory of Open Access Journals (Sweden)

    Hasumura Takahiro

    2012-08-01

    Full Text Available Abstract Background Visceral fat accumulation is one of the most important predictors of mortality in obese populations. Administration of green tea extract (GTE can reduce body fat and reduce the risk of obesity-related diseases in mammals. In this study, we investigated the effects and mechanisms of GTE on adiposity in diet-induced obese (DIO zebrafish. Methods Zebrafish at 3.5 to 4.5 months post-fertilization were allocated to four groups: non-DIO, DIO, DIO + 0.0025%GTE, and DIO + 0.0050%GTE. The non-DIO group was fed freshly hatched Artemia once daily (5 mg cysts/fish daily for 40 days. Zebrafish in the three DIO groups were fed freshly hatched Artemia three times daily (60 mg cysts/fish daily. Zebrafish in the DIO + 0.0025%GTE and DIO + 0.0050%GTE groups were exposed to GTE after the start of feeding three times daily for 40 days. Results Three-dimensional microcomputed tomography analysis showed that GTE exposure significantly decreased the volume of visceral but not subcutaneous fat tissue in DIO zebrafish. GTE exposure increased hepatic expression of the lipid catabolism genes ACOX1 (acyl-coenzyme A oxidase 1, palmitoyl, ACADM (acyl-coenzyme A dehydrogenase, c-4 to c-12 straight chain, and PPARA (peroxisome proliferator-activated receptor alpha. GTE exposure also significantly decreased the visceral fat expression of SOCS3 (suppressor of cytokine signaling 3b which inhibits leptin signaling. Conclusions The present results are consistent with those seen in mammals treated with GTE, supporting the validity of studying the effects of GTE in DIO zebrafish. Our results suggest that GTE exerts beneficial effects on adiposity, possibly by altering the expression of lipid catabolism genes and SOCS3.

  8. Famílias com um idoso dependente: avaliação da coesão e adaptação Las familias con un adulto mayor dependiente: evaluación de la cohesión y de la adaptación Families with a dependent elderly member: assessment of adaptation and cohesion

    Directory of Open Access Journals (Sweden)

    Isabel Araújo

    2012-03-01

    final del desarrollo del ciclo vital. Se les consideraron familias cohesivas y adaptadas aunque idealizan una mayor cohesión y adaptación.The aim of this study was to describe the typological profile and developmental stage of families that take care of an elderly dependent person; to identify the degree of dependency of the elderly person in the family context; and to evaluate cohesion and adaptation in families with a dependent elder. Methodologically, the study is positioned in the quantitative paradigm, and is a descriptive exploratory study. We administered a questionnaire, which included FACES III and the Barthel Index, to a group of families registered at health centers or family health clinics in a district in the North of Portugal. We chose a purposive sample of 108 families with an elderly dependent member. Data collection occurred from October 2007 to June 2008. From data analysis, we verified that families that include an elderly person at a high level of dependence are nuclear families and are in the final phase of development of the life cycle. They consider themselves cohesive and adapted families but ideally would like more cohesion and adaptation.

  9. Forbidding undesirable agreements: a dependence-based approach to the regulation of multi-agent systems

    NARCIS (Netherlands)

    P Turrini; D. Grossi; J. Broersen; J.-J.C. Meyer

    2010-01-01

    The purpose of this contribution is to set up a language to evaluate the results of concerted action among interdependent agents against predetermined properties that we can recognise as desirable from a deontic point of view. Unlike the standard view of logics to reason about coalitionally rational

  10. FINAL REMINDER EXTENSION/SUPPRESSION OF ALLOWANCE FOR A DEPENDENT CHILD AGED 18 AND ABOVE

    CERN Multimedia

    Social and Statutory Conditions; Tel. 72862-74474

    2000-01-01

    Members of the personnel with dependent children aged 18 or above (or reaching 18 during the 2000/2001 school year) who have not yet provided a SCHOOL CERTIFICATE must do so as soon as possible. If we have not received this certificate by November 28, 2000 at the latest, the child allowance will be withdrawn retroactively as from September 1,2000.

  11. Protein kinase A dependent phosphorylation activates Mg2+ efflux in the basolateral region of the liver.

    Science.gov (United States)

    Cefaratti, C; Ruse, Cristian

    2007-03-01

    Isolated hepatocytes in physiological [Na(+)]( 0 ) tightly maintain [Mg(2+)]( i ). Upon beta-adrenergic stimulation or in the presence of permeable cAMP, hepatocytes release 5-10% (1-3 mM Mg(2+)) of their total Mg(2+) content. However, isolated basolateral liver plasma membranes (bLPM), release Mg(2+) in the presence of [Na(+)]( o ) even in the absence of catecholamine stimulation. The data indicate that a physiological brake for Mg(2+) efflux is present in the hepatocyte and is removed upon cellular signaling. In contrast, this regulation "brake" is absent in purified bLPM thus rendering them fully active. The present study was carried out to reconstruct the missing regulatory component. Activation of Mg(2+) extrusion in intact cells is consistent with cAMP dependent phosphorylation of the transporter or a regulatory protein. Treatment of bLPM with a non-specific phosphatase such as alkaline phosphatase (AP), decreased Mg(2+) efflux by 70% compared to untreated bLPM. When AP-treated bLPM were loaded with protein kinase A (PKA), and stimulated with permeable cAMP, Mg(2+) transport fully recovered. These data suggest that phosphorylation of the Na(+)/Mg(2+) exchanger or a nearby protein activates the Mg(2+) transport mechanism in hepatocytes.

  12. Adoption and Business Value of Mobile Retail Channel: A Dependency Perspective on Mobile Commerce

    Science.gov (United States)

    Chou, Yen-Chun

    2013-01-01

    Forrest Research estimated that revenues derived from mobile devices will grow at an annual rate of 39% to reach $31 billion by 2016. With the tremendous market growth, mobile banking, mobile marketing, and mobile retailing have been recently introduced to satisfy customer needs. Academic and practical articles have widely discussed unique…

  13. THE MEANING OF BEING-A-CAREGIVER OF A DEPENDENT RELATIVE SUFFERING FROM CANCER: PALLIATIVE CONTRIBUTIONS

    Directory of Open Access Journals (Sweden)

    Joisy Aparecida Marchi

    2016-01-01

    Full Text Available Se tuvo como objetivo comprender el significado de ser-cuidador de un familiar con cáncer y con gran dependencia para las actividades diarias. Estudio fenomenológico fundamentado en Martín Heidegger realizado junto a tres núcleos integrados de salud en un municipio del noroeste de Paraná. La entrevista sucedió entre noviembre de 2012 y febrero de 2013 con 17 cuidadores familiares. Del análisis propuesto surgieron dos temáticas: “El ser-cuidador vivenciando distintos modos de disposición” y “Siendo-con-el: de la ocupación cotidiana a la preocupación libertadora”. Significó para el ser-cuidador aterrarse con el diagnóstico, horrorizarse con el tratamiento, aterrorizarse con los cuidados paliativos y ser-con-el-otro en la enfermedad. Se mostró ocupado con las cosas, pero también estuvo preocupado, evidenciando la solicitud en sus acciones. Esta base para un cuidado paliativo efectivo, debe permear la labor del enfermero visando que este profesional sea un verdadero ser-del-cuidado.

  14. Economic Value Added as a Dependence on the Corporate- and Market-life Cycle

    Directory of Open Access Journals (Sweden)

    Konečný Zdeněk

    2011-06-01

    Full Text Available Economic value added (EVA is an indicator which is widely used as the main tool for financial analysis. There are two methods of calculating it. The original method which was made by Stern & Stewart is defined as the net operating profit after taxes minus the cost of capital. The second method which was developed and used by the “Czech Ministry of Industry and Trade” indicates that, the economic value added is the difference between return on equity and the alternate cost of equity that is composed of separate risk rewards, and this “spread” is consequently multiplied by the equity. Economic value added depends on many factors. Whereas some of them are controllable by the company, others are not. This article is focused on the relationship between economic value added and the corporate- vs. market life cycle. This is because, there is an assumption that conditions for developing EVA changes depending on the actual phase of corporate- and market life cycle. In this research, the model by Reiners (2004 is used to identify the phases of corporate- and market life cycle and the method provided by the “Czech Ministry of Industry and Trade” is used to calculate EVA. However, there is a consideration of the relativity of EVA in the form of “spread” because of the intercompany comparison. The study found that, the highest spread is achieved by companies that are in the phase of expansion and phase of market expansion. On the contrary, companies in the phase of declension during market declension achieved the lowest and negative spread.

  15. Diversification of DnaA dependency for DNA replication in cyanobacterial evolution.

    Science.gov (United States)

    Ohbayashi, Ryudo; Watanabe, Satoru; Ehira, Shigeki; Kanesaki, Yu; Chibazakura, Taku; Yoshikawa, Hirofumi

    2016-05-01

    Regulating DNA replication is essential for all living cells. The DNA replication initiation factor DnaA is highly conserved in prokaryotes and is required for accurate initiation of chromosomal replication at oriC. DnaA-independent free-living bacteria have not been identified. The dnaA gene is absent in plastids and some symbiotic bacteria, although it is not known when or how DnaA-independent mechanisms were acquired. Here, we show that the degree of dependency of DNA replication on DnaA varies among cyanobacterial species. Deletion of the dnaA gene in Synechococcus elongatus PCC 7942 shifted DNA replication from oriC to a different site as a result of the integration of an episomal plasmid. Moreover, viability during the stationary phase was higher in dnaA disruptants than in wild-type cells. Deletion of dnaA did not affect DNA replication or cell growth in Synechocystis sp. PCC 6803 or Anabaena sp. PCC 7120, indicating that functional dependency on DnaA was already lost in some nonsymbiotic cyanobacterial lineages during diversification. Therefore, we proposed that cyanobacteria acquired DnaA-independent replication mechanisms before symbiosis and such an ancestral cyanobacterium was the sole primary endosymbiont to form a plastid precursor.

  16. Yersinia enterocolitica infection and tcaA-dependent killing of Caenorhabditis elegans.

    Science.gov (United States)

    Spanier, Britta; Starke, Mandy; Higel, Fabian; Scherer, Siegfried; Fuchs, Thilo M

    2010-09-01

    Caenorhabditis elegans is a validated model to study bacterial pathogenicity. We report that Yersinia enterocolitica strains W22703 (biovar 2, serovar O:9) and WA314 (biovar 1B, serovar O:8) kill C. elegans when feeding on the pathogens for at least 15 min before transfer to the feeding strain Escherichia coli OP50. The killing by Yersinia enterocolitica requires viable bacteria and, in contrast to that by Yersinia pestis and Yersinia pseudotuberculosis strains, is biofilm independent. The deletion of tcaA encoding an insecticidal toxin resulted in an OP50-like life span of C. elegans, indicating an essential role of TcaA in the nematocidal activity of Y. enterocolitica. TcaA alone is not sufficient for nematocidal activity because E. coli DH5alpha overexpressing TcaA did not result in a reduced C. elegans life span. Spatial-temporal analysis of C. elegans infected with green fluorescent protein-labeled Y. enterocolitica strains showed that Y. enterocolitica colonizes the nematode intestine, leading to an extreme expansion of the intestinal lumen. By low-dose infection with W22703 or DH5alpha followed by transfer to E. coli OP50, proliferation of Y. enterocolitica, but not E. coli, in the intestinal lumen of the nematode was observed. The titer of W22703 cells within the worm increased to over 10(6) per worm 4 days after infection while a significantly lower number of a tcaA knockout mutant was recovered. A strong expression of tcaA was observed during the first 5 days of infection. Y. enterocolitica WA314 (biovar 1B, serovar O:8) mutant strains lacking the yadA, inv, yopE, and irp1 genes known to be important for virulence in mammals were not attenuated or only slightly attenuated in their toxicity toward the nematode, suggesting that these factors do not play a significant role in the colonization and persistence of this pathogen in nematodes. In summary, this study supports the hypothesis that C. elegans is a natural host and nutrient source of Y. enterocolitica.

  17. Development of a Genetic Algorithm to Automate Clustering of a Dependency Structure Matrix

    Science.gov (United States)

    Rogers, James L.; Korte, John J.; Bilardo, Vincent J.

    2006-01-01

    Much technology assessment and organization design data exists in Microsoft Excel spreadsheets. Tools are needed to put this data into a form that can be used by design managers to make design decisions. One need is to cluster data that is highly coupled. Tools such as the Dependency Structure Matrix (DSM) and a Genetic Algorithm (GA) can be of great benefit. However, no tool currently combines the DSM and a GA to solve the clustering problem. This paper describes a new software tool that interfaces a GA written as an Excel macro with a DSM in spreadsheet format. The results of several test cases are included to demonstrate how well this new tool works.

  18. Protein kinase A-dependent step(s) in hepatitis C virus entry and infectivity.

    Science.gov (United States)

    Farquhar, Michelle J; Harris, Helen J; Diskar, Mandy; Jones, Sarah; Mee, Christopher J; Nielsen, Søren U; Brimacombe, Claire L; Molina, Sonia; Toms, Geoffrey L; Maurel, Patrick; Howl, John; Herberg, Friedrich W; van Ijzendoorn, Sven C D; Balfe, Peter; McKeating, Jane A

    2008-09-01

    Viruses exploit signaling pathways to their advantage during multiple stages of their life cycle. We demonstrate a role for protein kinase A (PKA) in the hepatitis C virus (HCV) life cycle. The inhibition of PKA with H89, cyclic AMP (cAMP) antagonists, or the protein kinase inhibitor peptide reduced HCV entry into Huh-7.5 hepatoma cells. Bioluminescence resonance energy transfer methodology allowed us to investigate the PKA isoform specificity of the cAMP antagonists in Huh-7.5 cells, suggesting a role for PKA type II in HCV internalization. Since viral entry is dependent on the host cell expression of CD81, scavenger receptor BI, and claudin-1 (CLDN1), we studied the role of PKA in regulating viral receptor localization by confocal imaging and fluorescence resonance energy transfer (FRET) analysis. Inhibiting PKA activity in Huh-7.5 cells induced a reorganization of CLDN1 from the plasma membrane to an intracellular vesicular location(s) and disrupted FRET between CLDN1 and CD81, demonstrating the importance of CLDN1 expression at the plasma membrane for viral receptor activity. Inhibiting PKA activity in Huh-7.5 cells reduced the infectivity of extracellular virus without modulating the level of cell-free HCV RNA, suggesting that particle secretion was not affected but that specific infectivity was reduced. Viral particles released from H89-treated cells displayed the same range of buoyant densities as did those from control cells, suggesting that viral protein association with lipoproteins is not regulated by PKA. HCV infection of Huh-7.5 cells increased cAMP levels and phosphorylated PKA substrates, supporting a model where infection activates PKA in a cAMP-dependent manner to promote virus release and transmission.

  19. Protein kinase A-dependent step(s) in hepatitis C virus entry and infectivity

    NARCIS (Netherlands)

    Farquhar, Michelle J.; Harris, Helen J.; Diskar, Mandy; Jones, Sarah; Mee, Christopher J.; Nielsen, Soren U.; Brimacombe, Claire L.; Molina, Sonia; Toms, Geoffrey L.; Maurel, Patrick; Howl, John; Herberg, Friedrich W.; van Ijzendoorn, Sven C. D.; Balfe, Peter; McKeating, Jane A.

    2008-01-01

    Viruses exploit signaling pathways to their advantage during multiple stages of their life cycle. We demonstrate a role for protein kinase A (PKA) in the hepatitis C virus (HCV) life cycle. The inhibition of PKA with H89, cyclic AMP (cAMP) antagonists, or the protein kinase inhibitor peptide reduced

  20. Environmental Degradation in a Dependent Region: The Rio Grande Valley of Mexico and Texas.

    Science.gov (United States)

    Jones, Richard C.

    1999-01-01

    Traces the interrelationships among dependence, environmental degradation, and human health in the Rio Grande Valley of Mexico and Texas. Presents a case study on environmental factors threatening family health in households located on both sides of the border; the health problems can be overcome by addressing restrictive zoning, health services,…

  1. Biological and environmental drivers of energy allocation in a dependent mammal, the Antarctic fur seal pup.

    Science.gov (United States)

    McDonald, Birgitte I; Goebel, Michael E; Crocker, Daniel E; Costa, Daniel P

    2012-01-01

    Little is known about how variation in the pattern and magnitude of parental effort influences allocation decisions in offspring. We determined the energy budget of Antarctic fur seal pups and examined the relative importance of timing of provisioning, pup traits (mass, condition, sex), and weather (wind chill and solar radiation) on allocation of energy obtained in milk by measuring milk energy intake, field metabolic rate (FMR), and growth rate in 48 Antarctic fur seal pups over three developmental stages (perinatal, premolt, and molt). The relative amount of milk energy used for growth was 59.1% ± 8.1% during the perinatal period but decreased to 23.4% ± 15.5% and 26.0% ± 13.9% during the premolt and molt. This decrease was associated with a greater amount of time spent fasting, along with an increase in pup activity while the mother was at sea foraging. Average daily milk intake, pup mass, and condition were all important in determining how much energy was available for growth, but the amount of energy obtained as milk was the single most important factor determining pup growth. While mean mass-specific FMR did not change with developmental stage (range = 1.74-1.77 mL O(2)/g/h), the factors that accounted for variation in FMR did. Weather (wind chill and solar radiation) and pup traits (mass and condition) influenced mass-specific FMR, but these impacts varied across development. This study provides information about the factors influencing how offspring allocate energy toward growth and maintenance and improves our predictions about how a changing environment may affect energy allocation in pups.

  2. Scarf’s State Reduction Method, Flexibility, and a Dependent Demand Inventory Model.

    Science.gov (United States)

    1983-04-01

    Microfounda- tions of Macroeconomics," American Economic Review , 72, 334-348. Brown, R. G., 1959. Statistical Forecasting for Inventory Control, McGraw-Hill...34Investment Decisions Under Uncertainty: The Irreversibility Effect," American Economic Review , 64, 1006-1012. Hillier, F. and G. Lieberman, 1980...Behavior of the Firm," American Economic Review , 72, 415-427. Rockefeller, R. T., 1970. Convex Analysis, Princeton University Press, Princeton, New Jersey

  3. Mount Athos, Wallachian princes (Voyvodes, John Kastriotis, and the Albanian tower, a dependency of Hilandar

    Directory of Open Access Journals (Sweden)

    Bojović Boško

    2006-01-01

    Full Text Available After the Ottoman conquest of the Balkan states, the princes of Moldavia and Wallachia, now the sultan's vassals, assumed responsibility for the Athonite monasteries. Reference in their donation charters to the founders of Hilandar Simeon Nemanja and St Sava ensured liturgical continuity, their names being added to a string of distinguished historical figures mentioned in prayers which contributed to the legitimacy and prestige of their power. The absence of such names from the charters to the Albanian Tower, or the Church of St Elias, two dependencies of Hilandar, may be explained by the fact that their founders were not that famous. Securing refuge at the time of the Ottoman invasion, John Kastriotis donated two villages to Hilandar in 1426, and in 1430 made an arrangement (adelphaton for lifetime use of the Tower on behalf of his four sons. One of them died on Mt Athos in 1431, and John himself, now monk Joachim, died in 1437. All the evidence testifies to close and long-lived connections between Albanian feudal lords and the Serbian imperial laura.

  4. A Dependence between Average Call Duration and Voice Transmission Quality: Measurement and applications

    NARCIS (Netherlands)

    Holub, J.; Beerends, J.G.; Smid, R.

    2004-01-01

    This contribution deals with the estimation of the relation between speech transmission quality and average call duration for a given network and portfolio of customers. It uses non-intrusive speech quality measurements on live speech calls. The basic idea behind this analysis is an expectation that

  5. Performance analysis of a dependable scheduling strategy based on a fault-tolerant grid model

    Institute of Scientific and Technical Information of China (English)

    WANG Yuanzhuo; LIN Chuang; YANG Yang; SHAN Zhiguang

    2007-01-01

    The grid provides an integrated computer platform composed of differentiated and distributed systems.These resources are dynamic and heterogeneous.In this paper,a novel fault-tolerant grid-scheduling model is pre sented based on Stochastic Petri Nets (SPN) to assure the heterogeneity and dynamism of the grid system.Also,a new grid-scheduling strategy,the dependable strategy for the shortest expected accomplishing time (DSEAT),is put forward,in which the dependability factor is introduced in the task-dispatching strategy.In the end,the performance of the scheduling strategy based on the fault-tolerant gridscheduling model is analyzed by an software package,named SPNP.The numerical results show that dynamic resources will increase the response time for all classes of tasks in differing degrees.Compared with shortest expected accomplishing time (SEAT) strategy,the DSEAT strategy can reduce the negative effects of dynamic and autonomic resources to some extent so as to guarantee a high quality of service (QoS).

  6. Metabolism of β-valine via a CoA-dependent ammonia lyase pathway

    NARCIS (Netherlands)

    Otzen, Marleen; Crismaru, Ciprian G.; Postema, Christiaan P.; Wijma, Hein J.; Heberling, Matthew M.; Szymanski, Wiktor; de Wildeman, Stefaan; Janssen, Dick B.

    2015-01-01

    Pseudomonas species strain SBV1 can rapidly grow on medium containing β-valine as a sole nitrogen source. The tertiary amine feature of β-valine prevents direct deamination reactions catalyzed by aminotransferases, amino acid dehydrogenases, and amino acid oxidases. However, lyase- or aminomutase-me

  7. Towards a dependable homogeneous many-processor system-on-chip

    NARCIS (Netherlands)

    Zhang, Xiao

    2014-01-01

    Nowadays, dependable computing systems are widely required in mission-critical applications. While the advance in CMOS technology enables smaller and faster circuits, the dependability of modern ICs has worsened as a result of the shrinking dimensions of MOS transistors and the increasing complexity

  8. Improving Regression Testing through Modified Ant Colony Algorithm on a Dependency Injected Test Pattern

    Directory of Open Access Journals (Sweden)

    G.Keerthi Lakshmi

    2012-03-01

    Full Text Available Performing regression testing on a pre production environment is often viewed by software practitioners as a daunting task since often the test execution shall by-pass the stipulated downtime or the test coverage would be non linear. Choosing the exact test cases to match this type of complexity not only needs prior knowledge of the system, but also a right use of calculations to set the goals right. On systems that are just entering the production environment after getting promoted from the staging phase, trade-offs are often needed to between time and the test coverage to ensure the maximum test cases are covered within the stipulated time. There arises a need to refine the test cases to accommodate the maximum test coverage it makes within the stipulated period of time since at most of the times, the most important test cases are often not deemed to qualify under the sanity test suite and any bugs that creped in them would go undetected until it is found out by the actual user at firsthand. Hence An attempt has been made in the paper to layout a testing framework to address the process of improving the regression suite by adopting a modified version of the Ant Colony Algorithm over and thus dynamically injecting dependency over the best route encompassed by the ant colony.

  9. A-dependence of hadron-nucleus massive lepton pair production

    Energy Technology Data Exchange (ETDEWEB)

    Ayala Filho, A.L.; Ducati, M.B. Gay [Rio Grande do Sul Univ., Porto Alegre, RS (Brazil). Inst. de Fisica; Epele, L.N.; Canal, C.A. Garcia [La Plata Univ. Nacional (Argentina). Lab. de Fisica Teorica

    1995-12-31

    The nuclear effects in the small x region of hadron-nucleus Drell-Yan processes at 800 GeV analyzed. We employ the parton recombination model to describe the suppression in the R{sup pA}{sub DY} (x{sub 2}) ratio as compared with R{sub EMC} (x), in the same kinematical region. A good agreement with experimental results is obtained. (author) 9 refs., 2 figs., 1 tab.

  10. [ital A] dependence of hadron-nucleus massive lepton pair production

    Energy Technology Data Exchange (ETDEWEB)

    Ayala F, A.L.; Gay Ducati, M.B. (Instituto de Fisica, Universidade Federal do Rio Grande do Sul, Caixa Postal 15051, 91500 Porto Alegre, RS (Brazil)); Epele, L.N.; Garcia Canal, C.A. (Laboratorio de Fisica Teorica, Universidad Nacional de La Plata, CC 67, 1900 La Plata (Argentina))

    1994-01-01

    Nuclear effects in the small [ital x] region of hadron-nucleus Drell-Yan processes at 800 GeV are analyzed. We employ the parton recombination model to describe the suppression in the [ital R][sub DY][sup [ital p][ital A

  11. Crank-Nicholson difference scheme for a stochastic parabolic equation with a dependent operator coefficient

    Science.gov (United States)

    Ashyralyev, Allaberen; Okur, Ulker

    2016-08-01

    In the present paper, the Crank-Nicolson difference scheme for the numerical solution of the stochastic parabolic equation with the dependent operator coefficient is considered. Theorem on convergence estimates for the solution of this difference scheme is established. In applications, convergence estimates for the solution of difference schemes for the numerical solution of three mixed problems for parabolic equations are obtained. The numerical results are given.

  12. História da enfermagem psiquiátrica e a dependência química no Brasil: atravessando a história para reflexão Historia de la enfermería psiquiátrica y la dependencia química en el Brasil: atravesando la historia para la reflexión History of the psychiatric nursing and chemical dependency in Brazil: crossing the history for reflection

    Directory of Open Access Journals (Sweden)

    Amanda Márcia dos Santos Reinaldo

    2007-12-01

    Full Text Available A formação do enfermeiro em Enfermagem Psiquiátrica e na área de dependências químicas norteia a discussão desse artigo, tendo em vista a complexidade dos problemas relacionados ao ensino de enfermagem, saúde mental, psiquiatria e álcool e drogas. Trata-se de um artigo de revisão de literatura onde as autoras compilaram fontes primárias e secundárias sobre o tema e, a partir da leitura do material bibliográfico, fizeram análises e reflexões acerca dos atravessamentos históricos que permeiam a história da Enfermagem Psiquiátrica e a dependência química no Brasil. Os resultados apontam para uma evolução do tema álcool e drogas dada a magnitude do problema na sociedade contemporânea. Em relação à Enfermagem Psiquiátrica, o ensino apresenta mudanças devidas à evolução histórica da psiquiatria que devem ser consideradas durante a formação do profissional enfermeiro. Ambas as temáticas encontram pontos de aproximação e distanciamento conforme o contexto em que são analisadas.La formación del enfermero en enfermería psiquiátrica y en el área de dependencias químicas, orienta la discusión de este artículo teniendo a la vista la complejidad de los problemas relacionados con la enseñanza de enfermería, salud mental, psiquiátrica, alcohol y drogas. Se trata de un artículo de revisión de la literatura donde las autoras compilaron fuentes primarias y secundarias sobre el tema y a partir de la lectura del material bibliográfico se realizaron análisis y reflexiones acerca de los acontecimientos históricos que permean la historia de la enfermería psiquiátrica y la dependencia química en el Brasil. Los resultados apuntan para una evolución del tema alcohol y drogas debido a la magnitud del problema en la sociedad contemporánea. En relación a la enfermería psiquiátrica, la enseñanza presenta cambios debido a la evolución histórica de la psiquiátrica que deben ser consideradas durante la formaci

  13. 小肠胆固醇吸收相关蛋白的研究进展%Several proteins involved in absorption of cholesterol in small intestine

    Institute of Scientific and Technical Information of China (English)

    袁敏; 徐东刚

    2015-01-01

    多种蛋白参与了小肠胆固醇的吸收,其中尼曼-匹克C1型类似蛋白1(Niemann-Pick C1 like 1,NPC1L1)主要介导小肠对胆固醇的吸收;小肠吸收的游离胆固醇在酰基辅酶A-胆固醇酰基转移酶2[acyl-coenzyme A(CoA)∶cholesterol acyltransferase 2,ACAT2]的催化下形成胆固醇酯并经淋巴系统进入血液循环,而未被酯化的胆固醇则通过ATP结合盒转运蛋白G5/G8[ATP-binding cassette(ABC) transporters G5/G8,ABCG5/ABCG8]分泌入肠腔,转录因子肝X受体( liver X receptor,LXR)在小肠胆固醇吸收过程中发挥了重要的调节作用。该文主要对小肠胆固醇吸收相关蛋白NPC1 L1、ABCG5/ABCG8、ACAT2和LXR的研究进展进行了综述。%Several proteins are involved in the absorption of cholesterol in small intestine.Niemann-Pick C1 like 1 (NPC1L1) mainly mediates the absorption of cholesterol, and acyl-coenzyme A ( CoA)∶cholesterol acyltransferase 2 (ACAT2) catalyzes the free cholesterol absorpted by intestine into cholesterol ester,while unesterified free cholesterol is secreted into intestinal lumen by ATP-binding cassette(ABC) transporters G5/G8(ABCG5/ABCG8).Transcription factor liver X receptor( LXR) plays an important role in the process of intestinal cholesterol absorption.The research progress in NPC1L1,ABCG5/ABCG8,ACAT2 and LXR is reviewed in this article.

  14. The minor C-allele of rs2014355 in ACADS is associated with reduced insulin release following an oral glucose load

    Directory of Open Access Journals (Sweden)

    Pisinger Charlotta

    2011-01-01

    Full Text Available Abstract Background A genome-wide association study (GWAS using metabolite concentrations as proxies for enzymatic activity, suggested that two variants: rs2014355 in the gene encoding short-chain acyl-coenzyme A dehydrogenase (ACADS and rs11161510 in the gene encoding medium-chain acyl-coenzyme A dehydrogenase (ACADM impair fatty acid β-oxidation. Chronic exposure to fatty acids due to an impaired β-oxidation may down-regulate the glucose-stimulated insulin release and result in an increased risk of type 2 diabetes (T2D. We aimed to investigate whether the two variants associate with altered insulin release following an oral glucose load or with T2D. Methods The variants were genotyped using KASPar® PCR SNP genotyping system and investigated for associations with estimates of insulin release and insulin sensitivity following an oral glucose tolerance test (OGTT in a random sample of middle-aged Danish individuals (nACADS = 4,324; nACADM = 4,337. The T2D-case-control study involved a total of ~8,300 Danish individuals (nACADS = 8,313; nACADM = 8,344. Results In glucose-tolerant individuals the minor C-allele of rs2014355 of ACADS associated with reduced measures of serum insulin at 30 min following an oral glucose load (per allele effect (β = -3.8% (-6.3%;-1.3%, P = 0.003, reduced incremental area under the insulin curve (β = -3.6% (-6.3%;-0.9%, P = 0.009, reduced acute insulin response (β = -2.2% (-4.2%;0.2%, P = 0.03, and with increased insulin sensitivity ISIMatsuda (β = 2.9% (0.5%;5.2%, P = 0.02. The C-allele did not associate with two other measures of insulin sensitivity or with a derived disposition index. The C-allele was not associated with T2D in the case-control analysis (OR 1.07, 95% CI 0.96-1.18, P = 0.21. rs11161510 of ACADM did not associate with any indices of glucose-stimulated insulin release or with T2D. Conclusions In glucose-tolerant individuals the minor C-allele of rs2014355 of ACADS was associated with reduced

  15. Membrane Stresses Induced by Overproduction of Free Fatty Acids in Escherichia coli.

    Energy Technology Data Exchange (ETDEWEB)

    Lennen, Rebecca M.; Kruziki, Max A.; Kumar, Kritika; Zinkel, Robert A.; Burnum, Kristin E.; Lipton, Mary S.; Hoover, Spencer W.; Ranatunga, Don Ruwan; Wittkopp, Tyler M.; Marner II, Wesley D.; Pfleger, Brian F.

    2011-11-01

    Microbially produced fatty acids are potential precursors to high energy density biofuels, including alkanes and alkyl ethyl esters by either catalytic conversion of free fatty acids (FFAs) or enzymatic conversions of acyl-acyl carrier protein or acyl-coenzyme A intermediates. Metabolic engineering efforts aimed at overproducing FFAs in Escherichia coli have achieved less than 30% of the maximum theoretical yield on the supplied carbon source. In this work, the viability, morphology, transcript levels, and protein levels of a strain of E. coli that overproduces medium chain length FFAs was compared to an engineered control strain. By early stationary phase, an 85% reduction in viable cell counts and exacerbated loss of inner membrane integrity were observed in the FFA overproducing strain. These effects were enhanced in strains endogenously producing FFAs compared to strains exposed to exogenously fed FFAs. Under two sets of cultivation conditions, long chain unsaturated fatty acid content greatly increased and the expression of genes and proteins required for unsaturated fatty acid biosynthesis were significantly decreased. Membrane stresses were further implicated by increased expression of genes and proteins of the phage shock response, the MarA/Rob/SoxS regulon, and the nuo and cyo operons of aerobic respiration. Gene deletion studies confirmed the importance of the phage shock proteins and Rob for maintaining cell viability, however little to no change in FFA titers was observed after 24 h cultivation. The results of this study serve as a baseline for future targeted attempts to improve FFA yields and titers in E. coli.

  16. Molecular cloning and nutrient regulation analysis of long chain acyl-CoA synthetase 1 gene in grass carp, Ctenopharyngodon idella L.

    Science.gov (United States)

    Cheng, Han-Liang; Chen, Shuai; Xu, Jian-He; Yi, Le-Fei; Peng, Yong-Xing; Pan, Qian; Shen, Xin; Dong, Zhi-Guo; Zhang, Xia-Qing; Wang, Wen-Xiang

    2017-02-01

    Long chain acyl-CoA synthetase 1 (ACSL1), a key regulatory enzyme of fatty acid metabolism, catalyzes the conversion of long-chain fatty acids to acyl-coenzyme A. The full-length cDNAs of ACSL1a and ACSL1b were cloned from the liver of a grass carp. Both cDNAs contained a 2094bp open reading frame encoding 697 amino acids. Amino acid sequence alignment showed that ACSL1a shared 73.5% sequence identity with ACSL1b. Each of the two ACSL1s proteins had a transmembrane domain, a P-loop domain, and L-, A-, and G-motifs, which were relatively conserved in comparison to other vertebrates. Relative expression profile of ACSL1 mRNAs in different tissues indicated that ACSL1a is highly expressed in heart, mesenteric adipose, and brain tissues, whereas ACSL1b is highly expressed in heart, white muscle, foregut, and liver tissues. Nutrient regulation research showed that the expression levels of ACSL1a and ACSL1b were significantly down-regulated when 3, 6, and 9% fish oil were added in diet of grass carp as compared to the control group. However, no significant difference in the levels of ACSL1 mRNA was observed between the experimental groups. This study demonstrated the relationship between ACSL1a and ACSL1b genes in grass carp and laid a foundation for further research on ACSL family members in other species.

  17. Purification of a jojoba embryo wax synthase, cloning of its cDNA, and production of high levels of wax in seeds of transgenic arabidopsis.

    Science.gov (United States)

    Lardizabal, K D; Metz, J G; Sakamoto, T; Hutton, W C; Pollard, M R; Lassner, M W

    2000-03-01

    Wax synthase (WS, fatty acyl-coenzyme A [coA]: fatty alcohol acyltransferase) catalyzes the final step in the synthesis of linear esters (waxes) that accumulate in seeds of jojoba (Simmondsia chinensis). We have characterized and partially purified this enzyme from developing jojoba embryos. A protein whose presence correlated with WS activity during chromatographic fractionation was identified and a cDNA encoding that protein was cloned. Seed-specific expression of the cDNA in transgenic Arabidopsis conferred high levels of WS activity on developing embryos from those plants. The WS sequence has significant homology with several Arabidopsis open reading frames of unknown function. Wax production in jojoba requires, in addition to WS, a fatty acyl-CoA reductase (FAR) and an efficient fatty acid elongase system that forms the substrates preferred by the FAR. We have expressed the jojoba WS cDNA in Arabidopsis in combination with cDNAs encoding the jojoba FAR and a beta-ketoacyl-CoA synthase (a component of fatty acid elongase) from Lunaria annua. (13)C-Nuclear magnetic resonance analysis of pooled whole seeds from transgenic plants indicated that as many as 49% of the oil molecules in the seeds were waxes. Gas chromatography analysis of transmethylated oil from individual seeds suggested that wax levels may represent up to 70% (by weight) of the oil present in those seeds.

  18. Changes in cardiac substrate transporters and metabolic proteins mirror the metabolic shift in patients with aortic stenosis.

    Directory of Open Access Journals (Sweden)

    Lisa C Heather

    Full Text Available In the hypertrophied human heart, fatty acid metabolism is decreased and glucose utilisation is increased. We hypothesized that the sarcolemmal and mitochondrial proteins involved in these key metabolic pathways would mirror these changes, providing a mechanism to account for the modified metabolic flux measured in the human heart. Echocardiography was performed to assess in vivo hypertrophy and aortic valve impairment in patients with aortic stenosis (n = 18. Cardiac biopsies were obtained during valve replacement surgery, and used for western blotting to measure metabolic protein levels. Protein levels of the predominant fatty acid transporter, fatty acid translocase (FAT/CD36 correlated negatively with levels of the glucose transporters, GLUT1 and GLUT4. The decrease in FAT/CD36 was accompanied by decreases in the fatty acid binding proteins, FABPpm and H-FABP, the β-oxidation protein medium chain acyl-coenzyme A dehydrogenase, the Krebs cycle protein α-ketoglutarate dehydrogenase and the oxidative phosphorylation protein ATP synthase. FAT/CD36 and complex I of the electron transport chain were downregulated, whereas the glucose transporter GLUT4 was upregulated with increasing left ventricular mass index, a measure of cardiac hypertrophy. In conclusion, coordinated downregulation of sequential steps involved in fatty acid and oxidative metabolism occur in the human heart, accompanied by upregulation of the glucose transporters. The profile of the substrate transporters and metabolic proteins mirror the metabolic shift from fatty acid to glucose utilisation that occurs in vivo in the human heart.

  19. Beneficial effects of curcumin on hyperlipidemia and insulin resistance in high-fat-fed hamsters.

    Science.gov (United States)

    Jang, Eun-Mi; Choi, Myung-Sook; Jung, Un Ju; Kim, Myung-Joo; Kim, Hye-Jin; Jeon, Seon-Min; Shin, Su-Kyung; Seong, Chi-Nam; Lee, Mi-Kyung

    2008-11-01

    This study investigated the effect of curcumin (0.05-g/100-g diet) supplementation on a high-fat diet (10% coconut oil, 0.2% cholesterol, wt/wt) fed to hamsters, one of the rodent species that are most closely related to humans in lipid metabolism. Curcumin significantly lowered the levels of free fatty acid, total cholesterol, triglyceride, and leptin and the homeostasis model assessment of insulin resistance index, whereas it elevated the levels of high-density lipoprotein cholesterol and apolipoprotein (apo) A-I and paraoxonase activity in plasma, compared with the control group. The levels of hepatic cholesterol and triglyceride were also lower in the curcumin group than in the control group. In the liver, fatty acid beta-oxidation activity was significantly higher in the curcumin group than in the control group, whereas fatty acid synthase, 3-hydroxy-3-methylglutaryl coenzyme A reductase, and acyl coenzyme A:cholesterol acyltransferase activities were significantly lower. Curcumin significantly lowered the lipid peroxide levels in the erythrocyte and liver compared with the control group. These results indicate that curcumin exhibits an obvious hypolipidemic effect by increasing plasma paraoxonase activity, ratios of high-density lipoprotein cholesterol to total cholesterol and of apo A-I to apo B, and hepatic fatty acid oxidation activity with simultaneous inhibition of hepatic fatty acid and cholesterol biosynthesis in high-fat-fed hamsters.

  20. The Golgi protein ACBD3 facilitates Enterovirus 71 replication by interacting with 3A

    Science.gov (United States)

    Lei, Xiaobo; Xiao, Xia; Zhang, Zhenzhen; Ma, Yijie; Qi, Jianli; Wu, Chao; Xiao, Yan; Zhou, Zhuo; He, Bin; Wang, Jianwei

    2017-01-01

    Enterovirus 71 (EV71) is a human pathogen that causes hand, foot, mouth disease and neurological complications. Although EV71, as well as other enteroviruses, initiates a remodeling of intracellular membrane for genomic replication, the regulatory mechanism remains elusive. By screening human cDNA library, we uncover that the Golgi resident protein acyl-coenzyme A binding domain-containing 3 (ACBD3) serves as a target of the 3A protein of EV71. This interaction occurs in cells expressing 3A or infected with EV71. Genetic inhibition or deletion of ACBD3 drastically impairs viral RNA replication and plaque formation. Such defects are corrected upon restoration of ACBD3. In infected cells, EV71 3A redirects ACBD3, to the replication sites. I44A or H54Y substitution in 3A interrupts the binding to ACBD3. As such, viral replication is impeded. These results reveal a mechanism of EV71 replication that involves host ACBD3 for viral replication. PMID:28303920

  1. VY6, a β-lactoglobulin-derived peptide, altered metabolic lipid pathways in the zebra fish liver.

    Science.gov (United States)

    Mohammed-Geba, K; Arrutia, F; Do-Huu, H; Borrell, Y J; Galal-Khallaf, A; Ardura, A; Riera, Francisco A; Garcia-Vazquez, Eva

    2016-04-01

    Today enormous research efforts are being focused on alleviating the massive, adverse effects of obesity. Short peptides are key targets for research as they can be generated from natural proteins, like milk. Here we conducted trypsinogen digestion of beta-lactoglobulin (β-lg), the major mammalian milk protein, to release the hexamer VY6. It was assayed in vivo for its activities on lipid metabolism using zebra fish as a vertebrate model. Zebra fish juveniles were injected with two different doses of the peptide: 100 and 800 μg per g fish and left for 5 days before sacrificing. Lipid measurements showed significant reduction in liver triglycerides and free cholesterol, as well as increased liver HDL cholesterol. Dose-dependent increases of the mRNA levels of the genes coding for the enzymes acyl coenzyme A oxidase 1 (acox1) and lipoprotein lipase (lpl) were also found. The complete results suggest significant anti-obesity activity of the β-lg-derived VY6 peptide. Its use as a nutraceutical has been discussed.

  2. Novel O-palmitolylated beta-E1 subunit of pyruvate dehydrogenase is phosphorylated during ischemia/reperfusion injury

    Directory of Open Access Journals (Sweden)

    Barr Amy J

    2010-07-01

    Full Text Available Abstract Background During and following myocardial ischemia, glucose oxidation rates are low and fatty acids dominate as a source of oxidative metabolism. This metabolic phenotype is associated with contractile dysfunction during reperfusion. To determine the mechanism of this reliance on fatty acid oxidation as a source of ATP generation, a functional proteomics approach was utilized. Results 2-D gel electrophoresis of mitochondria from working rat hearts subjected to 25 minutes of global no flow ischemia followed by 40 minutes of aerobic reperfusion identified 32 changes in protein abundance compared to aerobic controls. Of the five proteins with the greatest change in abundance, two were increased (long chain acyl-coenzyme A dehydrogenase (48 ± 1 versus 39 ± 3 arbitrary units, n = 3, P In silico analysis identified the putative kinases as the insulin receptor kinase for the more basic form and protein kinase Cζ or protein kinase A for the more acidic form. These modifications of pyruvate dehydrogenase are associated with a 35% decrease in glucose oxidation during reperfusion. Conclusions Cardiac ischemia/reperfusion induces significant changes to a number of metabolic proteins of the mitochondrial proteome. In particular, ischemia/reperfusion induced the post-translational modification of pyruvate dehydrogenase, the rate-limiting step of glucose oxidation, which is associated with a 35% decrease in glucose oxidation during reperfusion. Therefore these post-translational modifications may have important implications in the regulation of myocardial energy metabolism.

  3. Downregulation of miR-150 Expression by DNA Hypermethylation Is Associated with High 2-Hydroxy-(4-methylthio)butanoic Acid-Induced Hepatic Cholesterol Accumulation in Nursery Piglets.

    Science.gov (United States)

    Jia, Yimin; Ling, Mingfa; Zhang, Luchu; Jiang, Shuxia; Sha, Yusheng; Zhao, Ruqian

    2016-10-12

    Excess 2-hydroxy-(4-methylthio)butanoic acid (HMB) supplementation induces hyperhomocysteinemia, which contributes to hepatic cholesterol accumulation. However, it is unclear whether and how high levels of HMB break hepatic cholesterol homeostasis in nursery piglets. In this study, HMB oversupplementation suppressed food intake and decreased body weight in nursery piglets. Hyperhomocysteinemia and higher hepatic cholesterol accumulation were observed in HMB groups. Accordingly, HMB significantly increased the protein content of 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGCR) and glycine N-methyltransferase (GNMT) but decreased that of acyl-coenzyme A:cholesterol acyltransferase-1 (ACAT1). Significant downregulation of miR-150, miR-181d-5p, and miR-296-3p targeting the 3'-untranslated regions (UTRs) of GNMT and HMGCR was detected in the liver of HMB-treated piglets, and their functional validation was confirmed by dual-luciferase reporter assay. Furthermore, hypermethylation of miR-150 promoter was detected in association with suppressed miR-150 expression in the livers of HMB-treated piglets. This study indicated a new mechanism of hepatic cholesterol unhomeostasis by dietary methyl donor supplementation.

  4. 3,5-Diiodo-L-Thyronine Modifies the Lipid Droplet Composition in a Model of Hepatosteatosis

    Directory of Open Access Journals (Sweden)

    Elena Grasselli

    2014-02-01

    Full Text Available Background/Aims: Fatty acids are the main energy stores and the major membrane components of the cells. In the hepatocyte, fatty acids are esterified to triacylglycerols (TAGs and stored in lipid droplets (LDs. The lipid lowering action of 3,5-diiodo-L-thyronine (T2 on an in vitro model of hepatosteatosis was investigated in terms of fatty acid and protein content of LDs, lipid oxidation and secretion. Methods: FaO cells were exposed to oleate/palmitate, then treated with T2. Results: T2 reduced number and size of LDs, and modified their acyl composition by decreasing the content of saturated (SFA vs monounsaturated (MUFA fatty acids thus reversing the SFA/MUFA ratio. The expression of the LD-associated proteins adipose differentiation-related protein (ADRP, oxidative tissue-enriched PAT protein (OXPAT, and adipose triglyceride lipase (ATGL was increased in ‘steatotic' cells and further up-regulated by T2. Moreover, T2 stimulated the mitochondrial oxidation by up-regulating carnitine-palmitoyl-transferase (CPT1, uncoupling protein 2 (UCP2 and very long-chain acyl-coenzyme A dehydrogenase (VLCAD. Conclusions: T2 leads to mobilization of TAGs from LDs and stimulates mitochondrial oxidative metabolism of fatty acids, in particular of SFAs, and thus enriches of MUFAs the LDs. This action may protect the hepatocyte from excess of SFAs that are more toxic than MUFAs.

  5. Research for Developing Renewable Biofuels from Algae

    Energy Technology Data Exchange (ETDEWEB)

    Black, Paul N. [Univ. of Nebraska, Lincoln, NE (United States)

    2012-12-15

    Task A. Expansion of knowledge related to lipid production and secretion in algae A.1 Lipid biosynthesis in target algal species; Systems biology approaches are being used in combination with recent advances in Chlorella and Chlamydomonas genomics to address lipid accumulation in response to defined nutrient regimes. The UNL Algal Group continues screening additional species of Chlorella and other naturally occurring algae for those with optimal triglyceride production; Of the strains examined by the DOE's Aquatic Species Program, green algae, several species of Chlorella represent the largest group from which oleaginous candidates have been identified; A.1.1. Lipid profiling; Neutral lipid accumulation is routinely monitored by Nile red and BODIPY staining using high throughput strategies to screen for naturally occurring algae that accumulate triglyceride. These strategies complement those using spectrofluorometry to quantify lipid accumulation; Neutral lipid accumulation is routinely monitored by high performance thin-layer chromatography (HPTLC) and high performance liquid chromatography (HPLC) of lipid extracts in conjunction with; Carbon portioning experiments have been completed and the data currently are being analyzed and prepared for publication; Methods in the Black lab were developed to identify and quantify triacylglycerol (TAG), major membrane lipids [diacylglycerol trimethylhomoserine, phosphatidylethanolamine and chloroplast glycolipids], biosynthetic intermediates such as diacylglycerol, phosphatidic acid and lysophospholipids and different species of acyl-coenzyme A (acyl CoA).

  6. Cyclic vomiting syndrome masking a fatal metabolic disease.

    LENUS (Irish Health Repository)

    Fitzgerald, Marianne

    2013-05-01

    Disorders of fatty acid oxidation are rare but can be fatal. Hypoglycaemia with acidosis is a cardinal feature. Cases may present during early childhood or can be delayed into adolescence or beyond. We present a case of multiple acyl-coenzyme A dehydrogenase deficiency (MADD), an extremely rare disorder of fatty acid oxidation. Our 20-year-old patient presented with cardiovascular collapse, raised anion gap metabolic acidosis and non-ketotic hypoglycaemia. She subsequently developed multi-organ failure and sadly died. She had a previous diagnosis of cyclic vomiting syndrome (CVS) for more than 10 years, warranting frequent hospital admissions. The association between CVS and MADD has been made before though the exact relationship is unclear. All patients with persistent severe CVS should have metabolic investigations to exclude disorders of fatty acid oxidation. In case of non-ketotic hypoglycaemia with acidosis, the patient should be urgently referred to a specialist in metabolic diseases. All practitioners should be aware of these rare disorders as a cause of unexplained acidosis.

  7. Systematic Analysis of the 4-Coumarate:Coenzyme A Ligase (4CL Related Genes and Expression Profiling during Fruit Development in the Chinese Pear

    Directory of Open Access Journals (Sweden)

    Yunpeng Cao

    2016-10-01

    Full Text Available In plants, 4-coumarate:coenzyme A ligases (4CLs, comprising some of the adenylate-forming enzymes, are key enzymes involved in regulating lignin metabolism and the biosynthesis of flavonoids and other secondary metabolites. Although several 4CL-related proteins were shown to play roles in secondary metabolism, no comprehensive study on 4CL-related genes in the pear and other Rosaceae species has been reported. In this study, we identified 4CL-related genes in the apple, peach, yangmei, and pear genomes using DNATOOLS software and inferred their evolutionary relationships using phylogenetic analysis, collinearity analysis, conserved motif analysis, and structure analysis. A total of 149 4CL-related genes in four Rosaceous species (pear, apple, peach, and yangmei were identified, with 30 members in the pear. We explored the functions of several 4CL and acyl-coenzyme A synthetase (ACS genes during the development of pear fruit by quantitative real-time PCR (qRT-PCR. We found that duplication events had occurred in the 30 4CL-related genes in the pear. These duplicated 4CL-related genes are distributed unevenly across all pear chromosomes except chromosomes 4, 8, 11, and 12. The results of this study provide a basis for further investigation of both the functions and evolutionary history of 4CL-related genes.

  8. Altering small and medium alcohol selectivity in the wax ester synthase.

    Science.gov (United States)

    Barney, Brett M; Ohlert, Janet M; Timler, Jacobe G; Lijewski, Amelia M

    2015-11-01

    The bifunctional wax ester synthase/acyl-coenzyme A:diacylglycerol acyltransferase (WS/DGAT or wax ester synthase) catalyzes the terminal reaction in the bacterial wax ester biosynthetic pathway, utilizing a range of alcohols and fatty acyl-CoAs to synthesize the corresponding wax ester. The wild-type wax ester synthase Maqu_0168 from Marinobacter aquaeolei VT8 exhibits a preference for longer fatty alcohols, while applications with smaller alcohols would yield products with desired biotechnological properties. Small and medium chain length alcohol substrates are much poorer substrates for the native enzyme, which may hinder broad application of the wax ester synthase in many proposed biosynthetic schemes. Developing approaches to improve enzyme activity toward specific smaller alcohol substrates first requires a clear understanding of which amino acids of the primary sequences of these enzymes contribute to substrate specificity in the native enzyme. In this report, we surveyed a range of potential residues and identified the leucine at position 356 and methionine at position 405 in Maqu_0168 as residues that affected selectivity toward small, branched, and aromatic alcohols when substituted with different amino acids. This analysis provides evidence of residues that line the binding site for wax ester synthase, which will aid rational approaches to improve this enzyme with specific substrates.

  9. Differential effects of triclosan on the activation of mouse and human peroxisome proliferator-activated receptor alpha.

    Science.gov (United States)

    Wu, Yuanfeng; Wu, Qiangen; Beland, Frederick A; Ge, Peter; Manjanatha, Mugimane G; Fang, Jia-Long

    2014-11-18

    Triclosan is an anti-bacterial agent used in many personal care products, household items, medical devices, and clinical settings. Liver tumors occur in mice exposed to triclosan, a response attributed to peroxisome proliferator-activated receptor alpha (PPARα) activation; however, the effects of triclosan on mouse and human PPARα have not been fully evaluated. We compared the effects of triclosan on mouse and human PPARα using PPARα reporter assays and on downstream events of PPARα activation using mouse hepatoma Hepa1c1c7 cells and human hepatoma HepG2 cells. PPARα transcriptional activity was increased by triclosan in a mouse PPARα reporter assay and decreased in a human PPARα reporter assay. Concentrations of triclosan inhibiting 50% cell growth were similar in both human and mouse hepatoma cells. Western blotting analysis showed that triclosan increased acyl-coenzyme A oxidase (ACOX1), a PPARα target, in Hepa1c1c7 cells but decreased the level in HepG2 cells. Treatment of Hepa1c1c7 cells with triclosan enhanced DNA synthesis and suppressed transforming growth factor beta-mediated apoptosis. This did not occur in HepG2 cells. These data demonstrate that triclosan had similar cytotoxicity in Hepa1c1c7 and HepG2 cells, but differential effects on the activation of PPARα, the expression of ACOX1, and downstream events including DNA synthesis and apoptosis.

  10. Transcriptional analysis of sex pheromone biosynthesis signal genes in Bombyx mori

    Institute of Scientific and Technical Information of China (English)

    Shi-Heng An; Meng-Fang Du; Li-Juan Su; Xin-Ming Yin

    2012-01-01

    Six sex pheromone synthesis signal genes,including acyl coenzyme A (acylCoA) desaturase (desatl),fatty acyl reductase (FAR),pheromone biosynthesis activating neuropeptide receptor (PBANR),fatty acid transport protein (FATP),acyl-CoA binding protein (ACBP) and store-operated channel protein (OrailA),were studied for their transcriptional regulations.The expression profiles of these transcripts at different developmental stages (from-96 to 48 h) revealed that the genes are expressed in an age-dependent manner.The transcripts of these genes continued to increase despite decapitation,and compared with normally developmental females,decapitation significantly inhibited their expression.Further experiments with a methoprene,a juvenile hormone (JH) analogue,challenge showed that JH was not a key inhibiting factor in the expression of these genes,and mating was found to significantly inhibit the expression of these marker genes.Altogether,the results provide a reference for understanding the mechanism of sex pheromone synthesis.

  11. Exercise and obesity-induced insulin resistance in skeletal muscle

    Directory of Open Access Journals (Sweden)

    Hyo-Bum Kwak

    2013-12-01

    Full Text Available The skeletal muscle in our body is a major site for bioenergetics and metabolism during exercise. Carbohydrates and fats are the primary nutrients that provide the necessary energy required to maintain cellular activities during exercise. The metabolic responses to exercise in glucose and lipid regulation depend on the intensity and duration of exercise. Because of the increasing prevalence of obesity, recent studies have focused on the cellular and molecular mechanisms of obesity-induced insulin resistance in skeletal muscle. Accumulation of intramyocellular lipid may lead to insulin resistance in skeletal muscle. In addition, lipid intermediates (e.g., fatty acyl-coenzyme A, diacylglycerol, and ceramide impair insulin signaling in skeletal muscle. Recently, emerging evidence linking obesity-induced insulin resistance to excessive lipid oxidation, mitochondrial overload, and mitochondrial oxidative stress have been provided with mitochondrial function. This review will provide a brief comprehensive summary on exercise and skeletal muscle metabolism, and discuss the potential mechanisms of obesity-induced insulin resistance in skeletal muscle.

  12. Structure of a bifunctional alcohol dehydrogenase involved in bioethanol generation in Geobacillus thermoglucosidasius.

    Science.gov (United States)

    Extance, Jonathan; Crennell, Susan J; Eley, Kirstin; Cripps, Roger; Hough, David W; Danson, Michael J

    2013-10-01

    Bifunctional alcohol/aldehyde dehydrogenase (ADHE) enzymes are found within many fermentative microorganisms. They catalyse the conversion of an acyl-coenzyme A to an alcohol via an aldehyde intermediate; this is coupled to the oxidation of two NADH molecules to maintain the NAD(+) pool during fermentative metabolism. The structure of the alcohol dehydrogenase (ADH) domain of an ADHE protein from the ethanol-producing thermophile Geobacillus thermoglucosidasius has been determined to 2.5 Å resolution. This is the first structure to be reported for such a domain. In silico modelling has been carried out to generate a homology model of the aldehyde dehydrogenase domain, and this was subsequently docked with the ADH-domain structure to model the structure of the complete ADHE protein. This model suggests, for the first time, a structural mechanism for the formation of the large multimeric assemblies or `spirosomes' that are observed for this ADHE protein and which have previously been reported for ADHEs from other organisms.

  13. ATP-binding cassette transporters and sterol O-acyltransferases interact at membrane microdomains to modulate sterol uptake and esterification.

    Science.gov (United States)

    Gulati, Sonia; Balderes, Dina; Kim, Christine; Guo, Zhongmin A; Wilcox, Lisa; Area-Gomez, Estela; Snider, Jamie; Wolinski, Heimo; Stagljar, Igor; Granato, Juliana T; Ruggles, Kelly V; DeGiorgis, Joseph A; Kohlwein, Sepp D; Schon, Eric A; Sturley, Stephen L

    2015-11-01

    A key component of eukaryotic lipid homeostasis is the esterification of sterols with fatty acids by sterol O-acyltransferases (SOATs). The esterification reactions are allosterically activated by their sterol substrates, the majority of which accumulate at the plasma membrane. We demonstrate that in yeast, sterol transport from the plasma membrane to the site of esterification is associated with the physical interaction of the major SOAT, acyl-coenzyme A:cholesterol acyltransferase (ACAT)-related enzyme (Are)2p, with 2 plasma membrane ATP-binding cassette (ABC) transporters: Aus1p and Pdr11p. Are2p, Aus1p, and Pdr11p, unlike the minor acyltransferase, Are1p, colocalize to sterol and sphingolipid-enriched, detergent-resistant microdomains (DRMs). Deletion of either ABC transporter results in Are2p relocalization to detergent-soluble membrane domains and a significant decrease (53-36%) in esterification of exogenous sterol. Similarly, in murine tissues, the SOAT1/Acat1 enzyme and activity localize to DRMs. This subcellular localization is diminished upon deletion of murine ABC transporters, such as Abcg1, which itself is DRM associated. We propose that the close proximity of sterol esterification and transport proteins to each other combined with their residence in lipid-enriched membrane microdomains facilitates rapid, high-capacity sterol transport and esterification, obviating any requirement for soluble intermediary proteins.

  14. Taurine reduces the secretion of apolipoprotein B100 and lipids in HepG2 cells

    Directory of Open Access Journals (Sweden)

    Nagao Koji

    2008-10-01

    Full Text Available Abstract Background Higher concentrations of serum lipids and apolipoprotein B100 (apoB are major individual risk factors of atherosclerosis and coronary heart disease. Therefore ameliorative effects of food components against the diseases are being paid attention in the affluent countries. The present study was undertaken to investigate the effect of taurine on apoB secretion and lipid metabolism in human liver model HepG2 cells. Results The results demonstrated that an addition of taurine to the culture media reduces triacylglycerol (TG-mass in the cells and the medium. Similarly, cellular cholesterol-mass was decreased. Taurine inhibited the incorporation of [14C] oleate into cellular and medium TG, suggesting the inhibition of TG synthesis. In addition, taurine reduced the synthesis of cellular cholesterol ester and its secretion, suggesting the inhibition of acyl-coenzyme A:cholesterol acyltransferase activity. Furthermore, taurine reduced the secretion of apoB, which is a major protein component of very low-density lipoprotein. Conclusion This is a first report to demonstrate that taurine inhibits the secretion of apoB from HepG2 cells.

  15. Hybrid Structure of a Dynamic Single-Chain Carboxylase from Deinococcus radiodurans.

    Science.gov (United States)

    Hagmann, Anna; Hunkeler, Moritz; Stuttfeld, Edward; Maier, Timm

    2016-08-01

    Biotin-dependent acyl-coenzyme A (CoA) carboxylases (aCCs) are involved in key steps of anabolic pathways and comprise three distinct functional units: biotin carboxylase (BC), biotin carboxyl carrier protein (BCCP), and carboxyl transferase (CT). YCC multienzymes are a poorly characterized family of prokaryotic aCCs of unidentified substrate specificity, which integrate all functional units into a single polypeptide chain. We employed a hybrid approach to study the dynamic structure of Deinococcus radiodurans (Dra) YCC: crystal structures of isolated domains reveal a hexameric CT core with extended substrate binding pocket and a dimeric BC domain. Negative-stain electron microscopy provides an approximation of the variable positioning of the BC dimers relative to the CT core. Small-angle X-ray scattering yields quantitative information on the ensemble of Dra YCC structures in solution. Comparison with other carrier protein-dependent multienzymes highlights a characteristic range of large-scale interdomain flexibility in this important class of biosynthetic enzymes.

  16. ACAT1 deficiency increases cholesterol synthesis in mouse peritoneal macrophages.

    Science.gov (United States)

    Dove, Dwayne E; Su, Yan Ru; Swift, Larry L; Linton, MacRae F; Fazio, Sergio

    2006-06-01

    Acyl-coenzyme A:cholesterol acyltransferase (ACAT) esterifies free cholesterol and stores cholesteryl esters in lipid droplets. Macrophage ACAT1 deficiency results in increased atherosclerotic lesion area in hyperlipidemic mice via disrupted cholesterol efflux, increased lipoprotein uptake, accumulation of intracellular vesicles, and accelerated apoptosis. The objective of this study was to determine whether lipid synthesis is affected by ACAT1. The synthesis, esterification, and efflux of new cholesterol were measured in peritoneal macrophages from ACAT1(-/-) mice. Cholesterol synthesis was increased by 134% (p=0.001) in ACAT1(-/-) macrophages compared to wildtype macrophages. Increased synthesis resulted in a proportional increase in the efflux of newly synthesized cholesterol. Although the esterification of new cholesterol was reduced by 93% (pSREBP1a mRNA was increased 6-fold in ACAT1(-/-) macrophages compared to wildtype macrophages, suggesting an up-regulation of cholesterol and fatty acid synthesis in ACAT1(-/-) macrophages. Increased cholesterol synthesis and up-regulation of SREBP in ACAT1(-/-) macrophages suggests that ACAT1 affects the regulation of lipid metabolism in macrophages. This change in cholesterol homeostasis may contribute to the atherogenic potential of ACAT1(-/-) macrophages.

  17. An overview of the new frontiers in the treatment of atherogenic dyslipidemias.

    Science.gov (United States)

    Rached, F H; Chapman, M J; Kontush, A

    2014-07-01

    Cardiovascular diseases (CVDs) are the leading cause of morbidity/mortality worldwide. Dyslipidemia is a major risk factor for premature atherosclerosis and CVD. Lowering low-density-lipoprotein cholesterol (LDL-C) levels is well established as an intervention for the reduction of CVDs. Statins are the first-line drugs for treatment of dyslipidemia, but they do not address all CVD risk. Development of novel therapies is ongoing and includes the following: (i) reduction of LDL-C concentrations using antibodies to proprotein convertase subtilisin/kexin-9, antisense oligonucleotide inhibitors of apolipoprotein B production, microsomal transfer protein (MTP) inhibitors, and acyl-coenzyme A cholesterol acyl transferase inhibitors; (ii) reduction in levels of triglyceride-rich lipoproteins with ω-3 fatty acids, MTP inhibitors, and diacylglycerol acyl transferase-1 inhibitors; and (iii) increase of high-density-lipoprotein (HDL) cholesterol levels, HDL particle numbers, and/or HDL functionality using cholesteryl ester transfer protein inhibitors, HDL-derived agents, apolipoprotein AI mimetic peptides, and microRNAs. Large prospective outcome trials of several of these emerging therapies are under way, and thrilling progress in the field of lipid management is anticipated.

  18. Carnitine deficiency and its related diseases%肉碱缺乏及其相关疾病的研究进展

    Institute of Scientific and Technical Information of China (English)

    房玥晖; 蔡美琴

    2009-01-01

    L-carnitine plays an essential role in the beta-oxidation of fatty acids by transporting long chain acyl-coenzyme A into the mitochondrial matrix.Carnitine deficiency may lead to various diseases,including lipid storage myopathies,systemic carnitine deficiency syndrome,cardiomyopathy,obesity,and infertility.This article summarizes the causes of carnitine deficiency and elucidates the clinical features and treatment strategies of its related diseases.%肉碱是脂肪β-氧化过程中长链脂酰辅酶A透过线粒体内膜时的转运体,其缺乏导致脂质沉积性肌病、全身肉碱缺乏综合征、心肌病、肥胖、男性不育等疾病.本文主要综述肉碱缺乏的原因及所导致的相关疾病的主要临床表现和治疗手段.

  19. ACBD5 and VAPB mediate membrane associations between peroxisomes and the ER

    Science.gov (United States)

    Costello, Joseph L.; Hacker, Christian; Schrader, Tina A.; Zeuschner, Dagmar; Findeisen, Peter

    2017-01-01

    Peroxisomes (POs) and the endoplasmic reticulum (ER) cooperate in cellular lipid metabolism and form tight structural associations, which were first observed in ultrastructural studies decades ago. PO–ER associations have been suggested to impact on a diverse number of physiological processes, including lipid metabolism, phospholipid exchange, metabolite transport, signaling, and PO biogenesis. Despite their fundamental importance to cell metabolism, the mechanisms by which regions of the ER become tethered to POs are unknown, in particular in mammalian cells. Here, we identify the PO membrane protein acyl-coenzyme A–binding domain protein 5 (ACBD5) as a binding partner for the resident ER protein vesicle-associated membrane protein-associated protein B (VAPB). We show that ACBD5–VAPB interaction regulates PO–ER associations. Moreover, we demonstrate that loss of PO–ER association perturbs PO membrane expansion and increases PO movement. Our findings reveal the first molecular mechanism for establishing PO–ER associations in mammalian cells and report a new function for ACBD5 in PO–ER tethering. PMID:28108524

  20. Role of beta-oxidation enzymes in gamma-decalactone production by the yeast Yarrowia lipolytica.

    Science.gov (United States)

    Waché, Y; Aguedo, M; Choquet, A; Gatfield, I L; Nicaud, J M; Belin, J M

    2001-12-01

    Some microorganisms can transform methyl ricinoleate into gamma-decalactone, a valuable aroma compound, but yields of the bioconversion are low due to (i) incomplete conversion of ricinoleate (C(18)) to the C(10) precursor of gamma-decalactone, (ii) accumulation of other lactones (3-hydroxy-gamma-decalactone and 2- and 3-decen-4-olide), and (iii) gamma-decalactone reconsumption. We evaluated acyl coenzyme A (acyl-CoA) oxidase activity (encoded by the POX1 through POX5 genes) in Yarrowia lipolytica in lactone accumulation and gamma-decalactone reconsumption in POX mutants. Mutants with no acyl-CoA oxidase activity could not reconsume gamma-decalactone, and mutants with a disruption of pox3, which encodes the short-chain acyl-CoA oxidase, reconsumed it more slowly. 3-Hydroxy-gamma-decalactone accumulation during transformation of methyl ricinoleate suggests that, in wild-type strains, beta-oxidation is controlled by 3-hydroxyacyl-CoA dehydrogenase. In mutants with low acyl-CoA oxidase activity, however, the acyl-CoA oxidase controls the beta-oxidation flux. We also identified mutant strains that produced 26 times more gamma-decalactone than the wild-type parents.

  1. Role of β-Oxidation Enzymes in γ-Decalactone Production by the Yeast Yarrowia lipolytica

    Science.gov (United States)

    Waché, Yves; Aguedo, Mario; Choquet, Armelle; Gatfield, Ian L.; Nicaud, Jean-Marc; Belin, Jean-Marc

    2001-01-01

    Some microorganisms can transform methyl ricinoleate into γ-decalactone, a valuable aroma compound, but yields of the bioconversion are low due to (i) incomplete conversion of ricinoleate (C18) to the C10 precursor of γ-decalactone, (ii) accumulation of other lactones (3-hydroxy-γ-decalactone and 2- and 3-decen-4-olide), and (iii) γ-decalactone reconsumption. We evaluated acyl coenzyme A (acyl-CoA) oxidase activity (encoded by the POX1 through POX5 genes) in Yarrowia lipolytica in lactone accumulation and γ-decalactone reconsumption in POX mutants. Mutants with no acyl-CoA oxidase activity could not reconsume γ-decalactone, and mutants with a disruption of pox3, which encodes the short-chain acyl-CoA oxidase, reconsumed it more slowly. 3-Hydroxy-γ-decalactone accumulation during transformation of methyl ricinoleate suggests that, in wild-type strains, β-oxidation is controlled by 3-hydroxyacyl-CoA dehydrogenase. In mutants with low acyl-CoA oxidase activity, however, the acyl-CoA oxidase controls the β-oxidation flux. We also identified mutant strains that produced 26 times more γ-decalactone than the wild-type parents. PMID:11722925

  2. Coordinated gene expression in adipose tissue and liver differs between cows with high or low NEFA concentrations in early lactation.

    Science.gov (United States)

    van Dorland, H A; Sadri, H; Morel, I; Bruckmaier, R M

    2012-02-01

    Dairy cows with high and low plasma non-esterified fatty acid (NEFA) concentrations in early lactation were compared for plasma parameters and mRNA expression of genes in liver and subcutaneous adipose tissue. The study involved 16 multiparous dairy cows with a plasma NEFA concentration of >500 μmol/l [n = 8, high NEFA (HNEFA)] and Subcutaneous adipose and liver tissues were analysed for mRNA abundance by real-time qRT-PCR encoding parameters related to lipid metabolism. Results showed that mean daily milk yield and milk fat quantity were higher in HNEFA than in LNEFA cows (p carnitine palmitoyltransferase 2 and very long chain acyl-coenzyme A dehydrogenase) and ketogenesis (3-hydroxy-3-methylglutaryl-coenzyme A synthase 2) were lower in HNEFA than in LNEFA cows. No differences between the two groups were observed for mRNA expression of genes in adipose tissue. The number of calculated significant correlation coefficients (moderately strong) between parameters in the liver and in adipose tissue was nearly similar on +1d, and higher for HNEFA compared with LNEFA cows in +3wk. In conclusion, dairy cows with high compared with low plasma NEFA concentrations in early lactation show differentially synchronized mRNA expression of genes in adipose tissue and liver in +3wk that suggests a different orchestrated homeorhetic regulation of lipid metabolism.

  3. Key enzymes for biosynthesis of neutral lipid storage compounds in prokaryotes: properties, function and occurrence of wax ester synthases/acyl-CoA: diacylglycerol acyltransferases.

    Science.gov (United States)

    Wältermann, Marc; Stöveken, Tim; Steinbüchel, Alexander

    2007-02-01

    Triacylglycerols (TAGs) and wax esters (WEs) are beside polyhydroxyalkanoates (PHAs) important storage lipids in some groups of prokaryotes. Accumulation of these lipids occurs in cells when they are cultivated under conditions of unbalanced growth in the presence of high concentrations of a suitable carbon source, which can be used for fatty acid and storage lipid biosyntheses. The key enzymes, which mediate both WE and TAG formations from long-chain acyl-coenzyme A (CoA) as acyl donor and long-chain fatty alcohols or diacylglycerols as respective acyl acceptors in bacteria, are WE synthases/acyl-CoA:diacylglycerol acyltransferases (WS/DGATs). The WS/DGATs identified so far represent rather unspecific enzymes with broad spectra of possible substrates; this makes them interesting for many biotechnological applications. This review traces the molecular structure and biochemical properties including the probable regions responsible for acyltransferase properties, enzymatic activity and substrate specifities. The phylogenetic relationships based on amino acid sequence similarities of this unique class of enzymes were revealed. Furthermore, recent advances in understanding the physiological functions of WS/DGATs in their natural hosts including pathogenic Mycobacterium tuberculosis were discussed.

  4. Systematic Analysis of the 4-Coumarate:Coenzyme A Ligase (4CL) Related Genes and Expression Profiling during Fruit Development in the Chinese Pear.

    Science.gov (United States)

    Cao, Yunpeng; Han, Yahui; Li, Dahui; Lin, Yi; Cai, Yongping

    2016-10-19

    In plants, 4-coumarate:coenzyme A ligases (4CLs), comprising some of the adenylate-forming enzymes, are key enzymes involved in regulating lignin metabolism and the biosynthesis of flavonoids and other secondary metabolites. Although several 4CL-related proteins were shown to play roles in secondary metabolism, no comprehensive study on 4CL-related genes in the pear and other Rosaceae species has been reported. In this study, we identified 4CL-related genes in the apple, peach, yangmei, and pear genomes using DNATOOLS software and inferred their evolutionary relationships using phylogenetic analysis, collinearity analysis, conserved motif analysis, and structure analysis. A total of 149 4CL-related genes in four Rosaceous species (pear, apple, peach, and yangmei) were identified, with 30 members in the pear. We explored the functions of several 4CL and acyl-coenzyme A synthetase (ACS) genes during the development of pear fruit by quantitative real-time PCR (qRT-PCR). We found that duplication events had occurred in the 30 4CL-related genes in the pear. These duplicated 4CL-related genes are distributed unevenly across all pear chromosomes except chromosomes 4, 8, 11, and 12. The results of this study provide a basis for further investigation of both the functions and evolutionary history of 4CL-related genes.

  5. Micro method for determination of nonesterified fatty acid in whole blood obtained by fingertip puncture

    DEFF Research Database (Denmark)

    Hansen, Jesper S; Villadsen, Jens K; Gaster, Michael

    2006-01-01

    -related disorders. In the current study, we developed a sensitive and reproducible micro method for quantification of NEFA in less than 10 microl whole blood. The method involves only two steps: (i) conversion of NEFA to fatty acid acyl-coenzyme A (acyl-CoA) esters using an acyl-CoA synthetase and (ii......) quantification of the formed acyl-CoA esters with a fluorescent biosensor based on bovine acyl-CoA binding protein (ACBP). Lys50 of ACBP was mutagenized to a cysteine residue that was covalently modified with 6-bromoacetyl-2-dimethylaminonaphthalene to make a fluorescent acyl-CoA indicator (FACI-50). FACI-50...... exhibits high fluorescence emission yield with maximum at 490 nm in the presence of CoA when excited at 387 nm. The addition of palmitoyl-CoA to a CoA-saturated FACI-50 lowered fluorescence emission by eightfold. Ethanol extract from 1 microl whole blood was incubated with ATP, CoA, and FACI-50. Following...

  6. De novo biosynthesis of biodiesel by Escherichia coli in optimized fed-batch cultivation.

    Directory of Open Access Journals (Sweden)

    Yangkai Duan

    Full Text Available Biodiesel is a renewable alternative to petroleum diesel fuel that can contribute to carbon dioxide emission reduction and energy supply. Biodiesel is composed of fatty acid alkyl esters, including fatty acid methyl esters (FAMEs and fatty acid ethyl esters (FAEEs, and is currently produced through the transesterification reaction of methanol (or ethanol and triacylglycerols (TAGs. TAGs are mainly obtained from oilseed plants and microalgae. A sustainable supply of TAGs is a major bottleneck for current biodiesel production. Here we report the de novo biosynthesis of FAEEs from glucose, which can be derived from lignocellulosic biomass, in genetically engineered Escherichia coli by introduction of the ethanol-producing pathway from Zymomonas mobilis, genetic manipulation to increase the pool of fatty acyl-CoA, and heterologous expression of acyl-coenzyme A: diacylglycerol acyltransferase from Acinetobacter baylyi. An optimized fed-batch microbial fermentation of the modified E. coli strain yielded a titer of 922 mg L(-1 FAEEs that consisted primarily of ethyl palmitate, -oleate, -myristate and -palmitoleate.

  7. Current and future pharmacologic options for the management of patients unable to achieve low-density lipoprotein-cholesterol goals with statins.

    Science.gov (United States)

    El Harchaoui, Karim; Akdim, Fatima; Stroes, Erik S G; Trip, Mieke D; Kastelein, John J P

    2008-01-01

    Low-density lipoprotein-cholesterol (LDL-C) lowering is the mainstay of the current treatment guidelines in the management of cardiovascular risk. HMG-CoA reductase inhibitors (statins) are currently the most effective LDL-C-lowering drugs. However, a substantial number of patients do not reach treatment targets with statins. Therefore, an unmet medical need exists for lipid-lowering drugs with novel mechanisms of action to reach the recommended cholesterol target levels, either by monotherapy or combination therapy. Upregulation of the LDL receptor with squalene synthase inhibitors has shown promising results in animal studies but the clinical development of the lead compound lapaquistat (TAK-475) has recently been discontinued. Ezetimibe combined with statins allowed significantly more patients to reach their LDL-C targets. Other inhibitors of intestinal cholesterol absorption such as disodium ascorbyl phytostanol phosphate (FM-VP4) and bile acid transport inhibitors have shown positive results in early development trials, whereas the prospect of acyl coenzyme A: cholesterol acyltransferase inhibition in cardiovascular prevention is dire. Selective inhibition of messenger RNA (mRNA) by antisense oligonucleotides is a new approach to modify cholesterol levels. The inhibition of apolipoprotein B mRNA is in advanced development and mipomersen sodium (ISIS 301012) has shown striking results in phase II studies both as monotherapy as well as in combination with statins.

  8. Carnitine deficiency presenting with encephalopathy and hyperammonemia in a patient receiving chronic enteral tube feeding: a case report

    Directory of Open Access Journals (Sweden)

    Ling Peter

    2012-07-01

    Full Text Available Abstract Introduction Carnitine is an essential cofactor in mitochondrial fatty acid oxidation. Carnitine deficiency results in accumulation of non-oxidized fatty acyl-coenzyme A molecules, and this inhibits intra-mitochondrial degradation of ammonia. Hyperammonemia may lead to encephalopathy. This scenario has been previously reported. Case presentation We report the case of a 47-year-old Caucasian man who had sustained a remote motor vehicle accident injury and relied on long-term tube feeding with a commercial product that wascarnitine-free. He was also on phenytoin therapy for control of his chronic seizures. He developed significant acute psychological and behavioral changes superimposed on his chronic neurological impairment. His ammonia level was found to be elevated at 75 to 100μmol/L (normal Conclusion This case illustrates the importance of avoiding carnitine deficiency and anti-convulsant toxicity in tube-fed patients encountered in hospital wards and nursing homes. These patients should have their carnitine levels assessed regularly, and supplementation should be provided as necessary. Manufacturers of enteral feeds and formulas should consider adding carnitine to their product lines.

  9. High throughput de novo RNA sequencing elucidates novel responses in Penicillium chrysogenum under microgravity.

    Science.gov (United States)

    Sathishkumar, Yesupatham; Krishnaraj, Chandran; Rajagopal, Kalyanaraman; Sen, Dwaipayan; Lee, Yang Soo

    2016-02-01

    In this study, the transcriptional alterations in Penicillium chrysogenum under simulated microgravity conditions were analyzed for the first time using an RNA-Seq method. The increasing plethora of eukaryotic microbial flora inside the spaceship demands the basic understanding of fungal biology in the absence of gravity vector. Penicillium species are second most dominant fungal contaminant in International Space Station. Penicillium chrysogenum an industrially important organism also has the potential to emerge as an opportunistic pathogen for the astronauts during the long-term space missions. But till date, the cellular mechanisms underlying the survival and adaptation of Penicillium chrysogenum to microgravity conditions are not clearly elucidated. A reference genome for Penicillium chrysogenum is not yet available in the NCBI database. Hence, we performed comparative de novo transcriptome analysis of Penicillium chrysogenum grown under microgravity versus normal gravity. In addition, the changes due to microgravity are documented at the molecular level. Increased response to the environmental stimulus, changes in the cell wall component ABC transporter/MFS transporters are noteworthy. Interestingly, sustained increase in the expression of Acyl-coenzyme A: isopenicillin N acyltransferase (Acyltransferase) under microgravity revealed the significance of gravity in the penicillin production which could be exploited industrially.

  10. Moments of discovery.

    Science.gov (United States)

    Berg, Paul

    2008-01-01

    Devoted teachers and mentors during early childhood and adolescence nurtured my ambition to become a scientist, but it was not until I actually began doing experiments in college and graduate school that I was confident about that choice and of making it a reality. During my postdoctoral experiences and thereafter, I made several significant advances, most notably the discovery of the then novel acyl- and aminoacyl adenylates: the former as intermediates in fatty acyl coenzyme A (CoA) formation and the latter as precursors to aminoacyl tRNAs. In the early 1970s, my research changed from a focus on transcription and translation in Escherichia coli to the molecular genetics of mammalian cells. To that end, my laboratory developed a method for creating recombinant DNAs that led us and others, over the next two decades, to create increasingly sophisticated ways for introducing "foreign" DNAs into cultured mammalian cells and to target modifications of specific chromosomal loci. Circumstances surrounding that work drew me into the public policy debates regarding recombinant DNA practices. As an outgrowth of my commitment to teaching, I co-authored several textbooks on molecular genetics and a biography of George Beadle. The colleagues, students, and wealth of associates with whom I interacted have made being a scientist far richer than I can have imagined.

  11. Green tea catechins enhance norepinephrine-induced lipolysis via a protein kinase A-dependent pathway in adipocytes.

    Science.gov (United States)

    Chen, Shu; Osaki, Noriko; Shimotoyodome, Akira

    2015-05-22

    Green tea catechins have been shown to attenuate obesity in animals and humans. The catechins activate adenosine monophosphate-activated protein kinase (AMPK), and thereby increase fatty acid oxidation in liver and skeletal muscles. Green tea catechins have also been shown to reduce body fat in humans. However, the effect of the catechins on lipolysis in adipose tissue has not been fully understood. The aim of this study was to clarify the effect of green tea catechins on lipolysis in adipocytes and to elucidate the underlying mechanism. Differentiated mouse adipocyte cell line (3T3-L1) was stimulated with green tea catechins in the presence or absence of norepinephrine. Glycerol and free fatty acids in the media were measured. Phosphorylation of hormone-sensitive lipase (HSL) was determined by Western blotting, and the mRNA expression levels of HSL, adipose triglyceride lipase (ATGL), and perilipin were determined by quantitative RT-PCR. The cells were treated with inhibitors of protein kinase A (PKA), protein kinase C (PKC), protein kinase G (PKG), or mitogen-activated protein kinase (MAPK) to determine the responsible pathway. Treatment of 3T3-L1 adipocytes with green tea catechins increased the level of glycerol and free fatty acids released into the media in the presence, but not absence, of norepinephrine, and increased the level of phosphorylated HSL in the cells. The catechins also increased mRNA and protein levels of HSL and ATGL. PKA inhibitor (H89) attenuated the catechin-induced increase in glycerol release and HSL phosphorylation. The results demonstrate that green tea catechins enhance lipolysis in the presence of norepinephrine via a PKA-dependent pathway in 3T3-L1 adipocytes, providing a potential mechanism by which green tea catechins could reduce body fat.

  12. The RecA-Dependent SOS Response Is Active and Required for Processing of DNA Damage during Bacillus subtilis Sporulation.

    Science.gov (United States)

    Ramírez-Guadiana, Fernando H; Barajas-Ornelas, Rocío Del Carmen; Corona-Bautista, Saúl U; Setlow, Peter; Pedraza-Reyes, Mario

    2016-01-01

    The expression of and role played by RecA in protecting sporulating cells of Bacillus subtilis from DNA damage has been determined. Results showed that the DNA-alkylating agent Mitomycin-C (M-C) activated expression of a PrecA-gfpmut3a fusion in both sporulating cells' mother cell and forespore compartments. The expression levels of a recA-lacZ fusion were significantly lower in sporulating than in growing cells. However, M-C induced levels of ß-galactosidase from a recA-lacZ fusion ~6- and 3-fold in the mother cell and forespore compartments of B. subtilis sporangia, respectively. Disruption of recA slowed sporulation and sensitized sporulating cells to M-C and UV-C radiation, and the M-C and UV-C sensitivity of sporangia lacking the transcriptional repair-coupling factor Mfd was significantly increased by loss of RecA. We postulate that when DNA damage is encountered during sporulation, RecA activates the SOS response thus providing sporangia with the repair machinery to process DNA lesions that may compromise the spatio-temporal expression of genes that are essential for efficient spore formation.

  13. Thanks, but No Thanks: Women's Avoidance of Help-Seeking in the Context of a Dependency-Related Stereotype

    Science.gov (United States)

    Wakefield, Juliet R. H.; Hopkins, Nick; Greenwood, Ronni M.

    2012-01-01

    The stereotype that women are dependent on men is a commonly verbalized, potentially damaging aspect of benevolent sexism. We investigated how women may use behavioral disconfirmation of the personal applicability of the stereotype to negotiate such sexism. In an experiment (N = 86), we manipulated female college students' awareness that women may…

  14. The Mass-Metallicity relation explored with CALIFA: I. Is there a dependence on the star formation rate?

    CERN Document Server

    Sanchez, S F; Jungwiert, B; Iglesias-Paramo1, J; Vilchez, J M; Marino, R A; Walcher, C J; Husemann, B; Mast, D; Monreal-Ibero, A; Fernandes, R Cid; Perez, E; Delgado, R Gonzalez; Garcia-Benito, R; Galbany, L; van de Ven, G; Jahnke, K; Flores, H; Bland-Hawthorn, J; Lopez-Sánchez, A R; Stanishev, V; Miralles-Caballero, D; Diaz, A I; Sanchez-Blazquez, P; Molla, M; Gallazzi1, A; Papaderos, P; Gomes, J M; Gruel, N; Pérez, I; Ruiz-Lara, T; Florido, E; de Lorenzo-Cáceres, A; Mendez-Abreu, J; Kehrig, C; Roth, M M; Ziegler, B; Alves, J; Wisotzki, L; Kupko, D; Quirrenbach, A; Bomans, D

    2013-01-01

    We present the results on the study of the global and local M-Z relation based on the first data available from the CALIFA survey (150 galaxies). This survey provides integral field spectroscopy of the complete optical extent of each galaxy (up to 2-3 effective radii), with enough resolution to separate individual HII regions and/or aggregations. Nearly $\\sim$3000 individual HII regions have been detected. The spectra cover the wavelength range between [OII]3727 and [SII]6731, with a sufficient signal-to-noise to derive the oxygen abundance and star-formation rate associated with each region. In addition, we have computed the integrated and spatially resolved stellar masses (and surface densities), based on SDSS photometric data. We explore the relations between the stellar mass, oxygen abundance and star-formation rate using this dataset. We derive a tight relation between the integrated stellar mass and the gas-phase abundance, with a dispersion smaller than the one already reported in the literature ($\\sig...

  15. A-dependence for the charmed meson production; Dependencia em A para a producao de mesons charmosos

    Energy Technology Data Exchange (ETDEWEB)

    Alves, Gilvan Augusto

    1992-03-01

    A report is presented of a recent direct measurement of the nucleon number ({lambda}) dependence of the production cross sections for the charmed mesons D{sup 0} and D{sup +} using {pi}{sup +-} beams incident on a segmented target of Be, Al, Cu and W. The data derive from the experiment E769 - Hadroproduction of Charm - at Fermilab. The experimental apparatus is described together with the following analysis. Starting from a sample of {approx} 1500 D mesons in the range of O < X{sub F} < 1, the data are found to be well described by the parameterization {sigma}{sub A} = {sigma}{sub O} A{sup {alpha}}, with {alpha} = 0.99 {+-} 0.03. The X{sub F} dependence of {alpha} is examined and the results obtained are compared with those of other experiments and with theoretical expectations based on perturbative QCD and on an EMC like model of nuclear shadowing. (author). 85 refs, 61 figs, 22 tabs.

  16. The RecA-Dependent SOS Response Is Active and Required for Processing of DNA Damage during Bacillus subtilis Sporulation.

    Directory of Open Access Journals (Sweden)

    Fernando H Ramírez-Guadiana

    Full Text Available The expression of and role played by RecA in protecting sporulating cells of Bacillus subtilis from DNA damage has been determined. Results showed that the DNA-alkylating agent Mitomycin-C (M-C activated expression of a PrecA-gfpmut3a fusion in both sporulating cells' mother cell and forespore compartments. The expression levels of a recA-lacZ fusion were significantly lower in sporulating than in growing cells. However, M-C induced levels of ß-galactosidase from a recA-lacZ fusion ~6- and 3-fold in the mother cell and forespore compartments of B. subtilis sporangia, respectively. Disruption of recA slowed sporulation and sensitized sporulating cells to M-C and UV-C radiation, and the M-C and UV-C sensitivity of sporangia lacking the transcriptional repair-coupling factor Mfd was significantly increased by loss of RecA. We postulate that when DNA damage is encountered during sporulation, RecA activates the SOS response thus providing sporangia with the repair machinery to process DNA lesions that may compromise the spatio-temporal expression of genes that are essential for efficient spore formation.

  17. Modulation of HIV-1 infectivity and cyclophilin A-dependence by Gag sequence and target cell type

    Directory of Open Access Journals (Sweden)

    Dam Elisabeth

    2009-03-01

    Full Text Available Abstract Background HIV-1 Gag proteins are essential for virion assembly and viral replication in newly infected cells. Gag proteins are also strong determinants of viral infectivity; immune escape mutations in the Gag capsid (CA protein can markedly reduce viral fitness, and interactions of CA with host proteins such as cyclophilin A (CypA and TRIM5α can have important effects on viral infectivity. Little information, however, is available concerning the extent that different primary Gag proteins affect HIV-1 replication in different cell types, or the impact on viral replication of differences in the expression by target cells of proteins that interact with CA. To address these questions, we compared the infectivity of recombinant HIV-1 viruses expressing Gag-protease sequences from primary isolates in different target cells in the presence or absence of agents that disrupt cyclophilin A – CA interactions and correlated these results with the viral genotype and the expression of cyclophilin A and TRIM5α by the target cells. Results Viral infectivity was governed by the nature of the Gag proteins in a target cell-specific fashion. The treatment of target cells with agents that disrupt CypA-CA interactions often produced biphasic dose-response curves in which viral infectivity first increased and subsequently decreased as a function of the dose used. The extent that treatment of target cells with high-dose CypA inhibitors impaired viral infectivity was dependent on several factors, including the viral genotype, the nature of the target cell, and the extent that treatment with low-dose CypA inhibitors increased viral infectivity. Neither the presence of polymorphisms in the CA CypA-binding loop, the level of expression of CypA, or the level of TRIM5α expression could, alone, explain the differences in the shape of the dose-response curves observed or the extent that high-dose CypA inhibitors reduced viral infectivity. Conclusion Multiple interactions between host-cell factors and Gag can strongly affect HIV-1 infectivity, and these vary according to target cell type and the origin of the Gag sequence. Two of the cellular activities involved appear to be modulated in opposite directions by CypA-CA interactions, and Gag sequences determine the intrinsic sensitivity of a given virus to each of these cellular activities.

  18. Storage protein-2 as a dependable biochemical index for screening germplasm stocks of the silkworm Bombyx mori (L.

    Directory of Open Access Journals (Sweden)

    Jingade H. Anuradha

    2012-09-01

    Full Text Available Storage protein (SP-2 variation was investigated among selected silkworm germplasm stocks representing two major potential sericulture areas of India. The expression levels of storage protein varied among them, as seen in Sodium Dodecylsulfate Polyacrylamide Gel Electrophoresis (SDS-PAGE, which correlated with their geographical origin. The storage protein variation is an inter origin variability and this differential expression of the protein is helpful to tag the robustness of the breed/race associated with parentage and their origin. Present study revealed that silkworm races/breeds viz., LMO, Kolar Gold and A4e possess higher protein content among the races studied. This may be correlated with their robustness reflecting higher survival rate in the varied environments prevailing in the tropical zone. Such identified races can be conserved as storage protein rich genetic stocks for their maximal genetic potentials and high-grade silk productivity.

  19. VeriML: A Dependently-Typed, User-Extensible and Language-Centric Approach to Proof Assistants

    Science.gov (United States)

    2013-01-01

    else18 currewritemodule rewriter @e))19 idelem20 rewriters_list21 in22 if test_progress first_successful then23 let < e’ , pfe ’ > = first_successful...in24 let < e’’ , pfe ’’ > = rewriter @e’ in25 < @e’’ , @trans pfe ’ pfe ’’ >26 else27 first_successful)28 in29 rewriter ;;30 Code Listing 9.3: Building a...e ⇒2 holmatch @e with3 @(t1 : T’ → T) t2 7→4 let < t1’, pf1 > = recursive @t1 in5 let < e’, pfe ’ > =6 holmatch @t1’ as t1’’7 return ( e’ : @T

  20. Sphingosine 1-phosphate elicits RhoA-dependent proliferation and MRTF-A mediated gene induction in CPCs.

    Science.gov (United States)

    Castaldi, Alessandra; Chesini, Gino P; Taylor, Amy E; Sussman, Mark A; Brown, Joan Heller; Purcell, Nicole H

    2016-08-01

    Although c-kit(+) cardiac progenitor cells (CPCs) are currently used in clinical trials there remain considerable gaps in our understanding of the molecular mechanisms underlying their proliferation and differentiation. G-protein coupled receptors (GPCRs) play an important role in regulating these processes in mammalian cell types thus we assessed GPCR mRNA expression in c-kit(+) cells isolated from adult mouse hearts. Our data provide the first comprehensive overview of the distribution of this fundamental class of cardiac receptors in CPCs and reveal notable distinctions from that of adult cardiomyocytes. We focused on GPCRs that couple to RhoA activation in particular those for sphingosine-1-phosphate (S1P). The S1P2 and S1P3 receptors are the most abundant S1P receptor subtypes in mouse and human CPCs while cardiomyocytes express predominantly S1P1 receptors. Treatment of CPCs with S1P, as with thrombin and serum, increased proliferation through a pathway requiring RhoA signaling, as evidenced by significant attenuation when Rho was inhibited by treatment with C3 toxin. Further analysis demonstrated that both S1P- and serum-induced proliferation are regulated through the S1P2 and S1P3 receptor subtypes which couple to Gα12/13 to elicit RhoA activation. The transcriptional co-activator MRTF-A was activated by S1P as assessed by its nuclear accumulation and induction of a RhoA/MRTF-A luciferase reporter. In addition S1P treatment increased expression of cardiac lineage markers Mef2C and GATA4 and the smooth muscle marker GATA6 through activation of MRTF-A. In conclusion, we delineate an S1P-regulated signaling pathway in CPCs that introduces the possibility of targeting S1P2/3 receptors, Gα12/13 or RhoA to influence the proliferation and commitment of c-kit(+) CPCs and improve the response of the myocardium following injury.

  1. Sinupret activates CFTR and TMEM16A-dependent transepithelial chloride transport and improves indicators of mucociliary clearance.

    Directory of Open Access Journals (Sweden)

    Shaoyan Zhang

    Full Text Available INTRODUCTION: We have previously demonstrated that Sinupret, an established treatment prescribed widely in Europe for respiratory ailments including rhinosinusitis, promotes transepithelial chloride (Cl- secretion in vitro and in vivo. The present study was designed to evaluate other indicators of mucociliary clearance (MCC including ciliary beat frequency (CBF and airway surface liquid (ASL depth, but also investigate the mechanisms that underlie activity of this bioflavonoid. METHODS: Primary murine nasal septal epithelial (MNSE [wild type (WT and transgenic CFTR(-/-], human sinonasal epithelial (HSNE, WT CFTR-expressing CFBE and TMEM16A-expressing HEK cultures were utilized for the present experiments. CBF and ASL depth measurements were performed. Mechanisms underlying transepithelial Cl- transport were determined using pharmacologic manipulation in Ussing chambers, Fura-2 intracellular calcium [Ca(2+]i imaging, cAMP signaling, regulatory domain (R-D phosphorylation of CFTR, and excised inside out and whole cell patch clamp analysis. RESULTS: Sinupret-mediated Cl- secretion [ΔISC(µA/cm(2] was pronounced in WT MNSE (20.7+/-0.9 vs. 5.6+/-0.9(control, p<0.05, CFTR(-/- MNSE (10.1+/-1.0 vs. 0.9+/-0.3(control, p<0.05 and HSNE (20.7+/-0.3 vs. 6.4+/-0.9(control, p<0.05. The formulation activated Ca(2+ signaling and TMEM16A channels, but also increased CFTR channel open probability (Po without stimulating PKA-dependent pathways responsible for phosphorylation of the CFTR R-domain and resultant Cl- secretion. Sinupret also enhanced CBF and ASL depth. CONCLUSION: Sinupret stimulates CBF, promotes transepithelial Cl- secretion, and increases ASL depth in a manner likely to enhance MCC. Our findings suggest that direct stimulation of CFTR, together with activation of Ca(2+-dependent TMEM16A secretion account for the majority of anion transport attributable to Sinupret. These studies provide further rationale for using robust Cl- secretagogue based therapies as an emerging treatment modality for common respiratory diseases of MCC including acute and chronic bronchitis and CRS.

  2. General joint frailty model for recurrent event data with a dependent terminal event: Application to follicular lymphoma data.

    Science.gov (United States)

    Mazroui, Yassin; Mathoulin-Pelissier, Simone; Soubeyran, Pierre; Rondeau, Virginie

    2012-05-20

    Many biomedical studies focus on delaying disease relapses and on prolonging survival. Usual methods only consider one event, often the first recurrence or death. However, ignoring the other recurrences may lead to biased results. The whole history of the disease should be considered for each patient. In addition, some diseases involve recurrences that can increase the risk of death. In this case, the death time may be dependent on the recurrent event history. We propose a joint frailty model to analyze recurrences and death simultaneously. Two gamma-distributed frailties take into account both the inter-recurrences dependence and the dependence between the recurrences and the survival times. We estimate separate parameters for disease recurrent event times and survival times in the joint frailty model to distinguish treatment effects and prognostic factors on these two types of events. We show how maximum penalized likelihood estimation can be applied to semiparametric estimation of the continuous hazard functions in the proposed joint frailty model with right censoring. We also propose parametrical approach. We evaluate the model by simulation studies and illustrate through a study of patients with follicular lymphoma.

  3. On the A-dependence of {sigma}{sub L}/{sigma}{sub T}: Skeletons in the shadow

    Energy Technology Data Exchange (ETDEWEB)

    Milana, J. [Univ. of Maryland, College Park (United States)

    1994-04-01

    A most counter-intuitive dependence in the differential cross-section in the shadowing regime is shown to result from a higher-twist nuclear enhancement in R = {sigma}{sub L}/{sigma}{sub T} which severely complicates the unravelling from present data the corresponding dependence in Q{sup 2} of the nuclear structure functions, F{sub 2}{sup A}(x,Q{sup 2}). Indeed, until precision measurements close this loophole, the extrapolations of the structure functions to either higher Q{sup 2} or other processes (as is necessary to address present data at FNAL or future experiments at RHIC) must be considered problematic. The contribution CEBAF can make in this regard by providing systematic determination of R{sub A}(x, Q{sup 2}) is thus emphasized. The purpose of this talk is to motivate an experiment CEBAF can and should do, especially if upgraded to higher energies. While providing information on nuclear structure that is interesting in itself, the added motivation is that precision results will have important impact on other high-energy experiments involving nuclear targets that have been, and will continue to be done all over the world.

  4. Membrane association of PspA depends on activation of the phage-shock-protein response in Yersinia enterocolitica

    OpenAIRE

    YAMAGUCHI, Saori; Gueguen, Erwan; Horstman, N. Kaye; Andrew J. Darwin

    2010-01-01

    Regulation of the bacterial phage-shock-protein (Psp) system involves communication between integral (PspBC) and peripheral (PspA) cytoplasmic membrane proteins and a soluble transcriptional activator (PspF). In this study protein subcellular localization studies were used to distinguish between spatial models for this putative signal transduction pathway in Yersinia enterocolitica. In non-inducing conditions PspA and PspF were almost exclusively in the soluble fraction, consistent with them ...

  5. Long-chain acyl-CoA-dependent regulation of gene expression in bacteria, yeast and mammals

    DEFF Research Database (Denmark)

    Black, P N; Færgeman, Nils J.; DiRusso, C C

    2000-01-01

    signal that modulates gene expression. In the bacteria Escherichia coli, long-chain fatty acyl-CoA bind directly to the transcription factor FadR. Acyl-CoA binding renders the protein incapable of binding DNA, thus preventing transcription activation and repression of many genes and operons. In the yeast......). Both repression and activation are dependent upon the function of either of the acyl-CoA synthetases Faa1p or Faa4p. In mammals, purified hepatocyte nuclear transcription factor 4alpha (HNF-4alpha) like E. coli FadR, binds long chain acyl-CoA directly. Coexpression of HNF-4alpha and acyl-CoA synthetase...

  6. Is a mean machine better than a dependable drive? It’s geared towards your regulatory focus

    Directory of Open Access Journals (Sweden)

    Graham G. Scott

    2012-08-01

    Full Text Available While many studies have investigated the role of message-level valence in persuasive messages (i.e., how positive or negative message content affects attitudes, none of these have examined whether word-level valence can modulate such effects. We investigated whether emotional language used within persuasive messages influenced attitudes and whether the processing of such communications could be modulated by regulatory focus. Using a 2 (Message: Positive, Negative × 2 (Words: Positive, Negative design, participants read car reviews and rated each on a series of semantic differentials and product recommendations. While positive messages were always rated higher than negative ones, the valence of a message’s component words differentially impacted attitudes toward distinct aspects of the product. On promotion-focus features, messages containing negative words produced higher ratings; for prevention-focus aspects, those with positive words resulted in higher ratings. We argue that adopting a prevention- or promotion-focused stance can influence the interpretation of emotion words in relation to overall message comprehension.

  7. The electron transfer flavoprotein fixABCX gene products from Azospirillum brasilense show a NifA-dependent promoter regulation.

    Science.gov (United States)

    Sperotto, Raul Antonio; Gross, Jeferson; Vedoy, Cleber; Passaglia, Luciane Maria Pereira; Schrank, Irene Silveira

    2004-10-01

    The complete nucleotide sequence of the A. brasilense fixA, fixB, fixC, and fixX genes is reported here. Sequence similarities between the protein sequences deduced from fixABCX genes and many electron transfer flavoproteins (ETFs) have been noted. Comparison of the amino acid sequences of both subunits of ETF with the A. brasilense fixA and fixB gene products exhibits an identity of 30%. The amino acid sequence of the other two genes, fixC and fixX, revealed similarity with the membrane-bound electron transfer flavoprotein ubiquinone oxidoreductase (ETF-QO). Using site-directed mutagenesis, mutations were introduced in the fixA promoter element of the A. brasilense fixABCX operon and chimeric p fixA-lacZ reporter gene fusions were constructed. The activation of the fixA promoter of A. brasilense is dependent upon the presence of the NifA protein being approximately 7 times less active than the A. brasilense nifH promoter. These results indicate that NifA from Klebsiella pneumoniae activates the fix promoter of A. brasilense and provide further evidence in support of the regulatory model of NifA activation in A. brasilense. Although no specific function has been assigned to the fixABCX gene products they are apparently required for symbiotic nitrogen fixation. An electron-transferring capacity in the nitrogen fixation pathway has been suggested for the fix gene products based on sequence homologies to the ETFs and ETF-QO proteins and by the absence of orthologous electron transfer proteins NifJ and NifF in A. brasilense.

  8. A dependência da política: Fernando Henrique Cardoso e a sociologia no Brasil

    Directory of Open Access Journals (Sweden)

    Afrânio Garcia Jr.

    2004-06-01

    Full Text Available Este artigo analisa a trajetória social, intelectual e profissional de Fernando Henrique Cardoso para entender os diferentes recursos sociais e disposições pessoais utilizados em sua carreira como sociólogo e em suas atividades como especialista da política. Busca demonstrar que os capitais sociais e as disposições responsáveis pelo prestígio como pesquisador e professor de ciências sociais foram distintos dos aplicados no domínio da política, permitindo sua rápida ascensão à presidência da República. Depois de estudar suas origens familiares, focaliza seus investimentos escolares e a escolha do ofício de sociólogo, a carreira promissora sendo bloqueada pelo golpe de 1964. O exílio permitiu a extensão das atividades e o reconhecimento internacional, reinvestidos em novo concurso para a USP; o AI-5 o conduzirá à dupla condição de cientista social e de um dos líderes da frente de oposições aos militares. Por fim, analisa-se a reconversão de seus recursos sociais e pessoais na profissão política.This article analyzes Fernando Henrique Cardoso's social, intellectual and professional trajectory in order to understand the different social resources and personal dispositions carried out in his career as sociologist or in his activities as a professional politician. It seeks to prove that the social capitals and the dispositions that might explain his prestige as researcher and professor of social sciences were very different from those required in the political domain, the ones allowing his fast rise to be the president of the Republic. After having examined his family origins, it focuses his scholar investments and the choice of the sociologist's occupation, a promising career suddenly blocked by the 1964 military coup. The exile encouraged new initiatives and brought him international appraisal, this moment being crowned with his access to the chair of political science at the University of São Paulo; the AI-5 enforced by the military rulers will enable him to assume a double condition, as social scientist and as an important opposition leader facing the military. Finally, it analyzes how he was able to reconvert his social and personal resources into the political profession.

  9. BMP-SMAD-ID promotes reprogramming to pluripotency by inhibiting p16/INK4A-dependent senescence

    Science.gov (United States)

    Hayashi, Yohei; Hsiao, Edward C.; Sami, Salma; Lancero, Mariselle; Schlieve, Christopher R.; Nguyen, Trieu; Yano, Koyori; Nagahashi, Ayako; Ikeya, Makoto; Matsumoto, Yoshihisa; Nishimura, Ken; Fukuda, Aya; Hisatake, Koji; Tomoda, Kiichiro; Asaka, Isao; Toguchida, Junya; Conklin, Bruce R.; Yamanaka, Shinya

    2016-01-01

    Fibrodysplasia ossificans progressiva (FOP) patients carry a missense mutation in ACVR1 [617G > A (R206H)] that leads to hyperactivation of BMP-SMAD signaling. Contrary to a previous study, here we show that FOP fibroblasts showed an increased efficiency of induced pluripotent stem cell (iPSC) generation. This positive effect was attenuated by inhibitors of BMP-SMAD signaling (Dorsomorphin or LDN1931890) or transducing inhibitory SMADs (SMAD6 or SMAD7). In normal fibroblasts, the efficiency of iPSC generation was enhanced by transducing mutant ACVR1 (617G > A) or SMAD1 or adding BMP4 protein at early times during the reprogramming. In contrast, adding BMP4 at later times decreased iPSC generation. ID genes, transcriptional targets of BMP-SMAD signaling, were critical for iPSC generation. The BMP-SMAD-ID signaling axis suppressed p16/INK4A-mediated cell senescence, a major barrier to reprogramming. These results using patient cells carrying the ACVR1 R206H mutation reveal how cellular signaling and gene expression change during the reprogramming processes. PMID:27794120

  10. Computational analysis of a novel mutation in ETFDH gene highlights its long-range effects on the FAD-binding motif

    Directory of Open Access Journals (Sweden)

    Chang Jan-Gowth

    2011-10-01

    Full Text Available Abstract Background Multiple acyl-coenzyme A dehydrogenase deficiency (MADD is an autosomal recessive disease caused by the defects in the mitochondrial electron transfer system and the metabolism of fatty acids. Recently, mutations in electron transfer flavoprotein dehydrogenase (ETFDH gene, encoding electron transfer flavoprotein:ubiquinone oxidoreductase (ETF:QO have been reported to be the major causes of riboflavin-responsive MADD. To date, no studies have been performed to explore the functional impact of these mutations or their mechanism of disrupting enzyme activity. Results High resolution melting (HRM analysis and sequencing of the entire ETFDH gene revealed a novel mutation (p.Phe128Ser and the hotspot mutation (p.Ala84Thr from a patient with MADD. According to the predicted 3D structure of ETF:QO, the two mutations are located within the flavin adenine dinucleotide (FAD binding domain; however, the two residues do not have direct interactions with the FAD ligand. Using molecular dynamics (MD simulations and normal mode analysis (NMA, we found that the p.Ala84Thr and p.Phe128Ser mutations are most likely to alter the protein structure near the FAD binding site as well as disrupt the stability of the FAD binding required for the activation of ETF:QO. Intriguingly, NMA revealed that several reported disease-causing mutations in the ETF:QO protein show highly correlated motions with the FAD-binding site. Conclusions Based on the present findings, we conclude that the changes made to the amino acids in ETF:QO are likely to influence the FAD-binding stability.

  11. Separation of isomeric short-chain acyl-CoAs in plant matrices using ultra-performance liquid chromatography coupled with tandem mass spectrometry.

    Science.gov (United States)

    Purves, Randy W; Ambrose, Stephen J; Clark, Shawn M; Stout, Jake M; Page, Jonathan E

    2015-02-01

    Acyl coenzyme A (acyl-CoA) thioesters are important intermediates in cellular metabolism and being able to distinguish among them is critical to fully understanding metabolic pathways in plants. Although significant advances have been made in the identification and quantification of acyl-CoAs using liquid chromatography tandem mass spectrometry (LC-MS/MS), separation of isomeric species such as isobutyryl- and n-butyrl-CoA has remained elusive. Here we report an ultra-performance liquid chromatography (UPLC)-MS/MS method for quantifying short-chain acyl-CoAs including isomeric species n-butyryl-CoA and isobutyryl-CoA as well as n-valeryl-CoA and isovaleryl-CoA. The method was applied to the analysis of extracts of hop (Humulus lupulus) and provided strong evidence for the existence of an additional structural isomer of valeryl-CoA, 2-methylbutyryl-CoA, as well as an unexpected isomer of hexanoyl-CoA. The results showed differences in the acyl-CoA composition among varieties of Humulus lupulus, both in glandular trichomes and cone tissues. When compared with the analysis of hemp (Cannabis sativa) extracts, the contribution of isobutyryl-CoAs in hop was greater as would be expected based on the downstream polyketide products. Surprisingly, branched chain valeryl-CoAs (isovaleryl-CoA and 2-methylbutyryl-CoA) were the dominant form of valeryl-CoAs in both hop and hemp. The capability to separate these isomeric forms will help to understand biochemical pathways leading to specialized metabolites in plants.

  12. Effects of dietary supplementation of coriander oil, in canola oil diets, on the metabolism of [1-(14)C] 18:3n-3 and [1-(14)C] 18:2n-6 in rainbow trout hepatocytes.

    Science.gov (United States)

    Randall, K M; Drew, M D; Øverland, M; Østbye, T-K; Bjerke, M; Vogt, G; Ruyter, B

    2013-09-01

    The aim of this study was to investigate the effects of petroselinic acid, found in coriander oil, on the ability of rainbow trout hepatocytes to increase the production of eicosapentaenoic acid (20:5n-3; EPA) and docosahexaenoic acid (22:6n-3; DHA) from [1-(14)C] α-linolenic acid (18:3n-3; ALA) and to reduce the production of arachidonic acid (20:4n-6; ARA) from [1-(14)C] 18:2n-6. Addition of coriander oil increased the production of 22:6n-3, from [1-(14)C] 18:3n-3, at the 0.5 and 1.0% inclusion levels and reduced the conversion of [1-(14)C] 18:2n-6 to 20:4n-6. β-Oxidation was significantly increased at the 1.5% inclusion level for [1-(14)C] 18:2n-6, however β-oxidation for [1-(14)C] 18:3n-3 only showed an increasing trend. Acetate, a main breakdown product of fatty acids (FA) via peroxisomal β-oxidation, decreased three-fold for [1-(14)C] 18:2n-6 and nearly doubled for [1-(14)C] 18:3n-3 when coriander was added at a 1.5% inclusion level. Acyl coenzyme A oxidase (ACO) enzyme activity showed no significant differences between treatments. Relative gene expression of ∆6 desaturase decreased with addition of coriander oil compared to the control. The addition of petroselinic acid via coriander oil to vegetable oil (VO) based diets containing no fishmeal (FM) or fish oil (FO), significantly increased the production of anti-inflammatory precursor 22:6n-3 (P=0.011) and decreased pro-inflammatory precursor 20:4n-6 (P=0.023) in radiolabelled hepatocytes of rainbow trout.

  13. Neutral lipid biosynthesis in engineered Escherichia coli: jojoba oil-like wax esters and fatty acid butyl esters.

    Science.gov (United States)

    Kalscheuer, Rainer; Stöveken, Tim; Luftmann, Heinrich; Malkus, Ursula; Reichelt, Rudolf; Steinbüchel, Alexander

    2006-02-01

    Wax esters are esters of long-chain fatty acids and long-chain fatty alcohols which are of considerable commercial importance and are produced on a scale of 3 million tons per year. The oil from the jojoba plant (Simmondsia chinensis) is the main biological source of wax esters. Although it has a multitude of potential applications, the use of jojoba oil is restricted, due to its high price. In this study, we describe the establishment of heterologous wax ester biosynthesis in a recombinant Escherichia coli strain by coexpression of a fatty alcohol-producing bifunctional acyl-coenzyme A reductase from the jojoba plant and a bacterial wax ester synthase from Acinetobacter baylyi strain ADP1, catalyzing the esterification of fatty alcohols and coenzyme A thioesters of fatty acids. In the presence of oleate, jojoba oil-like wax esters such as palmityl oleate, palmityl palmitoleate, and oleyl oleate were produced, amounting to up to ca. 1% of the cellular dry weight. In addition to wax esters, fatty acid butyl esters were unexpectedly observed in the presence of oleate. The latter could be attributed to solvent residues of 1-butanol present in the medium component, Bacto tryptone. Neutral lipids produced in recombinant E. coli were accumulated as intracytoplasmic inclusions, demonstrating that the formation and structural integrity of bacterial lipid bodies do not require specific structural proteins. This is the first report on substantial biosynthesis and accumulation of neutral lipids in E. coli, which might open new perspectives for the biotechnological production of cheap jojoba oil equivalents from inexpensive resources employing recombinant microorganisms.

  14. A mycothiol synthase mutant of Mycobacterium smegmatis produces novel thiols and has an altered thiol redox status.

    Science.gov (United States)

    Newton, Gerald L; Ta, Philong; Fahey, Robert C

    2005-11-01

    Mycobacteria and other actinomycetes do not produce glutathione but make mycothiol (MSH; AcCys-GlcN-Ins) that has functions similar to those of glutathione and is essential for growth of Mycobacterium tuberculosis. Mycothiol synthase (MshD) catalyzes N acetylation of Cys-GlcN-Ins to produce MSH in Mycobacterium smegmatis mc2155, and Cys-GlcN-Ins is maintained at a low level. The mycothiol synthase mutant, the mshD::Tn5 mutant, produces high levels of Cys-GlcN-Ins along with two novel thiols, N-formyl-Cys-GlcN-Ins and N-succinyl-Cys-GlcN-Ins, and a small amount of MSH. The nonenzymatic reaction of acyl-coenzyme A (CoA) with Cys-GlcN-Ins to produce acyl-Cys-GlcN-Ins is a facile reaction under physiologic conditions, with succinyl-CoA being an order of magnitude more reactive than acetyl-CoA. The uncatalyzed reaction rates are adequate to account for the observed production of N-succinyl-Cys-GlcN-Ins and MSH under physiologic conditions. It was shown that the N-acyl-Cys-GlcN-Ins compounds are maintained in a substantially reduced state in the mutant but that Cys-GlcN-Ins exists in disulfide forms at 5 to 40% at different stages of growth. MSH was able to facilitate reduction of N-succinyl-Cys-GlcN-Ins disulfide through thiol-disulfide exchange, but N-formyl-Cys-GlcN-Ins was ineffective. The oxidized state of Cys-GlcN-Ins in cells appears to result from a high susceptibility to autoxidation and a low capacity of the cell to reduce its disulfide forms. The mutant exhibited no enhanced sensitivity to hydrogen peroxide, tert-butyl hydroperoxide, or cumene hydroperoxide relative to the parent strain, suggesting that the most abundant thiol, N-formyl-Cys-GlcN-Ins, functions as a substitute for MSH.

  15. Altered hyperlipidemia, hepatic steatosis, and hepatic peroxisome proliferator-activated receptors in rats with intake of tart cherry.

    Science.gov (United States)

    Seymour, E Mitchell; Singer, Andrew A M; Kirakosyan, Ara; Urcuyo-Llanes, Daniel E; Kaufman, Peter B; Bolling, Steven F

    2008-06-01

    Elevated plasma lipids, glucose, insulin, and fatty liver are among components of metabolic syndrome, a phenotypic pattern that typically precedes the development of Type 2 diabetes. Animal studies show that intake of anthocyanins reduces hyperlipidemia, obesity, and atherosclerosis and that anthocyanin-rich extracts may exert these effects in association with altered activity of tissue peroxisome proliferator-activated receptors (PPARs). However, studies are lacking to test this correlation using physiologically relevant, whole food sources of anthocyanins. Tart cherries are a rich source of anthocyanins, and whole cherry fruit intake may also affect hyperlipidemia and/or affect tissue PPARs. This hypothesis was tested in the Dahl Salt-Sensitive rat having insulin resistance and hyperlipidemia. For 90 days, Dahl rats were pair-fed AIN-76a-based diets supplemented with either 1% (wt:wt) freeze-dried whole tart cherry or with 0.85% additional carbohydrate to match macronutrient and calorie provision. After 90 days, the cherry-enriched diet was associated with reduced fasting blood glucose, hyperlipidemia, hyperinsulinemia, and reduced fatty liver. The cherry diet was also associated with significantly enhanced hepatic PPAR-alpha mRNA, enhanced hepatic PPAR-alpha target acyl-coenzyme A oxidase mRNA and activity, and increased plasma antioxidant capacity. In conclusion, physiologically relevant tart cherry consumption reduced several phenotypic risk factors that are associated with risk for metabolic syndrome and Type 2 diabetes. Tart cherries may represent a whole food research model of the health effects of anthocyanin-rich foods and may possess nutraceutical value against risk factors for metabolic syndrome and its clinical sequelae.

  16. High ACSL5 Transcript Levels Associate with Systemic Lupus Erythematosus and Apoptosis in Jurkat T Lymphocytes and Peripheral Blood Cells

    Science.gov (United States)

    2011-01-01

    Background Systemic lupus erythematosus (SLE) is a prototypical autoimmune disease in which increased apoptosis and decreased apoptotic cells removal has been described as most relevant in the pathogenesis. Long-chain acyl-coenzyme A synthetases (ACSLs) have been involved in the immunological dysfunction of mouse models of lupus-like autoimmunity and apoptosis in different in vitro cell systems. The aim of this work was to assess among the ACSL isoforms the involvement of ACSL2, ACSL4 and ACSL5 in SLE pathogenesis. Findings With this end, we determined the ACSL2, ACSL4 and ACSL5 transcript levels in peripheral blood mononuclear cells (PBMCs) of 45 SLE patients and 49 healthy controls by quantitative real time-PCR (q-PCR). We found that patients with SLE had higher ACSL5 transcript levels than healthy controls [median (range), healthy controls = 16.5 (12.3–18.0) vs. SLE = 26.5 (17.8–41.7), P = 3.9×10 E-5] but no differences were found for ACSL2 and ACSL4. In in vitro experiments, ACSL5 mRNA expression was greatly increased when inducing apoptosis in Jurkat T cells and PBMCs by Phorbol-Myristate-Acetate plus Ionomycin (PMA+Io). On the other hand, short interference RNA (siRNA)-mediated silencing of ACSL5 decreased induced apoptosis in Jurkat T cells up to the control levels as well as decreased mRNA expression of FAS, FASLG and TNF. Conclusions These findings indicate that ACSL5 may play a role in the apoptosis that takes place in SLE. Our results point to ACSL5 as a potential novel functional marker of pathogenesis and a possible therapeutic target in SLE. PMID:22163040

  17. Thiolation-enhanced substrate recognition by D-alanyl carrier protein ligase DltA from Bacillus cereus [v1; ref status: indexed, http://f1000r.es/3dx

    Directory of Open Access Journals (Sweden)

    Liqin Du

    2014-05-01

    Full Text Available D-alanylation of the lipoteichoic acid on Gram-positive cell wall is dependent on dlt gene-encoded proteins DltA, DltB, DltC and DltD. The D-alanyl carrier protein ligase DltA, as a remote homolog of acyl-(coenzyme A (CoA synthetase, cycles through two active conformations for the catalysis of adenylation and subsequent thiolation of D-alanine (D-Ala. The crystal structure of DltA in the absence of any substrate was observed to have a noticeably more disordered pocket for ATP which would explain why DltA has relatively low affinity for ATP in the absence of any D-alanyl carrier. We have previously enabled the thiolation of D-alanine in the presence of CoA as the mimic of D-alanyl carrier protein DltC which carries a 4’-phosphopantetheine group on a serine residue. Here we show that the resulting Michaelis constants in the presence of saturating CoA for both ATP and D-alanine were reduced more than 10 fold as compared to the values obtained in the absence of CoA. The presence of CoA also made DltA ~100-fold more selective on D-alanine over L-alanine. The CoA-enhanced substrate recognition further implies that the ATP and D-alanine substrates of the adenylation reaction are incorporated when the DltA enzyme cycles through its thiolation conformation.

  18. Structural and Functional Studies of Fatty Acyl Adenylate Ligases from E. coli and L. pneumophila

    Energy Technology Data Exchange (ETDEWEB)

    Z Zhang; R Zhou; J Sauder; P Tonge; S Burley; S Swaminathan

    2011-12-31

    Fatty acyl-AMP ligase (FAAL) is a new member of a family of adenylate-forming enzymes that were recently discovered in Mycobacterium tuberculosis. They are similar in sequence to fatty acyl-coenzyme A (CoA) ligases (FACLs). However, while FACLs perform a two-step catalytic reaction, AMP ligation followed by CoA ligation using ATP and CoA as cofactors, FAALs produce only the acyl adenylate and are unable to perform the second step. We report X-ray crystal structures of full-length FAAL from Escherichia coli (EcFAAL) and FAAL from Legionella pneumophila (LpFAAL) bound to acyl adenylate, determined at resolution limits of 3.0 and 1.85 {angstrom}, respectively. The structures share a larger N-terminal domain and a smaller C-terminal domain, which together resemble the previously determined structures of FAAL and FACL proteins. Our two structures occur in quite different conformations. EcFAAL adopts the adenylate-forming conformation typical of FACLs, whereas LpFAAL exhibits a unique intermediate conformation. Both EcFAAL and LpFAAL have insertion motifs that distinguish them from the FACLs. Structures of EcFAAL and LpFAAL reveal detailed interactions between this insertion motif and the interdomain hinge region and with the C-terminal domain. We suggest that the insertion motifs support sufficient interdomain motions to allow substrate binding and product release during acyl adenylate formation, but they preclude CoA binding, thereby preventing CoA ligation.

  19. Structural and Functional Studies of Fatty Acyl Adenylate Ligases from E. coli and L. pneumophila

    Energy Technology Data Exchange (ETDEWEB)

    Zhang, Z.; Swaminathan, S.; Zhou, R.; Sauder, J. M.; Tonge, P. J.; Burley, S. K.

    2011-02-18

    Fatty acyl-AMP ligase (FAAL) is a new member of a family of adenylate-forming enzymes that were recently discovered in Mycobacterium tuberculosis. They are similar in sequence to fatty acyl-coenzyme A (CoA) ligases (FACLs). However, while FACLs perform a two-step catalytic reaction, AMP ligation followed by CoA ligation using ATP and CoA as cofactors, FAALs produce only the acyl adenylate and are unable to perform the second step. We report X-ray crystal structures of full-length FAAL from Escherichia coli (EcFAAL) and FAAL from Legionella pneumophila (LpFAAL) bound to acyl adenylate, determined at resolution limits of 3.0 and 1.85 {angstrom}, respectively. The structures share a larger N-terminal domain and a smaller C-terminal domain, which together resemble the previously determined structures of FAAL and FACL proteins. Our two structures occur in quite different conformations. EcFAAL adopts the adenylate-forming conformation typical of FACLs, whereas LpFAAL exhibits a unique intermediate conformation. Both EcFAAL and LpFAAL have insertion motifs that distinguish them from the FACLs. Structures of EcFAAL and LpFAAL reveal detailed interactions between this insertion motif and the interdomain hinge region and with the C-terminal domain. We suggest that the insertion motifs support sufficient interdomain motions to allow substrate binding and product release during acyl adenylate formation, but they preclude CoA binding, thereby preventing CoA ligation.

  20. A novel technical approach for the measurement of individual ACAT-1 and ACAT-2 enzymatic activity in the testis.

    Science.gov (United States)

    Chen, Li; Lafond, Julie; Pelletier, R-Marc

    2009-01-01

    Acyl-coenzyme A:cholesterol acyltransferase (ACAT) is implicated in the esterification of cholesterol when the latter is present at concentrations exceeding metabolic demands. Thus, ACAT contributes to the maintenance of cholesterol homeostasis which in testis is essential for the production of fertile gametes. However, the role of individual isoform of the enzyme in the maintenance of cholesterol homeostasis in the gonads has not been addressed yet because approaches to measure the enzymatic activity of each isoform were lacking. Here, we used the selective ACAT-1 inhibitor, K-604, to measure the individual enzymatic activity of ACAT-1 and ACAT-2 in enriched fractions of mouse seminiferous tubules. K-604 inhibited adult mouse ACAT-1 much more than ACAT-2 with IC(50) values of 100 and 1,000 microM, respectively, in the tubules. Next, the inhibitor concentration (100 microM) that inhibits the activity of ACAT-1 but not the activity of ACAT-2 was determined and applied to measure ACAT-1 and ACAT-2 enzymatic activities in mouse seminiferous tubule-enriched fractions. ACAT-2 activity reached 2173 CPMB/200 microg protein, while ACAT-1 enzymatic activity was 713 CPMB/200 microg proteins in the tubules. We also compared the effect of another inhibitor Manassantin B with K-604. Increasing the concentration (0-1,000 microM) of Manassantin B resulted in the inhibition of the activity of both ACAT-1 and ACAT-2. The results show that only K-604 is a useful tool to determine the individual ACAT-1 and ACAT-2 enzymatic activities in the seminiferous tubules.

  1. Role of fatty acid binding proteins and long chain fatty acids in modulating nuclear receptors and gene transcription.

    Science.gov (United States)

    Schroeder, Friedhelm; Petrescu, Anca D; Huang, Huan; Atshaves, Barbara P; McIntosh, Avery L; Martin, Gregory G; Hostetler, Heather A; Vespa, Aude; Landrock, Danilo; Landrock, Kerstin K; Payne, H Ross; Kier, Ann B

    2008-01-01

    Abnormal energy regulation may significantly contribute to the pathogenesis of obesity, diabetes mellitus, cardiovascular disease, and cancer. For rapid control of energy homeostasis, allosteric and posttranslational events activate or alter activity of key metabolic enzymes. For longer impact, transcriptional regulation is more effective, especially in response to nutrients such as long chain fatty acids (LCFA). Recent advances provide insights into how poorly water-soluble lipid nutrients [LCFA; retinoic acid (RA)] and their metabolites (long chain fatty acyl Coenzyme A, LCFA-CoA) reach nuclei, bind their cognate ligand-activated receptors, and regulate transcription for signaling lipid and glucose catabolism or storage: (i) while serum and cytoplasmic LCFA levels are in the 200 mircroM-mM range, real-time imaging recently revealed that LCFA and LCFA-CoA are also located within nuclei (nM range); (ii) sensitive fluorescence binding assays show that LCFA-activated nuclear receptors [peroxisome proliferator-activated receptor-alpha (PPARalpha) and hepatocyte nuclear factor 4alpha (HNF4alpha)] exhibit high affinity (low nM KdS) for LCFA (PPARalpha) and/or LCFA-CoA (PPARalpha, HNF4alpha)-in the same range as nuclear levels of these ligands; (iii) live and fixed cell immunolabeling and imaging revealed that some cytoplasmic lipid binding proteins [liver fatty acid binding protein (L-FABP), acyl CoA binding protein (ACBP), cellular retinoic acid binding protein-2 (CRABP-2)] enter nuclei, bind nuclear receptors (PPARalpha, HNF4alpha, CRABP-2), and activate transcription of genes in fatty acid and glucose metabolism; and (iv) studies with gene ablated mice provided physiological relevance of LCFA and LCFA-CoA binding proteins in nuclear signaling. This led to the hypothesis that cytoplasmic lipid binding proteins transfer and channel lipidic ligands into nuclei for initiating nuclear receptor transcriptional activity to provide new lipid nutrient signaling pathways that

  2. Changes in esculeoside A content in different regions of the tomato fruit during maturation and heat processing.

    Science.gov (United States)

    Katsumata, Akiko; Kimura, Mizuki; Saigo, Hiromi; Aburaya, Kei; Nakano, Masako; Ikeda, Tsuyoshi; Fujiwara, Yukio; Nagai, Ryoji

    2011-04-27

    We previously demonstrated that esculeogenin A, a new aglycone of the tomato sapogenol esculeoside A, inhibits both acyl coenzyme A:cholesterol acyl-transferase (ACAT)-1 and -2 and ameliorates the pathogenesis of atherosclerosis in apoE deficient mice. Although we believe that daily intake of esculeoside A from tomato products can play a beneficial role in preventing the pathogenesis of atherosclerosis, the compound is not being used for preventive medicine due to the lack of information on methods for quantitative analysis and the content and stability of the compound in tomato products. In the present study, we report the development of a high-performance liquid chromatography (HPLC) method using an instrument equipped with a refractive index (RI) detector for esculeoside A quantification. We used this method to measure the changes in esculeoside A content during maturation, its distribution in the fruit body, and its stability during the heating process. The contents of esculeoside A in cherry tomatoes and Momotaro tomatoes were 21- and 9-fold, respectively, higher than that of lycopene, which is the most well-known compound in tomatoes. Furthermore, the esculeoside A content in pericarp wall was higher than in the whole tomato fruit and increased in a time-dependent manner during maturation. Although the melting point of purified esculeoside A was 225 °C, the esculeoside A in crude tomato extract decreased in a temperature-dependent manner. Degradation due to the heating process was inhibited under a pH of 9. These results demonstrated that the esculeoside A content differs in the various types of tomatoes, during maturation, and during the heating process used for preservation.

  3. Reducing isozyme competition increases target fatty acid accumulation in seed triacylglycerols of transgenic Arabidopsis.

    Science.gov (United States)

    van Erp, Harrie; Shockey, Jay; Zhang, Meng; Adhikari, Neil D; Browse, John

    2015-05-01

    One goal of green chemistry is the production of industrially useful fatty acids (FAs) in crop plants. We focus on hydroxy fatty acids (HFAs) and conjugated polyenoic FAs (α-eleostearic acids [ESAs]) using Arabidopsis (Arabidopsis thaliana) as a model. These FAs are found naturally in seed oils of castor (Ricinus communis) and tung tree (Vernicia fordii), respectively, and used for the production of lubricants, nylon, and paints. Transgenic oils typically contain less target FA than that produced in the source species. We hypothesized that competition between endogenous and transgenic isozymes for substrates limits accumulation of unique FAs in Arabidopsis seeds. This hypothesis was tested by introducing a mutation in Arabidopsis diacylglycerol acyltransferase1 (AtDGAT1) in a line expressing castor FA hydroxylase and acyl-Coenzyme A:RcDGAT2 in its seeds. This led to a 17% increase in the proportion of HFA in seed oil. Expression of castor phospholipid:diacylglycerol acyltransferase 1A in this line increased the proportion of HFA by an additional 12%. To determine if our observations are more widely applicable, we investigated if isozyme competition influenced production of ESA. Expression of tung tree FA conjugase/desaturase in Arabidopsis produced approximately 7.5% ESA in seed lipids. Coexpression of VfDGAT2 increased ESA levels to approximately 11%. Overexpression of VfDGAT2 combined with suppression of AtDGAT1 increased ESA accumulation to 14% to 15%. Our results indicate that isozyme competition is a limiting factor in the engineering of unusual FAs in heterologous plant systems and that reduction of competition through mutation and RNA suppression may be a useful component of seed metabolic engineering strategies.

  4. High ACSL5 transcript levels associate with systemic lupus erythematosus and apoptosis in Jurkat T lymphocytes and peripheral blood cells.

    Directory of Open Access Journals (Sweden)

    Antonio Catalá-Rabasa

    Full Text Available BACKGROUND: Systemic lupus erythematosus (SLE is a prototypical autoimmune disease in which increased apoptosis and decreased apoptotic cells removal has been described as most relevant in the pathogenesis. Long-chain acyl-coenzyme A synthetases (ACSLs have been involved in the immunological dysfunction of mouse models of lupus-like autoimmunity and apoptosis in different in vitro cell systems. The aim of this work was to assess among the ACSL isoforms the involvement of ACSL2, ACSL4 and ACSL5 in SLE pathogenesis. FINDINGS: With this end, we determined the ACSL2, ACSL4 and ACSL5 transcript levels in peripheral blood mononuclear cells (PBMCs of 45 SLE patients and 49 healthy controls by quantitative real time-PCR (q-PCR. We found that patients with SLE had higher ACSL5 transcript levels than healthy controls [median (range, healthy controls = 16.5 (12.3-18.0 vs. SLE = 26.5 (17.8-41.7, P = 3.9×10 E-5] but no differences were found for ACSL2 and ACSL4. In in vitro experiments, ACSL5 mRNA expression was greatly increased when inducing apoptosis in Jurkat T cells and PBMCs by Phorbol-Myristate-Acetate plus Ionomycin (PMA+Io. On the other hand, short interference RNA (siRNA-mediated silencing of ACSL5 decreased induced apoptosis in Jurkat T cells up to the control levels as well as decreased mRNA expression of FAS, FASLG and TNF. CONCLUSIONS: These findings indicate that ACSL5 may play a role in the apoptosis that takes place in SLE. Our results point to ACSL5 as a potential novel functional marker of pathogenesis and a possible therapeutic target in SLE.

  5. Unexpected functional diversity in the fatty acid desaturases of the flour beetle Tribolium castaneum and identification of key residues determining activity.

    Science.gov (United States)

    Haritos, Victoria S; Horne, Irene; Damcevski, Katherine; Glover, Karen; Gibb, Nerida

    2014-08-01

    Desaturases catalyse modifications to fatty acids which are essential to homeostasis and for pheromone and defensive chemical production. All desaturases of the flour beetle Tribolium castaneum were investigated via query of the sequenced genome which yielded 15 putative acyl-Coenzyme A genes. Eleven desaturase mRNA were obtained in full length and functionally expressed in yeast. Phylogenetic analysis separated the desaturases into 4 distinct clades; one clade contained conserved beetle Δ9 desaturases, second clade was Tribolium-specific having diverse activities including Δ5, Δ9 and Δ12 desaturation and the other 2 clades had mixed insect representatives. Three members of this clade contained unusual inserted sequences of ∼20 residues in the C-terminal region and were related to desaturases that all contained similar inserts. Deletion of the entirety of the insert in the flour beetle Δ12 desaturase abolished its activity but this was partially restored by the reintroduction of two histidine residues, suggesting the histidine(s) are required for activity but the full length insert is not. Five new desaturase activities were discovered: Δ9 desaturation of C12:0-C16:0 substrates; two unprecedented Δ5 enzymes acting on C18:0 and C16:0; Δ9 activity exclusively on C16:0 and a further stearate Δ9 desaturase. qPCR analysis ruled out a role in sex pheromone synthesis for the Δ5 and Δ9/C16:0 desaturases. The flour beetle genome has underpinned an examination of all transcribed desaturases in the organism and revealed a diversity of novel and unusual activities, an improved understanding of the evolutionary relationships among insect desaturases and sequence determinants of activity.

  6. Therapeutic potential of chalcones as cardiovascular agents.

    Science.gov (United States)

    Mahapatra, Debarshi Kar; Bharti, Sanjay Kumar

    2016-03-01

    Cardiovascular diseases are the leading cause of death affecting 17.3 million people across the globe and are estimated to affect 23.3 million people by year 2030. In recent years, about 7.3 million people died due to coronary heart disease, 9.4 million deaths due to high blood pressure and 6.2 million due to stroke, where obesity and atherosclerotic progression remain the chief pathological factors. The search for newer and better cardiovascular agents is the foremost need to manage cardiac patient population across the world. Several natural and (semi) synthetic chalcones deserve the credit of being potential candidates to inhibit various cardiovascular, hematological and anti-obesity targets like angiotensin converting enzyme (ACE), cholesteryl ester transfer protein (CETP), diacylglycerol acyltransferase (DGAT), acyl-coenzyme A: cholesterol acyltransferase (ACAT), pancreatic lipase (PL), lipoprotein lipase (LPL), calcium (Ca(2+))/potassium (K(+)) channel, COX-1, TXA2 and TXB2. In this review, a comprehensive study of chalcones, their therapeutic targets, structure activity relationships (SARs), mechanisms of actions (MOAs) have been discussed. Chemically diverse chalcone scaffolds, their derivatives including structural manipulation of both aryl rings, replacement with heteroaryl scaffold(s) and hybridization through conjugation with other pharmacologically active scaffold have been highlighted. Chalcones which showed promising activity and have a well-defined MOAs, SARs must be considered as prototype for the design and development of potential anti-hypertensive, anti-anginal, anti-arrhythmic and cardioprotective agents. With the knowledge of these molecular targets, structural insights and SARs, this review may be helpful for (medicinal) chemists to design more potent, safe, selective and cost effective chalcone derivatives as potential cardiovascular agents.

  7. Enhancement of human ACAT1 gene expression to promote the macrophage-derived foam cell formation by dexamethasone

    Institute of Scientific and Technical Information of China (English)

    Li YANG; Ta Yuan CHANG; Bo Liang LI; Jin Bo YANG; Jia CHEN; Guang Yao YU; Pei ZHOU; Lei LEI; Zhen Zhen WANG; Catherine CY CHANG; XinYing YANG

    2004-01-01

    In macrophages, the accumulation of cholesteryl esters synthesized by the activated acyl-coenzyme A:cholesterol acyltransferase-1 (ACAT1) results in the foam cell formation, a hallmark of early atherosclerotic lesions. In this study,with the treatment of a glucocorticoid hormone dexamethasone (Dex), lipid staining results clearly showed the large accumulation of lipid droplets containing cholesteryl esters in THP- 1-derived macrophages exposed to lower concentration of the oxidized low-density lipoprotein (ox-LDL). More notably, when treated together with specific anti-ACAT inhibitors, the abundant cholesteryl ester accumulation was markedly diminished in THP-l-derived macrophages, confirming that ACAT is the key enzyme responsible for intracellular cholesteryl ester synthesis. RT-PCR and Western blot results indicated that Dex caused up-regulation of human ACAT1 expression at both the mRNA and protein levels in THP-1 and THP- 1-derived macrophages. The luciferase activity assay demonstrated that Dex could enhance the activity of human ACAT1 gene P1 promoter, a major factor leading to the ACAT1 activation, in a cell-specific manner.Further experimental evidences showed that a glucocorticoid response element (GRE) located within human ACAT1gene P1 promoter to response to the elevation of human ACAT1 gene expression by Dex could be functionally bound with glucocorticoid receptor (GR) proteins. These data supported the hypothesis that the clinical treatment with Dex,which increased the incidence of atherosclerosis, may in part due to enhancing the ACAT1 expression to promote the accumulation of cholesteryl esters during the macrophage-derived foam cell formation, an early stage of atherosclerosis.

  8. ETHE1 mutations are specific to ethylmalonic encephalopathy.

    Science.gov (United States)

    Tiranti, V; Briem, E; Lamantea, E; Mineri, R; Papaleo, E; De Gioia, L; Forlani, F; Rinaldo, P; Dickson, P; Abu-Libdeh, B; Cindro-Heberle, L; Owaidha, M; Jack, R M; Christensen, E; Burlina, A; Zeviani, M

    2006-04-01

    Mutations in ETHE1, a gene located at chromosome 19q13, have recently been identified in patients affected by ethylmalonic encephalopathy (EE). EE is a devastating infantile metabolic disorder, characterised by widespread lesions in the brain, hyperlactic acidaemia, petechiae, orthostatic acrocyanosis, and high levels of ethylmalonic acid in body fluids. To investigate to what extent ETHE1 is responsible for EE, we analysed this gene in 29 patients with typical EE and in 11 patients presenting with early onset progressive encephalopathy with ethylmalonic aciduria (non-EE EMA). Frameshift, stop, splice site, and missense mutations of ETHE1 were detected in all the typical EE patients analysed. Western blot analysis of the ETHE1 protein indicated that some of the missense mutations are associated with the presence of the protein, suggesting that the corresponding wild type amino acid residues have a catalytic function. No ETHE1 mutations were identified in non-EE EMA patients. Experiments based on two dimensional blue native electrophoresis indicated that ETHE1 protein works as a supramolecular, presumably homodimeric, complex, and a three dimensional model of the protein suggests that it is likely to be a mitochondrial matrix thioesterase acting on a still unknown substrate. Finally, the 625G-->A single nucleotide polymorphism in the gene encoding the short chain acyl-coenzyme A dehydrogenase (SCAD) was previously proposed as a co-factor in the aetiology of EE and other EMA syndromes. SNP analysis in our patients ruled out a pathogenic role of SCAD variants in EE, but did show a highly significant prevalence of the 625A alleles in non-EE EMA patients.

  9. FadD Is Required for Utilization of Endogenous Fatty Acids Released from Membrane Lipids ▿ †

    Science.gov (United States)

    Pech-Canul, Ángel; Nogales, Joaquina; Miranda-Molina, Alfonso; Álvarez, Laura; Geiger, Otto; Soto, María José; López-Lara, Isabel M.

    2011-01-01

    FadD is an acyl coenzyme A (CoA) synthetase responsible for the activation of exogenous long-chain fatty acids (LCFA) into acyl-CoAs. Mutation of fadD in the symbiotic nitrogen-fixing bacterium Sinorhizobium meliloti promotes swarming motility and leads to defects in nodulation of alfalfa plants. In this study, we found that S. meliloti fadD mutants accumulated a mixture of free fatty acids during the stationary phase of growth. The composition of the free fatty acid pool and the results obtained after specific labeling of esterified fatty acids with a Δ5-desaturase (Δ5-Des) were in agreement with membrane phospholipids being the origin of the released fatty acids. Escherichia coli fadD mutants also accumulated free fatty acids released from membrane lipids in the stationary phase. This phenomenon did not occur in a mutant of E. coli with a deficient FadL fatty acid transporter, suggesting that the accumulation of fatty acids in fadD mutants occurs inside the cell. Our results indicate that, besides the activation of exogenous LCFA, in bacteria FadD plays a major role in the activation of endogenous fatty acids released from membrane lipids. Furthermore, expression analysis performed with S. meliloti revealed that a functional FadD is required for the upregulation of genes involved in fatty acid degradation and suggested that in the wild-type strain, the fatty acids released from membrane lipids are degraded by β-oxidation in the stationary phase of growth. PMID:21926226

  10. FadD is required for utilization of endogenous fatty acids released from membrane lipids.

    Science.gov (United States)

    Pech-Canul, Ángel; Nogales, Joaquina; Miranda-Molina, Alfonso; Álvarez, Laura; Geiger, Otto; Soto, María José; López-Lara, Isabel M

    2011-11-01

    FadD is an acyl coenzyme A (CoA) synthetase responsible for the activation of exogenous long-chain fatty acids (LCFA) into acyl-CoAs. Mutation of fadD in the symbiotic nitrogen-fixing bacterium Sinorhizobium meliloti promotes swarming motility and leads to defects in nodulation of alfalfa plants. In this study, we found that S. meliloti fadD mutants accumulated a mixture of free fatty acids during the stationary phase of growth. The composition of the free fatty acid pool and the results obtained after specific labeling of esterified fatty acids with a Δ5-desaturase (Δ5-Des) were in agreement with membrane phospholipids being the origin of the released fatty acids. Escherichia coli fadD mutants also accumulated free fatty acids released from membrane lipids in the stationary phase. This phenomenon did not occur in a mutant of E. coli with a deficient FadL fatty acid transporter, suggesting that the accumulation of fatty acids in fadD mutants occurs inside the cell. Our results indicate that, besides the activation of exogenous LCFA, in bacteria FadD plays a major role in the activation of endogenous fatty acids released from membrane lipids. Furthermore, expression analysis performed with S. meliloti revealed that a functional FadD is required for the upregulation of genes involved in fatty acid degradation and suggested that in the wild-type strain, the fatty acids released from membrane lipids are degraded by β-oxidation in the stationary phase of growth.

  11. Lipidomic and spatio-temporal imaging of fat by mass spectrometry in mice duodenum during lipid digestion.

    Science.gov (United States)

    Seyer, Alexandre; Cantiello, Michela; Bertrand-Michel, Justine; Roques, Véronique; Nauze, Michel; Bézirard, Valérie; Collet, Xavier; Touboul, David; Brunelle, Alain; Coméra, Christine

    2013-01-01

    Intestinal absorption of dietary fat is a complex process mediated by enterocytes leading to lipid assembly and secretion of circulating lipoproteins as chylomicrons, vLDL and intestinal HDL (iHDL). Understanding lipid digestion is of importance knowing the correlation between excessive fat absorption and atherosclerosis. By using time-of-flight secondary ion mass spectrometry (TOF-SIMS), we illustrated a spatio-temporal localization of fat in mice duodenum, at different times of digestion after a lipid gavage, for the first time. Fatty acids progressively increased in enterocytes as well as taurocholic acid, secreted by bile and engaged in the entero-hepatic re-absorption cycle. Cytosolic lipid droplets (CLD) from enterocytes were originally purified separating chylomicron-like, intermediate droplets and smaller HDL-like. A lipidomic quantification revealed their contents in triglycerides, free and esterified cholesterol, phosphatidylcholine, sphingomyelin and ceramides but also in free fatty acids, mono- and di-acylglycerols. An acyl-transferase activity was identified and the enzyme monoacylglycerol acyl transferase 2 (MGAT2) was immunodetected in all CLD. The largest droplets was also shown to contain the microsomal triglyceride transfer protein (MTTP), the acyl-coenzyme A-cholesterol acyltransferases (ACAT) 1 and 2, hormone sensitive lipase (HSL) and adipose triglyceride lipase (ATGL). This highlights the fact that during the digestion of fats, enterocyte CLD contain some enzymes involved in the different stages of the metabolism of diet fatty acids and cholesterol, in anticipation of the crucial work of endoplasmic reticulum in the process. The data further underlines the dual role of chylomicrons and iHDL in fat digestion which should help to efficiently complement lipid-lowering therapy.

  12. Two Human ACAT2 mRNA Variants Produced by Alternative Splicing and Coding for Novel Isoenzymes

    Institute of Scientific and Technical Information of China (English)

    Xiao-Min YAO; Bo-Liang LI; Can-Hua WANG; Bao-Liang SONG; Xin-Ying YANG; Zhen-Zhen WANG; Wei QI; Zhi-Xin LIN; Catherine C. Y. CHANG; Ta-Yuan CHANG

    2005-01-01

    Acyl coenzyme A:cholesterol acyltransferase 2 (ACAT2) plays an important role in cholesterol absorption. Human ACAT2 is highly expressed in small intestine and fetal liver, but its expression is greatly diminished in adult liver. The full-length human ACAT2 mRNA encodes a protein, designated ACAT2a, with 522 amino acids. We have previously reported the organization of the human ACAT2 gene and the differentiation-dependent promoter activity in intestinal Caco-2 cells. In the current work, two human ACAT2 mRNA variants produced by alternative splicing are cloned and predicted to encode two novel ACAT2 isoforms,named ACAT2b and ACAT2c, with 502 and 379 amino acids, respectively. These mRNA variants differ from ACAT2a mRNA by lack of the exon 4 (ACAT2b mRNA) and exons 4-5 plus 8-9-10 (ACAT2c mRNA).Significantly, comparable amounts of the alternatively spliced ACAT2 mRNA variants were detected by RTPCR, and Western blot analysis confirmed the presence of their corresponding proteins in human liver and intestine cells. Furthermore, phosphorylation and enzymatic activity analyses demonstrated that the novel isoenzymes ACAT2b and ACAT2c lacked the phosphorylatable site SLLD, and their enzymatic activities reduced to 25%-35% of that of ACAT2a. These evidences indicate that alternative splicing produces two human ACAT2 mRNA variants that encode the novel ACAT2 isoenzymes. Our findings might help to understand the regulation of the ACAT2 gene expression under certain physiological and pathological conditions.

  13. The Relevant Proteins in Intestinal Cholesterol Absorption%影响胆固醇在肠道吸收的相关蛋白

    Institute of Scientific and Technical Information of China (English)

    姜津; 唐朝克

    2011-01-01

    胆固醇在肠道吸收转运过程中,尼曼—匹克C1型类似蛋白1(Niemann-Pick C1 Like 1,NPC1 L1)介导了胆固醇的吸收,同时有协调ATP-结合盒转运体(ATP-binding cassette,ABC)G5和ABCG8的作用;胆固醇从质膜转运到内质网,通过酰基辅酶A-胆固醇酰基转移酶(acyl coenzyme A-cholesterol acyltransferase,ACAT)调节胆固醇的酯化速率,进而参与合成乳糜微粒,这个过程依赖脱辅基蛋白B(apoproteinB,apoB)、微粒体甘油三酯转移蛋白(microsomal triglyceride transfer protein,MTP)和内质网跨膜激酶(IRE1)β的活性.特异的小囊泡携带这些乳糜微粒到细胞的基底外侧膜,进入淋巴系统.另外未被ACAT酯化的胆固醇一部分通过ABCG5/G8返回肠道,另一部分通过ABCA1介导出基底外侧膜进入血浆.本文就调节胆固醇吸收相关蛋白及其调节机制的研究进展做一综述.

  14. Upper intestinal lipids trigger a gut-brain-liver axis to regulate glucose production.

    Science.gov (United States)

    Wang, Penny Y T; Caspi, Liora; Lam, Carol K L; Chari, Madhu; Li, Xiaosong; Light, Peter E; Gutierrez-Juarez, Roger; Ang, Michelle; Schwartz, Gary J; Lam, Tony K T

    2008-04-24

    Energy and glucose homeostasis are regulated by food intake and liver glucose production, respectively. The upper intestine has a critical role in nutrient digestion and absorption. However, studies indicate that upper intestinal lipids inhibit food intake as well in rodents and humans by the activation of an intestine-brain axis. In parallel, a brain-liver axis has recently been proposed to detect blood lipids to inhibit glucose production in rodents. Thus, we tested the hypothesis that upper intestinal lipids activate an intestine-brain-liver neural axis to regulate glucose homeostasis. Here we demonstrate that direct administration of lipids into the upper intestine increased upper intestinal long-chain fatty acyl-coenzyme A (LCFA-CoA) levels and suppressed glucose production. Co-infusion of the acyl-CoA synthase inhibitor triacsin C or the anaesthetic tetracaine with duodenal lipids abolished the inhibition of glucose production, indicating that upper intestinal LCFA-CoAs regulate glucose production in the preabsorptive state. Subdiaphragmatic vagotomy or gut vagal deafferentation interrupts the neural connection between the gut and the brain, and blocks the ability of upper intestinal lipids to inhibit glucose production. Direct administration of the N-methyl-d-aspartate ion channel blocker MK-801 into the fourth ventricle or the nucleus of the solitary tract where gut sensory fibres terminate abolished the upper-intestinal-lipid-induced inhibition of glucose production. Finally, hepatic vagotomy negated the inhibitory effects of upper intestinal lipids on glucose production. These findings indicate that upper intestinal lipids activate an intestine-brain-liver neural axis to inhibit glucose production, and thereby reveal a previously unappreciated pathway that regulates glucose homeostasis.

  15. Fatty acid biosynthesis in Pseudomonas aeruginosa is initiated by the FabY class of β-ketoacyl acyl carrier protein synthases.

    Science.gov (United States)

    Yuan, Yanqiu; Sachdeva, Meena; Leeds, Jennifer A; Meredith, Timothy C

    2012-10-01

    The prototypical type II fatty acid synthesis (FAS) pathway in bacteria utilizes two distinct classes of β-ketoacyl synthase (KAS) domains to assemble long-chain fatty acids, the KASIII domain for initiation and the KASI/II domain for elongation. The central role of FAS in bacterial viability and virulence has stimulated significant effort toward developing KAS inhibitors, particularly against the KASIII domain of the β-acetoacetyl-acyl carrier protein (ACP) synthase FabH. Herein, we show that the opportunistic pathogen Pseudomonas aeruginosa does not utilize a FabH ortholog but rather a new class of divergent KAS I/II enzymes to initiate the FAS pathway. When a P. aeruginosa cosmid library was used to rescue growth in a fabH downregulated strain of Escherichia coli, a single unannotated open reading frame, PA5174, complemented fabH depletion. While deletion of all four KASIII domain-encoding genes in the same P. aeruginosa strain resulted in a wild-type growth phenotype, deletion of PA5174 alone specifically attenuated growth due to a defect in de novo FAS. Siderophore secretion and quorum-sensing signaling, particularly in the rhl and Pseudomonas quinolone signal (PQS) systems, was significantly muted in the absence of PA5174. The defect could be repaired by intergeneric complementation with E. coli fabH. Characterization of recombinant PA5174 confirmed a preference for short-chain acyl coenzyme A (acyl-CoA) substrates, supporting the identification of PA5174 as the predominant enzyme catalyzing the condensation of acetyl coenzyme A with malonyl-ACP in P. aeruginosa. The identification of the functional role for PA5174 in FAS defines the new FabY class of β-ketoacyl synthase KASI/II domain condensation enzymes.

  16. Sex differences in global mRNA content of human skeletal muscle.

    Directory of Open Access Journals (Sweden)

    Amy C Maher

    Full Text Available Women oxidize more fat as compared to men during endurance exercise and several groups have shown that the mRNA content of selected genes related to fat oxidation are higher in women (e.g. hormone sensitive lipase, beta-hydroxyacyl-CoA dehydrogenase, CD36. One of the possible mechanisms is that women tend to have a higher area percentage of type I skeletal muscle fibers as compared with men. Consequently, we hypothesized that sex would influence the basal mRNA and protein content for genes involved in metabolism and the determination of muscle fiber type. Muscle biopsies from the vastus lateralis were collected from healthy men and women. We examined mRNA content globally using Affymetrix GeneChips, and selected genes were examined and/or confirmed by RT-PCR. Furthermore, we examined protein content by Western blot analysis. Stringent gene array analysis revealed 66 differentially expressed genes representing metabolism, mitochondrial function, transport, protein biosynthesis, cell proliferation, signal transduction pathways, transcription and translation. Stringent gene array analysis and RT-PCR confirmed that mRNA for; acyl-coenzyme A acyltransferase 2 (ACAA2, trifunctional protein beta (HADHB, catalase, lipoprotein lipase (LPL, and uncoupling protein-2 (UCP-2 were higher in women. Targeted gene analysis revealed that myosin heavy chain I (MHCI, peroxisome proliferator-activated receptor (PPARdelta were higher in women compared with men. Surprisingly, there were no significant sex based differences in protein content for HADHB, ACAA2, catalase, PPARdelta, and MHC1. In conclusion, the differences in the basal mRNA content in resting skeletal muscle suggest that men and women are transcriptionally "primed" for known physiological differences in metabolism however the mechanism behind sex differences in fiber type remains to be determined.

  17. The mechanisms underlying the hypolipidaemic effects of Grifola frondosa in the liver of rats

    Directory of Open Access Journals (Sweden)

    Yinrun Ding

    2016-08-01

    Full Text Available The present study investigated the hypolipidaemic effects of Grifola frondosa and its regulation mechanism involved in lipid metabolism in liver of rats fed a high-cholesterol diet. The body weights and serum lipid levels of control rats, of hyperlipidaemic rats and of hyperlipidaemic rats treated with oral Grifola frondosa were determined. mRNA expression and concentration of key lipid metabolism enzymes were investigated. Serum cholesterol, triacylglycerol and low-density lipoprotein cholesterol levels were markedly decreased in hyperlipidaemic rats treated with Grifola frondosa compared with untreated hyperlipidaemic rats. mRNA expression of 3-hydroxy-3-methylglutaryl-CoA reductase (HMGCR, acyl-coenzyme A: cholesterol acyltransferase (ACAT2, apolipoprotein B (ApoB, fatty acid synthase (FAS and acetyl-CoA carboxylase (ACC1 were significantly down-regulated, while expression of cholesterol 7-alpha-hydroxylase (CYP7A1 was significantly up-regulated in the livers of treated rats compared with untreated hyperlipidaemic rats. The concentrations of these enzymes also paralleled the observed changes in mRNA expression. Two-dimensional polyacrylamide gel electrophoresis (2-DE and Matrix-Assisted Laser Desorption/Ionization Time of Flight Mass Spectrometry (MALDI-TOF-MS were used to identify twenty proteins differentially expressed in livers of rats treated with Grifola frondosa compared with untreated hyperlipidemic rats. Of these twenty proteins, seven proteins were down-regulated and thirteen proteins were up-regulated. These findings indicate that the hypolipidaemic effects of Grifola frondosa reflected its modulation of key enzymes involved in cholesterol and triacylglycerol biosynthesis, absorption and catabolic pathways. Grifola frondosa may exert anti-atherosclerotic effects by inhibiting LDL oxidation through down-regulation and up-regulating proteins expression in the liver of rats. Therefore, Grifola frondosa may produce both hypolipidaemic

  18. Genetic examination of initial amino acid oxidation and glutamate catabolism in the hyperthermophilic archaeon Thermococcus kodakarensis.

    Science.gov (United States)

    Yokooji, Yuusuke; Sato, Takaaki; Fujiwara, Shinsuke; Imanaka, Tadayuki; Atomi, Haruyuki

    2013-05-01

    Amino acid catabolism in Thermococcales is presumed to proceed via three steps: oxidative deamination of amino acids by glutamate dehydrogenase (GDH) or aminotransferases, oxidative decarboxylation by 2-oxoacid:ferredoxin oxidoreductases (KOR), and hydrolysis of acyl-coenzyme A (CoA) by ADP-forming acyl-CoA synthetases (ACS). Here, we performed a genetic examination of enzymes involved in Glu catabolism in Thermococcus kodakarensis. Examination of amino acid dehydrogenase activities in cell extracts of T. kodakarensis KUW1 (ΔpyrF ΔtrpE) revealed high NADP-dependent GDH activity, along with lower levels of NAD-dependent activity. NADP-dependent activities toward Gln/Ala/Val/Cys and an NAD-dependent threonine dehydrogenase activity were also detected. In KGDH1, a gene disruption strain of T. kodakarensis GDH (Tk-GDH), only threonine dehydrogenase activity was detected, indicating that all other activities were dependent on Tk-GDH. KGDH1 could not grow in a medium in which growth was dependent on amino acid catabolism, implying that Tk-GDH is the only enzyme that can discharge the electrons (to NADP(+)/NAD(+)) released from amino acids in their oxidation to 2-oxoacids. In a medium containing excess pyruvate, KGDH1 displayed normal growth, but higher degrees of amino acid catabolism were observed compared to those for KUW1, suggesting that Tk-GDH functions to suppress amino acid oxidation and plays an anabolic role under this condition. We further constructed disruption strains of 2-oxoglutarate:ferredoxin oxidoreductase and succinyl-CoA synthetase. The two strains displayed growth defects in both media compared to KUW1. Succinate generation was not observed in these strains, indicating that the two enzymes are solely responsible for Glu catabolism among the multiple KOR and ACS enzymes in T. kodakarensis.

  19. α-Lipoic acid treatment increases mitochondrial biogenesis and promotes beige adipose features in subcutaneous adipocytes from overweight/obese subjects.

    Science.gov (United States)

    Fernández-Galilea, Marta; Pérez-Matute, Patricia; Prieto-Hontoria, Pedro L; Houssier, Marianne; Burrell, María A; Langin, Dominique; Martínez, J Alfredo; Moreno-Aliaga, María J

    2015-03-01

    α-Lipoic acid (α-Lip) is a natural occurring antioxidant with beneficial anti-obesity properties. The aim of this study was to investigate the putative effects of α-Lip on mitochondrial biogenesis and the acquirement of brown-like characteristics by subcutaneous adipocytes from overweight/obese subjects. Thus, fully differentiated human subcutaneous adipocytes were treated with α-Lip (100 and 250μM) for 24h for studies on mitochondrial content and morphology, mitochondrial DNA (mtDNA) copy number, fatty acid oxidation enzymes and brown/beige characteristic genes. The involvement of the Sirtuin1/Peroxisome proliferator-activated receptor gamma, coactivator 1 alpha (SIRT1/PGC-1α) pathway was also evaluated. Our results showed that α-Lip increased mitochondrial content in cultured human adipocytes as revealed by electron microscopy and by mitotracker green labeling. Moreover, an enhancement in mtDNA content was observed. This increase was accompanied by an up-regulation of SIRT1 protein levels, a decrease in PGC-1α acetylation and up-regulation of Nuclear respiratory factor 1 (Nrf1) and Mitochondrial transcription factor (Tfam) transcription factors. Enhanced oxygen consumption and fatty acid oxidation enzymes, Carnitine palmitoyl transferase 1 and Acyl-coenzyme A oxidase (CPT-1 and ACOX) were also observed. Mitochondria from α-Lip-treated adipocytes exhibited some morphological characteristics of brown mitochondria, and α-Lip also induced up-regulation of some brown/beige adipocytes markers such as cell death-inducing DFFA-like effector a (Cidea) and T-box 1 (Tbx1). Moreover, α-Lip up-regulated PR domain containing 16 (Prdm16) mRNA levels in treated adipocytes. Therefore, our study suggests the ability of α-Lip to promote mitochondrial biogenesis and brown-like remodeling in cultured white subcutaneous adipocytes from overweight/obese donors.

  20. Physiological characterization of lipid accumulation and in vivo ester formation in Gordonia sp. KTR9.

    Science.gov (United States)

    Eberly, Jed O; Ringelberg, David B; Indest, Karl J

    2013-02-01

    Previous work has demonstrated the feasibility of in vivo biodiesel synthesis in Escherichia coli, however, ethyl ester formation was dependent on an external fatty acid feedstock. In contrast to E. coli, actinomycetes may be ideal organisms for direct biodiesel synthesis because of their capacity to synthesize high levels of triacylglcerides (TAGs). In this study, we investigated the physiology and associated TAG accumulation along with the in vivo ability to catalyze ester formation from exogenous short chain alcohol sources in Gordonia sp. KTR9, a strain that possesses a large number of genes dedicated to fatty acid and lipid biosynthesis. Total lipid fatty acids content increased by 75 % and TAG content increased by 50 % under nitrogen starvation conditions in strain KTR9. Strain KTR9 tolerated the exogenous addition of up to 4 % methanol, 4 % ethanol and 2 % propanol in the media. Increasing alcohol concentrations resulted in a decrease in the degree of saturation of recovered fatty acid alcohol esters and a slight increase in the fatty acid chain length. A linear dose dependency in fatty alcohol ester synthesis was observed in the presence of 0.5-2 % methanol and ethanol compared to control KTR9 strains grown in the absence of alcohols. An inspection of the KTR9 genome revealed the presence of several putative wax ester synthase/acyl-coenzyme A : diacylglycerol acyltransferase (WS/DGAT) enzymes, encoded by atf gene homologs, that may catalyze the in vivo synthesis of fatty acid esters from short chain alcohols. Collectively, these results indicate that Gordonia sp. KTR9 may be a suitable actinomycete host strain for in vivo biodiesel synthesis.

  1. The Acyl Desaturase CER17 Is Involved in Producing Wax Unsaturated Primary Alcohols and Cutin Monomers1[OPEN

    Science.gov (United States)

    Yang, Xianpeng; Zhao, Huayan; Kosma, Dylan K.; Dyer, John M.; Li, Rongjun; Liu, Xiulin; Wang, Zhouya; Jenks, Matthew A.

    2017-01-01

    We report n-6 monounsaturated primary alcohols (C26:1, C28:1, and C30:1 homologs) in the cuticular waxes of Arabidopsis (Arabidopsis thaliana) inflorescence stem, a class of wax not previously reported in Arabidopsis. The Arabidopsis cer17 mutant was completely deficient in these monounsaturated alcohols, and CER17 was found to encode a predicted ACYL-COENZYME A DESATURASE LIKE4 (ADS4). Studies of the Arabidopsis cer4 mutant and yeast variously expressing CER4 (a predicted fatty acyl-CoA reductase) with CER17/ADS4, demonstrated CER4’s principal role in synthesis of these monounsaturated alcohols. Besides unsaturated alcohol deficiency, cer17 mutants exhibited a thickened and irregular cuticle ultrastructure and increased amounts of cutin monomers. Although unsaturated alcohols were absent throughout the cer17 stem, the mutation’s effects on cutin monomers and cuticle ultrastructure were much more severe in distal than basal stems, consistent with observations that the CER17/ADS4 transcript was much more abundant in distal than basal stems. Furthermore, distal but not basal stems of a double mutant deficient for both CER17/ADS4 and LONG-CHAIN ACYL-COA SYNTHETASE1 produced even more cutin monomers and a thicker and more disorganized cuticle ultrastructure and higher cuticle permeability than observed for wild type or either mutant parent, indicating a dramatic genetic interaction on conversion of very long chain acyl-CoA precursors. These results provide evidence that CER17/ADS4 performs n-6 desaturation of very long chain acyl-CoAs in both distal and basal stems and has a major function associated with governing cutin monomer amounts primarily in the distal segments of the inflorescence stem. PMID:28069670

  2. The Acyl Desaturase CER17 Is Involved in Producing Wax Unsaturated Primary Alcohols and Cutin Monomers.

    Science.gov (United States)

    Yang, Xianpeng; Zhao, Huayan; Kosma, Dylan K; Tomasi, Pernell; Dyer, John M; Li, Rongjun; Liu, Xiulin; Wang, Zhouya; Parsons, Eugene P; Jenks, Matthew A; Lü, Shiyou

    2017-02-01

    We report n-6 monounsaturated primary alcohols (C26:1, C28:1, and C30:1 homologs) in the cuticular waxes of Arabidopsis (Arabidopsis thaliana) inflorescence stem, a class of wax not previously reported in Arabidopsis. The Arabidopsis cer17 mutant was completely deficient in these monounsaturated alcohols, and CER17 was found to encode a predicted ACYL-COENZYME A DESATURASE LIKE4 (ADS4). Studies of the Arabidopsis cer4 mutant and yeast variously expressing CER4 (a predicted fatty acyl-CoA reductase) with CER17/ADS4, demonstrated CER4's principal role in synthesis of these monounsaturated alcohols. Besides unsaturated alcohol deficiency, cer17 mutants exhibited a thickened and irregular cuticle ultrastructure and increased amounts of cutin monomers. Although unsaturated alcohols were absent throughout the cer17 stem, the mutation's effects on cutin monomers and cuticle ultrastructure were much more severe in distal than basal stems, consistent with observations that the CER17/ADS4 transcript was much more abundant in distal than basal stems. Furthermore, distal but not basal stems of a double mutant deficient for both CER17/ADS4 and LONG-CHAIN ACYL-COA SYNTHETASE1 produced even more cutin monomers and a thicker and more disorganized cuticle ultrastructure and higher cuticle permeability than observed for wild type or either mutant parent, indicating a dramatic genetic interaction on conversion of very long chain acyl-CoA precursors. These results provide evidence that CER17/ADS4 performs n-6 desaturation of very long chain acyl-CoAs in both distal and basal stems and has a major function associated with governing cutin monomer amounts primarily in the distal segments of the inflorescence stem.

  3. Connecting the Molecular Structure of Cutin to Ultrastructure and Physical Properties of the Cuticle in Petals of Arabidopsis.

    Science.gov (United States)

    Mazurek, Sylwester; Garroum, Imène; Daraspe, Jean; De Bellis, Damien; Olsson, Vilde; Mucciolo, Antonio; Butenko, Melinka A; Humbel, Bruno M; Nawrath, Christiane

    2017-02-01

    The plant cuticle is laid down at the cell wall surface of epidermal cells in a wide variety of structures, but the functional significance of this architectural diversity is not yet understood. Here, the structure-function relationship of the petal cuticle of Arabidopsis (Arabidopsis thaliana) was investigated. Applying Fourier transform infrared microspectroscopy, the cutin mutants long-chain acyl-coenzyme A synthetase2 (lacs2), permeable cuticle1 (pec1), cyp77a6, glycerol-3-phosphate acyltransferase6 (gpat6), and defective in cuticular ridges (dcr) were grouped in three separate classes based on quantitative differences in the ν(C=O) and ν(C-H) band vibrations. These were associated mainly with the quantity of 10,16-dihydroxy hexadecanoic acid, a monomer of the cuticle polyester, cutin. These spectral features were linked to three different types of cuticle organization: a normal cuticle with nanoridges (lacs2 and pec1 mutants); a broad translucent cuticle (cyp77a6 and dcr mutants); and an electron-opaque multilayered cuticle (gpat6 mutant). The latter two types did not have typical nanoridges. Transmission electron microscopy revealed considerable variations in cuticle thickness in the dcr mutant. Different double mutant combinations showed that a low amount of C16 monomers in cutin leads to the appearance of an electron-translucent layer adjacent to the cuticle proper, which is independent of DCR action. We concluded that DCR is not only essential for incorporating 10,16-dihydroxy C16:0 into cutin but also plays a crucial role in the organization of the cuticle, independent of cutin composition. Further characterization of the mutant petals suggested that nanoridge formation and conical cell shape may contribute to the reduction of physical adhesion forces between petals and other floral organs during floral development.

  4. Connecting the Molecular Structure of Cutin to Ultrastructure and Physical Properties of the Cuticle in Petals of Arabidopsis1[OPEN

    Science.gov (United States)

    Mazurek, Sylwester; Garroum, Imène; Daraspe, Jean; De Bellis, Damien; Olsson, Vilde; Butenko, Melinka A.; Humbel, Bruno M.

    2017-01-01

    The plant cuticle is laid down at the cell wall surface of epidermal cells in a wide variety of structures, but the functional significance of this architectural diversity is not yet understood. Here, the structure-function relationship of the petal cuticle of Arabidopsis (Arabidopsis thaliana) was investigated. Applying Fourier transform infrared microspectroscopy, the cutin mutants long-chain acyl-coenzyme A synthetase2 (lacs2), permeable cuticle1 (pec1), cyp77a6, glycerol-3-phosphate acyltransferase6 (gpat6), and defective in cuticular ridges (dcr) were grouped in three separate classes based on quantitative differences in the ν(C=O) and ν(C-H) band vibrations. These were associated mainly with the quantity of 10,16-dihydroxy hexadecanoic acid, a monomer of the cuticle polyester, cutin. These spectral features were linked to three different types of cuticle organization: a normal cuticle with nanoridges (lacs2 and pec1 mutants); a broad translucent cuticle (cyp77a6 and dcr mutants); and an electron-opaque multilayered cuticle (gpat6 mutant). The latter two types did not have typical nanoridges. Transmission electron microscopy revealed considerable variations in cuticle thickness in the dcr mutant. Different double mutant combinations showed that a low amount of C16 monomers in cutin leads to the appearance of an electron-translucent layer adjacent to the cuticle proper, which is independent of DCR action. We concluded that DCR is not only essential for incorporating 10,16-dihydroxy C16:0 into cutin but also plays a crucial role in the organization of the cuticle, independent of cutin composition. Further characterization of the mutant petals suggested that nanoridge formation and conical cell shape may contribute to the reduction of physical adhesion forces between petals and other floral organs during floral development. PMID:27994007

  5. Risk stratification by residual enzyme activity after newborn screening for medium-chain acyl-CoA dehyrogenase deficiency: data from a cohort study

    Directory of Open Access Journals (Sweden)

    Touw Catharina M L

    2012-05-01

    Full Text Available Abstract Background Since the introduction of medium-chain acyl coenzyme A dehydrogenase (MCAD deficiency in population newborn bloodspot screening (NBS programs, subjects have been identified with variant ACADM (gene encoding MCAD enzyme genotypes that have never been identified in clinically ascertained patients. It could be hypothesised that residual MCAD enzyme activity can contribute in risk stratification of subjects with variant ACADM genotypes. Methods We performed a retrospective cohort study of all patients identified upon population NBS for MCAD deficiency in the Netherlands between 2007–2010. Clinical, molecular, and enzymatic data were integrated. Results Eighty-four patients from 76 families were identified. Twenty-two percent of the subjects had a variant ACADM genotype. In patients with classical ACADM genotypes, residual MCAD enzyme activity was significantly lower (median 0%, range 0-8% when compared to subjects with variant ACADM genotypes (range 0-63%; 4 cases with 0%, remainder 20-63%. Patients with (fatal neonatal presentations before diagnosis displayed residual MCAD enzyme activities Conclusions Determination of residual MCAD enzyme activity improves our understanding of variant ACADM genotypes and may contribute to risk stratification. Subjects with variant ACADM genotypes and residual MCAD enzyme activities ACADM genotypes. Parental instructions and an emergency regimen will remain principles of the treatment in any type of MCAD deficiency, as the effect of intercurrent illness on residual MCAD enzyme activity remains uncertain. There are, however, arguments in favour of abandoning the general advice to avoid prolonged fasting in subjects with variant ACADM genotypes and >10% residual MCAD enzyme activity.

  6. The Cirque du Soleil of Golgi membrane dynamics.

    Science.gov (United States)

    Bankaitis, Vytas A

    2009-07-27

    The role of lipid metabolic enzymes in Golgi membrane remodeling is a subject of intense interest. Now, in this issue, Schmidt and Brown (2009. J. Cell Biol. doi:10.1083/jcb.200904147) report that lysophosphatidic acid-specific acyltransferase, LPAAT3, contributes to Golgi membrane dynamics by suppressing tubule formation.

  7. Synthesis of Asymmetric Propanetriol Analogues

    Institute of Scientific and Technical Information of China (English)

    2002-01-01

    From natural tartaric acid, (R)-2-benzyloxy-3-(2-tetrahydropyranyloxy) propanol 3 was designed and synthesized, and (R)-2-benzyloxy-3-(4-methoxybenzyloxy) propanol 7 was prepared in a new method. They can be used as chiral synthons of lysophosphatidic acid and other compounds with asymmetric propanetriol backbone.

  8. Production of LPA by autotaxin and toxic phospholipases D : biochemical characterization and physiological implications

    NARCIS (Netherlands)

    Meeteren, Laurens Arend van

    2007-01-01

    Lysophosphatidic acid (LPA) is a lipid mediator with a wide variety of biological actions, particularly as an inducer of cell proliferation, migration and survival. LPA binds to at least five distinct G protein-coupled receptors and thereby activates multiple signal transduction pathways, including

  9. Volume-sensitive NADPH oxidase activity and taurine efflux in NIH3T3 mouse fibroblasts

    DEFF Research Database (Denmark)

    Friis, Martin Barfred; Vorum, Katrine Gribel; Lambert, Ian Henry

    2008-01-01

    +-mobilizing agonist ATP (10 microM) potentiates the release of taurine but has no effect on ROS production under hypotonic conditions. On the other hand, addition of the protein kinase C (PKC) activator phorbol 12-myristate 13-acetate (PMA, 100 nM) or the lipid messenger lysophosphatidic acid (LPA, 10 n...

  10. Genetic and Functional Evidence Supports LPAR1 as a Susceptibility Gene for Hypertension.

    Science.gov (United States)

    Xu, Ke; Ma, Lu; Li, Yang; Wang, Fang; Zheng, Gu-Yan; Sun, Zhijun; Jiang, Feng; Chen, Yundai; Liu, Huirong; Dang, Aimin; Chen, Xi; Chun, Jerold; Tian, Xiao-Li

    2015-09-01

    Essential hypertension is a complex disease affected by genetic and environmental factors and serves as a major risk factor for cardiovascular diseases. Serum lysophosphatidic acid correlates with an elevated blood pressure in rats, and lysophosphatidic acid interacts with 6 subtypes of receptors. In this study, we assessed the genetic association of lysophosphatidic acid receptors with essential hypertension by genotyping 28 single-nucleotide polymorphisms from genes encoding for lysophosphatidic acid receptors, LPAR1, LPAR2, LPAR3, LPAR4, LPAR5, and LPAR6 and their flanking sequences, in 3 Han Chinese cohorts consisting of 2630 patients and 3171 controls in total. We identified a single-nucleotide polymorphism, rs531003 in the 3'-flanking genomic region of LPAR1, associated with hypertension (the Bonferroni corrected P=1.09×10(-5), odds ratio [95% confidence interval]=1.23 [1.13-1.33]). The risk allele C of rs531003 is associated with the increased expression of LPAR1 and the susceptibility of hypertension, particularly in those with a shortage of sleep (P=4.73×10(-5), odds ratio [95% confidence interval]=1.75 [1.34-2.28]). We further demonstrated that blood pressure elevation caused by sleep deprivation and phenylephrine-induced vasoconstriction was both diminished in LPAR1-deficient mice. Together, we show that LPAR1 is a novel susceptibility gene for human essential hypertension and that stress, such as shortage of sleep, increases the susceptibility of patients with risk allele to essential hypertension.

  11. Accumulation of cyclin B1 requires E2F and cyclin-A-dependent rearrangement of the anaphase-promoting complex

    DEFF Research Database (Denmark)

    Lukas, C; Sørensen, Claus Storgaard; Kramer, E;

    1999-01-01

    genes beyond the G1/S transition is required for coordinating S-phase progression with cell division, a process driven by cyclin-B-dependent kinase and anaphase-promoting complex (APC)-mediated proteolysis. How E2F-dependent events at G1/S transition are orchestrated with cyclin B and APC activity...... in the timely accumulation of cyclin B1 and the coordination of cell-cycle progression during the post-restriction point period....

  12. The cyclase-associated protein FgCap1 has both protein kinase A-dependent and -independent functions during deoxynivalenol production and plant infection in Fusarium graminearum.

    Science.gov (United States)

    Yin, Tao; Zhang, Qiang; Wang, Jianhua; Liu, Huiquan; Wang, Chenfang; Xu, Jin-Rong; Jiang, Cong

    2017-01-31

    Fusarium graminearum is a causal agent of wheat scab and a producer of the trichothecene mycotoxin deoxynivalenol (DON). The expression of trichothecene biosynthesis (TRI) genes and DON production are mainly regulated by the cyclic adenosine monophosphate-protein kinase A (cAMP-PKA) pathway and two pathway-specific transcription factors (TRI6 and TRI10). Interestingly, deletion mutants of TRI6 show reduced expression of several components of cAMP signalling, including the FgCAP1 adenylate-binding protein gene that has not been functionally characterized in F. graminearum. In this study, we show that FgCap1 interacts with Fac1 adenylate cyclase and that deletion of FgCAP1 reduces the intracellular cAMP level and PKA activity. The Fgcap1 deletion mutant is defective in vegetative growth, conidiogenesis and plant infection. It also shows significantly reduced DON production and TRI gene expression, which can be suppressed by exogenous cAMP, indicating a PKA-dependent regulation of DON biosynthesis by FgCap1. The wild-type, but not tri6 mutant, shows increased levels of intracellular cAMP and FgCAP1 expression under DON-producing conditions. Furthermore, the promoter of FgCAP1 contains one putative Tri6-binding site that is important for its function during DON biosynthesis, but is dispensable for hyphal growth, conidiogenesis and pathogenesis. In addition, FgCap1 shows an actin-like localization to the cortical patches at the apical region of hyphal tips. Phosphorylation of FgCap1 at S353 was identified by phosphoproteomics analysis. The S353A mutation in FgCAP1 has no effect on its functions during vegetative growth, conidiation and DON production. However, expression of the FgCAP1(S353A) allele fails to complement the defects of the Fgcap1 mutant in plant infection, indicating the importance of the phosphorylation of FgCap1 at S353 during pathogenesis. Taken together, our results suggest that FgCAP1 is involved in the regulation of DON production via cAMP signalling and subjected to a feedback regulation by TRI6, but the phosphorylation of FgCap1 at S353 is probably unrelated to the cAMP-PKA pathway because the S353A mutation only affects plant infection.

  13. Protein kinase a dependent phosphorylation of apical membrane antigen 1 plays an important role in erythrocyte invasion by the malaria parasite.

    Directory of Open Access Journals (Sweden)

    Kerstin Leykauf

    Full Text Available Apicomplexan parasites are obligate intracellular parasites that infect a variety of hosts, causing significant diseases in livestock and humans. The invasive forms of the parasites invade their host cells by gliding motility, an active process driven by parasite adhesion proteins and molecular motors. A crucial point during host cell invasion is the formation of a ring-shaped area of intimate contact between the parasite and the host known as a tight junction. As the invasive zoite propels itself into the host-cell, the junction moves down the length of the parasite. This process must be tightly regulated and signalling is likely to play a role in this event. One crucial protein for tight-junction formation is the apical membrane antigen 1 (AMA1. Here we have investigated the phosphorylation status of this key player in the invasion process in the human malaria parasite Plasmodium falciparum. We show that the cytoplasmic tail of P. falciparum AMA1 is phosphorylated at serine 610. We provide evidence that the enzyme responsible for serine 610 phosphorylation is the cAMP regulated protein kinase A (PfPKA. Importantly, mutation of AMA1 serine 610 to alanine abrogates phosphorylation of AMA1 in vivo and dramatically impedes invasion. In addition to shedding unexpected new light on AMA1 function, this work represents the first time PKA has been implicated in merozoite invasion.

  14. β-Naphthoflavone protects mice from aristolochic acid-l-induced acute kidney injury in a CYP1A dependent mechanism

    Institute of Scientific and Technical Information of China (English)

    Ying XIAO; Xiang XUE; Yuan-feng WU; Guo-zhengXIN; Yong QIAN; Tian-pei XIE; Li-kun GONG; Jin REN

    2009-01-01

    Aim: The role of CYP1A in the protection of aristolochic acid (AA)l-induced nephrotoxicity has been suggested. In the present study we investigated the effects of P-naphthoflavone (BNF), a non-carcinogen CYP1A inducer, on Aal-induced kidney injury.Methods: Mice were pretreated with 80 mg/kg BNF by daily intraperitoneal injection (ip) for 3 days followed by a single ip of 10 mg/kg AAI. AAI and its major metabolites in blood, liver and kidney, the expression of CYP1A1 and CYP1A2 in microsomes of liver and kidney, as well as the nephrotoxicity were evaluated.Results: BNF pretreatment prevented Aal-induced renal damage by facilitating the disposal of AAI in liver. BNF pretreatment induced the expression of CYP1A1 in both liver and kidney; but the induction of CYP1A2 was only observed in liver. Conclusion: BNF prevents Aal-induced kidney toxicity primarily through CYP1A induction.

  15. The mitogen-activated protein kinase pathway mediates growth arrest or E1A-dependent apoptosis in SKBR3 human breast cancer cells.

    Science.gov (United States)

    Blagosklonny, M V

    1998-11-09

    Previously, we have shown that phorbol ester (PMA) induces p21(WAF1/CIP1)-dependent growth arrest in SKBr3 breast cancer and LNCaP prostate cancer cells. Here, I demonstrate that inhibition of Raf-1 kinase by dominant-negative Raf-1 or pharmacological depletion of Raf-1 prevented PMA-mediated induction of p21(WAF1/CIP1). Similarly, PD98059, a specific inhibitor of MEK, abolished p21(WAF1/CIP1) induction and PMA-induced growth arrest. Like PMA, the H-ras oncogene, another activator of the Raf-1/MEK/MAPK pathway, transactivated p21(WAF1/CIP1) in SKBr3 cells. I further investigated PMA-induced growth arrest following infection of SKBr3 cells with 12S E1A-expressing adenovirus. Although high levels of E1A oncoprotein prevented both PMA-induced p21(WAF1/CIP1) and growth arrest, smaller amounts of E1A abrogated growth arrest without down-regulation of p21(WAF1/CIP1). Therefore, E1A can stimulate proliferation downstream of p21(WAF1/CIP1). Albeit less effective than full activity, either Rb- or p300-binding activity of E1A was sufficient for the abrogation of PMA-mediated growth arrest. E1A-driven proliferation of PMA-treated SKBr3 cells was accompanied by apoptosis. New therapeutic approaches can be envisioned that would utilize stimulation of the Raf-1/MEK/MAPK pathway to inhibit growth of PMA-sensitive cancer cells.

  16. Group psycho-education in patients with bipolar disorder associated with a dependency of toxic substances in patients who are in abstinence

    Directory of Open Access Journals (Sweden)

    Patricia González Alegre

    2009-01-01

    Full Text Available The high comorbility that exists among psychiatric disorders and addictive is important. In the latest years it is produced an increase of the sensibility related to this problem. A great deal it is due to the demand of Mental Health Services and also due to drug dependency, as a consequence of the lack of an integral approach. Because of this fact and because of the mentioned demand, we though it should be pertinent developing a research project in order to check if the carrying the psycho-educative preventive group project out in patients with a diagnose of bipolar disorder with an abuse of drugs history and/or dependency of toxic substance in abstinence at the moment influents in a positive way in the course of the number of relapses in the toxic consumption during at least six months subsequent to the intervention. And at this way, these patients will purchase a greater consciousness of the important of healthy habits in the bipolar disorder and the recovery in the toxic substance abuse. The program will be developed in an experimental research where the patients will be randomly assigned in group control/ experimentally, the intervention will last twenty sessions, each session will be an hour and a half long and will be held weekly. In these sessions we will deal with topics related to the psychiatric disorder and the toxic consume. At the same time we will bank on the development of practical relaxation workshops on in some of the sessions with the object of providing a resource in view of stress situations.

  17. Measurements of observables in the pion-nucleon system, nuclear a- dependence of heavy quark production and rare decays of D and B mesons

    Energy Technology Data Exchange (ETDEWEB)

    Sadler, M.E.; Isenhower, L.D.

    1992-02-15

    This report discusses research on the following topics: pion-nucleon interactions; detector tomography facility; nuclear dependence of charm and beauty quark production and a study of two-prong decays of neutral D and B mesons; N* collaboration at CEBAF; and pilac experiments. (LSP)

  18. DNA binding of the cell cycle transcriptional regulator GcrA depends on N6-adenosine methylation in Caulobacter crescentus and other Alphaproteobacteria.

    Science.gov (United States)

    Fioravanti, Antonella; Fumeaux, Coralie; Mohapatra, Saswat S; Bompard, Coralie; Brilli, Matteo; Frandi, Antonio; Castric, Vincent; Villeret, Vincent; Viollier, Patrick H; Biondi, Emanuele G

    2013-05-01

    Several regulators are involved in the control of cell cycle progression in the bacterial model system Caulobacter crescentus, which divides asymmetrically into a vegetative G1-phase (swarmer) cell and a replicative S-phase (stalked) cell. Here we report a novel functional interaction between the enigmatic cell cycle regulator GcrA and the N6-adenosine methyltransferase CcrM, both highly conserved proteins among Alphaproteobacteria, that are activated early and at the end of S-phase, respectively. As no direct biochemical and regulatory relationship between GcrA and CcrM were known, we used a combination of ChIP (chromatin-immunoprecipitation), biochemical and biophysical experimentation, and genetics to show that GcrA is a dimeric DNA-binding protein that preferentially targets promoters harbouring CcrM methylation sites. After tracing CcrM-dependent N6-methyl-adenosine promoter marks at a genome-wide scale, we show that these marks recruit GcrA in vitro and in vivo. Moreover, we found that, in the presence of a methylated target, GcrA recruits the RNA polymerase to the promoter, consistent with its role in transcriptional activation. Since methylation-dependent DNA binding is also observed with GcrA orthologs from other Alphaproteobacteria, we conclude that GcrA is the founding member of a new and conserved class of transcriptional regulators that function as molecular effectors of a methylation-dependent (non-heritable) epigenetic switch that regulates gene expression during the cell cycle.

  19. DNA binding of the cell cycle transcriptional regulator GcrA depends on N6-adenosine methylation in Caulobacter crescentus and other Alphaproteobacteria.

    Directory of Open Access Journals (Sweden)

    Antonella Fioravanti

    2013-05-01

    Full Text Available Several regulators are involved in the control of cell cycle progression in the bacterial model system Caulobacter crescentus, which divides asymmetrically into a vegetative G1-phase (swarmer cell and a replicative S-phase (stalked cell. Here we report a novel functional interaction between the enigmatic cell cycle regulator GcrA and the N6-adenosine methyltransferase CcrM, both highly conserved proteins among Alphaproteobacteria, that are activated early and at the end of S-phase, respectively. As no direct biochemical and regulatory relationship between GcrA and CcrM were known, we used a combination of ChIP (chromatin-immunoprecipitation, biochemical and biophysical experimentation, and genetics to show that GcrA is a dimeric DNA-binding protein that preferentially targets promoters harbouring CcrM methylation sites. After tracing CcrM-dependent N6-methyl-adenosine promoter marks at a genome-wide scale, we show that these marks recruit GcrA in vitro and in vivo. Moreover, we found that, in the presence of a methylated target, GcrA recruits the RNA polymerase to the promoter, consistent with its role in transcriptional activation. Since methylation-dependent DNA binding is also observed with GcrA orthologs from other Alphaproteobacteria, we conclude that GcrA is the founding member of a new and conserved class of transcriptional regulators that function as molecular effectors of a methylation-dependent (non-heritable epigenetic switch that regulates gene expression during the cell cycle.

  20. Prolonged reversal of the phencyclidine-induced impairment in novel object recognition by a serotonin (5-HT)1A-dependent mechanism.

    Science.gov (United States)

    Horiguchi, Masakuni; Miyauchi, Masanori; Neugebauer, Nichole M; Oyamada, Yoshihiro; Meltzer, Herbert Y

    2016-03-15

    Many acute treatments transiently reverse the deficit in novel object recognition (NOR) produced by subchronic treatment with the N-methyl-d-aspartate receptor non-competitive antagonist, phencyclidine (PCP), in rodents. Treatments which restore NOR for prolonged periods after subchronic PCP treatment may have greater relevance for treating the cognitive impairment in schizophrenia than those which restore NOR transiently. We examined the ability of post-PCP subchronic lurasidone, an atypical APD with potent serotonin (5-HT)1A partial agonism and subchronic tandospirone, a selective 5-HT1A partial agonist, to enable prolonged reversal of the subchronic PCP-induced NOR deficit. Rats treated with subchronic PCP (2mg/kg, twice daily for 7 days) or vehicle, followed by a 7day washout period were subsequently administered lurasidone or tandospirone twice daily for 7 days (day 15-21), and tested for NOR weekly for up to two additional weeks. Subchronic lurasidone (1, but not 0.1mg/kg) or tandospirone (5, but not 0.6mg/kg) significantly reversed the PCP-induced NOR deficit at 24h and 7days after the last injection, respectively. The effect of lurasidone persisted for one more week (day 36, 14 days after the last lurasidone dose), while tandospirone-treated rats were able to perform NOR at 7, but not 14, days after the last tandospirone dose. Co-administration of WAY100635 (0.6mg/kg), a 5-HT1A antagonist, with lurasidone, blocked the ability of lurasidone to restore NOR, suggesting that 5-HT1A receptor stimulation is necessary for lurasidone to reverse the effects of PCP. The role of dopamine, GABA and the MAPK/ERK signalling pathway in the persistent, but not indefinite, restoration of NOR is discussed.

  1. NPM-ALK up-regulates iNOS expression through a STAT3/microRNA-26a-dependent mechanism.

    Science.gov (United States)

    Zhu, Haifeng; Vishwamitra, Deeksha; Curry, Choladda V; Manshouri, Roxsan; Diao, Lixia; Khan, Aarish; Amin, Hesham M

    2013-05-01

    NPM-ALK chimeric oncogene is aberrantly expressed in an aggressive subset of T-cell lymphomas that frequently occurs in children and young adults. The mechanisms underlying the oncogenic effects of NPM-ALK are not completely elucidated. Inducible nitric oxide synthase (iNOS) promotes the survival and maintains the malignant phenotype of cancer cells by generating NO, a highly active free radical. We tested the hypothesis that iNOS is deregulated in NPM-ALK(+) T-cell lymphoma and promotes the survival of this lymphoma. In line with this possibility, an iNOS inhibitor and NO scavenger decreased the viability, adhesion, and migration of NPM-ALK(+) T-cell lymphoma cells, and an NO donor reversed these effects. Moreover, the NO donor salvaged the viability of lymphoma cells treated with ALK inhibitors. In further support of an important role of iNOS, we found iNOS protein to be highly expressed in NPM-ALK(+) T-cell lymphoma cell lines and in 79% of primary tumours but not in human T lymphocytes. Although expression of iNOS mRNA was identified in NPM-ALK(+) T-cell lymphoma cell lines and tumours, iNOS mRNA was remarkably elevated in T lymphocytes, suggesting post-transcriptional regulation. Consistently, we found that miR-26a contains potential binding sites and interacts with the 3'-UTR of iNOS. In addition, miR-26a was significantly decreased in NPM-ALK(+) T-cell lymphoma cell lines and tumours compared with T lymphocytes and reactive lymph nodes. Restoration of miR-26a in lymphoma cells abrogated iNOS protein expression and decreased NO production and cell viability, adhesion, and migration. Importantly, the effects of miR-26a were substantially attenuated when the NO donor was simultaneously used to treat lymphoma cells. Our investigation of the mechanisms underlying the decrease in miR-26a in this lymphoma revealed novel evidence that STAT3, a major downstream substrate of NPM-ALK tyrosine kinase activity, suppresses MIR26A1 gene expression.

  2. Characterization of the gacA-dependent surface and coral mucus colonization by an opportunistic coral pathogen Serratia marcescens PDL100.

    Science.gov (United States)

    Krediet, Cory J; Carpinone, Emily M; Ritchie, Kim B; Teplitski, Max

    2013-05-01

    Opportunistic pathogens rely on global regulatory systems to assess the environment and to control virulence and metabolism to overcome host defenses and outcompete host-associated microbiota. In Gammaproteobacteria, GacS/GacA is one such regulatory system. GacA orthologs direct the expression of the csr (rsm) small regulatory RNAs, which through their interaction with the RNA-binding protein CsrA (RsmA), control genes with functions in carbon metabolism, motility, biofilm formation, and virulence. The csrB gene was controlled by gacA in Serratia marcescens PDL100. A disruption of the S. marcescens gacA gene resulted in an increased fitness of the mutant on mucus of the host coral Acropora palmata and its high molecular weight fraction, whereas the mutant was as competitive as the wild type on the low molecular weight fraction of the mucus. Swarming motility and biofilm formation were reduced in the gacA mutant. This indicates a critical role for gacA in the efficient utilization of specific components of coral mucus and establishment within the surface mucopolysaccharide layer. While significantly affecting early colonization behaviors (coral mucus utilization, swarming motility, and biofilm formation), gacA was not required for virulence of S. marcescens PDL100 in either a model polyp Aiptasia pallida or in brine shrimp Artemia nauplii.

  3. On-Chip Scan-Based Test Strategy for a Dependable Many-Core Processor Using a NoC as a Test Access Mechanism

    NARCIS (Netherlands)

    Zhang, Xiao; Kerkhoff, Hans G.; Vermeulen, Bart

    2010-01-01

    Periodic on-chip scan-based tests have to be applied to a many-core processor SoC to improve its dependability. An infrastructural IP module has been designed and incorporated into the SoC to function as an ATE. This paper introduces the reuse of a Network-on-Chip as a test access mechanism. Since t

  4. Tauroursodeoxycholate Protects Rat Hepatocytes from Bile Acid-Induced Apoptosis via β1-Integrin- and Protein Kinase A-Dependent Mechanisms

    Directory of Open Access Journals (Sweden)

    Annika Sommerfeld

    2015-05-01

    Full Text Available Background/Aims: Ursodeoxycholic acid, which in vivo is rapidly converted into its taurine conjugate, is frequently used for the treatment of cholestatic liver disease. Apart from its choleretic effects, tauroursodeoxycholate (TUDC can protect hepatocytes from bile acid-induced apoptosis, but the mechanisms underlying its anti-apoptotic effects are poorly understood. Methods: These mechanisms were investigated in perfused rat liver and isolated rat hepatocytes. Results: It was found that TUDC inhibited the glycochenodeoxycholate (GCDC-induced activation of the CD95 death receptor at the level of association between CD95 and the epidermal growth factor receptor. This was due to a rapid TUDC-induced β1-integrin-dependent cyclic AMP (cAMP signal with induction of the dual specificity mitogen-activated protein (MAP kinase phosphatase 1 (MKP-1, which prevented GCDC-induced phosphorylation of mitogen-activated protein kinase kinase 4 (MKK4 and c-jun-NH2-terminal kinase (JNK activation. Furthermore, TUDC induced a protein kinase A (PKA-mediated serine/threonine phosphorylation of the CD95, which was recently identified as an internalization signal for CD95. Furthermore, TUDC inhibited GCDC-induced CD95 targeting to the plasma membrane in a β1-integrin-and PKA-dependent manner. In line with this, the β1-integrin siRNA knockdown in sodium taurocholate cotransporting polypeptide (Ntcp-transfected HepG2 cells abolished the protective effect of TUDC against GCDC-induced apoptosis. Conclusion: TUDC exerts its anti-apoptotic effect via a β1-integrin-mediated formation of cAMP, which prevents CD95 activation by hydrophobic bile acids at the levels of JNK activation and CD95 serine/threonine phosphorylation.

  5. Salmonella enterica serotype Typhimurium DT104 ArtA-dependent modification of pertussis toxin-sensitive G proteins in the presence of [32P]NAD.

    Science.gov (United States)

    Uchida, Ikuo; Ishihara, Ryoko; Tanaka, Kiyoshi; Hata, Eiji; Makino, Sou-ichi; Kanno, Toru; Hatama, Shinichi; Kishima, Masato; Akiba, Masato; Watanabe, Atsushi; Kubota, Takayuki

    2009-11-01

    Salmonella enterica serotype Typhimurium (S. Typhimurium) definitive phage type (DT) 104 has become a widespread cause of human and other animal infections worldwide. The severity of clinical illness in S. Typhimurium DT104 outbreaks suggests that this strain possesses enhanced virulence. ArtA and ArtB - encoded by a prophage in S. Typhimurium DT104 - are homologues of components of pertussis toxin (PTX), including its ADP-ribosyltransferase subunit. Here, we show that exposing DT104 to mitomycin C, a DNA-damaging agent, induced production of prophage-encoded ArtA/ArtB. Pertussis-sensitive G proteins were labelled in the presence of [(32)P]NAD and ArtA, and the label was released by HgCl(2), which is known to cleave cysteine-ADP-ribose bonds. ADP-dependent modification of G proteins was markedly reduced in in vitro-synthesized ArtA(6Arg-Ala) and ArtA(115Glu-Ala), in which alanine was substituted for the conserved arginine at position 6 (necessary for NAD binding) and the predicted catalytic glutamate at position 115, respectively. A cellular ADP-ribosylation assay and two-dimensional electrophoresis showed that ArtA- and PTX-induced ADP-ribosylation in Chinese hamster ovary (CHO) cells occur with the same type of G proteins. Furthermore, exposing CHO cells to the ArtA/ArtB-containing culture supernatant of DT104 resulted in a clustered growth pattern, as is observed in PTX-exposed CHO cells. Hydrogen peroxide, an oxidative stressor, also induced ArtA/ArtB production, suggesting that these agents induce in vivo synthesis of ArtA/ArtB. These results, taken together, suggest that ArtA/ArtB is an active toxin similar to PTX.

  6. Tfap2a-dependent changes in mouse facial morphology result in clefting that can be ameliorated by a reduction in Fgf8 gene dosage.

    Science.gov (United States)

    Green, Rebecca M; Feng, Weiguo; Phang, Tzulip; Fish, Jennifer L; Li, Hong; Spritz, Richard A; Marcucio, Ralph S; Hooper, Joan; Jamniczky, Heather; Hallgrímsson, Benedikt; Williams, Trevor

    2015-01-01

    Failure of facial prominence fusion causes cleft lip and palate (CL/P), a common human birth defect. Several potential mechanisms can be envisioned that would result in CL/P, including failure of prominence growth and/or alignment as well as a failure of fusion of the juxtaposed epithelial seams. Here, using geometric morphometrics, we analyzed facial outgrowth and shape change over time in a novel mouse model exhibiting fully penetrant bilateral CL/P. This robust model is based upon mutations in Tfap2a, the gene encoding transcription factor AP-2α, which has been implicated in both syndromic and non-syndromic human CL/P. Our findings indicate that aberrant morphology and subsequent misalignment of the facial prominences underlies the inability of the mutant prominences to fuse. Exencephaly also occured in some of the Tfap2a mutants and we observed additional morphometric differences that indicate an influence of neural tube closure defects on facial shape. Molecular analysis of the CL/P model indicates that Fgf signaling is misregulated in the face, and that reducing Fgf8 gene dosage can attenuate the clefting pathology by generating compensatory changes. Furthermore, mutations in either Tfap2a or Fgf8 increase variance in facial shape, but the combination of these mutations restores variance to normal levels. The alterations in variance provide a potential mechanistic link between clefting and the evolution and diversity of facial morphology. Overall, our findings suggest that CL/P can result from small gene-expression changes that alter the shape of the facial prominences and uncouple their coordinated morphogenesis, which is necessary for normal fusion.

  7. Protein kinase A-dependent Neuronal Nitric Oxide Synthase Activation Mediates the Enhancement of Baroreflex Response by Adrenomedullin in the Nucleus Tractus Solitarii of Rats

    Directory of Open Access Journals (Sweden)

    Ho I-Chun

    2011-05-01

    Full Text Available Abstract Background Adrenomedullin (ADM exerts its biological functions through the receptor-mediated enzymatic mechanisms that involve protein kinase A (PKA, or neuronal nitric oxide synthase (nNOS. We previously demonstrated that the receptor-mediated cAMP/PKA pathway involves in ADM-enhanced baroreceptor reflex (BRR response. It remains unclear whether ADM may enhance BRR response via activation of nNOS-dependent mechanism in the nucleus tractus solitarii (NTS. Methods Intravenous injection of phenylephrine was administered to evoke the BRR before and at 10, 30, and 60 min after microinjection of the test agents into NTS of Sprague-Dawley rats. Western blotting analysis was used to measure the level and phosphorylation of proteins that involved in BRR-enhancing effects of ADM (0.2 pmol in NTS. The colocalization of PKA and nNOS was examined by immunohistochemical staining and observed with a laser confocal microscope. Results We found that ADM-induced enhancement of BRR response was blunted by microinjection of NPLA or Rp-8-Br-cGMP, a selective inhibitor of nNOS or protein kinase G (PKG respectively, into NTS. Western blot analysis further revealed that ADM induced an increase in the protein level of PKG-I which could be attenuated by co-microinjection with the ADM receptor antagonist ADM22-52 or NPLA. Moreover, we observed an increase in phosphorylation at Ser1416 of nNOS at 10, 30, and 60 min after intra-NTS administration of ADM. As such, nNOS/PKG signaling may also account for the enhancing effect of ADM on BRR response. Interestingly, biochemical evidence further showed that ADM-induced increase of nNOS phosphorylation was prevented by co-microinjection with Rp-8-Br-cAMP, a PKA inhibitor. The possibility of PKA-dependent nNOS activation was substantiated by immunohistochemical demonstration of co-localization of PKA and nNOS in putative NTS neurons. Conclusions The novel finding of this study is that the signal transduction cascade that underlies the enhancement of BRR response by ADM in NTS is composed sequentially of cAMP/PKA and nNOS/PKG pathways.

  8. Adiponectin promotes VEGF-A-dependent angiogenesis in human chondrosarcoma through PI3K, Akt, mTOR, and HIF-α pathway

    OpenAIRE

    Lee, Hsiang-Ping; Lin, Chih-Yang; Shih, Jhao-Sheng; Fong, Yi-Chin; Wang, Shih-Wei; Li, Te-Mao; Tang, Chih-Hsin

    2015-01-01

    Chondrosarcoma is a type of highly malignant tumor with a potent capacity to invade locally and cause distant metastasis. Adiponectin is a protein hormone secreted predominantly by differentiated adipocytes. On the other hand, angiogenesis is a critical step in tumor growth and metastasis. However, the relationship of adiponectin with vascular endothelial growth factor-A (VEGF-A) expression and angiogenesis in human chondrosarcoma is mostly unknown. In this study we first demonstrated that th...

  9. Protein Kinase A Dependent Phosphorylation of Apical Membrane Antigen 1 Plays an Important Role in Erythrocyte Invasion by the Malaria Parasite

    OpenAIRE

    Kerstin Leykauf; Moritz Treeck; Gilson, Paul R.; Thomas Nebl; Thomas Braulke; Cowman, Alan F; Gilberger, Tim W; Brendan S Crabb

    2010-01-01

    Apicomplexan parasites are obligate intracellular parasites that infect a variety of hosts, causing significant diseases in livestock and humans. The invasive forms of the parasites invade their host cells by gliding motility, an active process driven by parasite adhesion proteins and molecular motors. A crucial point during host cell invasion is the formation of a ring-shaped area of intimate contact between the parasite and the host known as a tight junction. As the invasive zoite propels i...

  10. HipA-triggered growth arrest and β-lactam tolerance in Escherichia coli are mediated by RelA-dependent ppGpp synthesis.

    Science.gov (United States)

    Bokinsky, Gregory; Baidoo, Edward E K; Akella, Swetha; Burd, Helcio; Weaver, Daniel; Alonso-Gutierrez, Jorge; García-Martín, Héctor; Lee, Taek Soon; Keasling, Jay D

    2013-07-01

    Persistence is a phenomenon whereby a subpopulation of bacterial cells enters a transient growth-arrested state that confers antibiotic tolerance. While entrance into persistence has been linked to the activities of toxin proteins, the molecular mechanisms by which toxins induce growth arrest and the persistent state remain unclear. Here, we show that overexpression of the protein kinase HipA in Escherichia coli triggers growth arrest by activating synthesis of the alarmone guanosine tetraphosphate (ppGpp) by the enzyme RelA, a signal typically associated with amino acid starvation. We further demonstrate that chemically suppressing ppGpp synthesis with chloramphenicol relieves inhibition of DNA replication initiation and RNA synthesis in HipA-arrested cells and restores vulnerability to β-lactam antibiotics. HipA-arrested cells maintain glucose uptake and oxygen consumption and accumulate amino acids as a consequence of translational inhibition. We harness the active metabolism of HipA-arrested cells to provide a bacteriophage-resistant platform for the production of biotechnologically relevant compounds, which may represent an innovative solution to the costly problem of phage contamination in industrial fermentations.

  11. Homozygous and heterozygous GH transgenesis alters fatty acid composition and content in the liver of Amago salmon (Oncorhynchus masou ishikawae

    Directory of Open Access Journals (Sweden)

    Manabu Sugiyama

    2012-08-01

    Growth hormone (GH transgenic Amago (Oncorhynchus masou ishikawae, containing the sockeye GH1 gene fused with metallothionein-B promoter from the same species, were generated and the physiological condition through lipid metabolism compared among homozygous (Tg/Tg and heterozygous GH transgenic (Tg/+ Amago and the wild type control (+/+. Previously, we have reported that the adipose tissue was generally smaller in GH transgenic fish compared to the control, and that the Δ-6 fatty acyl desaturase gene was down-regulated in the Tg/+ fish. However, fatty acid (FA compositions have not been measured previously in these fish. In this study we compared the FAs composition and content in the liver using gas chromatography. Eleven kinds of FA were detected. The composition of saturated and monounsaturated fatty acids (SFA and MUFA such as myristic acid (14:0, palmitoleic acid (16:1n-7, and cis-vaccenic acid (cis-18:1n-7 was significantly (P<0.05 decreased in GH transgenic Amago. On the other hand, the composition of polyunsaturated fatty acids (PUFAs such as linoleic acid (18:2n-6, arachidonic acid (20:4n-6, and docosapentaenoic acid (22:5n-3 was significantly (P<0.05 increased. Levels of serum glucose and triacylglycerol were significantly (P<0.05 decreased in the GH transgenics compared with +/+ fish. Furthermore, 3′-tag digital gene expression profiling was performed using liver tissues from Tg/Tg and +/+ fish, and showed that Mid1 interacting protein 1 (Mid1ip1, which is an important factor to activate Acetyl-CoA carboxylase (ACC, was down-regulated in Tg/Tg fish, while genes involved in FA catabolism were up-regulated, including long-chain-fatty-acid–CoA ligase 1 (ACSL1 and acyl-coenzyme A oxidase 3 (ACOX3. These data suggest that liver tissue from GH transgenic Amago showed starvation by alteration in glucose and lipid metabolism due to GH overexpression. The decrease of serum glucose suppressed Mid1ip1, and caused a decrease of de novo FA synthesis, resulting

  12. Alisol A 24-Acetate Prevents Hepatic Steatosis and Metabolic Disorders in HepG2 Cells

    Directory of Open Access Journals (Sweden)

    Lu Zeng

    2016-11-01

    Full Text Available Background: Non-alcoholic fatty liver disease (NAFLD is closely associated with metabolic disorders including hepatic lipid accumulation and inflammation. Alisol A 24-acetate, a triterpene from Alismatis rhizome, has multiple biologic activities such as hypolipidemic, anti-inflammatory and anti-diabetic. Thus we hypothesized that Alisol A 24 -acetate would have effect on NAFLD. The present study was conducted to investigate the therapeutic effects and potential mechanisms of Alisol A 24-acetate against hepatic steatosis in a free fatty acids (FFAs induced NAFLD cell model. Methods: This study was divided into four groups including Control group, Model group (FFA group, Alisol A 24-acetate (FFA+A group, Fenofibrate (FFA+F group. Preventive role of Alisol A 24-acetate was evaluated using 10µM Alisol A 24-acetate plus 1 mM FFA (oleate:palmitate=2:1 incubated with HepG2 cells for 24 h, which was determined by Oil Red O Staining, Oil Red O based colorimetric assay and intracellular triglyceride (TG content. Besides, the inflammatory cytokines tumor necrosis factor (TNF- α, interleukin (IL-6 levels as well as the protein and mRNA expressions that were involved in fatty acid synthesis and oxidation including Adiponectin, AMP-activated protein kinase (AMPK α, peroxisome proliferator-activated receptor (PPAR α, sterol regulatory element binding protein 1c (SREBP-1c, acetyl-CoA carboxylase (ACC, fatty acid synthase (FAS, carnitine palmitoyltransferase 1 (CPT1 and acyl coenzyme A oxidase 1 (ACOX1 were detected. Results: Alisol A 24-acetate significantly decreased the numbers of lipid droplets, Oil Red O lipid content, and intracellular TG content. Besides, inflammatory cytokines TNF-α, IL-6 levels were markedly inhibited by Alisol A 24-acetate. Furthermore, Alisol A 24-acetate effectively increased the protein and mRNA expressions of Adiponectin, the phosphorylation of AMPKα, CPT1 and ACOX1, whereas decreased SREBP-1c, the phosphorylation of ACC and

  13. Crystal Structures of Xanthomonas campestris OleA Reveal Features That Promote Head-to-Head Condensation of Two Long-Chain Fatty Acids

    Energy Technology Data Exchange (ETDEWEB)

    Goblirsch, Brandon R.; Frias, Janice A.; Wackett, Lawrence P.; Wilmot, Carrie M. (UMM)

    2012-10-25

    OleA is a thiolase superfamily enzyme that has been shown to catalyze the condensation of two long-chain fatty acyl-coenzyme A (CoA) substrates. The enzyme is part of a larger gene cluster responsible for generating long-chain olefin products, a potential biofuel precursor. In thiolase superfamily enzymes, catalysis is achieved via a ping-pong mechanism. The first substrate forms a covalent intermediate with an active site cysteine that is followed by reaction with the second substrate. For OleA, this conjugation proceeds by a nondecarboxylative Claisen condensation. The OleA from Xanthomonas campestris has been crystallized and its structure determined, along with inhibitor-bound and xenon-derivatized structures, to improve our understanding of substrate positioning in the context of enzyme turnover. OleA is the first characterized thiolase superfamily member that has two long-chain alkyl substrates that need to be bound simultaneously and therefore uniquely requires an additional alkyl binding channel. The location of the fatty acid biosynthesis inhibitor, cerulenin, that possesses an alkyl chain length in the range of known OleA substrates, in conjunction with a single xenon binding site, leads to the putative assignment of this novel alkyl binding channel. Structural overlays between the OleA homologues, 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) synthase and the fatty acid biosynthesis enzyme FabH, allow assignment of the two remaining channels: one for the thioester-containing pantetheinate arm and the second for the alkyl group of one substrate. A short {beta}-hairpin region is ordered in only one of the crystal forms, and that may suggest open and closed states relevant for substrate binding. Cys143 is the conserved catalytic cysteine within the superfamily, and the site of alkylation by cerulenin. The alkylated structure suggests that a glutamic acid residue (Glu117{beta}) likely promotes Claisen condensation by acting as the catalytic base. Unexpectedly

  14. The nonenzymatic reactivity of the acyl-linked metabolites of mefenamic acid toward amino and thiol functional group bionucleophiles.

    Science.gov (United States)

    Horng, Howard; Benet, Leslie Z

    2013-11-01

    Mefenamic acid (MFA), a carboxylic acid-containing nonsteroidal anti-inflammatory drug, is metabolized into the chemically-reactive MFA-1-O-acyl-glucuronide (MFA-1-O-G), MFA-acyl-adenylate (MFA-AMP), and the MFA-S-acyl-coenzyme A (MFA-CoA), all of which are electrophilic and capable of acylating nucleophilic sites on biomolecules. In this study, we investigate the nonenzymatic ability of each MFA acyl-linked metabolite to transacylate amino and thiol functional groups on the acceptor biomolecules Gly, Tau, l-glutathione (GSH), and N-acetylcysteine (NAC). In vitro incubations with each of the MFA acyl-linked metabolites (1 μM) in buffer under physiologic conditions with Gly, Tau, GSH, or NAC (10 mM) revealed that MFA-CoA was 11.5- and 19.5-fold more reactive than MFA-AMP toward the acylation of cysteine-sulfhydryl groups of GSH and NAC, respectively. However, MFA-AMP was more reactive toward both Gly and Tau, 17.5-fold more reactive toward the N-acyl-amidation of taurine than its corresponding CoA thioester, while MFA-CoA displayed little reactivity toward glycine. Additionally, mefenamic acid-S-acyl-glutathione (MFA-GSH) was 5.6- and 108-fold more reactive toward NAC than MFA-CoA and MFA-AMP, respectively. In comparison with MFA-AMP and MFA-CoA, MFA-1-O-G was not significantly reactive toward all four bionucleophiles. MFA-AMP, MFA-CoA, MFA-1-O-G, MFA-GSH, and mefenamic acid-taurine were also detected in rat in vitro hepatocyte MFA (100 μM) incubations, while mefenamic acid-glycine was not. These results demonstrate that MFA-AMP selectively reacts with the amino functional groups of glycine and lysine nonenzymatically, MFA-CoA selectively reacts nonenzymatically with the thiol functional groups of GSH and NAC, and MFA-GSH reacts with the thiol functional group of GSH nonenzymatically, all of which may potentially elicit an idiosyncratic toxicity in vivo.

  15. Sex Differences in Long Chain Fatty Acid Utilization and Fatty Acid Binding Protein Concentration in Rat Liver

    Science.gov (United States)

    Ockner, Robert K.; Burnett, David A.; Lysenko, Nina; Manning, Joan A.

    1979-01-01

    Female sex and estrogen administration are associated with increased hepatic production of triglyceride-rich lipoproteins; the basis for this has not been fully elucidated. Inasmuch as hepatic lipoprotein production is also influenced by FFA availability and triglyceride biosynthesis, we investigated sex differences in FFA utilization in rat hepatocyte suspensions and in the components of the triglyceride biosynthetic pathway. Isolated adult rat hepatocyte suspensions were incubated with albumin-bound [14C]oleate for up to 15 min. At physiological and low oleate concentrations, cells from females incorporated significantly more 14C into glycerolipids, especially triglycerides, and into oxidation products than did male cells, per milligram cell protein. At 0.44 mM oleate, incorporation into triglycerides in female cells was approximately twice that in male cells. Comparable sex differences were observed in cells from fasted animals and when [14C]-glycerol incorporation was measured. At higher oleate concentrations, i.e., fatty acid:albumin mole ratios in excess of 2:1, these sex differences were no longer demonstrable, suggesting that maximal rates of fatty acid esterification and oxidation were similar in female and male cells. In female and male hepatic microsomes, specific activities of long chain acyl coenzyme A synthetase, phosphatidate phosphohydrolase, and diglyceride acyltransferase were similar, but glycerol-3-phosphate acyltransferase activity was slightly greater in females at certain substrate concentrations. Microsomal incorporation of [14C]oleate into total glycerolipids was not significantly greater in females. In further contrast to intact cells, microsomal incorporation of [14C]oleate into triglycerides, although significantly greater in female microsomes, accounted for only a small fraction of the fatty acid esterified. The binding affinity and stoichiometry of partially purified female hepatic fatty acid binding protein (FABP) were similar to

  16. Neuroprotective effects of L-carnitine against oxygenglucose deprivation in rat primary cortical neurons

    Directory of Open Access Journals (Sweden)

    Yu Jin Kim

    2012-07-01

    Full Text Available &lt;b&gt;Purpose:&lt;/b&gt; Hypoxic-ischemic encephalopathy is an important cause of neonatal mortality, as this brain injury disrupts normal mitochondrial respiratory activity. Carnitine plays an essential role in mitochondrial fatty acid transport and modulates excess acyl coenzyme A levels. In this study, we investigated whether treatment of primary cultures of rat cortical neurons with L-carnitine was able to prevent neurotoxicity resulting from oxygen-glucose deprivation (OGD. &lt;b&gt;Methods:&lt;/b&gt; Cortical neurons were prepared from Sprague-Dawley rat embryos. L-Carnitine was applied to cultures just prior to OGD and subsequent reoxygenation. The numbers of cells that stained with acridine orange (AO and propidium iodide (PI were counted, and lactate dehydrogenase (LDH activity and reactive oxygen species (ROS levels were measured. The 3-(4,5-dimethylthiazol-2-yl-2,5- diphenyltetrazolium bromide assay and the terminal uridine deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling assay were performed to evaluate the effect of L-carnitine (1 μM, 10 μM, and 100 μM on OGD-induced neurotoxicity. &lt;B&gt;Results:&lt;/b&gt; Treatment of primary cultures of rat cortical neurons with L-carnitine significantly reduced cell necrosis and prevented apoptosis after OGD. L-Carnitine application significantly reduced the number of cells that died, as assessed by the PI/AO ratio, and also reduced ROS release in the OGD groups treated with 10 μM and 100 μM of L-carnitine compared with the untreated OGD group (P&lt;0.05. The application of L-carnitine at 100 μM significantly decreased cytotoxicity, LDH release, and inhibited apoptosis compared to the untreated OGD group (P&lt;0.05. &lt;B&gt;Conclusion:&lt;/b&gt; L-Carnitine has neuroprotective benefits against OGD in rat primary cortical neurons in vitro.

  17. Skeletal Muscle Magnetic Resonance Imaging of the Lower Limbs in Late-onset Lipid Storage Myopathy with Electron Transfer Flavoprotein Dehydrogenase Gene Mutations

    Institute of Scientific and Technical Information of China (English)

    Xin-Yi Liu; Ming Jin; Zhi-Qiang Wang; Dan-Ni Wang; Jun-Jie He; Min-Ting Lin; Hong-Xia Fu

    2016-01-01

    Background:Lipid storage myopathy (LSM) is a genetically heterogeneous group with variable clinical phenotypes.Late-onset multiple acyl-coenzyme A dehydrogenation deficiency (MADD) is a rather common form of LSM in China.Diagnosis and clinical management of it remain challenging,especially without robust muscle biopsy result and genetic detection.As the noninvasion and convenience,muscle magnetic resonance imaging (MRI) is a helpful assistant,diagnostic tool for neuromuscular disorders.However,the disease-specific MRI patterns of muscle involved and its diagnostic value in late-onset MADD have not been systematic analyzed.Methods:We assessed the MRI pattern and fat infiltration degree of the lower limb muscles in 28 late-onset MADD patients,combined with detailed clinical features and gene spectrum.Fat infiltration degree of the thigh muscle was scored while that ofgluteus was described as obvious or not.Associated muscular atrophy was defined as obvious muscle bulk reduction.Results:The mean scores were significantly different among the anterior,medial,and posterior thigh muscle groups.The mean of fat infiltration scores on posterior thigh muscle group was significantly higher than either anterior or medial thigh muscle group (P < 0.001).Moreover,the mean score on medial thigh muscle group was significantly higher than that of anterior thigh muscle group (P < 0.01).About half of the patients displayed fat infiltration and atrophy in gluteus muscles.Of 28 patients,12 exhibited atrophy in medial and/or posterior thigh muscle groups,especially in posterior thigh muscle group.Muscle edema pattern was not found in all the patients.Conclusions:Late-onset MADD patients show a typical muscular imaging pattern of fat infiltration and atrophy on anterior,posterior,and medial thigh muscle groups,with major involvement of posterior thigh muscle group and gluteus muscles and a sparing involvement of anterior thigh compartment.Our findings also suggest that muscle MRI of

  18. Mechanisms of toxicity of di(2-ethylhexyl) phthalate on the reproductive health of male zebrafish

    Energy Technology Data Exchange (ETDEWEB)

    Uren-Webster, Tamsyn M.; Lewis, Ceri; Filby, Amy L.; Paull, Gregory C. [Hatherly Laboratories, School of Biosciences, University of Exeter, Prince of Wales Road, Exeter, Devon EX4 4PS (United Kingdom); Santos, Eduarda M., E-mail: e.santos@exeter.ac.uk [Hatherly Laboratories, School of Biosciences, University of Exeter, Prince of Wales Road, Exeter, Devon EX4 4PS (United Kingdom)

    2010-09-01

    Phthalates are ubiquitous in the aquatic environment and are known to adversely affect male reproductive health in mammals through interactions with multiple receptor systems. However, little is known about the risks they pose to fish. This project investigated the effects of di(2-ethylhexyl) phthalate (DEHP), the most commonly used phthalate, on the reproductive health of male zebrafish (Danio rerio). Males were treated with 0.5, 50 and 5000 mg DEHP kg{sup -1} (body weight) for a period of 10 days via intraperitoneal injection. The effects of the exposure were assessed by analysing fertilisation success, testis histology, sperm DNA integrity and transcript profiles of the liver and testis. A significant increase in the hepatosomatic index and levels of hepatic vitellogenin transcript were observed following exposure to 5000 mg DEHP kg{sup -1}. Exposure to 5000 mg DEHP kg{sup -1} also resulted in a reduction in fertilisation success of oocytes spawned by untreated females. However, survival and development of the resulting embryos were unaffected by all treatments, and no evidence of DEHP-induced sperm DNA damage was observed. Exposure to 50 and 5000 mg DEHP kg{sup -1} caused alterations in the proportion of germ cells at specific stages of spermatogenesis in the testis, including a reduction in the proportion of spermatozoa and an increase in the proportion of spermatocytes, suggesting that DEHP may inhibit the progression of meiosis. In parallel, exposure to 5000 mg DEHP kg{sup -1} increased the levels of two peroxisome proliferator-activated receptor (PPAR) responsive genes (acyl-coenzyme A oxidase 1 (acox1) and enoyl-coenzyme A, hydratase/3-hydroxyacyl coenzyme A dehydrogenase (ehhadh). These data demonstrated that exposure to high concentrations of DEHP disrupts spermatogenesis in adult zebrafish with a consequent decrease in their ability to fertilise oocytes spawned by untreated females. Furthermore, our data suggest that the adverse effects caused by

  19. Effects of Selenium-Enriched Probiotics on Lipid Metabolism, Antioxidative Status, Histopathological Lesions, and Related Gene Expression in Mice Fed a High-Fat Diet.

    Science.gov (United States)

    Nido, Sonia Agostinho; Shituleni, Shituleni Andreas; Mengistu, Berhe Mekonnen; Liu, Yunhuan; Khan, Alam Zeb; Gan, Fang; Kumbhar, Shahnawaz; Huang, Kehe

    2016-06-01

    A total of 80 female albino mice were randomly allotted into five groups (n = 16) as follows: (A) normal control, (B) high-fat diet (HFD),; (C) HFD + probiotics (P), (D) HFD + sodium selenite (SS), and (E) HFD + selenium-enriched probiotics (SP). The selenium content of diets in groups A, B, C, D, and E was 0.05, 0.05, 0.05, 0.3, and 0.3 μg/g, respectively. The amount of probiotics contained in groups C and E was similar (Lactobacillus acidophilus 0.25 × 10(11)/mL and Saccharomyces cerevisiae 0.25 × 10(9)/mL colony-forming units (CFU)). The high-fat diet was composed of 15 % lard, 1 % cholesterol, 0.3 % cholic acid, and 83.7 % basal diet. At the end of the 4-week experiment, blood and liver samples were collected for the measurements of lipid metabolism, antioxidative status, histopathological lesions, and related gene expressions. The result shows that HFD significantly increased the body weights and liver damages compared to control, while P, SS, or SP supplementation attenuated the body weights and liver damages in mice. P, SS, or SP supplementation also significantly reversed the changes of alanine aminotransferase (AST), aspartate aminotransferase (ALT), total cholesterol (TC), triglyceride (TG), low-density lipoprotein (LDL), total protein (TP), high-density lipoprotein (HDL), glutathione peroxidase (GSH-Px), superoxide dismutase (SOD), catalasa (CAT), and malondialdehyde (MDA) levels induced by HFD. Generally, adding P, SS, or SP up-regulated mRNA expression of carnitine palmitoyltransferase-I (CPT1), carnitine palmitoyltransferase II (CPT2), acetyl-CoA acetyltransferase II (ACAT2), acyl-coenzyme A oxidase (ACOX2), and peroxisome proliferator-activated receptor alpha (PPARα) and down-regulated mRNA expression of fatty acid synthase (FAS), lipoprotein lipase (LPL), peroxisome proliferator-activated receptor gamma (PPARγ), and sterol regulatory element-binding protein-1 (SREBP1) involved in lipid metabolism. Among the group

  20. mRNA expression of genes regulating lipid metabolism in ringed seals (Pusa hispida) from differently polluted areas

    Energy Technology Data Exchange (ETDEWEB)

    Castelli, Martina Galatea [Norwegian Polar Institute, Fram Centre, 9296 Tromsø (Norway); University of Bergen, Department of Biology, 5020 Bergen (Norway); Rusten, Marte; Goksøyr, Anders [University of Bergen, Department of Biology, 5020 Bergen (Norway); Routti, Heli, E-mail: heli.routti@npolar.no [Norwegian Polar Institute, Fram Centre, 9296 Tromsø (Norway)

    2014-01-15

    Highlights: •Genes regulating lipid metabolism were studied in ringed seals. •We compared highly contaminated Baltic seals and less contaminated Svalbard seals. •mRNA expression of hepatic PPARγ was higher in the Baltic seals. •mRNA expression of adipose PPARγ target genes was higher in the Baltic seals. •Contaminant exposure may affect lipid metabolism in the Baltic ringed seals. -- Abstract: There is a growing concern about the ability of persistent organic pollutants (POPs) to influence lipid metabolism. Although POPs are found at high concentrations in some populations of marine mammals, for example in the ringed seal (Pusa hispida) from the Baltic Sea, little is known about the effects of POPs on their lipid metabolism. An optimal regulation of lipid metabolism is crucial for ringed seals during the fasting/molting season. This is a physiologically stressful period, during which they rely on the energy stored in their fat reserves. The mRNA expression levels for seven genes involved in lipid metabolism were analyzed in liver and/or blubber tissue from molting ringed seals from the polluted Baltic Sea and a less polluted reference location, Svalbard (Norway). mRNA expression of genes encoding peroxisome proliferator-activated receptors (PPAR) α and γ and their target genes acyl-coenzyme A oxidase 1 (ACOX1) and cluster of differentiation 36 (CD36) were analyzed in liver. mRNA expression level of genes encoding PPARβ, PPARγ and their target genes encoding fatty acid binding protein 4 (FABP4) and adiponectin (ADIPOQ) were measured in inner and middle blubber layers. In addition, we evaluated the influence of molting status on hepatic mRNA expression of genes encoding PPARs and their target genes in ringed seals from Svalbard. Our results show higher mRNA expression of genes encoding hepatic PPARγ and adipose PPARβ, FABP4, and ADIPOQ in the Baltic seals compared to the Svalbard seals. A positive relationship between mRNA expressions of genes

  1. Acyl-CoA:cholesterol acyltransferases (ACATs/SOATs): Enzymes with multiple sterols as substrates and as activators.

    Science.gov (United States)

    Rogers, Maximillian A; Liu, Jay; Song, Bao-Liang; Li, Bo-Liang; Chang, Catherine C Y; Chang, Ta-Yuan

    2015-07-01

    Cholesterol is essential to the growth and viability of cells. The metabolites of cholesterol include: steroids, oxysterols, and bile acids, all of which play important physiological functions. Cholesterol and its metabolites have been implicated in the pathogenesis of multiple human diseases, including: atherosclerosis, cancer, neurodegenerative diseases, and diabetes. Thus, understanding how cells maintain the homeostasis of cholesterol and its metabolites is an important area of study. Acyl-coenzyme A:cholesterol acyltransferases (ACATs, also abbreviated as SOATs) converts cholesterol to cholesteryl esters and play key roles in the regulation of cellular cholesterol homeostasis. ACATs are most unusual enzymes because (i) they metabolize diverse substrates including both sterols and certain steroids; (ii) they contain two different binding sites for steroidal molecules. In mammals, there are two ACAT genes that encode two different enzymes, ACAT1 and ACAT2. Both are allosteric enzymes that can be activated by a variety of sterols. In addition to cholesterol, other sterols that possess the 3-beta OH at C-3, including PREG, oxysterols (such as 24(S)-hydroxycholesterol and 27-hydroxycholesterol, etc.), and various plant sterols, could all be ACAT substrates. All sterols that possess the iso-octyl side chain including cholesterol, oxysterols, various plant sterols could all be activators of ACAT. PREG can only be an ACAT substrate because it lacks the iso-octyl side chain required to be an ACAT activator. The unnatural cholesterol analogs epi-cholesterol (with 3-alpha OH in steroid ring B) and ent-cholesterol (the mirror image of cholesterol) contain the iso-octyl side chain but do not have the 3-beta OH at C-3. Thus, they can only serve as activators and cannot serve as substrates. Thus, within the ACAT holoenzyme, there are site(s) that bind sterol as substrate and site(s) that bind sterol as activator; these sites are distinct from each other. These features form

  2. Overlapping repressor binding sites result in additive regulation of Escherichia coli FadH by FadR and ArcA.

    Science.gov (United States)

    Feng, Youjun; Cronan, John E

    2010-09-01

    Escherichia coli fadH encodes a 2,4-dienoyl reductase that plays an auxiliary role in beta-oxidation of certain unsaturated fatty acids. In the 2 decades since its discovery, FadH biochemistry has been studied extensively. However, the genetic regulation of FadH has been explored only partially. Here we report mapping of the fadH promoter and document its complex regulation by three independent regulators, the fatty acid degradation FadR repressor, the oxygen-responsive ArcA-ArcB two-component system, and the cyclic AMP receptor protein-cyclic AMP (CRP-cAMP) complex. Electrophoretic mobility shift assays demonstrated that FadR binds to the fadH promoter region and that this binding can be specifically reversed by long-chain acyl-coenzyme A (CoA) thioesters. In vivo data combining transcriptional lacZ fusion and real-time quantitative PCR (qPCR) analyses indicated that fadH is strongly repressed by FadR, in agreement with induction of fadH by long-chain fatty acids. Inactivation of arcA increased fadH transcription by >3-fold under anaerobic conditions. Moreover, fadH expression was increased 8- to 10-fold under anaerobic conditions upon deletion of both the fadR and the arcA gene, indicating that anaerobic expression is additively repressed by FadR and ArcA-ArcB. Unlike fadM, a newly reported member of the E. coli fad regulon that encodes another auxiliary beta-oxidation enzyme, fadH was activated by the CRP-cAMP complex in a manner similar to those of the prototypical fad genes. In the absence of the CRP-cAMP complex, repression of fadH expression by both FadR and ArcA-ArcB was very weak, suggesting a possible interplay with other DNA binding proteins.

  3. Validation of the Antidiabetic and Hypolipidemic Effects of Clitocybe nuda by Assessment of Glucose Transporter 4 and Gluconeogenesis and AMPK Phosphorylation in Streptozotocin-Induced Mice

    Directory of Open Access Journals (Sweden)

    Chun-Ching Shih

    2014-01-01

    Full Text Available The study was designed to investigate the effects of extract of Clitocybe nuda (CNE on type 1 diabetes mellitus and dyslipidemia in streptozotocin- (STZ- induced diabetic mice. Diabetes was induced by injection of STZ. Diabetic mice were randomly divided into five groups and given orally CNE (C1: 0.2, C2: 0.5, and C3: 1.0 g/kg body weight or metformin (Metf or vehicle for 4 weeks. STZ induction decreased in the levels of insulin, body weight, and the weight of skeletal muscle, whereas the levels of blood glucose, hemoglobin nonenzymatically (percent HbA1c, and circulating triglyceride (P < 0.001, P < 0.001, and P < 0.01, resp. were increased. CNE decreased the levels of blood glucose, HbA1c, and triglyceride levels, whereas it increased the levels of insulin and leptin compared with the vehicle-treated STZ group. STZ induction caused a decrease in the protein contents of skeletal muscular and hepatic phosphorylation of AMP-activated protein kinase (phospho-AMPK and muscular glucose transporter 4 (GLUT4. Muscular phospho-AMPK contents were increased in C2-, C3-, and Metf-treated groups. CNE and Metf significantly increased the muscular proteins of GLUT4. Liver phospho-AMPK showed an increase in all CNE- and Metf-treated groups combined with the decreased hepatic glucose production by decreasing phosphenolpyruvate carboxykinase (PEPCK, glucose-6-phosphatase (G6Pase, and 11beta hydroxysteroid dehydroxygenase (11β-HSD1 gene, which contributed to attenuating diabetic state. The study indicated that the hypoglycemic properties of CNE were related to both the increased muscular glucose uptake and the reduction in hepatic gluconeogenesis. CNE exerts hypolipidemic effect by increasing gene expressions of peroxisome proliferator-activated receptor α (PPARα and decreasing expressions of fatty acid synthesis, including acyl-coenzyme A: diacylglycerol acyltransferase (DGAT 2. Therefore, amelioration of diabetic and dyslipidemic state by CNE in STZ

  4. Phosphatidic acid signaling mediates lung cytokine expression and lung inflammatory injury after hemorrhage in mice.

    Science.gov (United States)

    Abraham, E; Bursten, S; Shenkar, R; Allbee, J; Tuder, R; Woodson, P; Guidot, D M; Rice, G; Singer, J W; Repine, J E

    1995-02-01

    Because phosphatidic acid (PA) pathway signaling may mediate many basic reactions involving cytokine-dependent responses, we investigated the effects of CT1501R, a functional inhibitor of the enzyme lysophosphatidic acid acyltransferase (LPAAT) which converts lysophosphatidic acid (Lyso-PA) to PA. We found that CT1501R treatment not only prevented hypoxia-induced PA increases and lyso-PA consumption in human neutrophils, but also prevented neutrophil chemotaxis and adherence in vitro, and lung injury and lung neutrophil accumulation in mice subjected to hemorrhage and resuscitation. In addition, CT1501R treatment prevented increases in mRNA levels and protein production of a variety of proinflammatory cytokines in multiple lung cell populations after blood loss and resuscitation. Our results indicate the fundamental role of PA metabolism in the development of acute inflammatory lung injury after blood loss.

  5. Structural basis for substrate discrimination and integrin binding by autotaxin

    OpenAIRE

    Hausmann, Jens; Kamtekar, Satwik; Christodoulou, Evangelos; Day, Jacqueline E.; Wu, Tao; Fulkerson, Zachary; Albers, Harald M. H. G.; van Meeteren, Laurens A.; Houben, Anna; Zeijl, Leonie van; Jansen, Silvia; Andries, Maria; Hall, Troii; Pegg, Lyle E.; Benson, Timothy E.

    2011-01-01

    Autotaxin (ATX) or ecto-nucleotide pyrophosphatase/phosphodiesterase-2 (ENPP2) is a secreted lysophospholipase D that generates the lipid mediator lysophosphatidic acid (LPA), a mitogen and chemo-attractant for many cell types. ATX-LPA signaling has roles in various pathologies including tumour progression and inflammation. However, the molecular basis of substrate recognition and catalysis, and the mechanism of interaction with target cells, has been elusive. Here we present the crystal stru...

  6. Gclust Server: 106666 [Gclust Server

    Lifescience Database Archive (English)

    Full Text Available 106666 HSA_7305013 Cluster Sequences Related Sequences(93) 351 NP_004711.2 endothel...ial differentiation, lysophosphatidic acid G-protein-coupled receptor, 4 ; no annotation 1 1.00e-80 0.0 0.0 ...0.0 0.0 0.0 12.5 Show 106666 Cluster ID 106666 Sequence ID HSA_7305013 Link to cluster sequences Cluster Seq

  7. Multidisciplinary Strategies in the Prevention and Early Detection of Ovarian Cancer

    Science.gov (United States)

    2001-09-01

    Pharmacol 1997;54:541-4. phosphatidic acid phosphatases (PAPs), sphingomyelinase, and 6. Bookman MA. Biological therapy of ovarian cancer: Current...smooth muscle cells in culture. Thrombosis Res WH. Lysophosphatidic acid induces neuronal shape changes via 1999;94:317-26. a novel receptor-mediated...2001 9 healing agenit--sph~igo~sylphosphorylcholinie. J Investig Derma- phosphatidic acid -stimulated mritogeii-activated protein kinase tol 1996;106:232

  8. Novel Methods for Imaging PET Biomarkers and Gene Therapy of Cancer

    Science.gov (United States)

    2009-05-01

    205- 975-5648; E-mail: flin@uab.edu. 4 The abbreviations used are: LPA, lysophosphatidic acid; GPCR , G protein- coupled receptor; 2-BP, 2...protein-coupled receptor ( GPCR )- mediated prosurvival signaling involves the coupling of ligand-bound receptors to heterotrimeric G proteins that...for 10 min and harvested. Endogenous LP A2 was immunoprecipitated with an anti-LPA2 rat antibody (a gift from Dr. Junken Aoki) or a control rat

  9. Autotaxin: Its Role in Biology of Melanoma Cells and as a Pharmacological Target

    Directory of Open Access Journals (Sweden)

    Maciej Jankowski

    2011-01-01

    Full Text Available Autotaxin (ATX is an extracellular lysophospholipase D (lysoPLD released from normal cells and cancer cells. Activity of ATX is detected in various biological fluids. The lysophosphatidic acid (LPA is the main product of ATX. LPA acting through specific G protein-coupled receptors (LPA1-LPA6 affects immunological response, normal development, and malignant tumors' formation and progression. In this review, the impact of autotoxin on biology of melanoma cells and potential treatment is discussed.

  10. Bioactive Lipids, LPC and LPA, are Novel Pro-metastatic Factors and Their Tissue Levels Increase in Response to Radio/Chemotherapy

    OpenAIRE

    Schneider, Gabriela; Sellers, Zachariah Payne; Abdel-Latif, Ahmed; Morris, Andrew J.; Mariusz Z. Ratajczak

    2014-01-01

    Bioactive lipids are fundamental mediators of a number of critical biological processes such as inflammation, proliferation, and apoptosis. Rhabdomyosarcoma (RMS) is common in adolescence with histological subtypes that favor metastasis. However, the factors that influence metastasis are not well appreciated. Here, it is shown that lysophosphatidylcholine (LPC) and its derivative, lysophosphatidic acid (LPA), strongly enhance motility and adhesion of human RMS cells. Importantly, these metast...

  11. Lysophospholipid receptors in drug discovery

    OpenAIRE

    Kihara, Yasuyuki; Mizuno, Hirotaka; Chun, Jerold

    2014-01-01

    Lysophospholipids (LPs), including lysophosphatidic acid (LPA), sphingosine 1-phospate (S1P), lysophosphatidylinositol (LPI), and lysophosphatidylserine (LysoPS), are bioactive lipids that transduce signals through their specific cell-surface G protein-coupled receptors, LPA1–6, S1P1–5, LPI1, and LysoPS1–3, respectively. These LPs and their receptors have been implicated in both physiological and pathophysiological processes such as autoimmune diseases, neurodegenerative diseases, fibrosis, p...

  12. A dependência pela prática de exercícios físicos e o uso de recursos ergogênicos = Physical exercise dependence and the use of ergogenic resources

    Directory of Open Access Journals (Sweden)

    José Luiz Lopes Vieira

    2010-01-01

    Full Text Available Objetivou-se Investigar a ocorrência de dependência por exercícios físicos quanto às características de praticantes de musculação e ginástica em academias, como uso de recursos ergogênicos, sexo e índice de massa corporal. Participaram do estudo 80 sujeitos (27,12 ± 6,60 anos, praticantes de ginástica e/ou musculação em academias, de ambos os sexos. Utilizou-se a Escala de Dependência por Exercícios Físicos, a listagem do tipo de suplemento alimentar utilizado como recurso ergogênico e o Índice de Massa Corporal – IMC (Kg/cm². A análise estatística foi realizada por meio da correlação de Spearman e o teste de Wilcoxon (p The aim of this study is to investigate the occurrence of physical exercise dependence in regards to the characteristics of participants in weight training and exercises at gyms, such as the use ofergogenic resources, gender and body mass index. Eighty subjects (27.12 ± 6.60 years from both genders took part in the study, all of whom practiced gymnastics and/or weight training in gyms. The study utilized the Exercise Dependence Scale, a check list of the kinds of nutritional supplementation used as ergogenic resources, and the Body Mass Index – BMI (Kg cm-². Statistical analysis was performed using Spearman's correlation and the Wilcoxon test (p < 0.05. The results showed that there was no statistically significant difference between physical exercise dependence in men (5.14 ± 1.28 and women (5.60 ± 1.45. The body mass index did not show statistically significant correlation with the scores of dependency, either. However, dependentmen showed high prevalence of use of ergogenic resources (63.63%, p = 0.01, while for dependent women there were no statistically significant results. The body mass index does not relate to the scores of exercise dependence. However, even with a normal BMI, the use of ergogenic resources presents high prevalence among dependent men. As a result, there is evidence that physical exercise dependence is a risk factor for the development of emotional disturbances related to exercise, such as muscle dysmorphia and overtraining.

  13. Gli2a protein localization reveals a role for Iguana/DZIP1 in primary ciliogenesis and a dependence of Hedgehog signal transduction on primary cilia in the zebrafish

    Directory of Open Access Journals (Sweden)

    van Eeden Freek

    2010-04-01

    Full Text Available Abstract Background In mammalian cells, the integrity of the primary cilium is critical for proper regulation of the Hedgehog (Hh signal transduction pathway. Whether or not this dependence on the primary cilium is a universal feature of vertebrate Hedgehog signalling has remained contentious due, in part, to the apparent divergence of the intracellular transduction pathway between mammals and teleost fish. Results Here, using a functional Gli2-GFP fusion protein, we show that, as in mammals, the Gli2 transcription factor localizes to the primary cilia of cells in the zebrafish embryo and that this localization is modulated by the activity of the Hh pathway. Moreover, we show that the Igu/DZIP1protein, previously implicated in the modulation of Gli activity in zebrafish, also localizes to the primary cilium and is required for its proper formation. Conclusion Our findings demonstrate a conserved role of the primary cilium in mediating Hedgehog signalling activity across the vertebrate phylum and validate the use of the zebrafish as a representative model for the in vivo analysis of vertebrate Hedgehog signalling.

  14. Measurements of observables in the pion-nucleon system, nuclear a- dependence of heavy quark production and rare decays of D and B mesons. Progress report, 1 December, 1990--15 February, 1992

    Energy Technology Data Exchange (ETDEWEB)

    Sadler, M.E.; Isenhower, L.D.

    1992-02-15

    This report discusses research on the following topics: pion-nucleon interactions; detector tomography facility; nuclear dependence of charm and beauty quark production and a study of two-prong decays of neutral D and B mesons; N* collaboration at CEBAF; and pilac experiments. (LSP)

  15. Genetic characterization of the CcpA-dependent, cellobiose-specific PTS system comprising CelB, PtcB and PtcA that transports lactose in Lactococcus lactis IL1403.

    Science.gov (United States)

    Aleksandrzak-Piekarczyk, Tamara; Polak, Jolanta; Jezierska, Beata; Renault, Pierre; Bardowski, Jacek

    2011-01-31

    Lactose metabolism is one of the most important areas of research on Lactic Acid Bacteria (LAB). In rapidly acidifying industrial Lactococcus lactis strains, lactose is transported by a lactose-specific phosphotransferase system (PTS) encoded by a plasmid. However, an alternative lactose catabolic pathway was evidenced in the plasmid-cured, and thus initially lactose-negative L. lactis IL1403. We showed that in this strain the chromosomally-encoded cellobiose-specific PTS system comprising the celB, ptcB and ptcA genes is also able to transport lactose. By expression studies in the wild type IL1403 strain and IBB550, its ccpA-deficient derivative, we demonstrated that celB, ptcB and ptcA are tightly regulated by the general catabolite repression system, whereas celB additionally requires the presence of cellobiose to be fully induced. The comparison of expression levels of sugar catabolic genes indicated that the efficiency of CcpA-mediated catabolic repression depends on conservation of the cre sequence, and that in the case of perfect matching with the cre consensus, CcpA still drives a strong repression even under non-repressing conditions.

  16. The transtheoretical model and substance dependence: theoretical and practical aspects O modelo transteórico e a dependência química: aspectos teóricos e práticos

    Directory of Open Access Journals (Sweden)

    Fabiana Andrioni De Biaze Vilela

    2009-12-01

    Full Text Available OBJECTIVE: This paper aims to present and discuss the Transtheoretical Model and its importance for the treatment of substance abuse disorders. METHOD: A literature review was made based on articles from the last 10 years in substance use with human subjects found in PubMed (Medline and the Scientific Electronic Library Online, as well as on the main books written by the creators of the model. From the initial collection of articles related to the Transtheoretical Model, the University of Rhode Island Assessment and substance abuse, those related to other health conditions were excluded. Although articles related to hospitalization were also excluded, as were those related to the Minnesota Model, treatment proposals were included. RESULTS: Although the TTM has been studied for over 20 years, new concerns regarding the initial idea continue to arise. Such concerns include the cross-sectional design of studies employing the model, as well as the prescriptive versus descriptive point of view. DISCUSSION: The review of the Transtheoretical Model brought intentional behavior change to light, which could broaden the understanding of addictive behaviors. Together with its concepts of processes and stages of change, the Transtheoretical Model provides professionals with the idea that the effectiveness of therapy is dependent upon the capability of the therapist to match the technique to the current motivational stage of the patient in the process of change. This demonstrates the importance of identifying the stage of change of the patient when they present for treatment. Here, we describe the principal elements of the Transtheoretical Model, as well as the instruments currently used to identify the stage of change. Finally, criticisms and limitations of the model are discussed.OBJETIVO: Este artigo tem como objetivos a apresentação e discussão do Modelo Transteórico e sua importância para o tratamento da dependência química. MÉTODO: Foi feita revisão de literatura baseada em artigos dos últimos 10 anos sobre abuso de substâncias com sujeitos humanos encontrados no PubMed (Medline e a Scientific Electronic Library Online, bem como as principais obras dos idealizadores da Teoria. Dos artigos encontrados inicialmente sobre o Modelo, University of Rhode Island Assessment e dependência química, aqueles relacionados a outras condições de saúde foram excluídos. Propostas de tratamento foram consideradas (exceto Modelo Minnesota e internação. RESULTADOS: Novas diretrizes surgem sobre a idéia inicial da teoria, apesar da mesma já ser estudada há mais de 20 anos: estudos transversais empregando o modelo, bem como a avaliação do potencial prescritivo ou descritivo do mesmo. DISCUSSÃO: Esta teoria enfoca a mudança intencional de comportamento, o que ampliou a compreensão dos comportamentos aditivos. O Modelo Transteórico sugere aos profissionais que a efetividade do tratamento depende da capacidade do mesmo de aplicar a técnica adequada à situação motivacional do paciente no processo de mudança. Isto demonstra porque é tão importante identificar o estágio de mudança do paciente quando este ingressa no tratamento. O artigo descreve os principais elementos da teoria bem como os principais instrumentos usados atualmente para identificar os estágios. Por fim, são discutidas as críticas e as limitações do modelo.

  17. An oxalyl-CoA dependent pathway of oxalate catabolism plays a role in regulating calcium oxalate crystal accumulation and defending against oxalate-secreting phytopathogens in Medicago truncatula

    Science.gov (United States)

    Considering the widespread occurrence of oxalate in nature and its broad impact on a host of organisms, it is surprising that so little is known about the turnover of this important acid. In plants, oxalate oxidase is the most well studied enzyme capable of degrading oxalate, but not all plants pos...

  18. 溶血磷脂酸受体3与相关疾病关系的研究进展%The Research on Relationship Between of Lysophospholipids Acid Receptor3 and The Related Diseases

    Institute of Scientific and Technical Information of China (English)

    木其日; 阿拉坦高勒

    2015-01-01

    Objective lysophosphatidic acid receptor 3 (lPa3), also known as endothelial differentiation gene receptor 7 (edG7), is one member of superfamily of G-protein coupled receptor (GPcr) or the endothelial differentiation family activated by lysophosphatidic acid. it has high afifnity to lysophosphatidic acid, and can mediate many kinds of cellular physiological responses. recent researches have shown that lPa3/edG7 is epressed in most major tissues.,but highly expressed in some speciifc tissue such as brain、heart and ovarian. in addition, it is closely connected with embryonic implantation、ovarian cancer, prostate cancer, lung cancer and atherosclerosis. so this review article will introduces the effect of lPa3 in embryo implantation process and how to participate in the development of tumor .%溶血磷脂酸受体3(lysophosphatidic acid receptor3,lPa3),又称内皮分化基因受体7(endothelial differentiation receptor 2.edG2),是G-蛋白偶联受体家族、edG亚家族成员(edG family),可在溶血磷脂酸(lPa)的诱导下产生诸多的细胞生理反应。lPa3在人和小鼠的组织中都有表达,但在诸如大脑、心脏、卵巢等特定的一些组织中高表达,同时研究发现lPa3也与胚胎着床、卵巢癌、前列腺癌、肺癌、动脉粥样硬化等疾病的发生和发展过程有着密切的关系。因此本文将概括的介绍lPa3在lPa介导下参与的胚胎着床以及如何参与肿瘤的发生、发展过程。

  19. Targeting GPR110 in HER2-Overexpressing Breast Cancers

    Science.gov (United States)

    2015-10-01

    modification on 8/24/2015. The US Army Medical Research and Materiel Command, Office of Research Protections , Human Research Protection Office...in France in January 2015. Because his PhD thesis focused on the study of a GPCR (lysophosphatidic acid receptor) in mediating bone metastasis of...Chemokine  receptor  CXCR4/5   Endothelin  receptor  ET(A)R   Estrogen  G  protein-­‐coupled  receptor  GPER   Lysophosphalipid

  20. Multiple Mechanisms are Responsible for Transactivation of the Epidermal Growth Factor Receptor in Mammary Epithelial Cells

    Energy Technology Data Exchange (ETDEWEB)

    Rodland, Karin D.; Bollinger, Nikki; Ippolito, Danielle L.; Opresko, Lee; Coffey, Robert J.; Zangar, Richard C.; Wiley, H. S.

    2008-11-14

    REVIEW ENTIRE DOCUMENT AT: https://pnlweb.pnl.gov/projects/bsd/ERICA%20Manuscripts%20for%20Review/KD%20Rodland%20D7E80/HMEC_transactivation_ms01_15+Figs.pdf ABSTRACT: Using a single nontransformed strain of human mammary epithelial cells, we found that the ability of multiple growth factors and cytokines to induce ERK phosphorylation was dependent on EGFR activity. These included lysophosphatidic acid (LPA), uridine triphosphate, growth hormone, vascular endothelial growth factor, insulin-like growth factor-1 (IGF-1), and tumor necrosis factoralpha. In contrast, hepatocyte growth factor could stimulate ERK phosphorylation independent of EGFR activity...

  1. The T Allele of the ACAT-2 734 C/T Polymorphism is Associated with the Changes of TG/HDLC, log (TG/HDLC) and LDLC/HDLC Induced by High-carbohydrate/ Low-Fat Diet in Healthy Young Women%酰基辅酶A:胆固醇酰基转移酶2基因734位点T等位基因与高糖低脂膳食诱导的健康青年女性TG/HDLC、log( TG/HDLC)及LDLC/HDLC改变相关联

    Institute of Scientific and Technical Information of China (English)

    姜喆; 龚仁蓉; 李元昊; 樊梅; 方定志

    2012-01-01

    Aim To investigate the role of the acyl-coenzyme A-' cholesterol acyltransferase-2 (ACAT-2) 734 C/T polymorphism on ratios of serum lipids and its interactions on the ratios with a high-carbohydrate/low-fat ( HC/LF) diet in a young healthy Chinese population. Methods After a washout diet for seven days, fifty six young healthy subjects were given the HC/LF diet for six days. The serum lipid profiles were analyzed using the twelve-hour fasting venous blood samples collected in the mornings of the first, the eighth and the fourteenth days. The ratios of TG/HDLC, log (TG/HDLC) , TC/HDLC and LDLC/HDLC were calculated. The ACAT-2 734 C/T polymorphism was analyzed by restriction fragments patterns after amplified by polymerase chain reactions. Results At baseline, the female carriers of the T allele had significantly lower log (TG/HDLC) than the female subjects with the CC genotype. The male subjects%目的 探讨酰基辅酶A:胆固醇酰基转移酶2(ACAT-2)基因734位点C/T多态性对健康青年血脂比值的影响及在高糖低脂膳食诱导的血脂比值变化中的作用.方法 给予56名健康青年志愿者7天平衡膳食和6天高糖低脂膳食,于第1天、第8天以及第14天清晨抽取12 h空腹静脉血,测定血脂,计算甘油三酯/高密度脂蛋白胆固醇(TG/HDLC)、log(TrG/HDLC)、总胆固醇/高密度脂蛋白胆固醇(TC/HDLC)和低密度脂蛋白胆固醇/高密度脂蛋白胆固醇(LDLC/HDLC)比值.提取基因组DNA,聚合酶链反应限制性酶切法分析ACAT-2基因734位点多态性.结果 女性T等位基因携带者log( TG/HDLC)显著低于CC纯合子受试者,男性CC纯合子受试者TC/HDLC显著高于女性CC纯合子受试者,男性T等位基因携带者log(TG/HDLC)显著高于女性.高糖低脂膳食前,男性T等位基因携带者TG/HDLC、log(TG/HDLC)显著高于女性.与高糖低脂膳食前相比,高糖低脂膳食后男性TC/HDLC、LDLC/HDLC显著降低,不受基因型影响.女性CC纯合子受试者

  2. Peroxisome proliferator-activated receptors, estrogenic responses and biotransformation system in the liver of salmon exposed to tributyltin and second messenger activator.

    Science.gov (United States)

    Pavlikova, Nela; Kortner, Trond M; Arukwe, Augustine

    2010-08-15

    The mechanisms by which organotin compounds produce modulations of the endocrine systems and other biological responses are not fully understood. In this study, juvenile salmon were force-fed diet containing TBT (0: solvent control, 0.1, 1 and 10mg/kg fish) for 72 h. Subsequently, fish exposed to solvent control and 10mg TBT were exposed to waterborne concentration (200 microg/l) of the adenylate cyclase (AC) stimulator, forskolin for 2 and 4h. The overall aim of the study was to explore whether TBT endocrine disruptive effects involve second messenger activation. Liver was sampled from individual fish (n=8) at the end of the exposures. The transcription patterns of peroxisome proliferator-activated receptor (PPAR) isotype and acyl-coenzyme A oxidase 1 (ACOX1), aromatase isoform, estrogen receptor-alpha (ER alpha), pregnane X receptor (PXR), CYP3A and glutathione S-transferase (GST) genes were measured by quantitative polymerase chain reaction (qPCR). Our data showed a consistent increase in PPAR alpha, PPAR beta and PPAR gamma mRNA and protein expression after TBT exposure that were inversely correlated with ACOX1 mRNA levels. Forskolin produced PPAR isotype-specific mRNA and protein effects that were modulated by TBT. ACOX1 expression was decreased (at 2h) and increased (at 4h) by forskolin and the presence of TBT potentiated these effects. TBT apparently increased mRNA and protein levels of cyp19a, compared to the solvent control, whereas cyp19b mRNA levels were unaffected by TBT treatment. Combined TBT and forskolin exposure produced respective decrease and increase of mRNA levels of cyp19a and cyp19b, compared with control. TBT decreased ER alpha mRNA at low dose (1mg/kg) and forskolin exposure alone produced a consistent decrease of ER alpha mRNA levels that were not affected by the presence of TBT. Interestingly, PXR and CYP3A mRNA levels were differentially affected, either decreased or increased, after exposure to TBT and forskolin, singly and also in

  3. Peroxisome proliferator-activated receptors, estrogenic responses and biotransformation system in the liver of salmon exposed to tributyltin and second messenger activator

    Energy Technology Data Exchange (ETDEWEB)

    Pavlikova, Nela [Department of Biology, Norwegian University of Science and Technology (NTNU), Hogskoleringen 5, 7491 Trondheim (Norway); RECETOX Research Centre for Environmental Chemistry and Ecotoxicology, Masaryk University, Kamenice 3, CZ62500 Brno (Czech Republic); Kortner, Trond M. [Department of Biology, Norwegian University of Science and Technology (NTNU), Hogskoleringen 5, 7491 Trondheim (Norway); Arukwe, Augustine, E-mail: arukwe@bio.ntnu.no [Department of Biology, Norwegian University of Science and Technology (NTNU), Hogskoleringen 5, 7491 Trondheim (Norway)

    2010-08-15

    The mechanisms by which organotin compounds produce modulations of the endocrine systems and other biological responses are not fully understood. In this study, juvenile salmon were force-fed diet containing TBT (0: solvent control, 0.1, 1 and 10 mg/kg fish) for 72 h. Subsequently, fish exposed to solvent control and 10 mg TBT were exposed to waterborne concentration (200 {mu}g/l) of the adenylate cyclase (AC) stimulator, forskolin for 2 and 4 h. The overall aim of the study was to explore whether TBT endocrine disruptive effects involve second messenger activation. Liver was sampled from individual fish (n = 8) at the end of the exposures. The transcription patterns of peroxisome proliferator-activated receptor (PPAR) isotype and acyl-coenzyme A oxidase 1 (ACOX1), aromatase isoform, estrogen receptor-{alpha} (ER{alpha}), pregnane X receptor (PXR), CYP3A and glutathione S-transferase (GST) genes were measured by quantitative polymerase chain reaction (qPCR). Our data showed a consistent increase in PPAR{alpha}, PPAR{beta} and PPAR{gamma} mRNA and protein expression after TBT exposure that were inversely correlated with ACOX1 mRNA levels. Forskolin produced PPAR isotype-specific mRNA and protein effects that were modulated by TBT. ACOX1 expression was decreased (at 2 h) and increased (at 4 h) by forskolin and the presence of TBT potentiated these effects. TBT apparently increased mRNA and protein levels of cyp19a, compared to the solvent control, whereas cyp19b mRNA levels were unaffected by TBT treatment. Combined TBT and forskolin exposure produced respective decrease and increase of mRNA levels of cyp19a and cyp19b, compared with control. TBT decreased ER{alpha} mRNA at low dose (1 mg/kg) and forskolin exposure alone produced a consistent decrease of ER{alpha} mRNA levels that were not affected by the presence of TBT. Interestingly, PXR and CYP3A mRNA levels were differentially affected, either decreased or increased, after exposure to TBT and forskolin, singly

  4. Expression of key lipid metabolism genes in adipose tissue is not altered by once-daily milking during a feed restriction of grazing dairy cows.

    Science.gov (United States)

    Grala, T M; Roche, J R; Phyn, C V C; Rius, A G; Boyle, R H; Snell, R G; Kay, J K

    2013-01-01

    The objective of this study was to investigate the effect of reduced milking frequency, at 2 feeding levels, on gene expression in adipose tissue of grazing dairy cows during early lactation. Multiparous Holstein-Friesian and Holstein-Friesian × Jersey cows (n=120) were grazed on pasture and milked twice daily (2×) from calving to 34±6d in milk (mean ± standard deviation). Cows were then allocated to 1 of 4 treatments in a 2×2 factorial arrangement. Treatments consisted of 2 milking frequencies (2× or once daily; 1×) and 2 feeding levels for 3 wk: adequately fed (AF), consuming 14.3 kg of dry matter/cow per day, or underfed (UF), consuming 8.3 kg of dry matter/cow per day. After the treatment period, all cows were fed to target grazing residuals ≥1,600 kg of DM/cow per day and milked 2× for 20 wk. Adipose tissue was collected from 12 cows per treatment by subcutaneous biopsy at -1, 3, and 5 wk relative to treatment start, RNA was extracted, and transcript abundance of genes involved in lipid metabolism was quantified using a linear mixed model. At the end of the 3-wk treatment period, transcript abundance of genes involved in fatty acid (FA) uptake into adipose tissue (LPL), FA synthesis [FA synthase (FASN) and stearoyl-coenzyme A desaturase (SCD)], FA oxidation [acyl-coenzyme A synthetase long-chain family member 1 (ACSL1) and carnitine palmitoyltransferase 2 (CPT2)], glyceroneogenesis [glycerol-3-phosphate dehydrogenase 1 (GPD1) and pyruvate carboxylase (PC)], and triacylglyceride synthesis [diacylglycerol O-acyltransferase 2 (DGAT2)] were greater in AF1× cows compared with all other treatments. However, when cows were underfed, no effects of milking frequency were observed on transcript abundance of genes involved in adipose lipid metabolism. Despite increases in plasma NEFA concentrations in UF cows, no effects of underfeeding were observed on the transcription of lipolytic genes. At 5 wk, after cows were returned to 2× milking and standard feed

  5. Autotaxin: A protein with two faces

    Energy Technology Data Exchange (ETDEWEB)

    Tania, Mousumi; Khan, Md. Asaduzzaman; Zhang, Huaiyuan; Li, Jinhua [Department of Biochemistry, School of Biological Science and Technology, Central South University, Changsha, Hunan 410013 (China); Song, Yuanda, E-mail: yuanda_song@hotmail.com [Department of Biochemistry, School of Biological Science and Technology, Central South University, Changsha, Hunan 410013 (China)

    2010-10-29

    Research highlights: {yields} Autotaxin (ATX) has lysophospholipase D activity. {yields} ATX catalyzes the formation of lysophosphatidic acid (LPA). {yields} LPA is a mitogen, and thus is responsible for cancer. {yields} ATX also catalyzes the formation of anti-cancerous cyclic phosphatidic acid. {yields} Autotaxin is a novel target of cancer therapy research. -- Abstract: Autotaxin (ATX) is a catalytic protein, which possesses lysophospholipase D activity, and thus involved in cellular membrane lipid metabolism and remodeling. Primarily, ATX was thought as a culprit protein for cancer, which potently stimulates cancer cell proliferation and tumor cell motility, augments the tumorigenicity and induces angiogenic responses. The product of ATX catalyzed reaction, lysophosphatidic acid (LPA) is a potent mitogen, which facilitates cell proliferation and migration, neurite retraction, platelet aggregation, smooth muscle contraction, actin stress formation and cytokine and chemokine secretion. In addition to LPA formation, later ATX has been found to catalyze the formation of cyclic phosphatidic acid (cPA), which have antitumor role by antimitogenic regulation of cell cycle, inhibition of cancer invasion and metastasis. Furthermore, the very attractive information to the scientists is that the LPA/cPA formation can be altered at different physiological conditions. Thus the dual role of ATX with the scope of product manipulation has made ATX a novel target for cancer treatment.

  6. Tumor-induced inflammation in mammary adipose tissue stimulates a vicious cycle of autotaxin expression and breast cancer progression.

    Science.gov (United States)

    Benesch, Matthew G K; Tang, Xiaoyun; Dewald, Jay; Dong, Wei-Feng; Mackey, John R; Hemmings, Denise G; McMullen, Todd P W; Brindley, David N

    2015-09-01

    Compared to normal tissues, many cancer cells overexpress autotaxin (ATX). This secreted enzyme produces extracellular lysophosphatidate, which signals through 6 GPCRs to drive cancer progression. Our previous work showed that ATX inhibition decreases 4T1 breast tumor growth in BALB/c mice by 60% for about 11 d. However, 4T1 cells do not produce significant ATX. Instead, the ATX is produced by adjacent mammary adipose tissue. We investigated the molecular basis of this interaction in human and mouse breast tumors. Inflammatory mediators secreted by breast cancer cells increased ATX production in adipose tissue. The increased lysophosphatidate signaling further increased inflammatory mediator production in adipose tissue and tumors. Blocking ATX activity in mice bearing 4T1 tumors with 10 mg/kg/d ONO-8430506 (a competitive ATX inhibitor, IC90 = 100 nM; Ono Pharma Co., Ltd., Osaka, Japan) broke this vicious inflammatory cycle by decreasing 20 inflammatory mediators by 1.5-8-fold in cancer-inflamed adipose tissue. There was no significant decrease in inflammatory mediator levels in fat pads that did not bear tumors. ONO-8430506 also decreased plasma TNF-α and G-CSF cytokine levels by >70% and leukocyte infiltration in breast tumors and adjacent adipose tissue by >50%. Hence, blocking tumor-driven inflammation by ATX inhibition is effective in decreasing tumor growth in breast cancers where the cancer cells express negligible ATX.

  7. A dependência pela prática de exercícios físicos e o uso de recursos ergogênicos - DOI: 10.4025/actascihealthsci.v32i1.4475 Physical exercise dependence and the use of ergogenic resources - DOI: 10.4025/actascihealthsci.v32i1.4475

    Directory of Open Access Journals (Sweden)

    Ricardo Aparecido Ferrarezi

    2009-12-01

    Full Text Available Objetivou-se Investigar a ocorrência de dependência por exercícios físicos quanto às características de praticantes de musculação e ginástica em academias, como uso de recursos ergogênicos, sexo e índice de massa corporal. Participaram do estudo 80 sujeitos (27,12 ± 6,60 anos, praticantes de ginástica e/ou musculação em academias, de ambos os sexos. Utilizou-se a Escala de Dependência por Exercícios Físicos, a listagem do tipo de suplemento alimentar utilizado como recurso ergogênico e o Índice de Massa Corporal – IMC (Kg/cm². A análise estatística foi realizada por meio da correlação de Spearman e o teste de Wilcoxon (p The aim of this study is to investigate the occurrence of physical exercise dependence in regards to the characteristics of participants in weight training and exercises at gyms, such as the use of ergogenic resources, gender and body mass index. Eighty subjects (27.12 ± 6.60 years from both genders took part in the study, all of whom practiced gymnastics and/or weight training in gyms. The study utilized the Exercise Dependence Scale, a check list of the kinds of nutritional supplementation used as ergogenic resources, and the Body Mass Index – BMI (Kg cm-². Statistical analysis was performed using Spearman's correlation and the Wilcoxon test (p < 0.05. The results showed that there was no statistically significant difference between physical exercise dependence in men (5.14 ± 1.28 and women (5.60 ± 1.45. The body mass index did not show statistically significant correlation with the scores of dependency, either. However, dependent men showed high prevalence of use of ergogenic resources (63.63%, p = 0.01, while for dependent women there were no statistically significant results. The body mass index does not relate to the scores of exercise dependence. However, even with a normal BMI, the use of ergogenic resources presents high prevalence among dependent men. As a result, there is evidence that physical exercise dependence is a risk factor for the development of emotional disturbances related to exercise, such as muscle dysmorphia and overtraining.

  8. O estresse físico e a dependência de exercício físico El estrés físico y la dependencia de ejercicio físico Physical stress and physical exercise dependence

    Directory of Open Access Journals (Sweden)

    Hanna K.M. Antunes

    2006-10-01

    Full Text Available O objetivo do presente estudo foi verificar os escores referentes à escala de dependência de exercício, qualidade de vida, bem como os escores indicativos de humor em atletas de corrida de aventura (CA. Participaram deste estudo 17 atletas de ambos os gêneros com histórico de prática da modalidade de pelo menos três anos, com experiência em provas nacionais e internacionais e que figuram nas primeiras posições do ranking brasileiro. A média (± desvio-padrão da idade, altura, massa corporal, índice de massa corpórea (IMC e consumo de oxigênio foram: 31,11 ± 6,30 anos; 1,73 ± 0,07cm; 70,75 ± 7,96kg; 23,48 ± 1,48kg/m² e 58,70 ± 6,63ml.min¹.kg¹, respectivamente. Os voluntários responderam aos seguintes questionários: Escala de Dependência de Exercício (EDE, Idate Traço e Estado, Profile of Mood States (POMS, SF-36 Pesquisa em Saúde e Questionário de Padrão Social. Os resultados revelaram que os escores observados na EDE foram indicativos de dependência de exercício; já os questionários de humor revelaram ansiedade moderada, enquanto o POMS não detectou escores indicativos de distúrbios de humor. Quanto à qualidade de vida, a média das oito dimensões referentes ao questionário SF-36 se mostrou acima de 85%, sugerindo que, apesar de haver dependência de exercício, parece que esse fato não foi capaz de promover alterações significativas no estado de humor e na qualidade de vida. Esses dados sugerem que atletas de CA apresentam dependência de exercício não associada aos distúrbios de humor.El objetivo del presente estudio ha sido el de verificar los escores referentes a la escala de dependencia de ejercicio, calidad de vida, así como los escores indicativos de humor en atletas de Carrera de Aventura (CA. Participaron de este estudio 17 atletas de ambos géneros con histórico de práctica de la modalidad de por lo menos tres años, con experiencia en pruebas nacionales e internacionales y que figuran en las primeras posiciones del ranking brasileño. La media (± desvío padrón de edad, altura, masa corporal, índice de masa corpórea (IMC y consumo de oxígeno fueron: 31,11 ± 6,30 años; 1,73 ± 0,07 cm; 70,75 ± 7,96 kg; 23,48 ± 1,48 kg/m² y 58,70 ± 6,63 ml.min-1.kg-1, respectivamente. Los voluntarios respondieron a los siguientes cuestionarios: Escala de Dependencia de Ejercicio (EDE, "Idate Traço" y Estado, Profile of Mood States (POMS, SF-36 Investigación e Salud y Cuestionario de Padrón Social. Los resultados revelaron que los escores observados en la EDE fueron indicativos de dependencia de ejercicio, mientras que los cuestionarios de humor revelaron ansiedad moderada, entretanto el POMS no detectó escores indicativos de disturbios de humor. En relación a la calidad de vida, la media de las 8 dimensiones referentes al cuestionario SF-36 se mostró por encima de 85%, lo que sugiere que a pesar de haber dependencia de ejercicio, parece que ese hecho no fue capaz de promover alteraciones significativas en el estado de humor y en la calidad de vida. Nuestros datos sugieren que atletas de CA presentan dependencia de ejercicio no asociada a los disturbios de humor.The aim of this study was to verify the referring scores of exercise dependence, quality of life as well as the mood indicators in adventure race (AR athletes. 17 athletes of both sexes participated in the study and all had a history of three years in this modality, with national and international experience, and figured in the first positions in the Brazilian ranking. The age, height, weight, body mass index (BMI and oxygen uptake averages ± standard deviations were: 31.11 ± 6.30 years; 1.73 ± 0.07 cm; 70.75 ± 7.96 kg; 23.48 ± 1.48 wt/ht² and 58.70 ± 6.63 ml.min-1.kg-1, respectively. The volunteers were given the following questionnaires: Exercise Dependency Scale (EDE, Idate Trait and State, Profile of Mood States (POMS, SF-36 Health Research and Social Patterns Questionnaire. The results showed that scores in EDE indicated exercise dependence, and the mood questionnaires revealed moderate anxiety, while the POMS did not detect any indicative scores of mood disorders. Concerning the quality of life, the average of 8 dimensions of the SF-36 was higher than 85%, suggesting that although there was exercise dependence, this fact alone did not promote significant alterations in mood and quality of life. Thus, our data suggested that athletes of AR have exercise dependence not associated to mood disorders.

  9. Regulation of insulin-like growth factor I transcription by cyclic adenosine 3',5'-monophosphate (cAMP) in fetal rat bone cells through an element within exon 1: protein kinase A-dependent control without a consensus AMP response element

    Science.gov (United States)

    McCarthy, T. L.; Thomas, M. J.; Centrella, M.; Rotwein, P.

    1995-01-01

    Insulin-like growth factor I (IGF-I) is a locally synthesized anabolic growth factor for bone. IGF-I synthesis by primary fetal rat osteoblasts (Ob) is stimulated by agents that increase the intracellular cAMP concentration, including prostaglandin E2 (PGE2). Previous studies with Ob cultures demonstrated that PGE2 enhanced IGF-I transcription through selective use of IGF-I promoter 1, with little effect on IGF-I messenger RNA half-life. Transient transfection of Ob cultures with an array of promoter 1-luciferase reporter fusion constructs has now allowed localization of a potential cis-acting promoter element(s) responsible for cAMP-stimulated gene expression to the 5'-untranslated region (5'-UTR) of IGF-I exon 1, within a segment lacking a consensus cAMP response element. Our evidence derives from three principal observations: 1) a transfection construct containing only 122 nucleotides (nt) of promoter 1 and 328 nt of the 5'-UTR retained full PGE2-stimulated reporter expression; 2) maximal PGE2-driven reporter expression required the presence of nt 196 to 328 of exon 1 when tested within the context of IGF-I promoter 1; 3) cotransfection of IGF-I promoter-luciferase-reporter constructs with a plasmid encoding the alpha-isoform of the catalytic subunit of murine cAMP-dependent protein kinase (PKA) produced results comparable to those seen with PGE2 treatment, whereas cotransfection with a plasmid encoding a mutant regulatory subunit of PKA that cannot bind cAMP blocked PGE2-induced reporter expression. Deoxyribonuclease I footprinting of the 5'-UTR of exon 1 demonstrated protected sequences at HS3A, HS3B, and HS3D, three of six DNA-protein binding sites previously characterized with rat liver nuclear extracts. Of these three regions, only the HS3D binding site is located within the functionally identified hormonally responsive segment of IGF-I exon 1. These results directly implicate PKA in the control of IGF-I gene transcription by PGE2 and identify a segment of IGF-I exon 1 as being essential for this hormonal regulation.

  10. Optimal Investment Strategy for an Insurer under Mean-Variance in a Dependent Risk Model%保险公司在风险相依模型中均值-方差准则下的最优投资策略*

    Institute of Scientific and Technical Information of China (English)

    谷爱玲; 李仲飞; 申曙光

    2013-01-01

    研究了具有两个业务部门的保险公司的最优投资问题,其中每个业务部门的盈余过程由二维的Lévy过程描述。保险公司可将其盈余投资于金融市场,其中金融市场由一个无风险资产和两个具有风险相关性的风险资产组成,而且风险资产的价格过程由二维的Lévy过程所驱动。文中讨论了两个优化问题。一个是基准问题,即选择适当的投资策略使保险公司的终端财富与一个基准值之差的平方期望最小;另一个是均值-方差(M-V)问题,即在保险公司终端财富给定的情形下,选择适当的投资策略使终端财富的方差最小。利用动态规划的方法,得到第一个优化问题的最优投资策略和最优值函数的解析式。结合第一个优化问题的结果,利用对偶定理得到第二个优化问题的最优投资策略和有效前沿。%Two optimal investment problems for an insurer with two business lines are considered,where each business line's risk process is modeled by two-dimensional Lévy process.It is assumed that the in-surer can invest its surplus in a risk-free asset and two risky assets,where the risky assets'price processes are described by a two-dimensional Lévy process.A benchmark problem and a mean-variance problem are discussed.The first problem is to choose the optimal investment strategy to minimize the expected quadratic distance of the risk reserve to a given benchmark;the second problem is to minimize the vari-ance of the terminal wealth when the expected terminal reserve is given.By employing stochastic dynamic programming approach,the explicit expressions of the optimal investment strategy and the optimal value function are derived for the first problem;with the results of the first problem and the duality theory,the optimal investment strategy and the efficient frontier for the second problem are derived.

  11. ¿La democracia es un sistema dependiente de valores como el conformismo y la obediencia? Un estudio comparado sobre las valoraciones que jóvenes y adultos españoles hacen de la democracia (Is democracy a dependent value such as conformity and obedience? A comparative study on the assestments that young and adult Spanish do of democracy

    Directory of Open Access Journals (Sweden)

    Liberto Carratalá Puertas

    2016-12-01

    Full Text Available Las investigaciones sobre la calidad de la democracia y la valoración que la ciudadanía realiza de ella han girado en torno a la influencia que el sistema económico o el político ejerce sobre las dimensiones que la componen. Los resultados han tenido un alcance explicativo limitado. Para superar este déficit se propone llevar a cabo un análisis de la cuestión desde una perspectiva que articule el nivel macro, la estructura política y económica, y micro, la subjetividad de la ciudadanía. Este trabajo abre una línea de investigación alternativa y complementaria a las ya existentes, con el objetivo de comprender cómo los valores humanos influyen en la valoración de la experiencia democrática de los jóvenes y adultos españoles. Los resultados, derivados del análisis de los datos con la técnica de los modelos de ecuaciones estructurales (SEM, ponen de relieve la diferencia existente entre jóvenes y adultos en sus respectivas valoraciones de la democracia, aunque coinciden en que es insatisfactoria. | The researches on democracy’s quality and the assessment citizens make of it have usually turned around the influence of economic or political systems applied on its dimensions. The results have had a limited explanatory range. In order to get over this shortfall, the proposal is the analysis from a perspective that will articulate macro and micro levels. This work will open an alternative and complementary research line to existing ones with the aim to understand how human values have influence on the assessment of the democratic experience from young and adult Spanish people. The results derived from data analysed with Structural Equation Models (SEM underline the difference between young’s and adult’s values of democracy even though they agree it is unsatisfying.

  12. Forty five years with membrane phospholipids, phospholipases and lipid mediators: A historical perspective.

    Science.gov (United States)

    Chap, Hugues

    2016-06-01

    Phospholipases play a key role in the metabolism of phospholipids and in cell signaling. They are also a very useful tool to explore phospholipid structure and metabolism as well as membrane organization. They are at the center of this review, covering a period starting in 1971 and focused on a number of subjects in which my colleagues and I have been involved. Those include determination of phospholipid asymmetry in the blood platelet membrane, biosynthesis of lysophosphatidic acid, biochemistry of platelet-activating factor, first attempts to define the role of phosphoinositides in cell signaling, and identification of novel digestive (phospho)lipases such as pancreatic lipase-related protein 2 (PLRP2) or phospholipase B. Besides recalling some of our contributions to those various fields, this review makes an appraisal of the impressive and often unexpected evolution of those various aspects of membrane phospholipids and lipid mediators. It is also the occasion to propose some new working hypotheses.

  13. DIFFERENTIAL EXPRESSION OF GENES INVOLVED IN METABOLISM BETWEEN TUMORIGENITIC HUMAN LEUKEMIA CELL LINES K562 AND K562-n

    Institute of Scientific and Technical Information of China (English)

    吕书晴; 许小平; 夏放; 居小萍; 李瑶; 应康; 毛裕民

    2003-01-01

    Objective: To study the molecular mechanism of different tumorigenicity in nude mice of human leukemia cell lines K562-n and K562. Methods: To analyze the genes differently expressed between K562 and K562-n cells by using cDNA microarray technique. Results: Among the 12800 genes detected, some genes involved in material metabolism and material transport were differently expressed between K562-n and K562 cells. These genes include homo sapiens placenta-specific ATP-binding cassette transporter gene, dihydrodiol dehydrogenase gene, hepatic dihydrodiol dehydrogenase gene, NAD-dependent methylene tetrahydrofolate dehydrogenase cyclohydrolase, lysophosphatidic acid acyltransferase, alpha gene, argininosuccinate lyase gene, mitochondrial isocitrtate dehydrogenase, adhesion protein SQM1 gene, dimethylarginine dimethylamino-hydrolase gene, M1 subunit of ribonucleotide reductase and farnesyl pyrophosphate synthetase gene. Conclusion: The high tumorigenicity of K562-n cells is related to the different expression of some genes concerned with cell metabolism and material transpoert.

  14. Dicty_cDB: Contig-U02102-1 [Dicty_cDB

    Lifescience Database Archive (English)

    Full Text Available subobscura Acph-1 gene ... 39 0.28 (A6H730) RecName: Full=Prostatic acid phosphatase; EC=3... 39 0.37 AY1344...ila virilis Acph gene for a... 38 0.63 NRL( 1RPA ) Prostatic acid phosphatase (EC 3.1.3.2) complexed with......295_53( FN357295 |pid:none) Schistosoma mansoni genome sequen... 35 5.3 (Q8CE08) RecName: Full=Prostatic aci...strain GB-M1 of... 36 3.1 (Q5R8C0) RecName: Full=Lysophosphatidic acid phosphatase type 6;... 36 3.1 ( P20646 ) RecName: Full=Prostat...ic acid phosphatase; EC=3... 35 4.1 BC073113_1( BC073113 |pid:none) Xenopus laevis

  15. Inhibitory effect of isothiocyanate derivant targeting AGPS by computer-aid drug design on proliferation of glioma and hepatic carcinoma cells.

    Science.gov (United States)

    Zhu, Yu; Li, Wen-Ming; Zhang, Ling; Xue, Jing; Zhao, Meng; Yang, Ping

    2015-01-01

    Lipids metabolism was involved in the process of many types of tumor and alkylglycerone phosphate synthase (AGPS) was considered implicated in tumor process. Benzyl isothiocyanate (BITC) showed the inhibitory effect of tumor and AGPS activity, therefore, we screened a group of small molecular compound based on BITC by computer-aid design targeting AGPS and the results showed that the derivants could suppress the proliferation, the expression of tumor related genes such as survivin and Bcl-2, and the level of ether lipids such as lysophosphatidic acid ether (LPAe) and platelet activating factor ether (PAFe); however, the activity of caspase-3/8 was improved in glioma U87MG and hepatic carcinoma HepG2 cells in vitro.

  16. LSD1 controls metastasis of androgen-independent prostate cancer cells through PXN and LPAR6.

    Science.gov (United States)

    Ketscher, A; Jilg, C A; Willmann, D; Hummel, B; Imhof, A; Rüsseler, V; Hölz, S; Metzger, E; Müller, J M; Schüle, R

    2014-10-06

    Lysine-specific demethylase 1 (LSD1) was shown to control gene expression and cell proliferation of androgen-dependent prostate cancer (PCa) cells, whereas the role of LSD1 in androgen-independent metastatic prostate cancer remains elusive. Here, we show that depletion of LSD1 leads to increased migration and invasion of androgen-independent PCa cells. Transcriptome and cistrome analyses reveal that LSD1 regulates expression of lysophosphatidic acid receptor 6 (LPAR6) and cytoskeletal genes including the focal adhesion adaptor protein paxillin (PXN). Enhanced LPAR6 signalling upon LSD1 depletion promotes migration with concomitant phosphorylation of PXN. In mice LPAR6 overexpression enhances, whereas knockdown of LPAR6 abolishes metastasis of androgen-independent PCa cells. Taken together, we uncover a novel mechanism of how LSD1 controls metastasis and identify LPAR6 as a promising therapeutic target to treat metastatic prostate cancer.

  17. Lysoglycerophospholipids in chronic inflammatory disorders: the PLA(2)/LPC and ATX/LPA axes.

    Science.gov (United States)

    Sevastou, Ioanna; Kaffe, Eleanna; Mouratis, Marios-Angelos; Aidinis, Vassilis

    2013-01-01

    Lysophosphatidylcholine (LPC) and lysophosphatidic acid (LPA), the most prominent lysoglycerophospholipids, are emerging as a novel class of inflammatory lipids, joining thromboxanes, leukotrienes and prostaglandins with which they share metabolic pathways and regulatory mechanisms. Enzymes that participate in LPC and LPA metabolism, such as the phospholipase A(2) superfamily (PLA(2)) and autotaxin (ATX, ENPP2), play central roles in regulating LPC and LPA levels and consequently their actions. LPC/LPA biosynthetic pathways will be briefly presented and LPC/LPA signaling properties and their possible functions in the regulation of the immune system and chronic inflammation will be reviewed. Furthermore, implications of exacerbated LPC and/or LPA signaling in the context of chronic inflammatory diseases, namely rheumatoid arthritis, multiple sclerosis, pulmonary fibrosis and hepatitis, will be discussed. This article is part of a Special Issue entitled Advances in Lysophospholipid Research.

  18. New insights into the autotaxin/LPA axis in cancer development and metastasis

    Energy Technology Data Exchange (ETDEWEB)

    Leblanc, Raphaël; Peyruchaud, Olivier, E-mail: olivier.peyruchaud@inserm.fr

    2015-05-01

    Lysophosphatidic acid (LPA) is a simple lipid with a single fatty acyl chain linked to a glycerophosphate backbone. Despite the simplicity of its structure but owing to its interactions with a series of at least six G protein-coupled receptors (LPA{sub 1–6}), LPA exerts pleiotropic bioactivities including stimulation of proliferation, migration and survival of many cell types. Autotaxin (ATX) is a unique enzyme with a lysophospholipase D (lysoPLD) activity that is responsible for the levels of LPA in the blood circulation. Both LPA receptor family members and ATX/LysoPLD are aberrantly expressed in many human cancers. This review will present the more striking as well as novel experimental evidences using cell lines, cancer mouse models and transgenic animals identifying the roles for ATX and LPA receptors in cancer progression, tumor cell invasion and metastasis.

  19. Antiphospholipid Antibody and Antiphospholipid Syndrome

    Institute of Scientific and Technical Information of China (English)

    吴竞生

    2008-01-01

    @@ Antiphospholipid antibodies (APA) APA is a big category for all kinds of negative charge phospholipid or lecithin - a protein complex autoantibodies or the same antibody, through its recognition of antigen (target protein) different, and phospholipids or lecithin - protein complex combination of various rely on the interference Phospholipid clotting and anti-coagulation factor, and promote endothelial cells, platelets, complement activation and play a role. APA including lupus anticoagulant(LA) and anticardiolipin antibody (ACA), In addition, there are anti-β2 glycoprotein-I (β2-GPI) antibody, anti-prothrombin (a- PT) antibody, anti-lysophosphatidic acid antibody and anti-phosphatidylserine antibody, and so on. APA as the main target of phospholipid-binding protein, including β2-GPI, prothrombin, annexin, protein C (PC) and protein S (PS), plasminogen, and so on.

  20. Non-cell autonomous and non-catalytic activities of ATX in the developing brain

    Directory of Open Access Journals (Sweden)

    Raanan eGreenman

    2015-03-01

    Full Text Available The intricate formation of the cerebral cortex requires a well-coordinated series of events, which are regulated at the level of cell-autonomous and non-cell autonomous mechanisms. Whereas cell-autonomous mechanisms that regulate cortical development are well-studied, the non cell-autonomous mechanisms remain poorly understood. A non-biased screen allowed us to identify Autotaxin (ATX as a non cell-autonomous regulator of neural stem cell proliferation. ATX (also known as ENPP2 is best known to catalyze lysophosphatidic acid (LPA production. Our results demonstrate that ATX affects the localization and adhesion of neuronal progenitors in a cell autonomous and non-cell autonomous manner, and strikingly, this activity is independent from its catalytic activity in producing LPA.

  1. Structure-Driven Pharmacology of Transient Receptor Potential Channel Vanilloid 1.

    Science.gov (United States)

    Díaz-Franulic, Ignacio; Caceres-Molina, Javier; Sepulveda, Romina V; Gonzalez-Nilo, Fernando; Latorre, Ramon

    2016-09-01

    The transient receptor potential vanilloid 1 (TRPV1) ion channel is a polymodal receptor that mediates the flux of cations across the membrane in response to several stimuli, including heat, voltage, and ligands. The best known agonist of TRPV1 channels is capsaicin, the pungent component of "hot" chili peppers. In addition, peptides found in the venom of poisonous animals, along with the lipids phosphatidylinositol 4,5-biphosphate, lysophosphatidic acid, and cholesterol, bind to TRPV1 with high affinity to modulate channel gating. Here, we discuss the functional evidence regarding ligand-dependent activation of TRPV1 channels in light of structural data recently obtained by cryoelectron microscopy. This review focuses on the mechanistic insights into ligand binding and allosteric gating of TRPV1 channels and the relevance of accurate polymodal receptor biophysical characterization for drug design in novel pain therapies.

  2. Migratory properties of cultured olfactory ensheathing cells by single-cell migration assay

    Institute of Scientific and Technical Information of China (English)

    Zhi-hui Huang; Ying Wang; Li Cao; Zhi-da Su; Yan-ling Zhu; Yi-zhang Chen; Xiao-bing Yuan; Cheng He

    2008-01-01

    Olfactory ensheathing cells (OECs) are a unique type of glial cells that have axonal growth-promoting properties. OEC transplantation has emerged as a promising experimental therapy of axonal injuries and demyelinating diseases. However, some fundamental cellular properties of OECs remain unclear. In this study, we found that the distinct OEC subpopulations exhibited different migratory properties based on time-lapse imaging of single isolated cells, possibly due to their different cytoskeletal organizations. Moreover, OEC subpopulations displayed different attractive migratory responses to a gradient of lysophosphatidic acid (LPA) in single-cell migration assays. Finally, we found that OEC subpopulations transformed into each other spontaneously. Together, these results demonstrate, for the first time to our knowledge, that distinct OEC subpopulations display different migratory properties in vitro and provide new evidence to support the notion of OECs as a single cell type with malleable functional phenotypes.

  3. The Peanut (Arachis hypogaea L. Gene AhLPAT2 Increases the Lipid Content of Transgenic Arabidopsis Seeds.

    Directory of Open Access Journals (Sweden)

    Silong Chen

    Full Text Available Lysophosphatidic acid acyltransferase (LPAT, which converts lysophosphatidic acid (LPA to phosphatidic acid (PA, catalyzes the addition of fatty acyl moieties to the sn-2 position of the LPA glycerol backbone in triacylglycerol (TAG biosynthesis. We recently reported the cloning and temporal-spatial expression of a peanut (Arachis hypogaea AhLPAT2gene, showing that an increase in AhLPAT2 transcript levels was closely correlated with an increase in seed oil levels. However, the function of the enzyme encoded by the AhLPAT2 gene remains unclear. Here, we report that AhLPAT2 transcript levels were consistently higher in the seeds of a high-oil cultivar than in those of a low-oil cultivar across different seed developmental stages. Seed-specific overexpression of AhLPAT2 in Arabidopsis results in a higher percentage of oil in the seeds and greater-than-average seed weight in the transgenic plants compared with the wild-type plants, leading to a significant increase in total oil yield per plant. The total fatty acid (FA content and the proportion of unsaturated FAs also increased. In the developing siliques of AhLPAT2-overexpressing plants, the expression levels of genes encoding crucial enzymes involved in de novo FA synthesis, acetyl-CoA subunit (AtBCCP2 and acyl carrier protein 1 (AtACP1 were elevated. AhLPAT2 overexpression also promoted the expression of several key genes related to TAG assembly, sucrose metabolism, and glycolysis. These results demonstrate that the expression of AhLPAT2 plays an important role in glycerolipid production in peanuts.

  4. Effect and Mechanism of Different Doses of Dehydroepiandrosterone on Lipid Metabolism in SD Rats%脱氢表雄酮对大鼠脂肪代谢的影响及机理

    Institute of Scientific and Technical Information of China (English)

    陈迪; 康健; 马海田

    2015-01-01

    目的:以雄性Sprague-Dawley (SD)大鼠为对象,探讨日粮中添加不同剂量的脱氢表雄酮(dehydroepiandrosterone,DHEA)对大鼠脂肪代谢常规生化指标及相关基因表达的影响.方法:将60只雄性SD大鼠随机分为对照组,DHEA低、中、高剂量组,各处理组大鼠分别灌胃0、25、50、100 mg/(kg·d)的DHEA(以体质量计,下同),连续灌胃8周,测定大鼠血脂、肝脂水平及肝脏脂肪代谢相关基因表达量.结果:DHEA中剂量组大鼠体质量显著低于对照组(P<0.05).中剂量的DHEA可极显著降低大鼠血清中甘油三酯和血糖含量(P<0.01),低、中剂量的DHEA可显著升高大鼠血清高密度脂蛋白胆固醇含量(P<0.05).脂肪代谢合成相关基因分析结果表明,低、高剂量的DHEA可显著或极显著降低大鼠肝脏组织中脂肪酸合成酶(fatty acid synthase,FAS) mRNA表达水平(P<0.05或P<0.01);与对照组相比,不同剂量的DHEA均可显著或极显著降低大鼠肝脏组织中固醇调节元件结合蛋白-1(sterol regulatory element binding protein-1,SREBP-1)mRNA和肝细胞核因子-4(hepatocyte nuclear factor-4,HNF-4)mRNA表达水平(P<0.05或P<0.01),但对乙酰辅酶A羧化酶(acetyl coenzyme A carboxylase,ACC) mRNA表达水平均无显著影响(P>0.05).脂肪代谢分解相关基因分析结果表明,不同剂量DHEA均可显著或极显著降低大鼠肝脏组织中酰基辅酶A氧化酶(acyl coenzyme A oxidase,ACO) mRNA表达水平(P<0.05或P<0.01);与对照组相比,低、高剂量的DHEA可显著或极显著降低大鼠肝脏组织中肉毒碱棕榈酰转移酶-1(carnitine palmitoyl transterase-1,LCPT-1)mRNA和过氧化物酶体增殖物激活受体α(peroxisome proliferator activated receptor-α,PPARα)mRNA表达水平(P<0.05或P<0.01);中、高剂量的DHEA可显著或极显著降低大鼠肝脏组织中脂肪三酰甘油脂肪酶(adipose triglyceride lipase,ATGL) mRNA表达水平(P<0.05或P<0.01).结论:长期灌胃DHEA

  5. Effects of berberine on expression of hepatic peroxisome proliferator-activated receptors and its target genes in type 2 diabetic Chinese hamsters%2型糖尿病中国地鼠模型构建与小檗碱对肝脏过氧化物酶体增殖体激活受体及其靶基因表达的影响

    Institute of Scientific and Technical Information of China (English)

    刘栩晗; 李国生; 黄澜; 朱华; 刘亚莉; 马春梅

    2011-01-01

    BACKGROUND: Although Berberine has been reported to treat type 2 diabetes, the underlying mechanisms of berberine on insulin resistance of type 2 diabetes, especially hepatic insulin resistance, remains not fully understood.OBJECTIVE: To study the effects of berberine on the expression of hepatic peroxisome proliferator-activated receptors (PPARs) and their target genes in type 2 diabetic Chinese hamsters.METHODS: The insulin-resistant and type 2 diabetic Chinese hamster models were induced by high-fat diet without or with low-dose streptozotocin. After the induction of models, the hamsters were randomly divided into normal control (standard food),insulin-resistant (high-fat diet), diabetic (high-fat diet and streptozotocin) and berberine-treated diabetic (high-fat diet and streptozotocin and berberine) groups. All groups were treated for 9 weeks.RESULTS AND CONCLUSION: Results of real-time quantitative PCR indicated that compared with normal control group, the expression of PPARα, PPARβ/δ, acyl-Coenzyme A oxidase (Acox), carnitine palmitoyltransferase 1 (Cpt1) and acetyl-Coenzyme A dehydrogenase, medium chain (Acadm) was decreased (P < 0.05) and the expression of sterol regulatory element binding factor 1 (SREBP1c), sterol regulatory element binding factor 2 (SREBP2), PPARγ, lipoprotein lipase (LPL), CD36/FA transporter (FAT/CD36) and adipocyte fatty acid-binding protein (ap2) was increased (P < 0.05) in the fatty liver of insulin-resistant and diabetic hamster groups. Berberine effectively improved insulin resistance, reversed the altered expression of PPARs and its target genes in diabetic hamsters. PPARs and its target genes involved in the therapeutic molecular mechanisms of berberine on fat-induced hepatic insulin resistance in type 2 diabetic hamsters.%背景:研究表明小檗碱可用于治疗2型糖尿病,但小檗碱治疗糖尿病胰岛素抵抗尤其是肝脏脂诱性胰岛素抵抗的分子机制仍不明确.目的:观察小檗碱对2型糖

  6. Structure and function of sterol carrier proteins in insects%昆虫固醇转运蛋白的结构与功能

    Institute of Scientific and Technical Information of China (English)

    张丽丽; 郭兴荣; 冯启理; 郑思春

    2011-01-01

    , cholesterol derivatives, fatty acids,acyl-coenzyme A and phospholipids. Over-expression of SlSCP-x and SlSCP-2 genes can increase the uptake of cholesterol into cells and RNAi inhibits the expression of SlSCP-x and SlSCP-2 genes in S. litura larvae,resulting in a decrease in cholesterol level in the hemolymph and a delay in larval growth and pupation.%在昆虫中,胆固醇不仅是细胞膜的重要成分之一,也是昆虫蜕皮激素生物合成的前体.由于昆虫体内缺少两种合成胆固醇所必需的关键性酶,所以昆虫不能自主地从简单的前体化合物从头合成胆同醇,而必须通过吸收食物中的甾醇转化为胆固醇来满足生长、发育和繁殖的需要.胆固醇在组织和细胞内的运输主要由固醇转运蛋白(sterol carrier proteins,SCPs)执行.因此,对同醇转运蛋白结构与功能的研究对于阐明昆虫中固醇运输具有重要的意义.本文对同醇转运蛋白的基因和蛋白结构、细胞内表达和定位、翻译后修饰、蛋白三维结构、底物特异性和可能的运输途径等方面的研究进展进行了综述,并对其作为害虫防治分子靶标的可能性进行了初步的讨论.研究发现,不同物种的SCP蛋白的基因编码形式和蛋白剪切形式不同;双翅目昆虫埃及伊蚊Aedes aegypti和黑腹果蝇Drosophila melanogaster除了SCP-x基因可编码SCP-x和SCP-2蛋白外,还有另外的SCP-2和类SCP-2(SCP-2L)基因编码SCP-2和类SCP-2蛋白;而鳞翅目昆虫棉贪夜蛾Spodoptera littoralis、斜纹夜蛾Spodoptera litura和家蚕Bombyx mori中SCP-x 基因的表达和转录方式与脊椎动物的SCP-x基因类似,通过转录和翻译后剪切形成SCP-2蛋白.SCP-x和SCP-2蛋白定位于过氧化物酶体.SCP-2蛋白由5个α-螺旋和5个β-折叠组成,其中α5-螺旋可影响蛋白与底物的结合.SCP-2蛋白以不同的亲和力与固醇、胆同醇衍生物、脂肪酸、脂酰辅酶A和磷脂等化合物结合.超表达斜纹夜蛾SlSCP-x和SlSCP-2

  7. Amyloid fibril formation of peptides derived from the C-terminus of CETP modulated by lipids

    Energy Technology Data Exchange (ETDEWEB)

    García-González, Victor [Instituto de Fisiología Celular, Universidad Nacional Autónoma de México, 04510 México, DF (Mexico); Mas-Oliva, Jaime, E-mail: jmas@ifc.unam.mx [Instituto de Fisiología Celular, Universidad Nacional Autónoma de México, 04510 México, DF (Mexico); División de Investigación, Facultad de Medicina, Universidad Nacional Autónoma de México, 04510 México, DF (Mexico)

    2013-04-26

    Highlights: •The secondary structure of a C-terminal peptide derived from CETP was studied. •Lipids modulate secondary structure changes of a C-terminal peptide derived from CETP. •Lysophosphatidic acid maintains a functional α-helix and prevents fibril formation. •Transfer of lipids by CETP is related to the presence of an α-helix at its C-end. -- Abstract: Cholesteryl-ester transfer protein (CETP) is a plasmatic protein involved in neutral lipid transfer between lipoproteins. Focusing on the last 12 C-terminus residues we have previously shown that mutation D{sub 470}N promotes a conformational change towards a β-secondary structure. In turn, this modification leads to the formation of oligomers and fibrillar structures, which cause cytotoxic effects similar to the ones provoked by amyloid peptides. In this study, we evaluated the role of specific lipid arrangements on the structure of peptide helix-Z (D{sub 470}N) through the use of thioflavin T fluorescence, peptide bond absorbance, circular dichroism and electron microscopy. The results indicate that the use of micelles formed with lysophosphatidylcholine and lysophosphatidic acid (LPA) under neutral pH induce a conformational transition of peptide helix-Z containing a β-sheet conformation to a native α-helix structure, therefore avoiding the formation of amyloid fibrils. In contrast, incubation with phosphatidic acid does not change the profile for the β-sheet conformation. When the electrostatic charge at the surface of micelles or vesicles is regulated through the use of lipids such as phospholipid and LPA, minimal changes and the presence of β-structures were recorded. Mixtures with a positive net charge diminished the percentage of β-structure and the amount of amyloid fibrils. Our results suggest that the degree of solvation determined by the presence of a free hydroxyl group on lipids such as LPA is a key condition that can modulate the secondary structure and the consequent formation of

  8. Anatomical location of LPA1 activation and LPA phospholipid precursors in rodent and human brain.

    Science.gov (United States)

    González de San Román, Estibaliz; Manuel, Iván; Giralt, María Teresa; Chun, Jerold; Estivill-Torrús, Guillermo; Rodríguez de Fonseca, Fernando; Santín, Luis Javier; Ferrer, Isidro; Rodríguez-Puertas, Rafael

    2015-08-01

    Lysophosphatidic acid (LPA) is a signaling molecule that binds to six known G protein-coupled receptors: LPA1 -LPA6 . LPA evokes several responses in the CNS, including cortical development and folding, growth of the axonal cone and its retraction process. Those cell processes involve survival, migration, adhesion proliferation, differentiation, and myelination. The anatomical localization of LPA1 is incompletely understood, particularly with regard to LPA binding. Therefore, we have used functional [(35) S]GTPγS autoradiography to verify the anatomical distribution of LPA1 binding sites in adult rodent and human brain. The greatest activity was observed in myelinated areas of the white matter such as corpus callosum, internal capsule and cerebellum. MaLPA1 -null mice (a variant of LPA1 -null) lack [(35) S]GTPγS basal binding in white matter areas, where the LPA1 receptor is expressed at high levels, suggesting a relevant role of the activity of this receptor in the most myelinated brain areas. In addition, phospholipid precursors of LPA were localized by MALDI-IMS in both rodent and human brain slices identifying numerous species of phosphatides and phosphatidylcholines. Both phosphatides and phosphatidylcholines species represent potential LPA precursors. The anatomical distribution of these precursors in rodent and human brain may indicate a metabolic relationship between LPA and LPA1 receptors. Lysophosphatidic acid (LPA) is a signaling molecule that binds to six known G protein-coupled receptors (GPCR), LPA1 to LPA6 . LPA evokes several responses in the central nervous system (CNS), including cortical development and folding, growth of the axonal cone and its retraction process. We used functional [(35) S]GTPγS autoradiography to verify the anatomical distribution of LPA1 -binding sites in adult rodent and human brain. The distribution of LPA1 receptors in rat, mouse and human brains show the highest activity in white matter myelinated areas. The basal and

  9. Comparison of glycerolipid biosynthesis in non-green plastids from sycamore (Acer pseudoplatanus) cells and cauliflower (Brassica oleracea) buds.

    Science.gov (United States)

    Alban, C; Joyard, J; Douce, R

    1989-05-01

    The availability of methods to fractionate non-green plastids and to prepare their limiting envelope membranes [Alban, Joyard & Douce (1988) Plant Physiol. 88, 709-717] allowed a detailed analysis of the biosynthesis of lysophosphatidic acid, phosphatidic acid, diacylglycerol and monogalactosyl-diacylglycerol (MGDG) in two different types of non-green starch-containing plastids: plastids isolated from cauliflower buds and amyloplasts isolated from sycamore cells. An enzyme [acyl-ACP (acyl carrier protein):sn-glycerol 3-phosphate acyltransferase) recovered in the soluble fraction of non-green plastids transfers oleic acid from oleoyl-ACP to the sn-1 position of sn-glycerol 3-phosphate to form lysophosphatidic acid. Then a membrane-bound enzyme (acyl-ACP:monoacyl-sn-glycerol 3-phosphate acyltransferase), localized in the envelope membrane, catalyses the acylation of the available sn-2 position of 1-oleoyl-sn-glycerol 3-phosphate by palmitic acid from palmitoyl-ACP. Therefore both the soluble phase and the envelope membranes are necessary for acylation of sn-glycerol 3-phosphate. The major difference between cauliflower (Brassica oleracea) and sycamore (Acer pseudoplatanus) membranes is the very low level of phosphatidate phosphatase activity in sycamore envelope membrane. Therefore, very little diacylglycerol is available for MGDG synthesis in sycamore, compared with cauliflower. These findings are consistent with the similarities and differences described in lipid metabolism of mature chloroplasts from 'C18:3' and 'C16:3' plants (those with MGDG containing C18:3 and C16:3 fatty acids). Sycamore contains only C18 fatty acids in MGDG, and the envelope membranes from sycamore amyloplasts have a low phosphatidate phosphatase activity and therefore the enzymes of the Kornberg-Pricer pathway have a low efficiency of incorporation of sn-glycerol 3-phosphate into MGDG. By contrast, cauliflower contains MGDG with C16:3 fatty acid, and the incorporation of sn-glycerol 3

  10. Noise-induced cochlear F-actin depolymerization is mediated via ROCK2/p-ERM signaling.

    Science.gov (United States)

    Han, Yu; Wang, Xianren; Chen, Jun; Sha, Su-Hua

    2015-06-01

    Our previous work has suggested that traumatic noise activates Rho-GTPase pathways in cochlear outer hair cells (OHCs), resulting in cell death and noise-induced hearing loss (NIHL). In this study, we investigated Rho effectors, Rho-associated kinases (ROCKs), and the targets of ROCKs, the ezrin-radixin-moesin (ERM) proteins, in the regulation of the cochlear actin cytoskeleton using adult CBA/J mice under conditions of noise-induced temporary threshold shift (TTS) and permanent threshold shift (PTS) hearing loss, which result in changes to the F/G-actin ratio. The levels of cochlear ROCK2 and p-ERM decreased 1 h after either TTS- or PTS-noise exposure. In contrast, ROCK2 and p-ERM in OHCs decreased only after PTS-, not after TTS-noise exposure. Treatment with lysophosphatidic acid, an activator of the Rho pathway, resulted in significant reversal of the F/G-actin ratio changes caused by noise exposure and attenuated OHC death and NIHL. Conversely, the down-regulation of ROCK2 by pretreatment with ROCK2 siRNA reduced the expression of ROCK2 and p-ERM in OHCs, exacerbated TTS to PTS, and worsened OHC loss. Additionally, pretreatment with siRNA against radixin, an ERM protein, aggravated TTS to PTS. Our results indicate that a ROCK2-mediated ERM-phosphorylation signaling cascade modulates noise-induced hair cell loss and NIHL by targeting the cytoskeleton. We propose the following cascade following noise trauma leading to alteration of the F-actin arrangement in the outer hair cell cytoskeleton: Noise exposure reduces the levels of GTP-RhoA and subsequently diminishes levels of RhoA effector ROCK2 (Rho-associated kinase 2). Phosphorylation of ezrin-radixin-moesin (ERM) by ROCK2 normally allows ERM to cross-link actin filaments with the plasma membrane. Noise-decreased levels of ROCK results in reduction of phosphorylation of ERM that leads to depolymerization of actin filaments. Lysophosphatidic acid (LPA), an agonist of RhoA, binds to the G-protein-coupled receptor

  11. YAP Induces Human Naive Pluripotency

    Directory of Open Access Journals (Sweden)

    Han Qin

    2016-03-01

    Full Text Available The human naive pluripotent stem cell (PSC state, corresponding to a pre-implantation stage of development, has been difficult to capture and sustain in vitro. We report that the Hippo pathway effector YAP is nuclearly localized in the inner cell mass of human blastocysts. Overexpression of YAP in human embryonic stem cells (ESCs and induced PSCs (iPSCs promotes the generation of naive PSCs. Lysophosphatidic acid (LPA can partially substitute for YAP to generate transgene-free human naive PSCs. YAP- or LPA-induced naive PSCs have a rapid clonal growth rate, a normal karyotype, the ability to form teratomas, transcriptional similarities to human pre-implantation embryos, reduced heterochromatin levels, and other hallmarks of the naive state. YAP/LPA act in part by suppressing differentiation-inducing effects of GSK3 inhibition. CRISPR/Cas9-generated YAP−/− cells have an impaired ability to form colonies in naive but not primed conditions. These results uncover an unexpected role for YAP in the human naive state, with implications for early human embryology.

  12. HIV-1 Tat Inhibits Autotaxin Lysophospholipase D Activity and Modulates Oligodendrocyte Differentiation

    Science.gov (United States)

    Wheeler, Natalie A.; Fuss, Babette; Knapp, Pamela E.

    2016-01-01

    White matter injury has been frequently reported in HIV+ patients. Previous studies showed that HIV-1 Tat (transactivator of transcription), a viral protein that is produced and secreted by HIV-infected cells, is toxic to young, immature oligodendrocytes (OLGs). Adding Tat to the culture medium reduced the viability of immature OLGs, and the surviving OLGs exhibited reduced process networks. OLGs produce and secrete autotaxin (ATX), an ecto-enzyme containing a lysophospholipase D (lysoPLD) activity that converts lysophosphatidylcholine (LPC) to lysophosphatidic acid (LPA), a lipid signaling molecule that stimulates OLG differentiation. We hypothesized that Tat affects OLG development by interfering with the ATX-LPA signaling pathway. Our data show that Tat treatment leads to changes in the expression of OLG differentiation genes and the area of OLG process networks, both of which can be rescued by LPA. Tat-treated OLGs showed no change in LPA receptor expression but significantly decreased extracellular ATX levels and lysoPLD activity. In Tat transgenic mice, expression of Tat in vivo leads to decreased OLG ATX secretion. Furthermore, co-immunoprecipitation experiments revealed a potential physical interaction between Tat and ATX. Together, these data strongly suggest two functional implications of Tat blocking ATX’s lysoPLD activity. On one hand, it attenuates OLG differentiation, and on the other hand it interferes with the protective effects of LPA on OLG process morphology. PMID:27659560

  13. Requirement of Osteopontin in the migration and protection against Taxol-induced apoptosis via the ATX-LPA axis in SGC7901 cells

    Directory of Open Access Journals (Sweden)

    Huang Zuhu

    2011-03-01

    Full Text Available Abstract Background Autotaxin (ATX possesses lysophospholipase D (lyso PLD activity, which converts lysophosphatidylcholine (LPC into lysophosphatidic acid (LPA. The ATX-LPA signaling axis has been implicated in angiogenesis, chronic inflammation and tumor progression. Osteopontin (OPN is an important chemokine involved in the survival, proliferation, migration, invasion and metastasis of gastric cancer cells. The focus of the present study was to investigate the relationship between the ATX-LPA axis and OPN. Results In comparison with non-treated cells, we found that the ATX-LPA axis up-regulated OPN expression by 1.92-fold in protein levels and 1.3-fold in mRNA levels. The ATX-LPA axis activates LPA2, Akt, ERK and ELK-1 and also protects SGC7901 cells from apoptosis induced by Taxol treatment. Conclusions This study provides the first evidence that expression of OPN induced by ATX-LPA axis is mediated by the activation of Akt and MAPK/ERK pathways through the LPA2 receptor. In addition, OPN is required for the protective effects of ATX-LPA against Taxol-induced apoptosis and ATX-LPA-induced migration of SGC7901 cells.

  14. Ikaros can enhance immune activity though the interaction with Autotaxin in LDIR exposed immune cells

    Energy Technology Data Exchange (ETDEWEB)

    Kim, Sung Jin; Kim, Min Young; Kim, Ji Young; Kim, Hee Sun; KIm, Cha Soon; Nam, Seon Young; Yang, Kwang Hee; Jin, Young Woo [Radiation Health Research Institute, Korea Hydro and Nuclear Power Co., Ltd, Seoul (Korea, Republic of)

    2009-04-15

    Ikaros, one of transcription factors, plays major roles in the differentiation and biology of leukocytes, including all classes of lymphocytes (NK, T, and B cells), monocytes/macrophages, and dendritic cells. Ikaros was also shown to regulate early neutrophils differentiation. Therefore, Ikaros appears to be a major determinant in the development and function of immune system. Autotaxin (ATX), which is also called nucleotide pyrophosphatase/phosphodiesterase 2 (NPP2), is an exo-enzyme originally identified as a tumor cell autocrine motility factor. ATX functions as a lysophospholipase D, converting lysophosphatidylcholine (LPC) into the lipid mediator lysophosphatidic acid (LPA). LPA bind together with specific G protein-coupled receptors, which elicit a wide range of cellular responses including the cell proliferation, migration and neurite remodeling. In the Recent report, ATX stimulate human endothelial cells (HUVECs) growth and cytokine production. In our previous study, we showed that low-dose ionizing radiation (LDIR) enhanced the cell proliferation cell coupled with Ikaros phosphorylation. In addition, we found that LDIR increased the expression level of cyclin E and cdk2 protein in IM-9 B lymphoblast cells. In this report, therefore, we try to find Ikaros binding proteins after LDIR in IM-9 lymphoblastic cell lines to examine whether the effects of LDIR induced cell proliferation are one of immune activation responses or not.

  15. Stimulation of human red blood cells leads to Ca2+-mediated intercellular adhesion

    CERN Document Server

    Steffen, Patrick; Nguyen, Duc Bach; Müller, Torsten; Bernhardt, Ingolf; Kaestner, Lars; Wagner, Christian

    2011-01-01

    Red blood cells (RBCs) are a major component of blood clots, which form physiologically as a response to injury or pathologically in thrombosis. The active participation of RBCs in thrombus solidification has been previously proposed but not yet experimentally proven. Holographic optical tweezers and single-cell force spectroscopy were used to study potential cell-cell adhesion between RBCs. Irreversible intercellular adhesion of RBCs could be induced by stimulation with lysophosphatidic acid (LPA), a compound known to be released by activated platelets. We identified Ca2+ as an essential player in the signaling cascade by directly inducing Ca2+ influx using A23187. Elevation of the internal Ca2+ concentration leads to an intercellular adhesion of RBCs similar to that induced by LPA stimulation. Using single-cell force spectroscopy, the adhesion of the RBCs was identified to be approximately 100 pN, a value large enough to be of significance inside a blood clot or in pathological situations like the vasco-occ...

  16. Anatomical Location of LPA1 Activation and LPA Phospholipid Precursors in Rodent and Human Brain

    Science.gov (United States)

    González de San Román, E; Manuel, I; Giralt, MT; Chun, J; Estivill-Torrús, G; Rodriguez de Fonseca, F; Santín, LJ; Ferrer, I; Rodriguez-Puertas, R

    2016-01-01

    Lysophosphatidic acid (LPA) is a signaling molecule that binds to six known G protein-coupled receptors (GPCRs): LPA1–LPA6. LPA evokes several responses in the CNS including cortical development and folding, growth of the axonal cone and its retraction process. Those cell processes involve survival, migration, adhesion proliferation, differentiation and myelination. The anatomical localization of LPA1 is incompletely understood, particularly with regard to LPA binding. Therefore, we have used functional [35S]GTPγS autoradiography to verify the anatomical distribution of LPA1 binding sites in adult rodent and human brain. The greatest activity was observed in myelinated areas of the white matter such as corpus callosum, internal capsule and cerebellum. MaLPA1-null mice (a variant of LPA1-null) lack [35S]GTPγS basal binding in white matter areas, where the LPA1 receptor is expressed at high levels, suggesting a relevant role of the activity of this receptor in the most myelinated brain areas. In addition, phospholipid precursors of LPA were localized by MALDI-IMS in both rodent and human brain slices identifying numerous species of phosphatides (PA) and phosphatidylcholines (PC). Both PA and PC species represent potential LPA precursors. The anatomical distribution of these precursors in rodent and human brain may indicate a metabolic relationship between LPA and LPA1 receptors. PMID:25857358

  17. LPA receptor expression in the central nervous system in health and following injury.

    Science.gov (United States)

    Goldshmit, Yona; Munro, Kathryn; Leong, Soo Yuen; Pébay, Alice; Turnley, Ann M

    2010-07-01

    Lysophosphatidic acid (LPA) is released from platelets following injury and also plays a role in neural development but little is known about its effects in the adult central nervous system (CNS). We have examined the expression of LPA receptors 1-3 (LPA(1-3)) in intact mouse spinal cord and cortical tissues and following injury. In intact and injured tissues, LPA(1) was expressed by ependymal cells in the central canal of the spinal cord and was upregulated in reactive astrocytes following spinal cord injury. LPA(2) showed low expression in intact CNS tissue, on grey matter astrocytes in spinal cord and in ependymal cells lining the lateral ventricle. Following injury, its expression was upregulated on astrocytes in both cortex and spinal cord. LPA(3) showed low expression in intact CNS tissue, viz. on cortical neurons and motor neurons in the spinal cord, and was upregulated on neurons in both regions after injury. Therefore, LPA(1-3) are differentially expressed in the CNS and their expression is upregulated in response to injury. LPA release following CNS injury may have different consequences for each cell type because of this differential expression in the adult nervous system.

  18. Structural basis of substrate discrimination and integrin binding by autotaxin

    Energy Technology Data Exchange (ETDEWEB)

    Hausmann, Jens; Kamtekar, Satwik; Christodoulou, Evangelos; Day, Jacqueline E.; Wu, Tao; Fulkerson, Zachary; Albers, Harald M.H.G.; van Meeteren, Laurens A.; Houben, Anna J.S.; van Zeijl, Leonie; Jansen, Silvia; Andries, Maria; Hall, Troii; Pegg, Lyle E.; Benson, Timothy E.; Kasiem, Mobien; Harlos, Karl; Vander Kooi, Craig W.; Smyth, Susan S.; Ovaa, Huib; Bollen, Mathieu; Morris, Andrew J.; Moolenaar, Wouter H.; Perrakis, Anastassis (Pfizer); (Leuven); (Oxford); (NCI-Netherlands); (Kentucky)

    2013-09-25

    Autotaxin (ATX, also known as ectonucleotide pyrophosphatase/phosphodiesterase-2, ENPP2) is a secreted lysophospholipase D that generates the lipid mediator lysophosphatidic acid (LPA), a mitogen and chemoattractant for many cell types. ATX-LPA signaling is involved in various pathologies including tumor progression and inflammation. However, the molecular basis of substrate recognition and catalysis by ATX and the mechanism by which it interacts with target cells are unclear. Here, we present the crystal structure of ATX, alone and in complex with a small-molecule inhibitor. We have identified a hydrophobic lipid-binding pocket and mapped key residues for catalysis and selection between nucleotide and phospholipid substrates. We have shown that ATX interacts with cell-surface integrins through its N-terminal somatomedin B-like domains, using an atypical mechanism. Our results define determinants of substrate discrimination by the ENPP family, suggest how ATX promotes localized LPA signaling and suggest new approaches for targeting ATX with small-molecule therapeutic agents.

  19. Inhibitory effects of LPA1 on cell motile activities stimulated by hydrogen peroxide and 2,3-dimethoxy-1,4-naphthoquinone in fibroblast 3T3 cells.

    Science.gov (United States)

    Hirane, Miku; Araki, Mutsumi; Dong, Yan; Honoki, Kanya; Fukushima, Nobuyuki; Tsujiuchi, Toshifumi

    2013-11-08

    Reactive oxygen species (ROS) are known to mediate a variety of biological responses, including cell motility. Recently, we indicated that lysophosphatidic acid (LPA) receptor-3 (LPA3) increased cell motile activity stimulated by hydrogen peroxide. In the present study, we assessed the role of LPA1 in the cell motile activity mediated by ROS in mouse fibroblast 3T3 cells. 3T3 cells were treated with hydrogen peroxide and 2,3-dimethoxy-1,4-naphthoquinone (DMNQ) at concentrations of 0.1 and 1 μM for 48 h. In cell motility assays with Cell Culture Inserts, the cell motile activities of 3T3 cells treated with hydrogen peroxide and DMNQ were significantly higher than those of untreated cells. 3T3 cells treated with hydrogen peroxide and DMNQ showed elevated expression levels of the Lpar3 gene, but not the Lpar1 and Lpar2 genes. To investigate the effects of LPA1 on the cell motile activity induced by hydrogen peroxide and DMNQ, Lpar1-overexpressing (3T3-a1) cells were generated from 3T3 cells and treated with hydrogen peroxide and DMNQ. The cell motile activities stimulated by hydrogen peroxide and DMNQ were markedly suppressed in 3T3-a1 cells. These results suggest that LPA signaling via LPA1 inhibits the cell motile activities stimulated by hydrogen peroxide and DMNQ in 3T3 cells.

  20. Hydrogen peroxide stimulates cell motile activity through LPA receptor-3 in liver epithelial WB-F344 cells.

    Science.gov (United States)

    Shibata, Ayano; Tanabe, Eriko; Inoue, Serina; Kitayoshi, Misaho; Okimoto, Souta; Hirane, Miku; Araki, Mutsumi; Fukushima, Nobuyuki; Tsujiuchi, Toshifumi

    2013-04-12

    Hydrogen peroxide which is one of reactive oxygen species (ROS) mediates a variety of biological responses, including cell proliferation and migration. In the present study, we investigated whether lysophosphatidic acid (LPA) signaling is involved in cell motile activity stimulated by hydrogen peroxide. The rat liver epithelial WB-F344 cells were treated with hydrogen peroxide at 0.1 or 1 μM for 48 h. In cell motility assays, hydrogen peroxide treated cells showed significantly high cell motile activity, compared with untreated cells. To measure the expression levels of LPA receptor genes, quantitative real time RT-PCR analysis was performed. The expressions of LPA receptor-3 (Lpar3) in hydrogen peroxide treated cells were significantly higher than those in control cells, but not Lpar1 and Lpar2 genes. Next, to assess the effect of LPA3 on cell motile activity, the Lpar3 knockdown cells from WB-F344 cells were also treated with hydrogen peroxide. The cell motile activity of the knockdown cells was not stimulated by hydrogen peroxide. Moreover, in liver cancer cells, hydrogen peroxide significantly activated cell motility of Lpar3-expressing cells, but not Lpar3-unexpressing cells. These results suggest that LPA signaling via LPA3 may be mainly involved in cell motile activity of WB-F344 cells stimulated by hydrogen peroxide.

  1. Autotaxin, Pruritus and Primary Biliary Cholangitis (PBC).

    Science.gov (United States)

    Sun, Ying; Zhang, Weici; Evans, Jilly F; Floreani, Annarosa; Zou, Zhengsheng; Nishio, Yukiko; Qi, Ruizhao; Leung, Patrick S C; Bowlus, Christopher L; Gershwin, M Eric

    2016-08-01

    Autotaxin (ATX) is a 125-kD type II ectonucleotide pyrophosphatase/phosphodiesterase (ENPP2 or NPP2) originally discovered as an unknown "autocrine motility factor" in human melanoma cells. In addition to its pyrophosphatase/phosphodiesterase activities ATX has lysophospholipase D (lysoPLD) activity, catalyzing the conversion of lysophosphatidylcholine (LPC) into lysophosphatidic acid (LPA). ATX is the only ENPP family member with lysoPLD activity and it produces most of the LPA in circulation. In support of this, ATX heterozygous mice have 50% of normal LPA plasma levels. The ATX-LPA signaling axis plays an important role in both normal physiology and disease pathogenesis and recently has been linked to pruritus in chronic cholestatic liver diseases, including primary biliary cholangitis (PBC). Several lines of evidence have suggested that a circulating puritogen is responsible, but the identification of the molecule has yet to be definitively identified. In contrast, plasma ATX activity is strongly associated with pruritus in PBC, suggesting a targetable molecule for treatment. We review herein the biochemistry of ATX and the rationale for its role in pruritus.

  2. Proteomic screening of human targets of viral microRNAs reveals functions associated with immune evasion and angiogenesis.

    Directory of Open Access Journals (Sweden)

    Amelia M Gallaher

    Full Text Available Kaposi's sarcoma (KS is caused by infection with Kaposi's sarcoma-associated herpesvirus (KSHV. The virus expresses unique microRNAs (miRNAs, but the targets and functions of these miRNAs are not completely understood. In order to identify human targets of viral miRNAs, we measured protein expression changes caused by multiple KSHV miRNAs using pulsed stable labeling with amino acids in cell culture (pSILAC in primary endothelial cells. This led to the identification of multiple human genes that are repressed at the protein level, but not at the miRNA level. Further analysis also identified that KSHV miRNAs can modulate activity or expression of upstream regulatory factors, resulting in suppressed activation of a protein involved in leukocyte recruitment (ICAM1 following lysophosphatidic acid treatment, as well as up-regulation of a pro-angiogenic protein (HIF1α, and up-regulation of a protein involved in stimulating angiogenesis (HMOX1. This study aids in our understanding of miRNA mechanisms of repression and miRNA contributions to viral pathogenesis.

  3. Identification and expression analysis of castor bean (Ricinus communis) genes encoding enzymes from the triacylglycerol biosynthesis pathway.

    Science.gov (United States)

    Cagliari, Alexandro; Margis-Pinheiro, Márcia; Loss, Guilherme; Mastroberti, Alexandra Antunes; de Araujo Mariath, Jorge Ernesto; Margis, Rogério

    2010-11-01

    Castor bean (Ricinus communis) oil contains ricinoleic acid-rich triacylglycerols (TAGs). As a result of its physical and chemical properties, castor oil and its derivatives are used for numerous bio-based products. In this study, we survey the Castor Bean Genome Database to report the identification of TAG biosynthesis genes. A set of 26 genes encoding six distinct classes of enzymes involved in TAGs biosynthesis were identified. In silico characterization and sequence analysis allowed the identification of plastidic isoforms of glycerol-3-phosphate acyltransferase and lysophosphatidate acyltransferase enzyme families, involved in the prokaryotic lipid biosynthesis pathway, that form a cluster apart from the cytoplasmic isoforms, involved in the eukaryotic pathway. In addition, two distinct membrane bound diacylglycerol acyltransferase enzymes were identified. Quantitative expression pattern analyses demonstrated variations in gene expressions during castor seed development. A tendency of maximum expression level at the middle of seed development was observed. Our results represent snapshots of global transcriptional activities of genes encompassing six enzyme families involved in castor bean TAG biosynthesis that are present during seed development. These genes represent potential targets for biotechnological approaches to produce nutritionally and industrially desirable oils.

  4. Protein kinase D is increased and activated in lung epithelial cells and macrophages in idiopathic pulmonary fibrosis.

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    Gan, Huachen; McKenzie, Raymond; Hao, Qin; Idell, Steven; Tang, Hua

    2014-01-01

    Idiopathic pulmonary fibrosis (IPF) is a relentlessly progressive and usually fatal lung disease of unknown etiology for which no effective treatments currently exist. Hence, there is a profound need for the identification of novel drugable targets to develop more specific and efficacious therapeutic intervention in IPF. In this study, we performed immunohistochemical analyses to assess the cell type-specific expression and activation of protein kinase D (PKD) family kinases in normal and IPF lung tissue sections. We also analyzed PKD activation and function in human lung epithelial cells. We found that PKD family kinases (PKD1, PKD2 and PKD3) were increased and activated in the hyperplastic and regenerative alveolar epithelial cells lining remodeled fibrotic alveolar septa and/or fibroblast foci in IPF lungs compared with normal controls. We also found that PKD family kinases were increased and activated in alveolar macrophages, bronchiolar epithelium, and honeycomb cysts in IPF lungs. Interestingly, PKD1 was highly expressed and activated in the cilia of IPF bronchiolar epithelial cells, while PKD2 and PKD3 were expressed in the cell cytoplasm and nuclei. In contrast, PKD family kinases were not apparently increased and activated in IPF fibroblasts or myofibroblasts. We lastly found that PKD was predominantly activated by poly-L-arginine, lysophosphatidic acid and thrombin in human lung epithelial cells and that PKD promoted epithelial barrier dysfunction. These findings suggest that PKD may participate in the pathogenesis of IPF and may be a novel target for therapeutic intervention in this disease.

  5. A FRET Biosensor for ROCK Based on a Consensus Substrate Sequence Identified by KISS Technology.

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    Li, Chunjie; Imanishi, Ayako; Komatsu, Naoki; Terai, Kenta; Amano, Mutsuki; Kaibuchi, Kozo; Matsuda, Michiyuki

    2017-01-11

    Genetically-encoded biosensors based on Förster/fluorescence resonance energy transfer (FRET) are versatile tools for studying the spatio-temporal regulation of signaling molecules within not only the cells but also tissues. Perhaps the hardest task in the development of a FRET biosensor for protein kinases is to identify the kinase-specific substrate peptide to be used in the FRET biosensor. To solve this problem, we took advantage of kinase-interacting substrate screening (KISS) technology, which deduces a consensus substrate sequence for the protein kinase of interest. Here, we show that a consensus substrate sequence for ROCK identified by KISS yielded a FRET biosensor for ROCK, named Eevee-ROCK, with high sensitivity and specificity. By treating HeLa cells with inhibitors or siRNAs against ROCK, we show that a substantial part of the basal FRET signal of Eevee-ROCK was derived from the activities of ROCK1 and ROCK2. Eevee-ROCK readily detected ROCK activation by epidermal growth factor, lysophosphatidic acid, and serum. When cells stably-expressing Eevee-ROCK were time-lapse imaged for three days, ROCK activity was found to increase after the completion of cytokinesis, concomitant with the spreading of cells. Eevee-ROCK also revealed a gradual increase in ROCK activity during apoptosis. Thus, Eevee-ROCK, which was developed from a substrate sequence predicted by the KISS technology, will pave the way to a better understanding of the function of ROCK in a physiological context.

  6. Ecdysone and insulin signaling play essential roles in readjusting the altered body size caused by the dGPAT4 mutation in Drosophila.

    Science.gov (United States)

    Yan, Yan; Wang, Hao; Chen, Hanqing; Lindström-Battle, Anya; Jiao, Renjie

    2015-09-20

    Body size is one of the features that distinguish one species from another in the biological world. Animals have developed mechanisms to control their body size during normal development. However, how animals cope with genetic alterations and/or environmental stresses to develop into normal-sized adults remain poorly understood. The ability of the animals to develop into a normal-sized adult after the challenges of genetic alterations and/or environmental stresses reveals a robustness of body size control. Here we show that the mutation of dGPAT4, a de novo synthase of lysophosphatidic acid, is a genetic alteration that triggers such a robust response of the animals to body size challenges in Drosophila. Loss of dGPAT4 leads to a severe delay of development, slow growth and resultant small-sized animals during the larval stages, but results in normal-sized adult flies. The robust body size adjustment of the dGPAT4 mutant is likely achieved by corresponding changes in ecdysone and insulin signaling, which is also manifested by compromised food intake. Thus, we propose that a strategy has been evolved by the animals to reach final body size when challenged by genetic alterations, which requires the coordinated ecdysone and insulin signaling.

  7. Quantitative Glycoproteomic Analysis Identifies Platelet-Induced Increase of Monocyte Adhesion via the Up-Regulation of Very Late Antigen 5.

    Science.gov (United States)

    Huang, Jiqing; Kast, Juergen

    2015-08-07

    Physiological stimuli, such as thrombin, or pathological stimuli, such as lysophosphatidic acid (LPA), activate platelets circulating in blood. Once activated, platelets bind to monocytes via P-selectin-PSGL-1 interactions but also release the stored contents of their granules. These platelet releasates, in addition to direct platelet binding, activate monocytes and facilitate their recruitment to atherosclerotic sites. Consequently, understanding the changes platelet releasates induce in monocyte membrane proteins is critical. We studied the glyco-proteome changes of THP-1 monocytic cells affected by LPA- or thrombin-induced platelet releasates. We employed lectin affinity chromatography combined with filter aided sample preparation to achieve high glyco- and membrane protein and protein sequence coverage. Using stable isotope labeling by amino acids in cell culture, we quantified 1715 proteins, including 852 membrane and 500 glycoproteins, identifying the up-regulation of multiple proteins involved in monocyte extracellular matrix binding and transendothelial migration. Flow cytometry indicated expression changes of integrin α5, integrin β1, PECAM-1, and PSGL-1. The observed increase in monocyte adhesion to fibronectin was determined to be mediated by the up-regulation of very late antigen 5 via a P-selectin-PSGL-1 independent mechanism. This novel aspect could be validated on CD14+ human primary monocytes, highlighting the benefits of the improved enrichment method regarding high membrane protein coverage and reliable quantification.

  8. LPA signaling initiates schizophrenia-like brain and behavioral changes in a mouse model of prenatal brain hemorrhage.

    Science.gov (United States)

    Mirendil, H; Thomas, E A; De Loera, C; Okada, K; Inomata, Y; Chun, J

    2015-04-07

    Genetic, environmental and neurodevelopmental factors are thought to underlie the onset of neuropsychiatric disorders such as schizophrenia. How these risk factors collectively contribute to pathology is unclear. Here, we present a mouse model of prenatal intracerebral hemorrhage--an identified risk factor for schizophrenia--using a serum-exposure paradigm. This model exhibits behavioral, neurochemical and schizophrenia-related gene expression alterations in adult females. Behavioral alterations in amphetamine-induced locomotion, prepulse inhibition, thigmotaxis and social interaction--in addition to increases in tyrosine hydroxylase-positive dopaminergic cells in the substantia nigra and ventral tegmental area and decreases in parvalbumin-positive cells in the prefrontal cortex--were induced upon prenatal serum exposure. Lysophosphatidic acid (LPA), a lipid component of serum, was identified as a key molecular initiator of schizophrenia-like sequelae induced by serum. Prenatal exposure to LPA alone phenocopied many of the schizophrenia-like alterations seen in the serum model, whereas pretreatment with an antagonist against the LPA receptor subtype LPA1 prevented many of the behavioral and neurochemical alterations. In addition, both prenatal serum and LPA exposure altered the expression of many genes and pathways related to schizophrenia, including the expression of Grin2b, Slc17a7 and Grid1. These findings demonstrate that aberrant LPA receptor signaling associated with fetal brain hemorrhage may contribute to the development of some neuropsychiatric disorders.

  9. [Regulatory role of mechanical stress response in cellular function: development of new drugs and tissue engineering].

    Science.gov (United States)

    Momose, Kazutaka; Matsuda, Takehisa; Oike, Masahiro; Obara, Kazuo; Laher, Ismail; Sugiura, Seiryo; Ohata, Hisayuki; Nakayama, Koichi

    2003-02-01

    The investigation of mechanotransduction in the cardiovascular system is essentially important for elucidating the cellular and molecular mechanisms involved in not only the maintenance of hemodynamic homeostasis but also etiology of cardiovascular diseases including arteriosclerosis. The present review summarizes the latest research performed by six academic groups, and presented at the 75th Annual Meeting of the Japanese Pharmacological Society. Technology of cellular biomechanics is also required for research and clinical application of a vascular hybrid tissue responding to pulsatile stress. 1) Vascular tissue engineering: Design of pulsatile stress-responsive scaffold and in vivo vascular wall reconstruction (T. Matsuda); 2) Cellular mechanisms of mechanosensitive calcium transients in vascular endothelium (M. Oike et al.); 3) Cross-talk of stimulation with fluid flow and lysophosphatidic acid in vascular endothelial cells (K. Momose et al.); 4) Mechanotransduction of vascular smooth muscles: Rate-dependent stretch-induced protein phosphorylations and contractile activation (K. Obara et al.); 5) Lipid mediators in vascular myogenic tone (I. Laher et al.); and 6) Caldiomyocyte regulates its mechanical output in response to mechanical load (S. Sugiura et al.).

  10. G alpha12 is targeted to the mitochondria and affects mitochondrial morphology and motility.

    Science.gov (United States)

    Andreeva, Alexandra V; Kutuzov, Mikhail A; Voyno-Yasenetskaya, Tatyana A

    2008-08-01

    G alpha12 constitutes, along with G alpha13, one of the four families of alpha subunits of heterotrimeric G proteins. We found that the N terminus of G alpha12, but not those of other G alpha subunits, contains a predicted mitochondrial targeting sequence. Using confocal microscopy and cell fractionation, we demonstrated that up to 40% of endogenous G alpha12 in human umbilical vein endothelial cells colocalize with mitochondrial markers. N-terminal sequence of G alpha12 fused to GFP efficiently targeted the fusion protein to mitochondria. G alpha12 with mutated mitochondrial targeting sequence was still located in mitochondria, suggesting the existence of additional mechanisms for mitochondrial localization. Lysophosphatidic acid, one of the known stimuli transduced by G alpha12/13, inhibited mitochondrial motility, while depletion of endogenous G alpha12 increased mitochondrial motility. G alpha12Q229L variants uncoupled from RhoGEFs (but not fully functional activated G alpha12Q229L) induced transformation of the mitochondrial network into punctate mitochondria and resulted in a loss of mitochondrial membrane potential. All examined G alpha12Q229L variants reduced phosphorylation of Bcl-2 at Ser-70, while only mutants unable to bind RhoGEFs also decreased cellular levels of Bcl-2. These G alpha12 mutants were also more efficient Hsp90 interactors. These findings are the first demonstration of a heterotrimeric G protein alpha subunit specifically targeted to mitochondria and involved in the control of mitochondrial morphology and dynamics.

  11. Edg8/S1P5: an oligodendroglial receptor with dual function on process retraction and cell survival.

    Science.gov (United States)

    Jaillard, C; Harrison, S; Stankoff, B; Aigrot, M S; Calver, A R; Duddy, G; Walsh, F S; Pangalos, M N; Arimura, N; Kaibuchi, K; Zalc, B; Lubetzki, C

    2005-02-09

    Endothelial differentiation gene (Edg) proteins are G-protein-coupled receptors activated by lysophospholipid mediators: sphingosine-1-phosphate (S1P) or lysophosphatidic acid. We show that in the CNS, expression of Edg8/S1P5, a high-affinity S1P receptor, is restricted to oligodendrocytes and expressed throughout development from the immature stages to the mature myelin-forming cell. S1P activation of Edg8/S1P5 on O4-positive pre-oligodendrocytes induced process retraction via a Rho kinase/collapsin response-mediated protein signaling pathway, whereas no retraction was elicited by S1P on these cells derived from Edg8/S1P5-deficient mice. Edg8/S1P5-mediated process retraction was restricted to immature cells and was no longer observed at later developmental stages. In contrast, S1P activation promoted the survival of mature oligodendrocytes but not of pre-oligodendrocytes. The S1P-induced survival of mature oligodendrocytes was mediated through a pertussis toxin-sensitive, Akt-dependent pathway. Our data demonstrate that Edg8/S1P5 activation on oligodendroglial cells modulates two distinct functional pathways mediating either process retraction or cell survival and that these effects depend on the developmental stage of the cell.

  12. PEDF and PEDF-derived peptide 44mer inhibit oxygen-glucose deprivation-induced oxidative stress through upregulating PPARγ via PEDF-R in H9c2 cells.

    Science.gov (United States)

    Zhuang, Wei; Zhang, Hao; Pan, Jiajun; Li, Zhimin; Wei, Tengteng; Cui, Huazhu; Liu, Zhiwei; Guan, Qiuhua; Dong, Hongyan; Zhang, Zhongming

    2016-04-08

    Pigment epithelial-derived factor (PEDF) is a glycoprotein with broad biological activities including inhibiting oxygen-glucose deprivation(OGD)-induced cardiomyocytes apoptosis through its anti-oxidative properties. PEDF derived peptide-44mer shows similar cytoprotective effect to PEDF. However, the molecular mechanisms mediating cardiomyocytes apoptosis have not been fully established. Here we found that PEDF and 44mer decreased the content of ROS. This content was abolished by either PEDF-R small interfering RNA (siRNA) or PPARγ antagonist. The level of Lysophosphatidic acid (LPA) and phospholipase A2 (PLA2) was observed as drawn from the ELISA assays. PEDF and 44mer sequentially induced PPARγ expression was observed both in qPCR and Western blot assays. The level of LPA and PLA2 and PPARγ expression increased by PEDF and 44mer was significantly attenuated by PEDF-R siRNA. However, PEDF and 44mer inhibited the H9c2 cells and cultured neonatal rat myocardial cells apoptosis rate. On the other hand, TUNEL assay and cleavage of procaspase-3 showed that PEDF-R siRNA or PPARγ antagonist increased the apoptosis again. We conclude that under OGD condition, PEDF and 44mer reduce H9c2 cells apoptosis and inhibit OGD-induced oxidative stress via its receptor PEDF-R and the PPARγ signaling pathway.

  13. Non-invasive imaging of tumors by monitoring autotaxin activity using an enzyme-activated near-infrared fluorogenic substrate.

    Directory of Open Access Journals (Sweden)

    Damian Madan

    Full Text Available Autotaxin (ATX, an autocrine motility factor that is highly upregulated in metastatic cancer, is a lysophospholipase D enzyme that produces the lipid second messenger lysophosphatidic acid (LPA from lysophosphatidylcholine (LPC. Dysregulation of the lysolipid signaling pathway is central to the pathophysiology of numerous cancers, idiopathic pulmonary fibrosis, rheumatoid arthritis, and other inflammatory diseases. Consequently, the ATX/LPA pathway has emerged as an important source of biomarkers and therapeutic targets. Herein we describe development and validation of a fluorogenic analog of LPC (AR-2 that enables visualization of ATX activity in vivo. AR-2 exhibits minimal fluorescence until it is activated by ATX, which substantially increases fluorescence in the near-infrared (NIR region, the optimal spectral window for in vivo imaging. In mice with orthotopic ATX-expressing breast cancer tumors, ATX activated AR-2 fluorescence. Administration of AR-2 to tumor-bearing mice showed high fluorescence in the tumor and low fluorescence in most healthy tissues with tumor fluorescence correlated with ATX levels. Pretreatment of mice with an ATX inhibitor selectively decreased fluorescence in the tumor. Together these data suggest that fluorescence directly correlates with ATX activity and its tissue expression. The data show that AR-2 is a non-invasive and selective tool that enables visualization and quantitation of ATX-expressing tumors and monitoring ATX activity in vivo.

  14. LPA is a chemorepellent for B16 melanoma cells: action through the cAMP-elevating LPA5 receptor.

    Directory of Open Access Journals (Sweden)

    Maikel Jongsma

    Full Text Available Lysophosphatidic acid (LPA, a lipid mediator enriched in serum, stimulates cell migration, proliferation and other functions in many cell types. LPA acts on six known G protein-coupled receptors, termed LPA(1-6, showing both overlapping and distinct signaling properties. Here we show that, unexpectedly, LPA and serum almost completely inhibit the transwell migration of B16 melanoma cells, with alkyl-LPA(18:1 being 10-fold more potent than acyl-LPA(18:1. The anti-migratory response to LPA is highly polarized and dependent on protein kinase A (PKA but not Rho kinase activity; it is associated with a rapid increase in intracellular cAMP levels and PIP3 depletion from the plasma membrane. B16 cells express LPA(2, LPA(5 and LPA(6 receptors. We show that LPA-induced chemorepulsion is mediated specifically by the alkyl-LPA-preferring LPA(5 receptor (GPR92, which raises intracellular cAMP via a noncanonical pathway. Our results define LPA(5 as an anti-migratory receptor and they implicate the cAMP-PKA pathway, along with reduced PIP3 signaling, as an effector of chemorepulsion in B16 melanoma cells.

  15. Calcium imaging of individual erythrocytes: problems and approaches.

    Science.gov (United States)

    Kaestner, Lars; Tabellion, Wiebke; Weiss, Erwin; Bernhardt, Ingolf; Lipp, Peter

    2006-01-01

    Although in erythrocytes calcium is thought to be important in homeostasis, measurements of this ion concentration are generally seen as rather problematic because of the auto-fluorescence or absorption properties of the intracellular milieu. Here, we describe experiments to assess the usability of popular calcium indicators such as Fura-2, Indo-1 and Fluo-4. In our experiments, Fluo-4 turned out to be the preferable indicator because (i) its excitation and emission properties were least influenced by haemoglobin and (ii) it was the only dye for which excitation light did not lead to significant auto-fluorescence of the erythrocytes. From these results, we conclude that the use of indicators such as Fura-2 together with red blood cells has to be revisited critically. We thus utilized Fluo-4 in erythrocytes to demonstrate a robust but heterogeneous calcium increase in these cells upon stimulation by prostaglandin E(2) and lysophosphatidic acid. For the latter stimulus, we recorded emission spectra of individual erythrocytes to confirm largely unaltered Fluo-4 emission. Our results emphasize that in erythrocytes measurements of intracellular calcium are reliably possible with Fluo-4 and that other indicators, especially those requiring UV-excitation, appear less favourable.

  16. Serum Autotaxin is a Marker of the Severity of Liver Injury and Overall Survival in Patients with Cholestatic Liver Diseases

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    Wunsch, Ewa; Krawczyk, Marcin; Milkiewicz, Malgorzata; Trottier, Jocelyn; Barbier, Olivier; Neurath, Markus F.; Lammert, Frank; Kremer, Andreas E.; Milkiewicz, Piotr

    2016-01-01

    Autotaxin (ATX) is involved in the synthesis of lysophosphatidic acid. Both have recently been linked to cholestatic pruritus and liver injury. We aimed to investigate whether ATX is an indicator of cholestatic liver injury, health-related quality of life (HRQoL) and prognosis based on a group of 233 patients, 118 with primary biliary cholangitis (PBC) and 115 with primary sclerosing cholangitis (PSC). Patients were followed for 1–60 months, cumulative survival rates were calculated. ATX activity was significantly higher in both groups than in the 103 controls, particularly in patients with cirrhosis and in patients with longer disease duration. Ursodeoxycholic acid (UDCA) non-responders with PBC exhibited increased ATX activity. ATX activity was correlated with liver biochemistry, MELD, Mayo Risk scores and was associated with worse disease-specific HRQoL aspects. In both groups, Cox model analysis indicated that ATX was a negative predictor of survival. Increased ATX levels were associated with a 4-fold higher risk of death/liver transplantation in patients with PBC and a 2.6-fold higher risk in patients with PSC. We conclude that in patients with cholestatic conditions, ATX is not only associated with pruritus but also indicates impairment of other HRQoL aspects, liver dysfunction, and can serve as a predictor of survival. PMID:27506882

  17. Pharmacological Characterization of a Potent Inhibitor of Autotaxin in Animal Models of Inflammatory Bowel Disease and Multiple Sclerosis.

    Science.gov (United States)

    Thirunavukkarasu, Kannan; Tan, Bailin; Swearingen, Craig A; Rocha, Guilherme; Bui, Hai H; McCann, Denis J; Jones, Spencer B; Norman, Bryan H; Pfeifer, Lance A; Saha, Joy K

    2016-10-01

    Autotaxin is a secreted enzyme that catalyzes the conversion of lysophosphatidyl choline into the bioactive lipid mediator lysophosphatidic acid (LPA). It is the primary enzyme responsible for LPA production in plasma. It is upregulated in inflammatory conditions and inhibition of autotaxin may have anti-inflammatory activity in a variety of inflammatory diseases. To determine the role of autotaxin and LPA in the pathophysiology of inflammatory disease states, we used a potent and orally bioavailable inhibitor of autotaxin that we have recently identified, and characterized it in mouse models of inflammation, inflammatory bowel disease (IBD), multiple sclerosis (MS), and visceral pain. Compound-1, a potent inhibitor of autotaxin with an IC50 of ∼2 nM, has good oral pharmacokinetic properties in mice and results in a substantial inhibition of plasma LPA that correlates with drug exposure levels. Treatment with the inhibitor resulted in significant anti-inflammatory and analgesic effects in the carrageenan-induced paw inflammation and acetic acid-induced visceral pain tests, respectively. Compound-1 also significantly inhibited disease activity score in the dextran sodium sulfate-induced model of IBD, and in the experimental autoimmune encephalomyelitis model of MS. In conclusion, the present study demonstrates the anti-inflammatory and analgesic properties of a novel inhibitor of autotaxin that may serve as a therapeutic option for IBD, MS, and pain associated with inflammatory states.

  18. Role of Ca2+-independent phospholipase A2 in cell growth and signaling.

    Science.gov (United States)

    Hooks, Shelley B; Cummings, Brian S

    2008-10-30

    Phospholipase A(2) (PLA(2)) are esterases that cleave glycerophospholipids to release fatty acids and lysophospholipids. Several studies demonstrate that PLA(2) regulate growth and signaling in several cell types. However, few of these studies have focused on Ca2+-independent phospholipase A(2) (iPLA(2) or Group VI PLA(2)). This class of PLA(2) was originally suggested to mediate phospholipid remodeling in several cell types including macrophages. As such, it was labeled as a housekeeping protein and thought not to play as significant of roles in cell growth as its older counterparts cytosolic PLA(2) (cPLA(2) or Group IV PLA(2)) and secretory PLA(2) (sPLA(2) or Groups I-III, V and IX-XIV PLA(2)). However, several recent studies demonstrate that iPLA(2) mediate cell growth, and do so by participating in signal transduction pathways that include epidermal growth factor receptors (EGFR), mitogen activated protein kinases (MAPK), mdm2, and even the tumor suppressor protein p53 and the cell cycle regulator p21. The exact mechanism by which iPLA(2) mediates these pathways are not known, but likely involve the generation of lipid signals such as arachidonic acid, lysophosphatidic acid (LPA) and lysophosphocholines (LPC). This review discusses the role of iPLA(2) in cell growth with special emphasis placed on their role in cell signaling. The putative lipid signals involved are also discussed.

  19. Alterations in lipid signaling underlie lipodystrophy secondary to AGPAT2 mutations.

    Science.gov (United States)

    Subauste, Angela R; Das, Arun K; Li, Xiangquan; Elliott, Brandon G; Elliot, Brandon; Evans, Charles; El Azzouny, Mahmoud; Treutelaar, Mary; Oral, Elif; Leff, Todd; Burant, Charles F

    2012-11-01

    Congenital generalized lipodystrophy (CGL), secondary to AGPAT2 mutation is characterized by the absence of adipocytes and development of severe insulin resistance. In the current study, we investigated the adipogenic defect associated with AGPAT2 mutations. Adipogenesis was studied in muscle-derived multipotent cells (MDMCs) isolated from vastus lateralis biopsies obtained from controls and subjects harboring AGPAT2 mutations and in 3T3-L1 preadipocytes after knockdown or overexpression of AGPAT2. We demonstrate an adipogenic defect using MDMCs from control and CGL human subjects with mutated AGPAT2. This defect was rescued in CGL MDMCs with a retrovirus expressing AGPAT2. Both CGL-derived MDMCs and 3T3-L1 cells with knockdown of AGPAT2 demonstrated an increase in cell death after induction of adipogenesis. Lack of AGPAT2 activity reduces Akt activation, and overexpression of constitutively active Akt can partially restore lipogenesis. AGPAT2 modulated the levels of phosphatidic acid, lysophosphatidic acid, phosphatidylinositol species, as well as the peroxisome proliferator-activated receptor γ (PPARγ) inhibitor cyclic phosphatidic acid. The PPARγ agonist pioglitazone partially rescued the adipogenic defect in CGL cells. We conclude that AGPAT2 regulates adipogenesis through the modulation of the lipome, altering normal activation of phosphatidylinositol 3-kinase (PI3K)/Akt and PPARγ pathways in the early stages of adipogenesis.

  20. Phosphatidic acid: biosynthesis, pharmacokinetics, mechanisms of action and effect on strength and body composition in resistance-trained individuals.

    Science.gov (United States)

    Bond, Peter

    2017-01-01

    The mechanistic target of rapamycin complex 1 (mTORC1) has received much attention in the field of exercise physiology as a master regulator of skeletal muscle hypertrophy. The multiprotein complex is regulated by various signals such as growth factors, energy status, amino acids and mechanical stimuli. Importantly, the glycerophospholipid phosphatidic acid (PA) appears to play an important role in mTORC1 activation by mechanical stimulation. PA has been shown to modulate mTOR activity by direct binding to its FKBP12-rapamycin binding domain. Additionally, it has been suggested that exogenous PA activates mTORC1 via extracellular conversion to lysophosphatidic acid and subsequent binding to endothelial differentiation gene receptors on the cell surface. Recent trials have therefore evaluated the effects of PA supplementation in resistance-trained individuals on strength and body composition. As research in this field is rapidly evolving, this review attempts to provide a comprehensive overview of its biosynthesis, pharmacokinetics, mechanisms of action and effect on strength and body composition in resistance-trained individuals.

  1. Fatty acid composition of muscle fat and enzymes of storage lipid synthesis in whole muscle from beef cattle.

    Science.gov (United States)

    Kazala, E Chris; Lozeman, Fred J; Mir, Priya S; Aalhus, Jennifer L; Schmutz, Sheila M; Weselake, Randall J

    2006-11-01

    Enhanced intramuscular fat content (i.e., marbling) in beef is a desirable trait, which can result in increased product value. This study was undertaken with the aim of revealing biochemical factors associated with the marbling trait in beef cattle. Samples of longissimus lumborum (LL) and pars costalis diaphragmatis (PCD) were taken from a group of intact crossbred males and females at slaughter, lipids extracted, and the resulting FAME examined for relationships with marbling fat deposition. For LL, significant associations were found between degree of marbling and myristic (14:0, r = 0.55, P muscle were assayed for diacylglycerol acyltransferase (DGAT), lysophosphatidic acid acyltransferase (LPAAT), and phosphatidic acid phosphatase-1 (PAP-1) activity, and the results examined for relationships with degree of intramuscular fat deposition. None of the enzyme activities from PCD displayed an association with marbling fat content, but DGAT specific activity showed significant positive associations with LPAAT (r = 0.54, P muscle tissues provide insight into possible enzyme action associated with the production of specific FA. The increased proportion of oleic acid associated with enhanced lipid content of whole muscle is noteworthy given the known health benefits of this FA.

  2. Microtubule Destabilizer KIF2A Undergoes Distinct Site-Specific Phosphorylation Cascades that Differentially Affect Neuronal Morphogenesis

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    Tadayuki Ogawa

    2015-09-01

    Full Text Available Neurons exhibit dynamic structural changes in response to extracellular stimuli. Microtubules (MTs provide rapid and dramatic cytoskeletal changes within the structural framework. However, the molecular mechanisms and signaling networks underlying MT dynamics remain unknown. Here, we have applied a comprehensive and quantitative phospho-analysis of the MT destabilizer KIF2A to elucidate the regulatory mechanisms of MT dynamics within neurons in response to extracellular signals. Interestingly, we identified two different sets of KIF2A phosphorylation profiles that accelerate (A-type and brake (B-type the MT depolymerization activity of KIF2A. Brain-derived neurotrophic factor (BDNF stimulates PAK1 and CDK5 kinases, which decrease the MT depolymerizing activity of KIF2A through B-type phosphorylation, resulting in enhanced outgrowth of neural processes. In contrast, lysophosphatidic acid (LPA induces ROCK2 kinase, which suppresses neurite outgrowth from round cells via A-type phosphorylation. We propose that these two mutually exclusive forms of KIF2A phosphorylation differentially regulate neuronal morphogenesis during development.

  3. Involvement of LPA Receptor 3 in LPA-induced BGC- 803 Cell Migration

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    Erdene Oyungerel

    2013-12-01

    Full Text Available Lysophosphatidic acid ˄ LPA ˅ is a bioactive phospholipid mediator, which elicits a variety of biological functions mainly through G-protein coupled receptors. Although LPA is shown to stimulate proliferation and motility via LPA receptors, LPAR1 and LPAR3 in several cancer cell lines, but the role of LPA receptors in gastric cancer cells is still being unknown. However, several researches reported that LPAR2 play an important role in the carcinogenesis of gastric cancer, but there is no report to show the LPAR3 involvement in the carcinogenesis. For this reason, we examined LPA receptors (LPAR1, LPAR2 and LPAR3 in BGC-803 cells along with real time PCR method. Real-time PCR analyses were used to evaluate the expression of LPA receptors in BGC-803 cells. Among these receptors, LPAR3 was shown to be highly expressed in BGC-803 cells, a human gastric cancer cell line. Transient transfection with LPAR3 siRNA was observed to reduce LPAR3 mRNA in BGC-803 cells and eliminate the LPA-induced cell migration. The results suggest that the LPAR3 regulates LPA-induced BGC-803 cell migration.

  4. Suppression of NADPH Oxidase Activity May Slow the Expansion of Osteolytic Bone Metastases

    Directory of Open Access Journals (Sweden)

    Mark F. McCarty

    2016-08-01

    Full Text Available Lysophosphatidic acid (LPA, generated in the microenvironment of cancer cells, can drive the proliferation, invasion, and migration of cancer cells by activating G protein-coupled LPA receptors. Moreover, in cancer cells that have metastasized to bone, LPA signaling can promote osteolysis by inducing cancer cell production of cytokines, such as IL-6 and IL-8, which can stimulate osteoblasts to secrete RANKL, a key promoter of osteoclastogenesis. Indeed, in cancers prone to metastasize to bone, LPA appears to be a major driver of the expansion of osteolytic bone metastases. Activation of NADPH oxidase has been shown to play a mediating role in the signaling pathways by which LPA, as well as RANKL, promote osteolysis. In addition, there is reason to suspect that Nox4 activation is a mediator of the feed-forward mechanism whereby release of TGF-beta from bone matrix by osteolysis promotes expression of PTHrP in cancer cells, and thereby induces further osteolysis. Hence, measures which can down-regulate NADPH oxidase activity may have potential for slowing the expansion of osteolytic bone metastases in cancer patients. Phycocyanin and high-dose statins may have utility in this regard, and could be contemplated as complements to bisphosphonates or denosumab for the prevention and control of osteolytic lesions. Ingestion of omega-3-rich flaxseed or fish oil may also have potential for controlling osteolysis in cancer patients.

  5. Prostaglandinsvis-à-vis bovine embryonic mortality:a review

    Institute of Scientific and Technical Information of China (English)

    Jerome A; Srivastava N

    2012-01-01

    Decline in fertility in bovines is attributed to various reproductive problems viz. anoestrus, repeat breeding, abortions and post parturient disorders.Among these, repeat breeding has been an important cause for reducing the animals’ fertility and life-time productivity.Many researchers have reported embryonic mortality as a major cause of repeat breeding arising due to premature corpus luteumlysis.ProstaglandinF2α released from the uterus causes alterations in luteal blood flow, induces luteal lysis, and hence reduces progesterone secretion from the bovine corpus luteum.Therefore various strategies have been tried to modulate prostaglandinF2α synthesis and secretion in order to prolong the lifespan ofCL.Administration of cyclooxygenase inhibitors which include non-steroidal anti-inflammatory drugs acting by competitive inhibition of key enzymes of prostaglandin synthesis is one such method.Feeding of diet rich in polyunsaturated fatty acids during critical period significantly reduces prostaglandin synthesis.Other drugs, which are potential candidates for reducing prostaglandin synthesis, include oxytocin receptor antagonist, recombinant bovine somatotropin, lysophosphatidic acid and prostaglandinF synthase inhibitors. To conclude, there is much scope of using various compounds to reduce prostaglandins synthesis during the critical period of pregnancy for improving the embryo survival rate.

  6. Regulation of T cell motility in vitro and in vivo by LPA and LPA2.

    Directory of Open Access Journals (Sweden)

    Sara A Knowlden

    Full Text Available Lysophosphatidic acid (LPA and the LPA-generating enzyme autotaxin (ATX have been implicated in lymphocyte trafficking and the regulation of lymphocyte entry into lymph nodes. High local concentrations of LPA are thought to be present in lymph node high endothelial venules, suggesting a direct influence of LPA on cell migration. However, little is known about the mechanism of action of LPA, and more work is needed to define the expression and function of the six known G protein-coupled receptors (LPA 1-6 in T cells. We studied the effects of 18∶1 and 16∶0 LPA on naïve CD4+ T cell migration and show that LPA induces CD4+ T cell chemorepulsion in a Transwell system, and also improves the quality of non-directed migration on ICAM-1 and CCL21 coated plates. Using intravital two-photon microscopy, lpa2-/- CD4+ T cells display a striking defect in early migratory behavior at HEVs and in lymph nodes. However, later homeostatic recirculation and LPA-directed migration in vitro were unaffected by loss of lpa2. Taken together, these data highlight a previously unsuspected and non-redundant role for LPA2 in intranodal T cell motility, and suggest that specific functions of LPA may be manipulated by targeting T cell LPA receptors.

  7. Lysophospholipids in coronary artery and chronic ischemic heart disease

    Science.gov (United States)

    Abdel-Latif, Ahmed; Heron, Paula M.; Morris, Andrew J.; Smyth, Susan S.

    2015-01-01

    Purpose of review The bioactive lysophospholipids, lysophosphatidic acid (LPA) and sphingosine 1 phosphate (S1P) have potent effects on blood and vascular cells. This review focuses their potential contributions to the development of atherosclerosis, acute complications, such as acute myocardial infarction, and chronic ischemic cardiac damage. Recent findings Exciting recent developments have provided insight into the molecular underpinnings of LPA and S1P receptor signaling. New lines of evidence suggest roles for these pathways in the development of atherosclerosis. In experimental animal models, the production, signaling and metabolism of LPA may be influenced by environmental factors in the diet that synergize to promote the progression of atherosclerotic vascular disease. This is supported by observations of human polymorphisms in the lysophospholipid metabolizing enzyme, PPAP2B, that are associated with risk of coronary artery disease and myocardial infarction. S1P signaling protects from myocardial damage that follows acute and chronic ischemia both by direct effects on cardiomyocytes and through stem cell recruitment to ischemic tissue. Summary This review will suggest novel strategies to prevent the complications of coronary artery disease by targeting LPA production and signaling. Additionally, ways in which S1P signaling pathways may be harnessed to attenuate ischemia-induced cardiac dysfunction will be explored. PMID:26270808

  8. Lipid composition of metacestodes of Taenia taeniaeformis and lipid changes during growth.

    Science.gov (United States)

    Mills, G L; Taylor, D C; Williams, J F

    1981-09-01

    A lipid analysis was performed on developing metacestodes of Taenia taeniaeformis removed from the livers of rats at times varying from 3 to 35 weeks post infection. Lipid accounted for 7-21% of the dry weight of the parasites. The highest proportions were found at the earlier stages. The distribution was as follows; neutral lipid 27-45%; glycolipid 5-11%; and phospholipid 50-61%. The major neutral lipid was cholesterol, and minor neutral lipids were sterol esters, triglycerides, diglycerides and monoglycerides. Hydrocarbons were present throughout development, but in the highest amounts at the earlier stages. Five different glycolipids were found, all of which were identified as glycosphingolipids. An increase in the proportion of more complex glycolipids was noted as parasites grew older. Ten different phospholipids were identified, with the major components being phosphatidylcholine, phosphatidylethanolamine, and phosphatidylserine. Other phospholipids were: lysophosphatides, phosphatidylinositol, phosphatidic acid, diphosphatidylglycerol, sphingomyelin, and an unknown phospholipid component. Changes in the relative amounts of the two major phospholipids were found when the early and late stages were compared. Two lipids found throughout development were identified as glycosylated dolichol phosphates, and they comprised between 1 and 3% of the total phospholipid fraction. Nineteen fatty acids were detected, and the fatty acid distribution for each lipid class at each stage was determined. Seven major fatty acids were common to each. These were: hexadecanoic, octadecanoic, oleic, linoleic, arachidonic, docosanoic, and docosahexaenoic.

  9. Glycerol-3-phosphate acyltransferase 4 gene is involved in mouse spermatogenesis

    Institute of Scientific and Technical Information of China (English)

    Qingming Qiu; Gang Liu; Weina Li; Qiuwen Shi; Fuxi Zhu; Guangxiu Lu

    2009-01-01

    Glycerol-3-phosphate acyltransferase (GPAT) catalyzes the first committed step of de novo triacylglycerol syn-thesis by converting glycerol-3-phosphate to lysopho-sphatidic acid (LPA). LPA is a mitogen that mediates multiple cellular processes including cell proliferation. Four GPAT isoforms have been cloned to date. GPAT4 is strongly expressed in the mouse testis. Reverse tran-scription-polymerase chain reaction (PCR), real-time PCR, and in situ hybridization (ISH) were used to analyze the GPAT4 expression and to localize the expressing cell types in the mouse testis during post-natal development. GPAT4 cDNA was inserted into pcDNA4/His to construct a recombinant vector, which was transfected into a mouse spermatogonial cell line (GC-lspg). GPAT4 was first expressed in mice at 2 weeks postnatally. Expression was abundant from the third week, plateaued at week 5-6 and then maintained at a high level in the adult. ISH revealed that GPAT4 gene was expressed abundantly in spermatocytes and around spermatids during meiosis but not in elongated spermatids during later spermiogenesis. GC-1spg cells showed a marked increase in proliferation after trans-fection with GPAT4; cell cycle analysis showed a decrease in the percentage of cells in the Go/G1 phase and an increase in the S phase. Thus, GPAT4 might play an important role in spermatogenesis, especially in mid-meiosis.

  10. Lack of Fas/CD95 surface expression in highly proliferative leukemic cell lines correlates with loss of CtBP/BARS and redirection of the protein toward giant lysosomal structures.

    Science.gov (United States)

    Monleón, Inmaculada; Iturralde, María; Martínez-Lorenzo, María José; Monteagudo, Luis; Lasierra, Pilar; Larrad, Luis; Piñeiro, Andrés; Naval, Javier; Alava, María Angeles; Anel, Alberto

    2002-07-01

    Fas/CD95 is a type-I membrane glycoprotein, which inducesapoptotic cell death when ligated by its physiological ligand. We generated previously hyperproliferative sublines derived from the human T-cell leukemia Jurkat, Jurkat-ws and Jurkat-hp, which lost Fas/CD95 surface expression. We have now observed that the total amount of Fas protein is similar in the sublines and in the parental cells, indicating that in the sublines Fas remains in an intracellular compartment. We have found that the protein is directed toward lysosomes in the sublines, where it is degraded. This defect in the secretory pathway correlates with loss of polyunsaturated fatty acids from cellular lipids, and with the lack of expression of endophilin-I and CtBP/BARS, enzymes that regulate vesicle fission by catalyzing the acylation of arachidonate into lysophosphatidic acid. In addition, great multillamer bodies, which contained acid phosphatase activity, absent in the parental Jurkat cells, were observed by transmission electron microscopy in the sublines.

  11. Expression of Castor LPAT2 Enhances Ricinoleic Acid Content at the sn-2 Position of Triacylglycerols in Lesquerella Seed

    Directory of Open Access Journals (Sweden)

    Grace Q. Chen

    2016-04-01

    Full Text Available Lesquerella is a potential industrial oilseed crop that makes hydroxy fatty acid (HFA. Unlike castor its seeds are not poisonous but accumulate lesquerolic acid mostly at the sn-1 and sn-3 positions of triacylglycerol (TAG, whereas castor contains ricinoleic acid (18:1OH at all three positions. To investigate whether lesquerella can be engineered to accumulate HFAs in the sn-2 position, multiple transgenic lines were made that express castor lysophosphatidic acid acyltransferase 2 (RcLPAT2 in the seed. RcLPAT2 increased 18:1OH at the sn-2 position of TAGs from 2% to 14%–17%, which resulted in an increase of tri-HFA-TAGs from 5% to 13%–14%. Our result is the first example of using a LPAT to increase ricinoleic acid at the sn-2 position of seed TAG. This work provides insights to the mechanism of HFA-containing TAG assembly in lesquerella and directs future research to optimize this plant for HFA production.

  12. Targeting the myofibroblast genetic switch: inhibitors of myocardin-related transcription factor/serum response factor-regulated gene transcription prevent fibrosis in a murine model of skin injury.

    Science.gov (United States)

    Haak, Andrew J; Tsou, Pei-Suen; Amin, Mohammad A; Ruth, Jeffrey H; Campbell, Phillip; Fox, David A; Khanna, Dinesh; Larsen, Scott D; Neubig, Richard R

    2014-06-01

    Systemic sclerosis (SSc), or scleroderma, similar to many fibrotic disorders, lacks effective therapies. Current trials focus on anti-inflammatory drugs or targeted approaches aimed at one of the many receptor mechanisms initiating fibrosis. In light of evidence that a myocardin-related transcription factor (MRTF)-and serum response factor (SRF)-regulated gene transcriptional program induced by Rho GTPases is essential for myofibroblast activation, we explored the hypothesis that inhibitors of this pathway may represent novel antifibrotics. MRTF/SRF-regulated genes show spontaneously increased expression in primary dermal fibroblasts from patients with diffuse cutaneous SSc. A novel small-molecule inhibitor of MRTF/SRF-regulated transcription (CCG-203971) inhibits expression of connective tissue growth factor (CTGF), α-smooth muscle actin (α-SMA), and collagen 1 (COL1A2) in both SSc fibroblasts and in lysophosphatidic acid (LPA)-and transforming growth factor β (TGFβ)-stimulated fibroblasts. In vivo treatment with CCG-203971 also prevented bleomycin-induced skin thickening and collagen deposition. Thus, targeting the MRTF/SRF gene transcription pathway could provide an efficacious new approach to therapy for SSc and other fibrotic disorders.

  13. Sphingolipids in human synovial fluid--a lipidomic study.

    Directory of Open Access Journals (Sweden)

    Marta Krystyna Kosinska

    Full Text Available Articular synovial fluid (SF is a complex mixture of components that regulate nutrition, communication, shock absorption, and lubrication. Alterations in its composition can be pathogenic. This lipidomic investigation aims to quantify the composition of sphingolipids (sphingomyelins, ceramides, and hexosyl- and dihexosylceramides and minor glycerophospholipid species, including (lysophosphatidic acid, (lysophosphatidylglycerol, and bis(monoacylglycerophosphate species, in the SF of knee joints from unaffected controls and from patients with early (eOA and late (lOA stages of osteoarthritis (OA, and rheumatoid arthritis (RA. SF without cells and cellular debris from 9 postmortem donors (control, 18 RA, 17 eOA, and 13 lOA patients were extracted to measure lipid species using electrospray ionization tandem mass spectrometry--directly or coupled with hydrophilic interaction liquid chromatography. We provide a novel, detailed overview of sphingolipid and minor glycerophospholipid species in human SF. A total of 41, 48, and 50 lipid species were significantly increased in eOA, lOA, and RA SF, respectively when compared with normal SF. The level of 21 lipid species differed in eOA SF versus SF from lOA, an observation that can be used to develop biomarkers. Sphingolipids can alter synovial inflammation and the repair responses of damaged joints. Thus, our lipidomic study provides the foundation for studying the biosynthesis and function of lipid species in health and most prevalent joint diseases.

  14. Untargeted metabolomics approach for unraveling robust biomarkers of nutritional status in fasted gilthead sea bream (Sparus aurata

    Directory of Open Access Journals (Sweden)

    Ruben Gil-Solsona

    2017-01-01

    Full Text Available A metabolomic study has been performed to identify sensitive and robust biomarkers of malnutrition in farmed fish, using gilthead sea bream (Sparus aurata as a model. The metabolomic fingerprinting of serum from fasted fish was assessed by means of ultra-high performance liquid chromatography coupled to quadrupole time-of-flight mass spectrometry. More than 15,000 different m/z ions were detected and Partial Least Squares–Discriminant analysis allowed a clear differentiation between the two experimental groups (fed and 10-day fasted fish with more than 90% of total variance explained by the two first components. The most significant metabolites (up to 45 were elucidated on the basis of their tandem mass spectra with a broad representation of amino acids, oligopeptides, urea cycle metabolites, L-carnitine-related metabolites, glutathione-related metabolites, fatty acids, lysophosphatidic acids, phosphatidylcholines as well as biotin- and noradrenaline-related metabolites. This untargeted approach highlighted important adaptive responses in energy and oxidative metabolism, contributing to identify robust and nutritionally-regulated biomarkers of health and metabolic condition that will serve to assess the welfare status of farmed fish.

  15. Role of mitochondrial lipids in guiding fission and fusion.

    Science.gov (United States)

    Frohman, Michael A

    2015-03-01

    Clinically important links have been established between mitochondrial function and cardiac physiology and disease in the context of signaling mechanisms, energy production, and muscle cell development. The proteins and processes that drive mitochondrial fusion and fission are now known to have emergent functions in intracellular calcium homeostasis, apoptosis, vascular smooth muscle cell proliferation, myofibril organization, and Notch-driven cell differentiation, all key issues in cardiac disease. Moreover, decreasing fission may confer protection against ischemic heart disease, particularly in the setting of obesity, diabetes, and heart failure. The importance of lipids in controlling mitochondrial fission and fusion is increasingly becoming appreciated. Roles for the bulk and signaling lipids cardiolipin, phosphatidylethanolamine, phosphatidic acid, diacylglycerol, and lysophosphatidic acid and the enzymes that synthesize or metabolize them in the control of mitochondrial shape and function are reviewed here. A number of diseases have been linked to loss-of-function alleles for a subset of the enzymes, emphasizing the importance of the lipid environment in this context.

  16. Compartmentation of hepatic fatty-acid-binding protein in liver cells and its effect on microsomal phosphatidic acid biosynthesis.

    Science.gov (United States)

    Bordewick, U; Heese, M; Börchers, T; Robenek, H; Spener, F

    1989-03-01

    Fatty-acid-binding proteins are known to occur in the cytosol of mammalian cells and to bind fatty acids and their CoA-esters. Application of the postembedding protein A-gold labeling method with antibody against the hepatic type fatty-acid-binding protein (hFABP) to cross-sections of liver cells and a newly developed gel-chromatographic immunofluorescence assay established qualitatively (1) that hFABP in mitochondria was confined to outer mitochondrial membranes, (2) the presence of this protein in microsomes and (3) that nuclei were also filled with hFABP. Quantitative data elaborated with a non-competitive ELISA confirmed these results. A significant difference to the distribution of cardiac FABP in heart muscle cells, where this type of protein was found in cytosol, matrix and nuclei, was observed (Börchers et al. (1989) Biochim. Biophys. Acta, in the press). hFABP-containing rat liver microsomes were incubated with long-chain acyl-CoAs in the presence of hFABP (isolated from rat liver cytosol) in a study on the acylation of sn-glycerol-3-phosphate and lysophosphatidic acid. Both acyltransferases were stimulated by addition of hFABP to the incubation medium. The morphological, immunochemical as well as kinetic data infer a direct interaction of hFABP with microsomal membranes in liver cells.

  17. Characterization of a small acyl-CoA-binding protein (ACBP) from Helianthus annuus L. and its binding affinities.

    Science.gov (United States)

    Aznar-Moreno, Jose A; Venegas-Calerón, Mónica; Du, Zhi-Yan; Garcés, Rafael; Tanner, Julian A; Chye, Mee-Len; Martínez-Force, Enrique; Salas, Joaquín J

    2016-05-01

    Acyl-CoA-binding proteins (ACBPs) bind to acyl-CoA esters and promote their interaction with other proteins, lipids and cell structures. Small class I ACBPs have been identified in different plants, such as Arabidopsis thaliana (AtACBP6), Brassica napus (BnACBP) and Oryza sativa (OsACBP1, OsACBP2, OsACBP3), and they are capable of binding to different acyl-CoA esters and phospholipids. Here we characterize HaACBP6, a class I ACBP expressed in sunflower (Helianthus annuus) tissues, studying the specificity of its corresponding recombinant HaACBP6 protein towards various acyl-CoA esters and phospholipids in vitro, particularly using isothermal titration calorimetry and protein phospholipid binding assays. This protein binds with high affinity to de novo synthetized derivatives palmitoly-CoA, stearoyl-CoA and oleoyl-CoA (Kd 0.29, 0.14 and 0.15 μM respectively). On the contrary, it showed lower affinity towards linoleoyl-CoA (Kd 5.6 μM). Moreover, rHaACBP6 binds to different phosphatidylcholine species (dipalmitoyl-PC, dioleoyl-PC and dilinoleoyl-PC), yet it displays no affinity towards other phospholipids like lyso-PC, phosphatidic acid and lysophosphatidic acid derivatives. In the light of these results, the possible involvement of this protein in sunflower oil synthesis is considered.

  18. Autotaxin-mediated lipid signaling intersects with LIF and BMP signaling to promote the naive pluripotency transcription factor program

    Science.gov (United States)

    Kime, Cody; Sakaki-Yumoto, Masayo; Goodrich, Leeanne; Hayashi, Yohei; Sami, Salma; Derynck, Rik; Asahi, Michio; Panning, Barbara; Yamanaka, Shinya; Tomoda, Kiichiro

    2016-01-01

    Developmental signaling molecules are used for cell fate determination, and understanding how their combinatorial effects produce the variety of cell types in multicellular organisms is a key problem in biology. Here, we demonstrate that the combination of leukemia inhibitory factor (LIF), bone morphogenetic protein 4 (BMP4), lysophosphatidic acid (LPA), and ascorbic acid (AA) efficiently converts mouse primed pluripotent stem cells (PSCs) into naive PSCs. Signaling by the lipid LPA through its receptor LPAR1 and downstream effector Rho-associated protein kinase (ROCK) cooperated with LIF signaling to promote this conversion. BMP4, which also stimulates conversion to naive pluripotency, bypassed the need for exogenous LPA by increasing the activity of the extracellular LPA-producing enzyme autotaxin (ATX). We found that LIF and LPA-LPAR1 signaling affect the abundance of signal transducer and activator of transcription 3 (STAT3), which induces a previously unappreciated Kruppel-like factor (KLF)2-KLF4-PR domain 14 (PRDM14) transcription factor circuit key to establish naive pluripotency. AA also affects this transcription factor circuit by controlling PRDM14 expression. Thus, our study reveals that ATX-mediated autocrine lipid signaling promotes naive pluripotency by intersecting with LIF and BMP4 signaling. PMID:27738243

  19. Enhancement of migration and invasion of hepatoma cells via a Rho GTPase signaling pathway

    Institute of Scientific and Technical Information of China (English)

    De-Sheng Wang; Ke-Feng Dou; Kai-Zong Li; Zhen-Shun Song

    2004-01-01

    AIM: Intrahepatic extension is the main cause of liver failure and death in hepatocellular carcinoma patients. The small GTPase Rho and one of its effector molecules ROCK regulate cytoskeleton and actomyosin contractility, and play a crucial role in cell adhesion and motility. We investigated the role of small GTPase Rho in biological behaviors of hepatocellular carcinoma to demonstrate the importance of Rho in cancer invasion and metastasis.METHODS: Using Western blotting, we quantitated Rho protein expression in SMMC-7721 cells induced by Lysophosphatidic acid (LPA). Furthermore, we examined the role of Rho signaling in regulating the motile and invasiveproperties of tumor cells.RESULTS: Rho protein expression was stimulated by LPA.Using the Rhotekin binding assay to assess Rho activation,we observed that the level of GTP-bound Rho was elevated transiently after the addition of LPA, and Y-27632 decreased the level of active Rho. LPA enhanced the motility of tumor cells and facilitated their invasion. Rho played an essential role in the migratory process, as evidenced by the inhibition of migration and motility of cancer cells by a specific inhibitor of ROCK, Y-27632.CONCLUSION: The finding that invasiveness of hepatocellular carcinoma is facilitated by the Rho/Rho-kinase pathway is likely to be relevant to tumor progression and Y-27632 may be a new potential effective agent for the prevention of intrahepatic extension of human liver cancer.

  20. Cellular responses to chlorin-based photosensitizer DH-II-24 under darkness in human gastric adenocarcinoma AGS cells.

    Science.gov (United States)

    Lim, Young-Cheol; Yoo, Je-Ok; Kang, Seong-Sik; Kim, Young-Myeong; Ha, Kwon-Soo

    2011-03-01

    We investigated cellular responses to chlorin-based photosensitizer DH-II-24 under darkness in human gastric adenocarcinoma AGS cells. Cells were loaded with 0.5-10 μg/mL DH-II-24 for 12 h, and intracellular reactive oxygen species (ROS) and intracellular Ca(2+) levels, in situ tissue transglutaminase (tTGase) activity, cell viability, cell morphology and cell cycle were examined. DH-II-24 treatment had no effect on intracellular ROS production or cell morphology, and did not induce cell detachment at any concentrations tested. In addition, cell viability and cell cycle progression were not altered by the photosensitizer. However, DH-II-24 treatment elevated the basal level of intracellular Ca(2+) in a dose-dependent manner and inhibited tTGase activity without affecting tTGase expression levels. Furthermore, DH-II-24 inhibited lysophosphatidic acid-induced activation of tTGase in a dose-dependent manner. In contrast, photodynamic therapy (PDT) with 1 μg/mL DH-II-24 significantly elevated intracellular ROS and in situ tTGase activity in parallel with a rapid and large increase in intracellular Ca(2+) levels. DH-II-24-mediated PDT decreased cell viability and induced cell detachment. These results demonstrate that DH-II-24 treatment alone under darkness induced different cellular responses to DH-II-24-mediated PDT.

  1. Fc-receptor-mediated phagocytosis is regulated by mechanical properties of the target

    Science.gov (United States)

    Beningo, Karen A.; Wang, Yu-li

    2002-01-01

    Phagocytosis is an actin-based process used by macrophages to clear particles greater than 0.5 microm in diameter. In addition to its role in immunological responses, phagocytosis is also necessary for tissue remodeling and repair. To prevent catastrophic autoimmune reactions, phagocytosis must be tightly regulated. It is commonly assumed that the recognition/selection of phagocytic targets is based solely upon receptor-ligand binding. Here we report an important new criterion, that mechanical parameters of the target can dramatically affect the efficiency of phagocytosis. When presented with particles of identical chemical properties but different rigidity, macrophages showed a strong preference to engulf rigid objects. Furthermore, phagocytosis of soft particles can be stimulated with the microinjection of constitutively active Rac1 but not RhoA, and with lysophosphatidic acid, an agent known to activate the small GTP-binding proteins of the Rho family. These data suggest a Rac1-dependent mechanosensory mechanism for phagocytosis, which probably plays an important role in a number of physiological and pathological processes from embryonic development to autoimmune diseases.

  2. 内皮分化基因受体介导溶血磷脂酸:肿瘤靶向治疗的新靶点%Edg/LPAR mediated LPA: A novel target for tumor targeting therapy

    Institute of Scientific and Technical Information of China (English)

    龚涌灵; Frank FANG

    2011-01-01

    溶血磷脂酸(lysophosphatidic acid,LPA)是一种对多种细胞具有不同生物学活性的磷脂介质,在组织中广泛存在.从细胞形态学改变到细胞功能的影响,LPA可产生如促进细胞增殖、迁移和耐药等生物学效应.如其他生物递质一样,LPA与细胞表面特定的G蛋白偶合受体(Gprotein-coupled receptor,GPCR)发生交联作用,这些受体主要有Edg-2/LPA1、Edg-4/LPA2和Edg-7/LPA3等,它们被命名为内皮分化基因或溶血磷脂受体亚家族(endothelial differentiation gene or lysophospholipid receptor subfamily,Edg/LPAR subfamily).LPA在体内外参与细胞增殖以及血管生成等病理生理过程,LPA代谢和Edg/LPA受体功能的异常与肿瘤的发生、发展相关,可能是肿瘤临床诊治的潜在靶点.本文阐述了LPA通过Edg/LPA受体介导在肿瘤发生和发展中的作用及其机制,并就其在胰腺癌临床诊治中的意义进行了评价.%Lysophosphatidic acid (LPA) is a naturally occurring phospholipid with diverse effects on various cells,ranging from cellular morphology alterations to cellular function changes such as induction of cell proliferation, survival, drug resistance and motility. Like many other biomediators, LPA interacts with cells through specific cell surface receptors (G protein-coupled receptors). Edg-2/LPA1, Edg-4/LPA2 and Edg-7/LPA3, named as endothelial differentiation gene or lysophospholipidic receptor subfamily (Edg/LPA subfamily) , are three most common LPA receptors. LPA plays a critical role as a general growth, survival and pro-angiogenic factor in the regulation of pathophysiological processes in vivo and in vitro. Recent reports in the literature suggest that abnormalities in LPA metabolism and Edg/LPA receptors function in cancer patients may contribute to the development and progression of the disease. Thus, LPA and its receptors might be potential targets for clinical cancer diagnosis and therapy. Herein we review the function and mechanism

  3. Fear extinction and acute stress reactivity reveal a role of LPA(1) receptor in regulating emotional-like behaviors.

    Science.gov (United States)

    Pedraza, C; Sánchez-López, J; Castilla-Ortega, E; Rosell-Valle, C; Zambrana-Infantes, E; García-Fernández, M; Rodriguez de Fonseca, F; Chun, J; Santín, L J; Estivill-Torrús, G

    2014-09-01

    LPA1 receptor is one of the six characterized G protein-coupled receptors (LPA1-6) through which lysophosphatidic acid acts as an intercellular signaling molecule. It has been proposed that this receptor has a role in controlling anxiety-like behaviors and in the detrimental consequences of stress. Here, we sought to establish the involvement of the LPA1 receptor in emotional regulation. To this end, we examined fear extinction in LPA1-null mice, wild-type and LPA1 antagonist-treated animals. In LPA1-null mice we also characterized the morphology and GABAergic properties of the amygdala and the medial prefrontal cortex. Furthermore, the expression of c-Fos protein in the amygdala and the medial prefrontal cortex, and the corticosterone response following acute stress were examined in both genotypes. Our data indicated that the absence of the LPA1 receptor significantly inhibited fear extinction. Treatment of wild-type mice with the LPA1 antagonist Ki16425 mimicked the behavioral phenotype of LPA1-null mice, revealing that the LPA1 receptor was involved in extinction. Immunohistochemistry studies revealed a reduction in the number of neurons, GABA+ cells, calcium-binding proteins and the volume of the amygdala in LPA1-null mice. Following acute stress, LPA1-null mice showed increased corticosterone and c-Fos expression in the amygdala. In conclusion, LPA1 receptor is involved in emotional behaviors and in the anatomical integrity of the corticolimbic circuit, the deregulation of which may be a susceptibility factor for anxiety disorders and a potential therapeutic target for the treatment of these diseases.

  4. Review of the Third Domain Receptor Binding Fragment of Alpha-fetoprotein (AFP): Plausible Binding of AFP to Lysophospholipid Receptor Targets.

    Science.gov (United States)

    Mizejewski, G J

    2016-01-31

    Alpha-fetoprotein (AFP) is a 69 kD fetal- and tumor-associated single-chain glycoprotein belonging to the albuminoid gene family. AFP functions as a carrier/transport molecule as well as a growth regulator and has been utilized as a clinical biomarker for both fetal defects and cancer growth. Lysophospholipids (LPLs) are plasma membrane-derived bioactive lipid signaling mediators composed of a small molecular weight single acyl carbon chain (palmitic, oleic acid) attached to a polar headgroup; they range in molecular mass from 250-750 daltons. The LPLs consist of either sphingosine-1-phosphate or lysophosphatidic acid, and mostly their choline, ethanolamine, serine or inositol derivatives. They are present only in vertebrates. These bioactive paracrine lipid mediators are ubiquitously distributed in tissues and are released from many different cell types (platelets, macrophages, monocytes, etc.) involved in developmental, physiological, and pathological processes. The LPLs bind to four different classes of G-protein coupled receptors described herein which transduce a multiple of cell effects encompassing activities such as morphogenesis, neural development, angiogenesis, and carcinogenesis. The identification of potential binding sites of LPL receptors on the AFP third domain receptor binding fragment were derived by computer modeling analysis. It is conceivable, but not proven, that AFP might bind not only to the LPL receptors, but also to LPLs themselves since AFP binds medium and long chain fatty acids. It is proposed that some of the activities ascribed to AFP in the past might be due in part to the presence of bound LPLs and/or their receptors.

  5. Kaempferol suppresses lipid accumulation by inhibiting early adipogenesis in 3T3-L1 cells and zebrafish.

    Science.gov (United States)

    Lee, Yeon-Joo; Choi, Hyeon-Son; Seo, Min-Jung; Jeon, Hui-Jeon; Kim, Kui-Jin; Lee, Boo-Yong

    2015-08-01

    Kaempferol is a flavonoid present in Kaempferia galanga and Opuntia ficus indica var. saboten. Recent studies have suggested that it has anti-oxidant, anti-inflammatory, anti-cancer, and anti-obesity effects. In this study, we focused on the anti-adipogenic effects of kaempferol during adipocyte differentiation. The results showed that kaempferol inhibits lipid accumulation in adipocytes and zebrafish. Oil Red O and Nile Red staining showed that the number of intracellular lipid droplets decreased in adipocytes and zebrafish treated with kaempferol. LPAATθ (lysophosphatidic acid acyltransferase), lipin1, and DGAT1 (triglyceride synthetic enzymes) and FASN and SREBP-1C (fatty acid synthetic proteins) showed decreased expression levels in the presence of kaempferol. In addition, treatment of kaempferol showed an inhibitory activity on cell cycle progression. Kaempferol delayed cell cycle progression from the S to G2/M phase through the regulation of cyclins in a dose-dependent manner. Kaempferol blocked the phosphorylation of AKT (protein kinase B) and mammalian target of rapamycin (mTOR) signaling pathway during the early stages of adipogenesis. In addition, kaempferol down-regulated pro-early adipogenic factors such as CCAAT-enhancer binding proteins β (C/EBPβ), and Krüppel-like factors (KLFs) 4 and 5, while anti-early adipogenic factors, such as KLF2 and pref-1(preadipocyte factor-1), were upregulated. These kaempferol-mediated regulations of early adipogenic factors resulted in the attenuation of late adipogenic factors such as C/EBPα and peroxisome proliferator-activated receptor γ (PPARγ). These results were supported in zebrafish based on the decrease in lipid accumulation and expression of adipogenic factors. Our results indicated that kaempferol might have an anti-obesity effect by regulating lipid metabolism.

  6. Receptor-mediated stimulation of lipid signalling pathways in CHO cells elicits the rapid transient induction of the PDE1B isoform of Ca2+/calmodulin-stimulated cAMP phosphodiesterase.

    Science.gov (United States)

    Spence, S; Rena, G; Sullivan, M; Erdogan, S; Houslay, M D

    1997-01-01

    Chinese hamster ovary cells (CHO cells) do not exhibit any Ca2+/calmodulin-stimulated cAMP phosphodiesterase (PDE1) activity. Challenge of CHO cells with agonists for endogenous P2-purinoceptors, lysophosphatidic acid receptors and thrombin receptors caused a similar rapid transient induction of PDE1 activity in each instance. This was also evident on noradrenaline challenge of a cloned CHO cell line transfected so as to overexpress alpha 1B-adrenoceptors. This novel PDE1 activity appeared within about 15 min of exposure to ligands, rose to a maximum value within 30 min to 1 h and then rapidly decreased. In each case, the expression of novel PDE1 activity was blocked by the transcriptional inhibitor actinomycin D. Challenge with insulin of either native CHO cells or a CHO cell line transfected so as to overexpress the human insulin receptor failed to induce PDE1 activity. Reverse transcriptase-PCR analyses, using degenerate primers able to detect the PDE1C isoform, did not amplify any fragment from RNA preparations of CHO cells expressing PDE1 activity, although they did so from the human thyroid carcinoma FTC133 cell line. Reverse transcriptase-PCR analyses, using degenerate primers able to detect the PDE1A and PDE1B isoforms, successfully amplified a fragment of the predicted size from RNA preparations of both CHO cells expressing PDE1 activity and human Jurkat T-cells. Sequencing of the PCR products, generated using the PDE1A/B primers, yielded a novel sequence which, by analogy with sequences reported for bovine and murine PDE1B forms, suggests that the PDE1 species induced in CHO cells through protein kinase C activation and that expressed in Jurkat T-cells are PDE1B forms.

  7. Association between Promoter Hypomethylation and Overexpression of Autotaxin with Outcome Parameters in Biliary Atresia

    Science.gov (United States)

    Udomsinprasert, Wanvisa; Kitkumthorn, Nakarin; Mutirangura, Apiwat; Chongsrisawat, Voranush; Poovorawan, Yong; Honsawek, Sittisak

    2017-01-01

    Objective Biliary atresia (BA) is a progressive fibroinflammatory liver disease. Autotaxin (ATX) has a profibrotic effect resulting from lysophosphatidic acid activity. The purpose of this study was to examine ATX expression and ATX promoter methylation in peripheral blood leukocytes and liver tissues from BA patients and controls and investigate their associations with outcome parameters in BA patients. Methods A total of 130 subjects (65 BA patients and 65 age-matched controls) were enrolled. DNA was extracted from circulating leukocytes and liver tissues of BA patients and from and age-matched controls. ATX promoter methylation status was determined by bisulfite pyrosequencing. ATX expression was analyzed using quantitative real-time polymerase chain reaction and enzyme-linked immunosorbent assay. Results Decreased methylation of specific CpGs were observed at the ATX promoter in BA patients. Subsequent analysis revealed that BA patients with advanced stage had lower methylation levels of ATX promoter than those with early stage. ATX promoter methylation levels were found to be associated with hepatic dysfunction in BA. In addition, ATX expression was significantly elevated and correlated with a decrease in ATX promoter methylation in BA patients compared to the controls. Furthermore, promoter hypomethylation and overexpression of ATX were inversely associated with jaundice status, hepatic dysfunction, and liver stiffness in BA patients. Conclusion Accordingly, it has been hypothesized that ATX promoter methylation and ATX expression in peripheral blood may serve as possible biomarkers reflecting the progression of liver fibrosis in postoperative BA. These findings suggest that the promoter hypomethylation and overexpression of ATX might play a contributory role in the pathogenesis of liver fibrosis in BA. PMID:28052132

  8. Lysophospholipid Growth Factors and Their G Protein-Coupled Receptors in Immunity, Coronary Artery Disease, and Cancer

    Directory of Open Access Journals (Sweden)

    Edward J. Goetzl

    2002-01-01

    Full Text Available The physiological lysophospholipids (LPLs, exemplified by lysophosphatidic acid (LPA and sphingosine 1-phosphate (S1P, are omnific mediators of normal cellular proliferation, survival, and functions. Although both LPA and S1P attain micromolar concentrations in many biological fluids, numerous aspects of their biosynthesis, transport, and metabolic degradation are unknown. Eight members of a new subfamily of G protein-coupled LPA/S1P receptors, originally termed Edg Rs, bind either LPA or S1P with high affinity and transduce a series of growth-related and/or cytoskeleton-based functional responses. The most critical areas of LPL biology and pathobiology are neural development and neurodegeneration, immunity, atherosclerosis and myocardial injury, and cancer. Data from analyses of T cells established two basic points: (1 the plasticity and adaptability of expression of LPA/S1P Rs by some cells as a function of activation, and (2 the role of opposing signals from two different receptors for the same ligand as a mechanism for fine control of effects of LPLs. In the heart, LPLs may promote coronary atherosclerosis, but are effectively cytoprotective for hypoxic cardiac myocytes and those exposed to oxygen free radicals. The findings of production of LPA by some types of tumor cells, overexpression of selected sets of LPA receptors by the same tumor cells, and augmentation of the effects of protein growth factors by LPA have suggested pathogenetic roles for the LPLs in cancer. The breadth of physiologic and pathologic activities of LPLs emphasizes the importance of developing bioavailable nonlipid agonists and antagonists of the LPA/S1P receptors for diverse therapeutic applications.

  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. Experimental studies of relationship between phospholipid level and Yangxueqingnao particles in transient cerebral ischemia attack%短暂性脑缺血发作磷脂水平与养血清脑颗粒相关的实验研究

    Institute of Scientific and Technical Information of China (English)

    牛建平; 薛萍; 伍期专; 李丹; 姚存姗; 王燕华; 包鹤丘

    2003-01-01

    AIM: To investigate the effect of transient ischemia attack(TIA) on phospho-lipid level and the effect of traditional Chinese herb - Yangxueqingnao par-ticles. METHODS: To establish the animal model of TIA in mice by injectingPeroxide through caudal vein, and then feed the mice with Yangxueqingnaoparticles in different dose. After evoking 4 times of TIA and cut off the headthe other day, then measure the level of lysophosphatidic acid(LPA), generalphospholipidin blood plasma and brain tissue. RESULTS: Score of the groupwithout treatment was 1.0±0.8, while that of group administered withYangxueqingnao particles(2 g/kg) was 0.5±0.4(P<0.05) LPA level ofTIA mice were 6. 1:1±0.4and 2.1±1.3 and increased significantly com-pared with that of the control.This changes can be reversed by treatment as canbe seen the result 4.9±1.6. general phospholipid level of TIA mice in braintissue decreased significantly compared with that of the control as the result:(2.3±1.1) and (6.6±2.8) μmol/g, respectively. After treatment, generalphopholipid level can reach (4.3±2.0) μmol/g.CONCLUSION: LPA andgeneral phospholipid levehin blood plasma and brain tissue are abnorma] inexperimental TIA mice, but can be reversed by Yangxueqingnao particles andit can be used in treatment.

  11. Ectodomain cleavage of the EGF ligands HB-EGF, neuregulin1-beta, and TGF-alpha is specifically triggered by different stimuli and involves different PKC isoenzymes.

    Science.gov (United States)

    Herrlich, Andreas; Klinman, Eva; Fu, Jonathan; Sadegh, Cameron; Lodish, Harvey

    2008-12-01

    Metalloproteinase cleavage of transmembrane proteins (ectodomain cleavage), including the epidermal growth factor (EGF) ligands heparin-binding EGF-like growth factor (HB-EGF), neuregulin (NRG), and transforming growth factor-alpha (TGF-alpha), is important in many cellular signaling pathways and is disregulated in many diseases. It is largely unknown how physiological stimuli of ectodomain cleavage--hypertonic stress, phorbol ester, or activation of G-protein-coupled receptors [e.g., by lysophosphatidic acid (LPA)]--are molecularly connected to metalloproteinase activation. To study this question, we developed a fluorescence-activated cell sorting (FACS)- based assay that measures cleavage of EGF ligands in single living cells. EGF ligands expressed in mouse lung epithelial cells are differentially and specifically cleaved depending on the stimulus. Inhibition of protein kinase C (PKC) isoenzymes or metalloproteinase inhibition by batimastat (BB94) showed that different regulatory signals are used by different stimuli and EGF substrates, suggesting differential effects that act on the substrate, the metalloproteinase, or both. For example, hypertonic stress led to strong cleavage of HB-EGF and NRG but only moderate cleavage of TGF-alpha. HB-EGF, NRG, and TGF-alpha cleavage was not dependent on PKC, and only HB-EGF and NRG cleavage were inhibited by BB94. In contrast, phorbol 12-myristate-13-acetate (TPA) -induced cleavage of HB-EGF, NRG, and TGF-alpha was dependent on PKC and sensitive to BB94 inhibition. LPA led to significant cleavage of only NRG and TGF-alpha and was inhibited by BB94; only LPA-induced NRG cleavage required PKC. Surprisingly, specific inhibition of atypical PKCs zeta and iota [not activated by diacylglycerol (DAG) and calcium] significantly enhanced TPA-induced NRG cleavage. Employed in a high-throughput cloning strategy, our cleavage assay should allow the identification of candidate proteins involved in signal transduction of different

  12. Epidermal growth factor (EGF) ligand release by substrate-specific a disintegrin and metalloproteases (ADAMs) involves different protein kinase C (PKC) isoenzymes depending on the stimulus.

    Science.gov (United States)

    Dang, Michelle; Dubbin, Karen; D'Aiello, Antonio; Hartmann, Monika; Lodish, Harvey; Herrlich, Andreas

    2011-05-20

    The dysregulation of EGF family ligand cleavage has severe consequences for the developing as well as the adult organism. Therefore, their production is highly regulated. The limiting step is the ectodomain cleavage of membrane-bound precursors by one of several a disintegrin and metalloprotease (ADAM) metalloproteases, and understanding the regulation of cleavage is an important goal of current research. We have previously reported that in mouse lung epithelial cells, the pro-EGF ligands TGFα, neuregulin 1β (NRG), and heparin-binding EGF are differentially cleaved depending on the cleavage stimulus (Herrlich, A., Klinman, E., Fu, J., Sadegh, C., and Lodish, H. (2008) FASEB J.). In this study in mouse embryonic fibroblasts that lack different ADAMs, we show that induced cleavage of EGF ligands can involve the same substrate-specific metalloprotease but does require different stimulus-dependent signaling pathways. Cleavage was stimulated by phorbol ester (12-O-tetradecanoylphorbol-13-acetate (TPA), a mimic of diacylglycerol and PKC activator), hypertonic stress, lysophosphatidic acid (LPA)-induced G protein-coupled receptor activation, or by ionomycin-induced intracellular calcium release. Although ADAMs showed substrate preference (ADAM17, TGFα and heparin-binding EGF; and ADAM9, NRG), substrate cleavage differed substantially with the stimulus, and cleavage of the same substrate depended on the presence of different, sometimes multiple, PKC isoforms. For instance, classical PKC was required for TPA-induced but not hypertonic stress-induced cleavage of all EGF family ligands. Inhibition of PKCζ enhanced NRG release upon TPA stimulation, but it blocked NRG release in response to hypertonic stress. Our results suggest a model in which substantial regulation of ectodomain cleavage occurs not only on the metalloprotease level but also on the level of the substrate or of a third protein.

  13. Enhancement of endothelial cell migration by constitutively active LPA{sub 1}-expressing tumor cells

    Energy Technology Data Exchange (ETDEWEB)

    Kitayoshi, Misaho; Kato, Kohei; Tanabe, Eriko; Yoshikawa, Kyohei; Fukui, Rie [Division of Cancer Biology and Bioinformatics, Department of Life Science, Faculty of Science and Engineering, Kinki University, 3-4-1, Kowakae, Higashiosaka, Osaka 577-8502 (Japan); Fukushima, Nobuyuki [Division of Molecular Neurobiology, Department of Life Science, Faculty of Science and Engineering, Kinki University, 3-4-1, Kowakae, Higashiosaka, Osaka 577-8502 (Japan); Tsujiuchi, Toshifumi, E-mail: ttujiuch@life.kindai.ac.jp [Division of Cancer Biology and Bioinformatics, Department of Life Science, Faculty of Science and Engineering, Kinki University, 3-4-1, Kowakae, Higashiosaka, Osaka 577-8502 (Japan)

    2012-06-01

    Highlights: Black-Right-Pointing-Pointer Mutated LPA{sub 1} stimulates cell migration of endothelial cells. Black-Right-Pointing-Pointer VEGF expressions are increased by mutated LPA{sub 1}. Black-Right-Pointing-Pointer LPA signaling via mutated LPA{sub 1} is involved in angiogenesis. Black-Right-Pointing-Pointer Mutated LPA{sub 1} promotes cancer cell progression. -- Abstract: Lysophosphatidic acid (LPA) receptors belong to G protein-coupled transmembrane receptors (LPA receptors; LPA{sub 1} to LPA{sub 6}). They indicate a variety of cellular response by the interaction with LPA, including cell proliferation, migration and differentiation. Recently, we have reported that constitutive active mutated LPA{sub 1} induced the strong biological effects of rat neuroblastoma B103 cells. In the present study, we examined the effects of mutated LPA{sub 1} on the interaction between B103 cells and endothelial F-2 cells. Each LPA receptor expressing B103 cells were maintained in serum-free DMEM and cell motility assay was performed with a Cell Culture Insert. When F-2 cells were cultured with conditioned medium from Lpar1 and Lpar3-expressing cells, the cell motility of F-2 cells was significantly higher than control cells. Interestingly, the motile activity of F-2 cells was strongly induced by mutated LPA{sub 1} than other cells, correlating with the expression levels of vascular endothelial growth factor (Vegf)-A and Vegf-C. Pretreatment of LPA signaling inhibitors inhibited F-2 cell motility stimulated by mutated LPA{sub 1}. These results suggest that activation of LPA signaling via mutated LPA{sub 1} may play an important role in the promotion of angiogenesis in rat neuroblastoma cells.

  14. Platelet lipidomic.

    Science.gov (United States)

    Dolegowska, B; Lubkowska, A; De Girolamo, L

    2012-01-01

    Lipids account for 16-19 percent dry platelet matter and includes 65 percent phospholipids, 25 percent neutral lipids and about 8 percent glycosphingolipids. The cell membrane that surrounds platelets is a bilayer that contains different types phospholipids symmetrically distributed in resting platelets, such as phosphatidylserine (PS), phosphatidylethanolamine (PE), phosphatidylcholine, and sphingomyelin. The collapse of lipid asymmetry is exposure of phosphatidylserine in the external leaflet of the plasma bilayer, where it is known to serve at least two major functions: providing a platform for development of the blood coagulation cascade and presenting the signal that induces phagocytosis of apoptotic cells. During activation, this asymmetrical distribution becomes disrupted, and PS and PE become exposed on the cell surface. The transbilayer movement of phosphatidylserine is responsible for the platelet procoagulant activity. Exposure of phosphatidylserine is a flag for macrophage recognition and clearance from the circulation. Platelets, stored at room temperature for transfusion for more than 5 days, undergo changes collectively known as platelet storage lesions. Thus, the platelet lipid composition and its possible modifications over time are crucial for efficacy of platelet rich plasma therapy. Moreover, a number of substances derived from lipids are contained into platelets. Eicosanoids are lipid signaling mediators generated by the action of lipoxygenase and include prostaglandins, thromboxane A2, 12-hydroxyeicosatetraenoic acid. Isoprostanes have a chemical structure similar to this of prostanoids, but are differently produced into the particle, and are ligands for prostaglandins receptors, exhibiting biological activity like thromboxane A2. Endocannabinoids are derivatives from arachidonic acid which could reduce local pain. Phospholipids growth factors (sphingolipids, lysophosphatidic acid, platelet-activating factor) are involved in tissue

  15. Krüppel-like factor 5 incorporates into the β-catenin/TCF complex in response to LPA in colon cancer cells.

    Science.gov (United States)

    Guo, Leilei; He, Peijian; No, Yi Ran; Yun, C Chris

    2015-05-01

    Lysophosphatidic acid (LPA) is a simple phospholipid with potent mitogenic effects on various cells including colon cancer cells. LPA stimulates proliferation of colon cancer cells by activation of β-catenin or Krüppel-like factor 5 (KLF5), but the functional relationship between these two transcription factors is not clear. Hence, we sought to investigate the mechanism of β-catenin activation by LPA and the role of KLF5 in the regulation of β-catenin by LPA. We found that LPA and Wnt3 additively activated the β-catenin/TCF (T cell factor) reporter activity in HCT116 cells. In addition to phosphorylating glycogen synthase kinase 3β (GSK-3β) at Ser9, LPA resulted in phosphorylation of β-catenin at Ser552 and Ser675. Mutation of Ser552 and Ser675 ablated LPA-induced β-catenin/TCF transcriptional activity. Knockdown of KLF5 significantly attenuated activation of β-catenin/TCF reporter activity by LPA but not by Wnt3. However, nuclear accumulation of β-catenin by LPA was not altered by knockdown of KLF5. β-catenin, TCF, and KLF5 were present in a 250-300kDa macro-complex, and their presence was enhanced by LPA. LPA simulated the interaction of β-catenin with TCF4, and depletion of KLF5 decreased β-catenin-TCF4 association and the transcriptional activity. In summary, LPA activates β-catenin by multiple pathways involving phosphorylation of GSK-3 and β-catenin, and enhancing β-catenin interaction with TCF4. KLF5 plays a critical role in β-catenin activation by increasing the β-catenin-TCF4 interaction.

  16. Targeting melanoma growth and viability reveals dualistic functionality of the phosphonothionate analogue of carba cyclic phosphatidic acid

    Directory of Open Access Journals (Sweden)

    Prestwich Glenn D

    2010-06-01

    Full Text Available Abstract Background Although the incidence of melanoma in the U.S. is rising faster than any other cancer, the FDA-approved chemotherapies lack efficacy for advanced disease, which results in poor overall survival. Lysophosphatidic acid (LPA, autotaxin (ATX, the enzyme that produces LPA, and the LPA receptors represent an emerging group of therapeutic targets in cancer, although it is not known which of these is most effective. Results Herein we demonstrate that thio-ccPA 18:1, a stabilized phosphonothionate analogue of carba cyclic phosphatidic acid, ATX inhibitor and LPA1/3 receptor antagonist, induced a marked reduction in the viability of B16F10 metastatic melanoma cells compared with PBS-treated control by 80-100%. Exogenous LPA 18:1 or D-sn-1-O-oleoyl-2-O-methylglyceryl-3-phosphothioate did not reverse the effect of thio-ccPA 18:1. The reduction in viability mediated by thio-ccPA 18:1 was also observed in A375 and MeWo melanoma cell lines, suggesting that the effects are generalizable. Interestingly, siRNA to LPA3 (siLPA3 but not other LPA receptors recapitulated the effects of thio-ccPA 18:1 on viability, suggesting that inhibition of the LPA3 receptor is an important dualistic function of the compound. In addition, siLPA3 reduced proliferation, plasma membrane integrity and altered morphology of A375 cells. Another experimental compound designed to antagonize the LPA1/3 receptors significantly reduced viability in MeWo cells, which predominantly express the LPA3 receptor. Conclusions Thus the ability of thio-ccPA 18:1 to inhibit the LPA3 receptor and ATX are key to its molecular mechanism, particularly in melanoma cells that predominantly express the LPA3 receptor. These observations necessitate further exploration and exploitation of these targets in melanoma.

  17. Reassessing the Potential Activities of Plant CGI-58 Protein.

    Science.gov (United States)

    Khatib, Abdallah; Arhab, Yani; Bentebibel, Assia; Abousalham, Abdelkarim; Noiriel, Alexandre

    2016-01-01

    Comparative Gene Identification-58 (CGI-58) is a widespread protein found in animals and plants. This protein has been shown to participate in lipolysis in mice and humans by activating Adipose triglyceride lipase (ATGL), the initial enzyme responsible for the triacylglycerol (TAG) catabolism cascade. Human mutation of CGI-58 is the cause of Chanarin-Dorfman syndrome, an orphan disease characterized by a systemic accumulation of TAG which engenders tissue disorders. The CGI-58 protein has also been shown to participate in neutral lipid metabolism in plants and, in this case, a mutation again provokes TAG accumulation. Although its roles as an ATGL coactivator and in lipid metabolism are quite clear, the catalytic activity of CGI-58 is still in question. The acyltransferase activities of CGI-58 have been speculated about, reported or even dismissed and experimental evidence that CGI-58 expressed in E. coli possesses an unambiguous catalytic activity is still lacking. To address this problem, we developed a new set of plasmids and site-directed mutants to elucidate the in vivo effects of CGI-58 expression on lipid metabolism in E. coli. By analyzing the lipid composition in selected E. coli strains expressing CGI-58 proteins, and by reinvestigating enzymatic tests with adequate controls, we show here that recombinant plant CGI-58 has none of the proposed activities previously described. Recombinant plant and mouse CGI-58 both lack acyltransferase activity towards either lysophosphatidylglycerol or lysophosphatidic acid to form phosphatidylglycerol or phosphatidic acid and recombinant plant CGI-58 does not catalyze TAG or phospholipid hydrolysis. However, expression of recombinant plant CGI-58, but not mouse CGI-58, led to a decrease in phosphatidylglycerol in all strains of E. coli tested, and a mutation of the putative catalytic residues restored a wild-type phenotype. The potential activities of plant CGI-58 are subsequently discussed.

  18. Design of serum-free medium for suspension culture of CHO cells on the basis of general commercial media.

    Science.gov (United States)

    Miki, Hideo; Takagi, Mutsumi

    2015-08-01

    The design of serum-free media for suspension culture of genetically engineered Chinese hamster ovary (CHO) cells using general commercial media as a basis was investigated. Subcultivation using a commercial serum-free medium containing insulin-like growth factor (IGF)-1 with or without FCS necessitated additives other than IGF-1 to compensate for the lack of FCS and improve cell growth. Suspension culture with media containing several combinations of growth factors suggested the effectiveness of addition of both IGF-1 and the lipid signaling molecule lysophosphatidic acid (LPA) for promoting cell growth. Subcultivation of CHO cells in suspension culture using the commercial serum-free medium EX-CELL™302, which contained an IGF-1 analog, supplemented with LPA resulted in gradually increasing specific growth rate comparable to the serum-containing medium and in almost the same high antibody production regardless of the number of generations. The culture with EX-CELL™302 supplemented with LPA in a jar fermentor with pH control at 6.9 showed an apparently higher cell growth rate than the cultures without pH control and with pH control at 6.8. The cell growth in the medium supplemented with aurintricarboxylic acid (ATA), which was much cheaper than IGF-1, in combination with LPA was synergistically promoted similarly to that in the medium supplemented with IGF-1 and LPA. In conclusion, the serum-free medium designed on the basis of general commercial media could support the growth of CHO cells and antibody production comparable to serum-containing medium in suspension culture. Moreover, the possibility of cost reduction by the substitution of IGF-1 with ATA was also shown.

  19. Melanoma cells break down LPA to establish local gradients that drive chemotactic dispersal.

    Directory of Open Access Journals (Sweden)

    Andrew J Muinonen-Martin

    2014-10-01

    Full Text Available The high mortality of melanoma is caused by rapid spread of cancer cells, which occurs unusually early in tumour evolution. Unlike most solid tumours, thickness rather than cytological markers or differentiation is the best guide to metastatic potential. Multiple stimuli that drive melanoma cell migration have been described, but it is not clear which are responsible for invasion, nor if chemotactic gradients exist in real tumours. In a chamber-based assay for melanoma dispersal, we find that cells migrate efficiently away from one another, even in initially homogeneous medium. This dispersal is driven by positive chemotaxis rather than chemorepulsion or contact inhibition. The principal chemoattractant, unexpectedly active across all tumour stages, is the lipid agonist lysophosphatidic acid (LPA acting through the LPA receptor LPAR1. LPA induces chemotaxis of remarkable accuracy, and is both necessary and sufficient for chemotaxis and invasion in 2-D and 3-D assays. Growth factors, often described as tumour attractants, cause negligible chemotaxis themselves, but potentiate chemotaxis to LPA. Cells rapidly break down LPA present at substantial levels in culture medium and normal skin to generate outward-facing gradients. We measure LPA gradients across the margins of melanomas in vivo, confirming the physiological importance of our results. We conclude that LPA chemotaxis provides a strong drive for melanoma cells to invade outwards. Cells create their own gradients by acting as a sink, breaking down locally present LPA, and thus forming a gradient that is low in the tumour and high in the surrounding areas. The key step is not acquisition of sensitivity to the chemoattractant, but rather the tumour growing to break down enough LPA to form a gradient. Thus the stimulus that drives cell dispersal is not the presence of LPA itself, but the self-generated, outward-directed gradient.

  20. Gossypol increases expression of the pro-apoptotic BH3-only protein NOXA through a novel mechanism involving phospholipase A2, cytoplasmic calcium, and endoplasmic reticulum stress.

    Science.gov (United States)

    Soderquist, Ryan S; Danilov, Alexey V; Eastman, Alan

    2014-06-06

    Gossypol is a putative BH3 mimetic proposed to inhibit BCL2 and BCLXL based on cell-free assays. We demonstrated previously that gossypol failed to directly inhibit BCL2 in cells or induce apoptosis in chronic lymphocytic leukemia (CLL) cells or platelets, which require BCL2 or BCLXL, respectively, for survival. Here, we demonstrate that gossypol rapidly increased activity of phospholipase A2 (PLA2), which led to an increase in cytoplasmic calcium, endoplasmic reticulum (ER) stress, and up-regulation of the BH3-only protein NOXA. Pretreatment with the PLA2 inhibitor, aristolochic acid, abrogated the increase in calcium, ER stress, and NOXA. Calcium chelation also abrogated the gossypol-induced increase in calcium, ER stress, and NOXA, but not the increase in PLA2 activity, indicating that PLA2 is upstream of these events. In addition, incubating cells with the two products of PLA2 (lysophosphatidic acid and arachidonic acid) mimicked treatment with gossypol. NOXA is a pro-apoptotic protein that functions by binding the BCL2 family proteins MCL1 and BFL1. The BCL2 inhibitor ABT-199 is currently in clinical trials for CLL. Resistance to ABT-199 can occur from up-regulation of other BCL2 family proteins, and this resistance can be mimicked by culturing CLL cells on CD154(+) stroma cells. We report here that AT-101, a derivative of gossypol in clinical trials, overcomes stroma-mediated resistance to ABT-199 in primary CLL cells, suggesting that a combination of these drugs may be efficacious in the clinic.

  1. To the Root of the Curl: A Signature of a Recent Selective Sweep Identifies a Mutation That Defines the Cornish Rex Cat Breed.

    Directory of Open Access Journals (Sweden)

    Barbara Gandolfi

    Full Text Available The cat (Felis silvestris catus shows significant variation in pelage, morphological, and behavioral phenotypes amongst its over 40 domesticated breeds. The majority of the breed specific phenotypic presentations originated through artificial selection, especially on desired novel phenotypic characteristics that arose only a few hundred years ago. Variations in coat texture and color of hair often delineate breeds amongst domestic animals. Although the genetic basis of several feline coat colors and hair lengths are characterized, less is known about the genes influencing variation in coat growth and texture, especially rexoid - curly coated types. Cornish Rex is a cat breed defined by a fixed recessive curly coat trait. Genome-wide analyses for selection (di, Tajima's D and nucleotide diversity were performed in the Cornish Rex breed and in 11 phenotypically diverse breeds and two random bred populations. Approximately 63K SNPs were used in the analysis that aimed to localize the locus controlling the rexoid hair texture. A region with a strong signature of recent selective sweep was identified in the Cornish Rex breed on chromosome A1, as well as a consensus block of homozygosity that spans approximately 3 Mb. Inspection of the region for candidate genes led to the identification of the lysophosphatidic acid receptor 6 (LPAR6. A 4 bp deletion in exon 5, c.250_253_delTTTG, which induces a premature stop codon in the receptor, was identified via Sanger sequencing. The mutation is fixed in Cornish Rex, absent in all straight haired cats analyzed, and is also segregating in the German Rex breed. LPAR6 encodes a G protein-coupled receptor essential for maintaining the structural integrity of the hair shaft; and has mutations resulting in a wooly hair phenotype in humans.

  2. Association of genetic loci with sleep apnea in European Americans and African-Americans: the Candidate Gene Association Resource (CARe.

    Directory of Open Access Journals (Sweden)

    Sanjay R Patel

    Full Text Available Although obstructive sleep apnea (OSA is known to have a strong familial basis, no genetic polymorphisms influencing apnea risk have been identified in cross-cohort analyses. We utilized the National Heart, Lung, and Blood Institute (NHLBI Candidate Gene Association Resource (CARe to identify sleep apnea susceptibility loci. Using a panel of 46,449 polymorphisms from roughly 2,100 candidate genes on a customized Illumina iSelect chip, we tested for association with the apnea hypopnea index (AHI as well as moderate to severe OSA (AHI≥15 in 3,551 participants of the Cleveland Family Study and two cohorts participating in the Sleep Heart Health Study.Among 647 African-Americans, rs11126184 in the pleckstrin (PLEK gene was associated with OSA while rs7030789 in the lysophosphatidic acid receptor 1 (LPAR1 gene was associated with AHI using a chip-wide significance threshold of p-value<2×10(-6. Among 2,904 individuals of European ancestry, rs1409986 in the prostaglandin E2 receptor (PTGER3 gene was significantly associated with OSA. Consistency of effects between rs7030789 and rs1409986 in LPAR1 and PTGER3 and apnea phenotypes were observed in independent clinic-based cohorts.Novel genetic loci for apnea phenotypes were identified through the use of customized gene chips and meta-analyses of cohort data with replication in clinic-based samples. The identified SNPs all lie in genes associated with inflammation suggesting inflammation may play a role in OSA pathogenesis.

  3. Amyloid beta protein inhibits cellular MTT reduction not by suppression of mitochondrial succinate dehydrogenase but by acceleration of MTT formazan exocytosis in cultured rat cortical astrocytes.

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    Abe, K; Saito, H

    1998-08-01

    Alzheimer's disease amyloid beta protein (Abeta) inhibits cellular reduction of the dye 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT). Kaneko et al. have previously hypothesized that Abeta works by suppressing mitochondrial succinate dehydrogenase (SDH), but Liu and Schubert have recently demonstrated that Abeta decreases cellular MTT reduction by accelerating the exocytosis of MTT formazan in neuronal cells. To ask which is the case in astrocytes, we compared the effects of Abeta and 3-nitropropionic acid (3-NP), a specific SDH inhibitor, on MTT reduction in cultured rat cortical astrocytes. Treatment with 3-NP (10 mM) decreased cellular activity of MTT reduction, regardless of the time of incubation with MTT. On the other hand. Abeta-induced inhibition of cellular MTT reduction was dependent on the time of incubation with MTT. The cells treated with Abeta (0.1-1000 nM) exhibited normal capacity for MTT reduction at an early stage of incubation ( 1 h). Microscopic examination revealed that Abeta treatment accelerated the appearance of needle-like MTT formazan crystals at the cell surface. These observations support that Abeta accelerates the exocytosis of MTT formazan in astrocytes. In addition to inhibition of MTT reduction, Abeta is known to induce morphological changes in astrocytes. Following addition of Abeta (20 microM), polygonal astrocytes changed into process-bearing stellate cells. To explore a possible linkage between these two effects of Abeta, we tested if astrocyte stellation is induced by agents that mimic the effect of Abeta on MTT reduction. Cholesterol (5 5000 nM) and lysophosphatidic acid (0.2-20 microg/ml) were found to accelerate the exocytosis of MTT formazan in a similar manner to Abeta, but failed to induce astrocyte stellation. Therefore, Abeta-induced inhibition of MTT reduction is unlikely to be directly linked to its effect on astrocyte morphology.

  4. C3 toxin activates the stress signaling pathways, JNK and p38, but antagonizes the activation of AP-1 in rat-1 cells.

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    Beltman, J; Erickson, J R; Martin, G A; Lyons, J F; Cook, S J

    1999-02-05

    Lysophosphatidic acid (LPA) stimulates the c-Fos serum response element (SRE) by activating two distinct signal pathways regulated by the small GTPases, Ras and RhoA. Ras activates the ERK cascade leading to phosphorylation of the transcription factors Elk-1 and Sap1a at the Ets/TCF site. RhoA regulates an undefined pathway required for the activation of the SRF/CArG site. Here we have examined the role of the Ras and RhoA pathways in activation of the SRE and c-Fos expression in Rat-1 cells. Pertussis toxin and PD98059 strongly inhibited LPA-stimulated c-Fos expression and activation of a SRE:Luc reporter. C3 toxin completely inhibited RhoA function, partially inhibited SRE:Luc activity, but had no effect on LPA-stimulated c-Fos expression. Thus, in a physiological context the Ras-Raf-MEK-ERK pathway, but not RhoA, is required for LPA-stimulated c-Fos expression in Rat-1 cells. C3 toxin stimulated the stress-activated protein kinases JNK and p38 and potentiated c-Jun expression and phosphorylation; these properties were shared by another cellular stress agonist the protein kinase C inhibitor Ro-31-8220. However, C3 toxin alone or in combination with growth factors did not stimulate AP-1:Luc activity and actually antagonized the synergistic activation of AP-1:Luc observed in response to co-stimulation with growth factors and Ro-31-8220. These data indicate that C3 toxin is a cellular stress which antagonizes activation of AP-1 at a point downstream of stress-activated kinase activation or immediate-early gene induction.

  5. 24R,25-dihydroxyvitamin D3 [24R,25(OH)2D3] controls growth plate development by inhibiting apoptosis in the reserve zone and stimulating response to 1alpha,25(OH)2D3 in hypertrophic cells.

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    Boyan, B D; Hurst-Kennedy, J; Denison, T A; Schwartz, Z

    2010-07-01

    Previously we showed that costochondral growth plate resting zone (RC) chondrocytes response primarily to 24R,25(OH)2D3 whereas prehypertrophic and hypertrophic (GC) cells respond to 1alpha,25(OH)2D3. 24R,25(OH)2D3 increases RC cell proliferation and inhibits activity of matrix processing enzymes, suggesting it stabilizes cells in the reserve zone, possibly by inhibiting the matrix degradation characteristic of apoptotic hypertrophic GC cells. To test this, apoptosis was induced in rat RC cells by treatment with exogenous inorganic phosphate (Pi). 24R,25(OH)2D3 blocked apoptotic effects in a dose-dependent manner. Similarly, apoptosis was induced in ATDC5 cell cultures and 24R,25(OH)2D3 blocked this effect. Further studies indicated that 24R,25(OH)2D3 acts via at least two independent pathways. 24R,25(OH)2D3 increases LPA receptor-1 (LPA R1) expression and production of lysophosphatidic acid (LPA), and subsequent LPA R1/3-dependent signaling, thereby decreasing p53 abundance. LPA also increases the Bcl-2/Bax ratio. In addition, 24R,25(OH)2D3 acts by increasing PKC activity. 24R,25(OH)2D3 stimulates 1-hydroxylase activity, resulting in increased levels of 1,25(OH)2D3, and it increases levels of phospholipase A2 activating protein, which is required for rapid 1alpha,25(OH)2D3-dependent activation of PKC in GC cells. These results suggest that 24R,25(OH)2D3 modulates growth plate development by controlling the rate and extent of RC chondrocyte transition to a GC chondrocyte phenotype.

  6. Phospholipase C-η1 is activated by intracellular Ca(2+) mobilization and enhances GPCRs/PLC/Ca(2+) signaling.

    Science.gov (United States)

    Kim, Jung Kuk; Choi, Jung Woong; Lim, Seyoung; Kwon, Ohman; Seo, Jeong Kon; Ryu, Sung Ho; Suh, Pann-Ghill

    2011-06-01

    Phospholipase C-η1 (PLC-η1) is the most recently identified PLC isotype and is primarily expressed in nerve tissue. However, its functional role is unclear. In the present study, we report for the first time that PLC-η1 acts as a signal amplifier in G protein-coupled receptor (GPCR)-mediated PLC and Ca(2+) signaling. Short-hairpin RNA (shRNA)-mediated knockdown of endogenous PLC-η1 reduced lysophosphatidic acid (LPA)-, bradykinin (BK)-, and PACAP-induced PLC activity in mouse neuroblastoma Neuro2A (N2A) cells, indicating that PLC-η1 participates in GPCR-mediated PLC activation. Interestingly, ionomycin-induced PLC activity was significantly decreased by PLC-η1, but not PLC-η2, knockdown. In addition, we found that intracellular Ca(2+) source is enough for PLC-η1 activation. Furthermore, the IP(3) receptor inhibitor, 2-APB, inhibited LPA-induced PLC activity in control N2A cells, whereas this effect was not observed in PLC-η1 knockdown N2A cells, suggesting a pivotal role of intracellular Ca(2+) mobilization in PLC-η1 activation. Finally, we found that LPA-induced ERK1/2 phosphorylation and expression of the downstream target gene, krox-24, were significantly decreased by PLC-η1 knockdown, and these knockdown effects were abolished by 2-APB. Taken together, our results strongly suggest that PLC-η1 is activated via intracellular Ca(2+) mobilization from the ER, and therefore amplifies GPCR-mediated signaling.<