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Sample records for acetyl-coa carboxylases accs

  1. Inhibition of Acetyl-CoA Carboxylase 1 (ACC1) and 2 (ACC2) Reduces Proliferation and De Novo Lipogenesis of EGFRvIII Human Glioblastoma Cells

    PubMed Central

    Jones, Jessica E. C.; Esler, William P.; Patel, Rushi; Lanba, Adhiraj; Vera, Nicholas B.; Pfefferkorn, Jeffrey A.; Vernochet, Cecile

    2017-01-01

    Tumor cell proliferation and migration processes are regulated by multiple metabolic pathways including glycolysis and de novo lipogenesis. Since acetyl-CoA carboxylase (ACC) is at the junction of lipids synthesis and oxidative metabolic pathways, we investigated whether use of a dual ACC inhibitor would provide a potential therapy against certain lipogenic cancers. The impact of dual ACC1/ACC2 inhibition was investigated using a dual ACC1/ACC2 inhibitor as well as dual siRNA knock down on the cellular viability and metabolism of two glioblastoma multiform cancer cell lines, U87 and a more aggressive form, U87 EGFRvIII. We first demonstrated that while ACCi inhibited DNL in both cell lines, ACCi preferentially blunted the U87 EGFRvIII cellular proliferation capacity. Metabolically, chronic treatment with ACCi significantly upregulated U87 EGFRvIII cellular respiration and extracellular acidification rate, a marker of glycolytic activity, but impaired mitochondrial health by reducing maximal respiration and decreasing mitochondrial ATP production efficiency. Moreover, ACCi treatment altered the cellular lipids content and increased apoptotic caspase activity in U87 EGFRvIII cells. Collectively these data indicate that ACC inhibition, by reducing DNL and increasing cellular metabolic rate, may have therapeutic utility for the suppression of lipogenic tumor growth and warrants further investigation. PMID:28081256

  2. The glossyhead1 Allele of ACC1 Reveals a Principal Role for Multidomain Acetyl-Coenzyme A Carboxylase in the Biosynthesis of Cuticular Waxes by Arabidopsis

    SciTech Connect

    Lu, S.; Xu, C.; Zhao, H.; Parsons, E. P.; Kosma, D. K.; Xu, X.; Chao, D.; Lohrey, G.; Bangarusamy, D. K.; Wang, G.; Bressan, R. A.; Jenks, M. A.

    2011-11-01

    A novel mutant of Arabidopsis (Arabidopsis thaliana), having highly glossy inflorescence stems, postgenital fusion in floral organs, and reduced fertility, was isolated from an ethyl methanesulfonate-mutagenized population and designated glossyhead1 (gsd1). The gsd1 locus was mapped to chromosome 1, and the causal gene was identified as a new allele of Acetyl-Coenzyme A Carboxylase1 (ACC1), a gene encoding the main enzyme in cytosolic malonyl-coenzyme A synthesis. This, to our knowledge, is the first mutant allele of ACC1 that does not cause lethality at the seed or early germination stage, allowing for the first time a detailed analysis of ACC1 function in mature tissues. Broad lipid profiling of mature gsd1 organs revealed a primary role for ACC1 in the biosynthesis of the very-long-chain fatty acids (C{sub 20:0} or longer) associated with cuticular waxes and triacylglycerols. Unexpectedly, transcriptome analysis revealed that gsd1 has limited impact on any lipid metabolic networks but instead has a large effect on environmental stress-responsive pathways, especially senescence and ethylene synthesis determinants, indicating a possible role for the cytosolic malonyl-coenzyme A-derived lipids in stress response signaling.

  3. Potential Functional Replacement of the Plastidic Acetyl-CoA Carboxylase Subunit (accD) Gene by Recent Transfers to the Nucleus in Some Angiosperm Lineages1[W][OA

    PubMed Central

    Rousseau-Gueutin, Mathieu; Huang, Xun; Higginson, Emily; Ayliffe, Michael; Day, Anil; Timmis, Jeremy N.

    2013-01-01

    Eukaryotic cells originated when an ancestor of the nucleated cell engulfed bacterial endosymbionts that gradually evolved into the mitochondrion and the chloroplast. Soon after these endosymbiotic events, thousands of ancestral prokaryotic genes were functionally transferred from the endosymbionts to the nucleus. This process of functional gene relocation, now rare in eukaryotes, continues in angiosperms. In this article, we show that the chloroplastic acetyl-CoA carboxylase subunit (accD) gene that is present in the plastome of most angiosperms has been functionally relocated to the nucleus in the Campanulaceae. Surprisingly, the nucleus-encoded accD transcript is considerably smaller than the plastidic version, consisting of little more than the carboxylase domain of the plastidic accD gene fused to a coding region encoding a plastid targeting peptide. We verified experimentally the presence of a chloroplastic transit peptide by showing that the product of the nuclear accD fused to green fluorescent protein was imported in the chloroplasts. The nuclear gene regulatory elements that enabled the erstwhile plastidic gene to become functional in the nuclear genome were identified, and the evolution of the intronic and exonic sequences in the nucleus is described. Relocation and truncation of the accD gene is a remarkable example of the processes underpinning endosymbiotic evolution. PMID:23435694

  4. Comparison of the A-Cc curve fitting methods in determining maximum ribulose 1.5-bisphosphate carboxylase/oxygenase carboxylation rate, potential light saturated electron transport rate and leaf dark respiration.

    PubMed

    Miao, Zewei; Xu, Ming; Lathrop, Richard G; Wang, Yufei

    2009-02-01

    A review of the literature revealed that a variety of methods are currently used for fitting net assimilation of CO2-chloroplastic CO2 concentration (A-Cc) curves, resulting in considerable differences in estimating the A-Cc parameters [including maximum ribulose 1.5-bisphosphate carboxylase/oxygenase (Rubisco) carboxylation rate (Vcmax), potential light saturated electron transport rate (Jmax), leaf dark respiration in the light (Rd), mesophyll conductance (gm) and triose-phosphate utilization (TPU)]. In this paper, we examined the impacts of fitting methods on the estimations of Vcmax, Jmax, TPU, Rd and gm using grid search and non-linear fitting techniques. Our results suggested that the fitting methods significantly affected the predictions of Rubisco-limited (Ac), ribulose 1,5-bisphosphate-limited (Aj) and TPU-limited (Ap) curves and leaf photosynthesis velocities because of the inconsistent estimate of Vcmax, Jmax, TPU, Rd and gm, but they barely influenced the Jmax : Vcmax, Vcmax : Rd and Jmax : TPU ratio. In terms of fitting accuracy, simplicity of fitting procedures and sample size requirement, we recommend to combine grid search and non-linear techniques to directly and simultaneously fit Vcmax, Jmax, TPU, Rd and gm with the whole A-Cc curve in contrast to the conventional method, which fits Vcmax, Rd or gm first and then solves for Vcmax, Jmax and/or TPU with V(cmax), Rd and/or gm held as constants.

  5. Structure and function of biotin-dependent carboxylases

    PubMed Central

    Tong, Liang

    2012-01-01

    Biotin-dependent carboxylases include acetyl-CoA carboxylase (ACC), propionyl-CoA carboxylase (PCC), 3-methylcrotonyl-CoA carboxylase (MCC), geranyl-CoA carboxylase (GCC), pyruvate carboxylase (PC), and urea carboxylase (UC). They contain biotin carboxylase (BC), carboxyltransferase (CT) and biotin-carboxyl carrier protein (BCCP) components. These enzymes are widely distributed in nature and have important functions in fatty acid metabolism, amino acid metabolism, carbohydrate metabolism, polyketide biosynthesis, urea utilization, and other cellular processes. ACCs are also attractive targets for drug discovery against type 2 diabetes, obesity, cancer, microbial infections, and other diseases, and the plastid ACC of grasses is the target of action of three classes of commercial herbicides. Deficiencies in the activities of PCC, MCC or PC are linked to serious diseases in humans. Our understanding of these enzymes has been greatly enhanced over the past few years by the crystal structures of the holoenzymes of PCC, MCC, PC, and UC. The structures reveal unanticipated features in the architectures of the holoenzymes, including the presence of previously unrecognized domains, and provide a molecular basis for understanding their catalytic mechanism as well as the large collection of disease-causing mutations in PCC, MCC and PC. This review will summarize the recent advances in our knowledge on the structure and function of these important metabolic enzymes. PMID:22869039

  6. Acc homoeoloci and the evolution of wheat genomes

    Technology Transfer Automated Retrieval System (TEKTRAN)

    We analyzed the DNA sequences of BACs from many wheat libraries containing the Acc-1 and Acc-2 loci, encoding the plastid and cytosolic forms of the enzyme acetyl-CoA carboxylase, to gain understanding of the evolution of these genes and the origin of the three genomes in modern hexaploid wheat. Mor...

  7. The dynamic organization of fungal acetyl-CoA carboxylase

    PubMed Central

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

    2016-01-01

    Acetyl-CoA carboxylases (ACCs) catalyse the committed step in fatty-acid biosynthesis: the ATP-dependent carboxylation of acetyl-CoA to malonyl-CoA. They are important regulatory hubs for metabolic control and relevant drug targets for the treatment of the metabolic syndrome and cancer. Eukaryotic ACCs are single-chain multienzymes characterized by a large, non-catalytic central domain (CD), whose role in ACC regulation remains poorly characterized. Here we report the crystal structure of the yeast ACC CD, revealing a unique four-domain organization. A regulatory loop, which is phosphorylated at the key functional phosphorylation site of fungal ACC, wedges into a crevice between two domains of CD. Combining the yeast CD structure with intermediate and low-resolution data of larger fragments up to intact ACCs provides a comprehensive characterization of the dynamic fungal ACC architecture. In contrast to related carboxylases, large-scale conformational changes are required for substrate turnover, and are mediated by the CD under phosphorylation control. PMID:27073141

  8. The dynamic organization of fungal acetyl-CoA carboxylase

    NASA Astrophysics Data System (ADS)

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

    2016-04-01

    Acetyl-CoA carboxylases (ACCs) catalyse the committed step in fatty-acid biosynthesis: the ATP-dependent carboxylation of acetyl-CoA to malonyl-CoA. They are important regulatory hubs for metabolic control and relevant drug targets for the treatment of the metabolic syndrome and cancer. Eukaryotic ACCs are single-chain multienzymes characterized by a large, non-catalytic central domain (CD), whose role in ACC regulation remains poorly characterized. Here we report the crystal structure of the yeast ACC CD, revealing a unique four-domain organization. A regulatory loop, which is phosphorylated at the key functional phosphorylation site of fungal ACC, wedges into a crevice between two domains of CD. Combining the yeast CD structure with intermediate and low-resolution data of larger fragments up to intact ACCs provides a comprehensive characterization of the dynamic fungal ACC architecture. In contrast to related carboxylases, large-scale conformational changes are required for substrate turnover, and are mediated by the CD under phosphorylation control.

  9. Adaptive Cruise Control (ACC)

    NASA Astrophysics Data System (ADS)

    Reif, Konrad

    Die adaptive Fahrgeschwindigkeitsregelung (ACC, Adaptive Cruise Control) ist eine Weiterentwicklung der konventionellen Fahrgeschwindigkeitsregelung, die eine konstante Fahrgeschwindigkeit einstellt. ACC überwacht mittels eines Radarsensors den Bereich vor dem Fahrzeug und passt die Geschwindigkeit den Gegebenheiten an. ACC reagiert auf langsamer vorausfahrende oder einscherende Fahrzeuge mit einer Reduzierung der Geschwindigkeit, sodass der vorgeschriebene Mindestabstand zum vorausfahrenden Fahrzeug nicht unterschritten wird. Hierzu greift ACC in Antrieb und Bremse ein. Sobald das vorausfahrende Fahrzeug beschleunigt oder die Spur verlässt, regelt ACC die Geschwindigkeit wieder auf die vorgegebene Sollgeschwindigkeit ein (Bild 1). ACC steht somit für eine Geschwindigkeitsregelung, die sich dem vorausfahrenden Verkehr anpasst.

  10. Inhibitors of Pyruvate Carboxylase

    PubMed Central

    Zeczycki, Tonya N.; Maurice, Martin St.; Attwood, Paul V.

    2010-01-01

    This review aims to discuss the varied types of inhibitors of biotin-dependent carboxylases, with an emphasis on the inhibitors of pyruvate carboxylase. Some of these inhibitors are physiologically relevant, in that they provide ways of regulating the cellular activities of the enzymes e.g. aspartate and prohibitin inhibition of pyruvate carboxylase. Most of the inhibitors that will be discussed have been used to probe various aspects of the structure and function of these enzymes. They target particular parts of the structure e.g. avidin – biotin, FTP – ATP binding site, oxamate – pyruvate binding site, phosphonoacetate – binding site of the putative carboxyphosphate intermediate. PMID:22180764

  11. Synthesis and structure-activity relationships of N-{3-[2-(4-alkoxyphenoxy)thiazol-5-yl]-1- methylprop-2-ynyl}carboxy derivatives as selective acetyl-CoA carboxylase 2 inhibitors.

    PubMed

    Gu, Yu Gui; Weitzberg, Moshe; Clark, Richard F; Xu, Xiangdong; Li, Qun; Zhang, Tianyuan; Hansen, T Matthew; Liu, Gang; Xin, Zhili; Wang, Xiaojun; Wang, Rongqi; McNally, Teresa; Zinker, Bradley A; Frevert, Ernst U; Camp, Heidi S; Camp, Heidi; Beutel, Bruce A; Sham, Hing L

    2006-06-29

    A structurally novel acetyl-CoA carboxylase (ACC) inhibitor is identified from high-throughput screening. A preliminary structure-activity relationship study led to the discovery of potent dual ACC1/ACC2 and ACC2 selective inhibitors against human recombinant ACC1 and ACC2. Selective ACC2 inhibitors exhibited IC50<20 nM and >1000-fold selectivity against ACC1. (S)-Enantiomer 9p exhibited high ACC2 activity and lowered muscle malonyl-CoA dose-dependently in acute rodent studies, whereas (R)-enantiomer 9o was weak and had no effect on the malonyl-CoA level.

  12. Piperazine oxadiazole inhibitors of acetyl-CoA carboxylase.

    PubMed

    Bourbeau, Matthew P; Siegmund, Aaron; Allen, John G; Shu, Hong; Fotsch, Christopher; Bartberger, Michael D; Kim, Ki-Won; Komorowski, Renee; Graham, Melissa; Busby, James; Wang, Minghan; Meyer, James; Xu, Yang; Salyers, Kevin; Fielden, Mark; Véniant, Murielle M; Gu, Wei

    2013-12-27

    Acetyl-CoA carboxylase (ACC) is a target of interest for the treatment of metabolic syndrome. Starting from a biphenyloxadiazole screening hit, a series of piperazine oxadiazole ACC inhibitors was developed. Initial pharmacokinetic liabilities of the piperazine oxadiazoles were overcome by blocking predicted sites of metabolism, resulting in compounds with suitable properties for further in vivo studies. Compound 26 was shown to inhibit malonyl-CoA production in an in vivo pharmacodynamic assay and was advanced to a long-term efficacy study. Prolonged dosing with compound 26 resulted in impaired glucose tolerance in diet-induced obese (DIO) C57BL6 mice, an unexpected finding.

  13. The influence of AccD5 on AccD6 carboxyltransferase essentiality in pathogenic and non-pathogenic Mycobacterium

    PubMed Central

    Pawelczyk, Jakub; Viljoen, Albertus; Kremer, Laurent; Dziadek, Jaroslaw

    2017-01-01

    Malonyl-coenzyme A (CoA) is a crucial extender unit for the synthesis of mycolic and other fatty acids in mycobacteria, generated in a reaction catalyzed by acetyl-CoA carboxylase. We previously reported on the essentiality of accD6Mtb encoding the functional acetyl-CoA carboxylase subunit in Mycobacterium tuberculosis. Strikingly, the homologous gene in the fast-growing, non-pathogenic Mycobacterium smegmatis - (accD6Msm) appeared to be dispensable, and its deletion did not influence the cell lipid content. Herein, we demonstrate that, despite the difference in essentiality, accD6Msm and accD6Mtb encode proteins of convergent catalytic activity in vivo. To identify an alternative, AccD6-independent, malonyl-CoA synthesis pathway in M. smegmatis, a complex genetic approach combined with lipid analysis was applied to screen all five remaining carboxyltransferase genes (accD1-accD5) with respect to their involvement in mycolic acid biosynthesis and ability to utilize acetyl-CoA as the substrate for carboxylation. This approach revealed that AccD1Msm, AccD2Msm and AccD3Msm are not essential for mycolic acid biosynthesis. Furthermore, we confirmed in vivo the function of AccD4Msm as an essential, long-chain acyl-CoA carboxyltransferase, unable to carboxylate short-chain substrate. Finally, our comparative studies unambiguously demonstrated between-species difference in in vivo ability of AccD5 carboxyltransferase to utilize acetyl-CoA that influences AccD6 essentiality in pathogenic and non-pathogenic mycobacteria. PMID:28205597

  14. Discovery of Small Molecule Isozyme Non-specific Inhibitors of Mammalian Acetyl-CoA Carboxylase 1 and 2

    SciTech Connect

    Corbett, J.; Freeman-Cook, K; Elliott, R; Vajdos, F; Rajamohan, F; Kohls, D; Marr, E; Harwood Jr., H; Esler, W; et al.

    2010-01-01

    Screening Pfizer's compound library resulted in the identification of weak acetyl-CoA carboxylase inhibitors, from which were obtained rACC1 CT-domain co-crystal structures. Utilizing HTS hits and structure-based drug discovery, a more rigid inhibitor was designed and led to the discovery of sub-micromolar, spirochromanone non-specific ACC inhibitors. Low nanomolar, non-specific ACC-isozyme inhibitors that exhibited good rat pharmacokinetics were obtained from this chemotype.

  15. Discovery of small molecule isozyme non-specific inhibitors of mammalian acetyl-CoA carboxylase 1 and 2.

    PubMed

    Corbett, Jeffrey W; Freeman-Cook, Kevin D; Elliott, Richard; Vajdos, Felix; Rajamohan, Francis; Kohls, Darcy; Marr, Eric; Zhang, Hailong; Tong, Liang; Tu, Meihua; Murdande, Sharad; Doran, Shawn D; Houser, Janet A; Song, Wei; Jones, Christopher J; Coffey, Steven B; Buzon, Leanne; Minich, Martha L; Dirico, Kenneth J; Tapley, Susan; McPherson, R Kirk; Sugarman, Eliot; Harwood, H James; Esler, William

    2010-04-01

    Screening Pfizer's compound library resulted in the identification of weak acetyl-CoA carboxylase inhibitors, from which were obtained rACC1 CT-domain co-crystal structures. Utilizing HTS hits and structure-based drug discovery, a more rigid inhibitor was designed and led to the discovery of sub-micromolar, spirochromanone non-specific ACC inhibitors. Low nanomolar, non-specific ACC-isozyme inhibitors that exhibited good rat pharmacokinetics were obtained from this chemotype.

  16. A unified molecular mechanism for the regulation of acetyl-CoA carboxylase by phosphorylation

    PubMed Central

    Wei, Jia; Zhang, Yixiao; Yu, Tai-Yuan; Sadre-Bazzaz, Kianoush; Rudolph, Michael J; Amodeo, Gabriele A; Symington, Lorraine S; Walz, Thomas; Tong, Liang

    2016-01-01

    Acetyl-CoA carboxylases (ACCs) are crucial metabolic enzymes and attractive targets for drug discovery. Eukaryotic acetyl-CoA carboxylases are 250 kDa single-chain, multi-domain enzymes and function as dimers and higher oligomers. Their catalytic activity is tightly regulated by phosphorylation and other means. Here we show that yeast ACC is directly phosphorylated by the protein kinase SNF1 at residue Ser1157, which potently inhibits the enzyme. Crystal structure of three ACC central domains (AC3–AC5) shows that the phosphorylated Ser1157 is recognized by Arg1173, Arg1260, Tyr1113 and Ser1159. The R1173A/R1260A double mutant is insensitive to SNF1, confirming that this binding site is crucial for regulation. Electron microscopic studies reveal dramatic conformational changes in the holoenzyme upon phosphorylation, likely owing to the dissociation of the biotin carboxylase domain dimer. The observations support a unified molecular mechanism for the regulation of ACC by phosphorylation as well as by the natural product soraphen A, a potent inhibitor of eukaryotic ACC. These molecular insights enhance our understanding of acetyl-CoA carboxylase regulation and provide a basis for drug discovery. PMID:27990296

  17. Dimerization of the Bacterial Biotin Carboxylase Subunit Is Required for Acetyl Coenzyme A Carboxylase Activity In Vivo

    PubMed Central

    Smith, Alexander C.

    2012-01-01

    Acetyl coenzyme A (acteyl-CoA) carboxylase (ACC) is the first committed enzyme of the fatty acid synthesis pathway. Escherichia coli ACC is composed of four different proteins. The first enzymatic activity of the ACC complex, biotin carboxylase (BC), catalyzes the carboxylation of the protein-bound biotin moiety of another subunit with bicarbonate in an ATP-dependent reaction. Although BC is found as a dimer in cell extracts and the carboxylase activities of the two subunits of the dimer are interdependent, mutant BC proteins deficient in dimerization are reported to retain appreciable activity in vitro (Y. Shen, C. Y. Chou, G. G. Chang, and L. Tong, Mol. Cell 22:807–818, 2006). However, in vivo BC must interact with the other proteins of the complex, and thus studies of the isolated BC may not reflect the intracellular function of the enzyme. We have tested the abilities of three BC mutant proteins deficient in dimerization to support growth and report that the two BC proteins most deficient in dimerization fail to support growth unless expressed at high levels. In contrast, the wild-type protein supports growth at low expression levels. We conclude that BC must be dimeric to fulfill its physiological function. PMID:22037404

  18. Wheat acetyl-coenzyme A carboxylase: cDNA and protein structure.

    PubMed Central

    Gornicki, P; Podkowinski, J; Scappino, L A; DiMaio, J; Ward, E; Haselkorn, R

    1994-01-01

    cDNA fragments encoding part of wheat (Triticum aestivum) acetyl-CoA carboxylase (ACC; EC 6.4.1.2) were cloned by PCR using primers based on the alignment of several biotin-dependent carboxylases. A set of overlapping clones encoding the entire wheat ACC was then isolated by using these fragments as probes. The cDNA sequence contains a 2257-amino acid reading frame encoding a 251-kDa polypeptide. The amino acid sequence of the most highly conserved domain, corresponding to the biotin carboxylases of prokaryotes, is 52-55% identical to ACC of yeast, rat, and diatom. Identity with the available C-terminal amino acid sequence of maize ACC is 66%. The biotin attachment site has the typical eukaryotic EVMKM sequence. The cDNA does not encode an obvious chloroplast targeting sequence. Various cDNA fragments hybridize in Northern blots to a 7.9-kb mRNA. Southern analysis with cDNA probes revealed multiple hybridizing fragments in hexaploid wheat DNA. Some of the wheat cDNA probes also hybridize with ACC-specific DNA from other plants, indicating significant conservation among plant ACCs. Images PMID:7913745

  19. Discovery and optimization of antibacterial AccC inhibitors

    SciTech Connect

    Cheng, Cliff C.; Shipps, Jr., Gerald W.; Yang, Zhiwei; Sun, Binyuan; Kawahata, Noriyuki; Soucy, Kyle A.; Soriano, Aileen; Orth, Peter; Xiao, Li; Mann, Paul; Black, Todd

    2010-09-17

    The biotin carboxylase (AccC) is part of the multi-component bacterial acetyl coenzyme-A carboxylase (ACCase) and is essential for pathogen survival. We describe herein the affinity optimization of an initial hit to give 2-(2-chlorobenzylamino)-1-(cyclohexylmethyl)-1H-benzo[d]imidazole-5-carboxamide (1), which was identified using our proprietary Automated Ligand Identification System (ALIS). The X-ray co-crystal structure of 1 was solved and revealed several key interactions and opportunities for further optimization in the ATP site of AccC. Structure Based Drug Design (SBDD) and parallel synthetic approaches resulted in a novel series of AccC inhibitors, exemplified by (R)-2-(2-chlorobenzylamino)-1-(2,3-dihydro-1H-inden-1-yl)-1H-imidazo[4,5-b]pyridine-5-carboxamide (40). This compound is a potent and selective inhibitor of bacterial AccC with an IC{sub 50} of 20 nM and a MIC of 0.8 {micro}g/mL against a sensitized strain of Escherichia coli (HS294 E. coli).

  20. Requirement for Acetyl-CoA Carboxylase in Trypanosoma brucei is Dependent Upon the Growth Environment

    PubMed Central

    Vigueira, Patrick A.; Paul, Kimberly S.

    2013-01-01

    Summary Trypanosoma brucei, the causative agent of human African trypanosomiasis, possesses two fatty acid synthesis pathways: a major de novo synthesis pathway in the ER and a mitochondrial pathway. The 2-carbon donor for both pathways is malonyl-CoA, which is synthesized from acetyl-CoA by Acetyl-CoA Carboxylase (ACC). Here, we show that T. brucei ACC shares the same enzyme architecture and moderate ~30% identity with yeast and human ACCs. ACC is cytoplasmic and appears to be distributed throughout the cell in numerous puncta distinct from glycosomes and other organelles. ACC is active in both bloodstream and procyclic forms. Reduction of ACC activity by RNA interference (RNAi) resulted in a stage-specific phenotype. In procyclic forms, ACC RNAi resulted in 50-75% reduction in fatty acid elongation and a 64% reduction in growth in low lipid media. In bloodstream forms, ACC RNAi resulted in a minor 15% decrease in fatty acid elongation and no growth defect in culture, even in low lipid media. However, ACC RNAi did attenuate virulence in a mouse model of infection. Thus the requirement for ACC in T. brucei is dependent upon the growth environment in two different life cycle stages. PMID:21306439

  1. Is Dimerization Required for the Catalytic Activity of Bacterial Biotin Carboxylase?

    SciTech Connect

    Shen,Y.; Chou, C.; Chang, G.; Tong, L.

    2006-01-01

    Acetyl-coenzyme A carboxylases (ACCs) have crucial roles in fatty acid metabolism. The biotin carboxylase (BC) subunit of Escherichia coli ACC is believed to be active only as a dimer, although the crystal structure shows that the active site of each monomer is 25 Angstroms from the dimer interface. We report here biochemical, biophysical, and structural characterizations of BC carrying single-site mutations in the dimer interface. Our studies demonstrate that two of the mutants, R19E and E23R, are monomeric in solution but have only a 3-fold loss in catalytic activity. The crystal structures of the E23R and F363A mutants show that they can still form the correct dimer at high concentrations. Our data suggest that dimerization is not an absolute requirement for the catalytic activity of the E. coli BC subunit, and we propose a new model for the molecular mechanism of action for BC in multisubunit and multidomain ACCs.

  2. Cloning of human acetyl-CoA carboxylase-beta and its unique features.

    PubMed Central

    Ha, J; Lee, J K; Kim, K S; Witters, L A; Kim, K H

    1996-01-01

    Acetyl-CoA carboxylase, which has a molecular mass of 265 kDa (ACC-alpha), catalyzes the rate-limiting step in the biosynthesis of long-chain fatty acids. In this study we report the complete amino acid sequence and unique features of an isoform of ACC with a molecular mass of 275 kDa (ACC-beta), which is primarily expressed in heart and skeletal muscles. In these tissues, ACC-beta may be involved in the regulation of fatty acid oxidation, rather than fatty acid biosynthesis. ACC-beta contains an amino acid sequence at the N terminus which is about 200 amino acids long and may be uniquely related to the role of ACC-beta in controlling carnitine palmitoyltransferase I activity and fatty acid oxidation by mitochondria. If we exclude this unique sequence at the N terminus the two forms of ACC show about 75% amino acid identity. All of the known functional domains of ACC are found in the homologous regions. Human ACC-beta cDNA has an open reading frame of 7,343 bases, encoding a protein of 2,458 amino acids, with a calculated molecular mass of 276,638 Da. The mRNA size of human ACC-beta is approximately 10 kb and is primarily expressed in heart and skeletal muscle tissues, whereas ACC-alpha mRNA is detected in all tissues tested. A fragment of ACC-beta cDNA was expressed in Escherichia coli and antibodies against the peptide were generated to establish that the cDNA sequence that we cloned is that for ACC-beta. Images Fig. 2 Fig. 3 Fig. 4 Fig. 5 PMID:8876158

  3. AMPK activation represses the human gene promoter of the cardiac isoform of acetyl-CoA carboxylase: Role of nuclear respiratory factor-1

    SciTech Connect

    Adam, Tasneem; Opie, Lionel H.; Essop, M. Faadiel

    2010-07-30

    Research highlights: {yields} AMPK inhibits acetyl-CoA carboxylase beta gene promoter activity. {yields} Nuclear respiratory factor-1 inhibits acetyl-CoA carboxylase beta promoter activity. {yields} AMPK regulates acetyl-CoA carboxylase beta at transcriptional level. -- Abstract: The cardiac-enriched isoform of acetyl-CoA carboxylase (ACC{beta}) produces malonyl-CoA, a potent inhibitor of carnitine palmitoyltransferase-1. AMPK inhibits ACC{beta} activity, lowering malonyl-CoA levels and promoting mitochondrial fatty acid {beta}-oxidation. Previously, AMPK increased promoter binding of nuclear respiratory factor-1 (NRF-1), a pivotal transcriptional modulator controlling gene expression of mitochondrial proteins. We therefore hypothesized that NRF-1 inhibits myocardial ACC{beta} promoter activity via AMPK activation. A human ACC{beta} promoter-luciferase construct was transiently transfected into neonatal cardiomyocytes {+-} a NRF-1 expression construct. NRF-1 overexpression decreased ACC{beta} gene promoter activity by 71 {+-} 4.6% (p < 0.001 vs. control). Transfections with 5'-end serial promoter deletions revealed that NRF-1-mediated repression of ACC{beta} was abolished with a pPII{beta}-18/+65-Luc deletion construct. AMPK activation dose-dependently reduced ACC{beta} promoter activity, while NRF-1 addition did not further decrease it. We also investigated NRF-1 inhibition in the presence of upstream stimulatory factor 1 (USF1), a known transactivator of the human ACC{beta} gene promoter. Here NRF-1 blunted USF1-dependent induction of ACC{beta} promoter activity by 58 {+-} 7.5% (p < 0.001 vs. control), reversed with a dominant negative NRF-1 construct. NRF-1 also suppressed endogenous USF1 transcriptional activity by 55 {+-} 6.2% (p < 0.001 vs. control). This study demonstrates that NRF-1 is a novel transcriptional inhibitor of the human ACC{beta} gene promoter in the mammalian heart. Our data extends AMPK regulation of ACC{beta} to the transcriptional level.

  4. Expression, purification, and characterization of human acetyl-CoA carboxylase 2.

    PubMed

    Kim, Ki Won; Yamane, Harvey; Zondlo, James; Busby, James; Wang, Minghan

    2007-05-01

    The full-length human acetyl-CoA carboxylase 1 (ACC1) was expressed and purified to homogeneity by two separate groups (Y.G. Gu, M. Weitzberg, R.F. Clark, X. Xu, Q. Li, T. Zhang, T.M. Hansen, G. Liu, Z. Xin, X. Wang, T. McNally, H. Camp, B.A. Beutel, H.I. Sham, Synthesis and structure-activity relationships of N-{3-[2-(4-alkoxyphenoxy)thiazol-5-yl]-1-methylprop-2-ynyl}carboxy derivatives as selective acetyl-CoA carboxylase 2 inhibitors, J. Med. Chem. 49 (2006) 3770-3773; D. Cheng, C.H. Chu, L. Chen, J.N. Feder, G.A. Mintier, Y. Wu, J.W. Cook, M.R. Harpel, G.A. Locke, Y. An, J.K. Tamura, Expression, purification, and characterization of human and rat acetyl coenzyme A carboxylase (ACC) isozymes, Protein Expr. Purif., in press). However, neither group was successful in expressing the full-length ACC2 due to issues of solubility and expression levels. The two versions of recombinant human ACC2 in these reports are either truncated (lacking 1-148 aa) or have the N-terminal 275 aa replaced with the corresponding ACC1 region (1-133 aa). Despite the fact that ACC activity was observed in both cases, these constructs are not ideal because the N-terminal region of ACC2 could be important for the correct folding of the catalytic domains. Here, we report the high level expression and purification of full-length human ACC2 that lacks only the N-terminal membrane attachment sequence (1-20 and 1-26 aa, respectively) in Trichoplusia ni cells. In addition, we developed a sensitive HPLC assay to analyze the kinetic parameters of the recombinant enzyme. The recombinant enzyme is a soluble protein and has a K(m) value of 2 microM for acetyl-CoA, almost 30-fold lower than that reported for the truncated human ACC2. Our recombinant enzyme also has a lower K(m) value for ATP (K(m)=52 microM). Although this difference could be ascribed to different assay conditions, our data suggest that the longer human ACC2 produced in our system may have higher affinities for the substrates and could

  5. Identification of dual Acetyl-CoA carboxylases 1 and 2 inhibitors by pharmacophore based virtual screening and molecular docking approach.

    PubMed

    Bhadauriya, Anuseema; Dhoke, Gaurao V; Gangwal, Rahul P; Damre, Mangesh V; Sangamwar, Abhay T

    2013-02-01

    Acetyl-CoA carboxylase (ACC) is a crucial metabolic enzyme that plays a vital role in obesity-induced type 2 diabetes and fatty acid metabolism. To identify dual inhibitors of Acetyl-CoA carboxylase1 and Acetyl-CoA carboxylase2, a pharmacophore modelling approach has been employed. The best HypoGen pharmacophore model for ACC2 inhibitors (Hypo1_ACC2) consists of one hydrogen bond acceptor, one hydrophobic aliphatic and one hydrophobic aromatic feature, whereas the best pharmacophore (Hypo1_ACC1) for ACC1 consists of one additional hydrogen-bond donor (HBD) features. The best pharmacophore hypotheses were validated by various methods such as test set, decoy set and Cat-Scramble methodology. The validated pharmacophore models were used to screen several small-molecule databases, including Specs, NCI, ChemDiv and Natural product databases to identify the potential dual ACC inhibitors. The virtual hits were then subjected to several filters such as estimated [Formula: see text] value, quantitative estimation of drug-likeness and molecular docking analysis. Finally, three novel compounds with diverse scaffolds were selected as potential starting points for the design of novel dual ACC inhibitors.

  6. Glucose and fat metabolism in adipose tissue of acetyl-CoA carboxylase 2 knockout mice

    PubMed Central

    Oh, WonKeun; Abu-Elheiga, Lutfi; Kordari, Parichher; Gu, Zeiwei; Shaikenov, Tattym; Chirala, Subrahmanyam S.; Wakil, Salih J.

    2005-01-01

    Acc2-/- mutant mice, when fed a high-fat/high-carbohydrate (HF/HC) diet, were protected against diet-induced obesity and diabetes. To investigate the role of acetyl-CoA carboxylase 2 (ACC2) in the regulation of energy metabolism in adipose tissues, we studied fatty acid and glucose oxidation in primary cultures of adipocytes isolated from wild-type and Acc2-/- mutant mice fed either normal chow or a HF/HC diet. When fed normal chow, oxidation of [14C]palmitate in adipocytes of Acc2-/- mutant mice was ≈80% higher than in adipocytes of WT mice, and it remained significantly higher in the presence of insulin. Interestingly, in addition to increased fatty acid oxidation, we also observed increased glucose oxidation in adipocytes of Acc2-/- mutant mice compared with that of WT mice. When fed a HF/HC diet for 4-5 months, adipocytes of Acc2-/- mutant mice maintained a 25% higher palmitate oxidation and a 2-fold higher glucose oxidation than WT mice. The mRNA level of glucose transporter 4 (GLUT4) decreased several fold in the adipose tissue of WT mice fed a HF/HC diet; however, in the adipose tissue of Acc2-/- mutant mice, it was 7-fold higher. Moreover, lipolysis activity was higher in adipocytes of Acc2-/- mutant mice compared with that in WT mice. These findings suggest that continuous fatty acid oxidation in the adipocytes of Acc2-/- mutant mice, combined with a higher level of glucose oxidation and a higher rate of lipolysis, are major factors leading to efficient maintenance of insulin sensitivity and leaner Acc2-/- mutant mice. PMID:15677334

  7. Overexpression of ACC gene from oleaginous yeast Lipomyces starkeyi enhanced the lipid accumulation in Saccharomyces cerevisiae with increased levels of glycerol 3-phosphate substrates.

    PubMed

    Wang, Jiancai; Xu, Ronghua; Wang, Ruling; Haque, Mohammad Enamul; Liu, Aizhong

    2016-06-01

    The conversion of acetyl-CoA to malonyl-CoA by acetyl-CoA carboxylase (ACC) is the rate-limiting step in fatty acid biosynthesis. In this study, a gene coding for ACC was isolated and characterized from an oleaginous yeast, Lipomyces starkeyi. Real-time quantitative PCR (qPCR) analysis of L. starkeyi acetyl-CoA carboxylase gene (LsACC1) showed that the expression levels were upregulated with the fast accumulation of lipids. The LsACC1 was co-overexpressed with the glycerol 3-phosphate dehydrogenase gene (GPD1), which regulates lipids biosynthesis by supplying another substrates glycerol 3-phosphate for storage lipid assembly, in the non-oleaginous yeast Saccharomyces cerevisiae. Further, the S. cerevisiae acetyl-CoA carboxylase (ScACC1) was transferred with GPD1 and its function was analyzed in comparison with LsACC1. The results showed that overexpressed LsACC1 and GPD1 resulted in a 63% increase in S. cerevisiae. This study gives new data in understanding of the molecular mechanisms underlying the regulation of fatty acids and lipid biosynthesis in yeasts.

  8. Expression and characterization of recombinant fungal acetyl-CoA carboxylase and isolation of a soraphen-binding domain.

    PubMed

    Weatherly, Stephanie C; Volrath, Sandra L; Elich, Tedd D

    2004-05-15

    Acetyl-CoA carboxylase (ACC) catalyses the first step in fatty-acid biosynthesis. Owing to its role in primary metabolism, ACC has been exploited as a commercial herbicide target and identified as a chemically validated fungicide target. In animals, ACC is also a key regulator of fat metabolism. This function has made ACC a prime target for the development of anti-obesity and anti-Type II diabetes therapeutics. Despite its economic importance, there is a lack of published information on recombinant expression of ACC. We report here the expression of enzymically active fungal (Ustilago maydis ) ACC in Escherichia coli. The recombinant enzyme exhibited Km values of 0.14+/-0.013 mM and 0.19+/-0.041 mM for acetyl-CoA and ATP respectively, which are comparable with those reported for the endogenous enzyme. The polyketide natural product soraphen is a potent inhibitor of the BC (biotin carboxylase) domain of endogenous fungal ACC. Similarly, recombinant ACC activity was inhibited by soraphen with a K(i) of 2.1+/-0.9 nM. A truncated BC domain that included amino acids 2-560 of the full-length protein was also expressed in E. coli. The isolated BC domain was expressed to higher levels, and was more stable than full-length ACC. Although incapable of enzymic turnover, the BC domain exhibited high-affinity soraphen binding (Kd 1.1+/-0.3 nM), demonstrating a native conformation. Additional BC domains from the phytopathogenic fungi Magnaporthe grisea and Phytophthora infestans were also cloned and expressed, and were shown to exhibit high-affinity soraphen binding. Together, these reagents will be useful for structural studies and assay development.

  9. Genetic inhibition of hepatic acetyl-CoA carboxylase activity increases liver fat and alters global protein acetylationa

    PubMed Central

    Chow, Jenny D.Y.; Lawrence, Robert T.; Healy, Marin E.; Dominy, John E.; Liao, Jason A.; Breen, David S.; Byrne, Frances L.; Kenwood, Brandon M.; Lackner, Carolin; Okutsu, Saeko; Mas, Valeria R.; Caldwell, Stephen H.; Tomsig, Jose L.; Cooney, Gregory J.; Puigserver, Pere B.; Turner, Nigel; James, David E.; Villén, Judit; Hoehn, Kyle L.

    2014-01-01

    Lipid deposition in the liver is associated with metabolic disorders including fatty liver disease, type II diabetes, and hepatocellular cancer. The enzymes acetyl-CoA carboxylase 1 (ACC1) and ACC2 are powerful regulators of hepatic fat storage; therefore, their inhibition is expected to prevent the development of fatty liver. In this study we generated liver-specific ACC1 and ACC2 double knockout (LDKO) mice to determine how the loss of ACC activity affects liver fat metabolism and whole-body physiology. Characterization of LDKO mice revealed unexpected phenotypes of increased hepatic triglyceride and decreased fat oxidation. We also observed that chronic ACC inhibition led to hyper-acetylation of proteins in the extra-mitochondrial space. In sum, these data reveal the existence of a compensatory pathway that protects hepatic fat stores when ACC enzymes are inhibited. Furthermore, we identified an important role for ACC enzymes in the regulation of protein acetylation in the extra-mitochondrial space. PMID:24944901

  10. Abundance and diversity of archaeal accA gene in hot springs in Yunnan Province, China.

    PubMed

    Song, Zhao-Qi; Wang, Li; Wang, Feng-Ping; Jiang, Hong-Chen; Chen, Jin-Quan; Zhou, En-Min; Liang, Feng; Xiao, Xiang; Li, Wen-Jun

    2013-09-01

    It has been suggested that archaea carrying the accA gene, encoding the alpha subunit of the acetyl CoA carboxylase, autotrophically fix CO2 using the 3-hydroxypropionate/4-hydroxybutyrate pathway in low-temperature environments (e.g., soils, oceans). However, little new information has come to light regarding the occurrence of archaeal accA genes in high-temperature ecosystems. In this study, we investigated the abundance and diversity of archaeal accA gene in hot springs in Yunnan Province, China, using DNA- and RNA-based phylogenetic analyses and quantitative polymerase chain reaction. The results showed that archaeal accA genes were present and expressed in the investigated Yunnan hot springs with a wide range of temperatures (66-96 °C) and pH (4.3-9.0). The majority of the amplified archaeal accA gene sequences were affiliated with the ThAOA/HWCG III [thermophilic ammonia-oxidizing archaea (AOA)/hot water crenarchaeotic group III]. The archaeal accA gene abundance was very close to that of AOA amoA gene, encoding the alpha subunit of ammonia monooxygenase. These data suggest that AOA in terrestrial hot springs might acquire energy from ammonia oxidation coupled with CO2 fixation using the 3-hydroxypropionate/4-hydroxybutyrate pathway.

  11. Inhibition of acetyl-CoA carboxylase suppresses fatty acid synthesis and tumor growth of non-small cell lung cancer in preclinical models

    PubMed Central

    Svensson, Robert U.; Parker, Seth J.; Eichner, Lillian J.; Kolar, Matthew J.; Wallace, Martina; Brun, Sonja N.; Lombardo, Portia S.; Van Nostrand, Jeanine L.; Hutchins, Amanda; Vera, Lilliana; Gerken, Laurie; Greenwood, Jeremy; Bhat, Sathesh; Harriman, Geraldine; Westlin, William F.; Harwood, H. James; Saghatelian, Alan; Kapeller, Rosana; Metallo, Christian M.; Shaw, Reuben J.

    2016-01-01

    Continuous de novo fatty acid synthesis is a common feature of cancer required to meet the biosynthetic demands of a growing tumor. This process is controlled by the rate-limiting enzyme acetyl-CoA carboxylase (ACC), an attractive but traditionally intractable drug target. Here, we provide genetic and pharmacological evidence that in preclinical models ACC is required to maintain de novo fatty acid synthesis needed for growth and viability of non-small cell lung cancer (NSCLC). We describe the ability of ND-646—an allosteric inhibitor of the ACC enzymes ACC1 and ACC2 that prevents ACC subunit dimerization—to suppress fatty acid synthesis in vitro and in vivo. Chronic ND-646 treatment of xenograft and genetically engineered mouse models of NSCLC inhibited tumor growth. When administered as a single agent or in combination with the standard-of-care drug carboplatin, ND-646 markedly suppressed lung tumor growth in the Kras;Trp53−/− (also known as KRAS p53) and Kras;Stk11−/− (also known as KRAS Lkb1) mouse models of NSCLC. These findings demonstrate that ACC mediates a metabolic liability of NSCLC and that ACC inhibition by ND-646 is detrimental to NSCLC growth, supporting further examination of the use of ACC inhibitors in oncology. PMID:27643638

  12. Crystal Structure of the alpha6beta6 Holoenzyme of propionyl-coenzyme A Carboxylase

    SciTech Connect

    Huang, C.; Sadre-Bazzaz, K; Shen, Y; Deng, B; Zhou, Z; Tong, L

    2010-01-01

    Propionyl-coenzyme A carboxylase (PCC), a mitochondrial biotin-dependent enzyme, is essential for the catabolism of the amino acids Thr, Val, Ile and Met, cholesterol and fatty acids with an odd number of carbon atoms. Deficiencies in PCC activity in humans are linked to the disease propionic acidaemia, an autosomal recessive disorder that can be fatal in infants. The holoenzyme of PCC is an {alpha}{sub 6}{beta}{sub 6} dodecamer, with a molecular mass of 750 kDa. The {alpha}-subunit contains the biotin carboxylase (BC) and biotin carboxyl carrier protein (BCCP) domains, whereas the {beta}-subunit supplies the carboxyltransferase (CT) activity. Here we report the crystal structure at 3.2-{angstrom} resolution of a bacterial PCC {alpha}{sub 6}{beta}{sub 6} holoenzyme as well as cryo-electron microscopy (cryo-EM) reconstruction at 15-{angstrom} resolution demonstrating a similar structure for human PCC. The structure defines the overall architecture of PCC and reveals unexpectedly that the {alpha}-subunits are arranged as monomers in the holoenzyme, decorating a central {beta}{sub 6} hexamer. A hitherto unrecognized domain in the {alpha}-subunit, formed by residues between the BC and BCCP domains, is crucial for interactions with the {beta}-subunit. We have named it the BT domain. The structure reveals for the first time the relative positions of the BC and CT active sites in the holoenzyme. They are separated by approximately 55 {angstrom}, indicating that the entire BCCP domain must translocate during catalysis. The BCCP domain is located in the active site of the {beta}-subunit in the current structure, providing insight for its involvement in the CT reaction. The structural information establishes a molecular basis for understanding the large collection of disease-causing mutations in PCC and is relevant for the holoenzymes of other biotin-dependent carboxylases, including 3-methylcrotonyl-CoA carboxylase (MCC) and eukaryotic acetyl-CoA carboxylase (ACC).

  13. Evaluating Performance Portability of OpenACC

    SciTech Connect

    Sabne, Amit J; Sakdhnagool, Putt; Lee, Seyong; Vetter, Jeffrey S

    2015-01-01

    Accelerator-based heterogeneous computing is gaining momentum in High Performance Computing arena. However, the increased complexity of the accelerator architectures demands more generic, high-level programming models. OpenACC is one such attempt to tackle the problem. While the abstraction endowed by OpenACC offers productivity, it raises questions on its portability. This paper evaluates the performance portability obtained by OpenACC on twelve OpenACC programs on NVIDIA CUDA, AMD GCN, and Intel MIC architectures. We study the effects of various compiler optimizations and OpenACC program settings on these architectures to provide insights into the achieved performance portability.

  14. Acetyl-CoA carboxylase inhibition by ND-630 reduces hepatic steatosis, improves insulin sensitivity, and modulates dyslipidemia in rats

    PubMed Central

    Harriman, Geraldine; Greenwood, Jeremy; Bhat, Sathesh; Huang, Xinyi; Wang, Ruiying; Paul, Debamita; Tong, Liang; Saha, Asish K.; Westlin, William F.; Kapeller, Rosana; Harwood, H. James

    2016-01-01

    Simultaneous inhibition of the acetyl-CoA carboxylase (ACC) isozymes ACC1 and ACC2 results in concomitant inhibition of fatty acid synthesis and stimulation of fatty acid oxidation and may favorably affect the morbidity and mortality associated with obesity, diabetes, and fatty liver disease. Using structure-based drug design, we have identified a series of potent allosteric protein–protein interaction inhibitors, exemplified by ND-630, that interact within the ACC phosphopeptide acceptor and dimerization site to prevent dimerization and inhibit the enzymatic activity of both ACC isozymes, reduce fatty acid synthesis and stimulate fatty acid oxidation in cultured cells and in animals, and exhibit favorable drug-like properties. When administered chronically to rats with diet-induced obesity, ND-630 reduces hepatic steatosis, improves insulin sensitivity, reduces weight gain without affecting food intake, and favorably affects dyslipidemia. When administered chronically to Zucker diabetic fatty rats, ND-630 reduces hepatic steatosis, improves glucose-stimulated insulin secretion, and reduces hemoglobin A1c (0.9% reduction). Together, these data suggest that ACC inhibition by representatives of this series may be useful in treating a variety of metabolic disorders, including metabolic syndrome, type 2 diabetes mellitus, and fatty liver disease. PMID:26976583

  15. Assay of ribulose bisphosphate carboxylase

    SciTech Connect

    Pike, C.; Berry, J.

    1987-04-01

    Assays of ribulose bisphosphate carboxylase (rubisco) can be used to illustrate many properties of photosynthetic systems. Many different leaves have been assayed with this standard procedure. The tissue is ground with a mortar and pestle in extraction buffer. The supernatant after centrifugation is used as the source of enzyme. Buffer, RuBP, (/sup 14/C)-NaHCO/sub 3/, and enzyme are combined in a scintillation vial; the reaction is run for 1 min at 30/sup 0/. The acid-stable products are counted. Reproducibility in student experiments has been excellent. The assay data can be combined with analyses of leaf properties such as fresh and dry weight, chlorophyll and protein content, etc. Students have done projects such as the response of enzyme to temperature and to various inhibitors. They also report on the use of a transition state analog, carboxyarabinitol bisphosphate, to titrate the molar concentration of rubisco molecules (active sites) in an enzyme sample. Thus, using crude extracts the catalytic activity of a sample can be compared to the absolute quantity of enzyme or to the turnover number.

  16. Airborne Cloud Computing Environment (ACCE)

    NASA Technical Reports Server (NTRS)

    Hardman, Sean; Freeborn, Dana; Crichton, Dan; Law, Emily; Kay-Im, Liz

    2011-01-01

    Airborne Cloud Computing Environment (ACCE) is JPL's internal investment to improve the return on airborne missions. Improve development performance of the data system. Improve return on the captured science data. The investment is to develop a common science data system capability for airborne instruments that encompasses the end-to-end lifecycle covering planning, provisioning of data system capabilities, and support for scientific analysis in order to improve the quality, cost effectiveness, and capabilities to enable new scientific discovery and research in earth observation.

  17. Accumulation fatty acids of in Chlorella vulgaris under heterotrophic conditions in relation to activity of acetyl-CoA carboxylase, temperature, and co-immobilization with Azospirillum brasilense

    NASA Astrophysics Data System (ADS)

    Leyva, Luis A.; Bashan, Yoav; Mendoza, Alberto; de-Bashan, Luz E.

    2014-10-01

    The relation between fatty acid accumulation, activity of acetyl-CoA carboxylase (ACC), and consequently lipid accumulation was studied in the microalgae Chlorella vulgaris co-immobilized with the plant growth-promoting bacterium Azospirillum brasilense under dark heterotrophic conditions with Na acetate as a carbon source. In C. vulgaris immobilized alone, cultivation experiments for 6 days showed that ACC activity is directly related to fatty acid accumulation, especially in the last 3 days. In co-immobilization experiments, A. brasilense exerted a significant positive effect over ACC activity, increased the quantity in all nine main fatty acids, increased total lipid accumulation in C. vulgaris, and mitigated negative effects of nonoptimal temperature for growth. No correlation between ACC activity and lipid accumulation in the cells was established for three different temperatures. This study demonstrated that the interaction between A. brasilense and C. vulgaris has a significant effect on fatty acid and lipid accumulation in the microalgae.

  18. Improving Production of Malonyl Coenzyme A-Derived Metabolites by Abolishing Snf1-Dependent Regulation of Acc1

    PubMed Central

    Shi, Shuobo; Chen, Yun; Siewers, Verena

    2014-01-01

    ABSTRACT Acetyl coenzyme A (acetyl-CoA) carboxylase (ACCase) plays a central role in carbon metabolism and has been the site of action for the development of therapeutics or herbicides, as its product, malonyl-CoA, is a precursor for production of fatty acids and other compounds. Control of Acc1 activity in the yeast Saccharomyces cerevisiae occurs mainly at two levels, i.e., regulation of transcription and repression by Snf1 protein kinase at the protein level. Here, we demonstrate a strategy for improving the activity of ACCase in S. cerevisiae by abolishing posttranslational regulation of Acc1 via site-directed mutagenesis. It was found that introduction of two site mutations in Acc1, Ser659 and Ser1157, resulted in an enhanced activity of Acc1 and increased total fatty acid content. As Snf1 regulation of Acc1 is particularly active under glucose-limited conditions, we evaluated the effect of the two site mutations in chemostat cultures. Finally, we showed that our modifications of Acc1 could enhance the supply of malonyl-CoA and therefore successfully increase the production of two industrially important products derived from malonyl-CoA, fatty acid ethyl esters and 3-hydroxypropionic acid. PMID:24803522

  19. The human ACC2 CT-domain C-terminus is required for full functionality and has a novel twist

    SciTech Connect

    Madauss, Kevin P.; Burkhart, William A.; Consler, Thomas G.; Cowan, David J.; Gottschalk, William K.; Miller, Aaron B.; Short, Steven A.; Tran, Thuy B.; Williams, Shawn P.

    2009-05-01

    The use of biophysical assays permitted the identification of a specific human ACC2 carboxyl transferase (CT) domain mutant that binds inhibitors and crystallizes in their presence. This mutant led to determination of the human ACC2 CT domain–CP-640186 complex crystal structure, which revealed differences in the inhibitor conformation from the yeast protein complex that are caused by differing residues in the binding pocket. Inhibition of acetyl-CoA carboxylase (ACC) may prevent lipid-induced insulin resistance and type 2 diabetes, making the enzyme an attractive pharmaceutical target. Although the enzyme is highly conserved amongst animals, only the yeast enzyme structure is available for rational drug design. The use of biophysical assays has permitted the identification of a specific C-terminal truncation of the 826-residue human ACC2 carboxyl transferase (CT) domain that is both functionally competent to bind inhibitors and crystallizes in their presence. This C-terminal truncation led to the determination of the human ACC2 CT domain–CP-640186 complex crystal structure, which revealed distinctions from the yeast-enzyme complex. The human ACC2 CT-domain C-terminus is comprised of three intertwined α-helices that extend outwards from the enzyme on the opposite side to the ligand-binding site. Differences in the observed inhibitor conformation between the yeast and human structures are caused by differing residues in the binding pocket.

  20. A Different Mechanism for the Inhibition of the Carboxyltransferase Domain of Acetyl-coenzyme A Carboxylase by Tepraloxydim

    SciTech Connect

    Xiang, S.; Callaghan, M; Watson, K; Tong, L

    2009-01-01

    Acetyl-CoA carboxylases (ACCs) are crucial metabolic enzymes and are attractive targets for drug discovery. Haloxyfop and tepraloxydim belong to two distinct classes of commercial herbicides and kill sensitive plants by inhibiting the carboxyltransferase (CT) activity of ACC. Our earlier structural studies showed that haloxyfop is bound near the active site of the CT domain, at the interface of its dimer, and a large conformational change in the dimer interface is required for haloxyfop binding. We report here the crystal structure at 2.3 {angstrom} resolution of the CT domain of yeast ACC in complex with tepraloxydim. The compound has a different mechanism of inhibiting the CT activity compared to haloxyfop, as well as the mammalian ACC inhibitor CP-640186. Tepraloxydim probes a different region of the dimer interface and requires only small but important conformational changes in the enzyme, in contrast to haloxyfop. The binding mode of tepraloxydim explains the structure-activity relationship of these inhibitors, and provides a molecular basis for their distinct sensitivity to some of the resistance mutations, as compared to haloxyfop. Despite the chemical diversity between haloxyfop and tepraloxydim, the compounds do share two binding interactions to the enzyme, which may be important anchoring points for the development of ACC inhibitors

  1. ACC Effectiveness Review, 1999-2002.

    ERIC Educational Resources Information Center

    Wallace, Roslyn, Ed.

    2002-01-01

    These newsletters on Institutional Effectiveness (IE) at Austin Community College (ACC) in Texas include the following articles: (1) "The 'Fast Track'...Students Say It Works!" (2) "Are Students Successfully Completing Distance Learning Courses at ACC?" (3) "Tracking Transfers"; (4) "Math Pilot: Study Skills…

  2. Gene encoding acetyl-coenzyme A carboxylase

    DOEpatents

    Roessler, P.G.; Ohlrogge, J.B.

    1996-09-24

    A DNA encoding an acetyl-coenzyme A carboxylase (ACCase) from a photosynthetic organism and functional derivatives are disclosed which are resistant to inhibition from certain herbicides. This gene can be placed in organisms to increase their fatty acid content or to render them resistant to certain herbicides. 5 figs.

  3. Gene encoding acetyl-coenzyme A carboxylase

    DOEpatents

    Roessler, Paul G.; Ohlrogge, John B.

    1996-01-01

    A DNA encoding an acetyl-coenzyme A carboxylase (ACCase) from a photosynthetic organism and functional derivatives thereof which are resistant to inhibition from certain herbicides. This gene can be placed in organisms to increase their fatty acid content or to render them resistant to certain herbicides.

  4. Functional reconstitution of the Mycobacterium tuberculosis long-chain acyl-CoA carboxylase from multiple acyl-CoA subunits.

    PubMed

    Bazet Lyonnet, Bernardo; Diacovich, Lautaro; Gago, Gabriela; Spina, Lucie; Bardou, Fabienne; Lemassu, Anne; Quémard, Annaïk; Gramajo, Hugo

    2017-02-21

    Mycobacterium tuberculosis produces a large number of structurally diverse lipids that have been implicated in the pathogenicity, persistence and antibiotic resistance of this organism. Most building blocks involved in the biosynthesis of all these lipids are generated by acyl-CoA carboxylases (ACCase) whose subunit composition and physiological roles have not yet been clearly established. A rather controversial data in the literature refers to the exact protein composition and substrate specificity of the enzyme complex that produces the long-chain α-carboxy-acyl-CoAs; one of the substrates involved in the last step of condensation mediated by the polyketide synthase Pks13 to synthesize mature mycolic acids. Here we have successfully reconstituted the so called long-chain acyl-CoA carboxylase complex (LCC) from its purified components: the α-subunit AccA3, the ε-subunit AccE5 and the two β-subunits AccD4 and AccD5, and demonstrated that the four subunits are essential for its LCC activity. Furthermore, we also showed by substrate competition experiments and the use of a specific inhibitor of the AccD5 subunit, that its role in the carboxylation of the long acyl-CoAs, as part of the LCC complex, was structural rather than catalytic. Moreover, AccD5 was also able to carboxylate its natural substrates, acetyl-CoA and propionyl-CoA, in the context of the LCC enzyme complex. Thus, the supercomplex formed by these four subunits has the potential to generate the main substrates, malonyl-CoA, methylmalonyl-CoA and α-carboxy-C24-26 -CoA, used as condensing units for the biosynthesis of all the lipids present in this pathogen. This article is protected by copyright. All rights reserved.

  5. A Chemogenomic Screen Reveals Novel Snf1p/AMPK Independent Regulators of Acetyl-CoA Carboxylase.

    PubMed

    Bozaquel-Morais, Bruno L; Madeira, Juliana B; Venâncio, Thiago M; Pacheco-Rosa, Thiago; Masuda, Claudio A; Montero-Lomeli, Monica

    2017-01-01

    Acetyl-CoA carboxylase (Acc1p) is a key enzyme in fatty acid biosynthesis and is essential for cell viability. To discover new regulators of its activity, we screened a Saccharomyces cerevisiae deletion library for increased sensitivity to soraphen A, a potent Acc1p inhibitor. The hits identified in the screen (118 hits) were filtered using a chemical-phenotype map to exclude those associated with pleiotropic drug resistance. This enabled the identification of 82 ORFs that are genetic interactors of Acc1p. The main functional clusters represented by these hits were "transcriptional regulation", "protein post-translational modifications" and "lipid metabolism". Further investigation of the "transcriptional regulation" cluster revealed that soraphen A sensitivity is poorly correlated with ACC1 transcript levels. We also studied the three top unknown ORFs that affected soraphen A sensitivity: SOR1 (YDL129W), SOR2 (YIL092W) and SOR3 (YJR039W). Since the C18/C16 ratio of lipid acyl lengths reflects Acc1p activity levels, we evaluated this ratio in the three mutants. Deletion of SOR2 and SOR3 led to reduced acyl lengths, suggesting that Acc1p is indeed down-regulated in these strains. Also, these mutants showed no differences in Snf1p/AMPK activation status and deletion of SNF1 in these backgrounds did not revert soraphen A sensitivity completely. Furthermore, plasmid maintenance was reduced in sor2Δ strain and this trait was shared with 18 other soraphen A sensitive hits. In summary, our screen uncovered novel Acc1p Snf1p/AMPK-independent regulators.

  6. A Chemogenomic Screen Reveals Novel Snf1p/AMPK Independent Regulators of Acetyl-CoA Carboxylase

    PubMed Central

    Bozaquel-Morais, Bruno L.; Madeira, Juliana B.; Venâncio, Thiago M.; Pacheco-Rosa, Thiago; Masuda, Claudio A.; Montero-Lomeli, Monica

    2017-01-01

    Acetyl-CoA carboxylase (Acc1p) is a key enzyme in fatty acid biosynthesis and is essential for cell viability. To discover new regulators of its activity, we screened a Saccharomyces cerevisiae deletion library for increased sensitivity to soraphen A, a potent Acc1p inhibitor. The hits identified in the screen (118 hits) were filtered using a chemical-phenotype map to exclude those associated with pleiotropic drug resistance. This enabled the identification of 82 ORFs that are genetic interactors of Acc1p. The main functional clusters represented by these hits were “transcriptional regulation”, “protein post-translational modifications” and “lipid metabolism”. Further investigation of the “transcriptional regulation” cluster revealed that soraphen A sensitivity is poorly correlated with ACC1 transcript levels. We also studied the three top unknown ORFs that affected soraphen A sensitivity: SOR1 (YDL129W), SOR2 (YIL092W) and SOR3 (YJR039W). Since the C18/C16 ratio of lipid acyl lengths reflects Acc1p activity levels, we evaluated this ratio in the three mutants. Deletion of SOR2 and SOR3 led to reduced acyl lengths, suggesting that Acc1p is indeed down-regulated in these strains. Also, these mutants showed no differences in Snf1p/AMPK activation status and deletion of SNF1 in these backgrounds did not revert soraphen A sensitivity completely. Furthermore, plasmid maintenance was reduced in sor2Δ strain and this trait was shared with 18 other soraphen A sensitive hits. In summary, our screen uncovered novel Acc1p Snf1p/AMPK-independent regulators. PMID:28076367

  7. Effects of wintertime fasting and seasonal adaptation on AMPK and ACC in hypothalamus, adipose tissue and liver of the raccoon dog (Nyctereutes procyonoides).

    PubMed

    Kinnunen, Sanni; Mänttäri, Satu; Herzig, Karl-Heinz; Nieminen, Petteri; Mustonen, Anne-Mari; Saarela, Seppo

    2016-02-01

    The raccoon dog (Nyctereutes procyonoides) is a canid with autumnal fattening and passive wintering strategy. We examined the effects of wintertime fasting and seasonality on AMP-activated protein kinase (AMPK), a regulator of metabolism, and its target, acetyl-CoA carboxylase (ACC) on the species. Twelve farmed raccoon dogs (eleven females/one male) were divided into two groups: half were fasted for ten weeks in December-March (winter fasted) and the others were fed ad libitum (winter fed). A third group (autumn fed, eight females) was fed ad libitum and sampled in December. Total AMPK, ACC and their phosphorylated forms (pAMPK, pACC) were measured from hypothalamus, liver, intra-abdominal (iWAT) and subcutaneous white adipose tissues (sWAT). The fasted animals lost 32% and the fed 20% of their body mass. Hypothalamic AMPK expression was lower and pACC levels higher in the winter groups compared to the autumn fed group. Liver pAMPK was lower in the winter fasted group, with consistently decreased ACC and pACC. AMPK and pAMPK were down-regulated in sWAT and iWAT of both winter groups, with a parallel decline in pACC in sWAT. The responses of AMPK and ACC to fasting were dissimilar to the effects observed previously in non-seasonal mammals and hibernators. Differences between the winter fed and autumn fed groups indicate that the functions of AMPK and ACC could be regulated in a season-dependent manner. Furthermore, the distinctive effects of prolonged fasting and seasonal adaptation on AMPK-ACC pathway could contribute to the wintering strategy of the raccoon dog.

  8. Synthesis of 7-oxo-dihydrospiro[indazole-5,4'-piperidine] acetyl-CoA carboxylase inhibitors.

    PubMed

    Bagley, Scott W; Southers, James A; Cabral, Shawn; Rose, Colin R; Bernhardson, David J; Edmonds, David J; Polivkova, Jana; Yang, Xiaojing; Kung, Daniel W; Griffith, David A; Bader, Scott J

    2012-02-03

    Synthesis of oxo-dihydrospiroindazole-based acetyl-CoA carboxylase (ACC) inhibitors is reported. The dihydrospiroindazoles were assembled in a regioselective manner in six steps from substituted hydrazines and protected 4-formylpiperidine. Enhanced regioselectivity in the condensation between a keto enamine and substituted hydrazines was observed when using toluene as the solvent, leading to selective formation of 1-substituted spiroindazoles. The 2-substituted spiroindazoles were formed selectively from alkyl hydrazones by ring closure with Vilsmeier reagent. The key step in the elaboration to the final products is the conversion of an intermediate olefin to the desired ketone through elimination of HBr from an O-methyl bromohydrin. This methodology enabled the synthesis of each desired regioisomer on 50-75 g scale with minimal purification. Acylation of the resultant spirocyclic amines provided potent ACC inhibitors.

  9. Evolutionary history and biotechnological future of carboxylases.

    PubMed

    Schada von Borzyskowski, Lennart; Rosenthal, Raoul G; Erb, Tobias J

    2013-11-01

    Carbon dioxide (CO2) is a potent greenhouse gas whose presence in the atmosphere is a critical factor for global warming. At the same time atmospheric CO2 is also a cheap and readily available carbon source that can in principle be used to synthesize value-added products. However, as uncatalyzed chemical CO2-fixation reactions usually require quite harsh conditions to functionalize the CO2 molecule, not many processes have been developed that make use of CO2. In contrast to synthetical chemistry, Nature provides a multitude of different carboxylating enzymes whose carboxylating principle(s) might be exploited in biotechnology. This review focuses on the biochemical features of carboxylases, highlights possible evolutionary scenarios for the emergence of their reactivity, and discusses current, as well as potential future applications of carboxylases in organic synthesis, biotechnology and synthetic biology.

  10. Studies of vitamin K-dependent carboxylase

    SciTech Connect

    Wood, G.M.

    1986-01-01

    Carboxylase was studied in detergent solubilized rat liver microsomes, using the peptide substrate Phe-Leu-(..gamma..-/sup 3/H)-Glu-Glu-Leu. Cleavage of the ..gamma..-C-H bond in Glu was measured as the release of /sup 3/H from this peptide to water, carboxylation was measured as the incorporation of H/sup 14/CO/sub 3/-into the peptide, and KO formation was measured by an HPLC assay. All three products could be measured simultaneously, and this system was used to examine the effects of cyanide, manganese, tetrachloropyridinol, and Boc-SerP-SerP-Leu-OMe on the separate steps of the carboxylase reaction. Vitamin K-epoxide formation was studied separately from the other reactions, and it was found that in the absence of a Glu-containing substrate, carboxylase catalyzed the uncoupled formation of KO from KH/sub 2/ and O/sub 2/. The stoichiometry of product formation (GLa, KO, and ..gamma..-protons) was measured, and the results obtained were all in agreement with the values predicted from the proposed mechanism. When all of the substrates were saturating, the stoichiometry of ..gamma..-C-H bond cleavage, carboxylation, and KO formation was 1:1:1.

  11. PHD3 Loss in Cancer Enables Metabolic Reliance on Fatty Acid Oxidation via Deactivation of ACC2.

    PubMed

    German, Natalie J; Yoon, Haejin; Yusuf, Rushdia Z; Murphy, J Patrick; Finley, Lydia W S; Laurent, Gaëlle; Haas, Wilhelm; Satterstrom, F Kyle; Guarnerio, Jlenia; Zaganjor, Elma; Santos, Daniel; Pandolfi, Pier Paolo; Beck, Andrew H; Gygi, Steven P; Scadden, David T; Kaelin, William G; Haigis, Marcia C

    2016-09-15

    While much research has examined the use of glucose and glutamine by tumor cells, many cancers instead prefer to metabolize fats. Despite the pervasiveness of this phenotype, knowledge of pathways that drive fatty acid oxidation (FAO) in cancer is limited. Prolyl hydroxylase domain proteins hydroxylate substrate proline residues and have been linked to fuel switching. Here, we reveal that PHD3 rapidly triggers repression of FAO in response to nutrient abundance via hydroxylation of acetyl-coA carboxylase 2 (ACC2). We find that PHD3 expression is strongly decreased in subsets of cancer including acute myeloid leukemia (AML) and is linked to a reliance on fat catabolism regardless of external nutrient cues. Overexpressing PHD3 limits FAO via regulation of ACC2 and consequently impedes leukemia cell proliferation. Thus, loss of PHD3 enables greater utilization of fatty acids but may also serve as a metabolic and therapeutic liability by indicating cancer cell susceptibility to FAO inhibition.

  12. MEDICA 16 inhibits hepatic acetyl-CoA carboxylase and reduces plasma triacylglycerol levels in insulin-resistant JCR: LA-cp rats.

    PubMed

    Atkinson, Laura L; Kelly, Sandra E; Russell, James C; Bar-Tana, Jacob; Lopaschuk, Gary D

    2002-05-01

    Intracellular triacylglycerol (TG) content of liver and skeletal muscle contributes to insulin resistance, and a significant correlation exists between TG content and the development of insulin resistance. Because acetyl-CoA carboxylase (ACC) is the rate-limiting enzyme for liver fatty acid biosynthesis and a key regulator of muscle fatty acid oxidation, we examined whether ACC plays a role in the accumulation of intracellular TG. We also determined the potential role of 5'-AMP-activated protein kinase (AMPK) in this process, since it can phosphorylate and inhibit ACC activity in both liver and muscle. TG content, ACC, and AMPK were examined in the liver and skeletal muscle of insulin-resistant JCR:LA-cp rats during the time frame when insulin resistance develops. At 12 weeks of age, there was a threefold elevation in liver TG content and a sevenfold elevation in skeletal muscle TG content. Hepatic ACC activity was significantly elevated in 12-week-old JCR:LA-cp rats compared with lean age-matched controls (8.75 +/- 0.53 vs. 3.30 +/- 0.18 nmol. min(-1). mg(-1), respectively), even though AMPK activity was also increased. The observed increase in hepatic ACC activity was accompanied by a 300% increase in ACC protein expression. There were no significant differences in ACC activity, ACC protein expression, or AMPK activity in the skeletal muscle of the 12-week JCR:LA-cp rats. Treatment of 12-week JCR:LA-cp rats with MEDICA 16 (an ATP-citrate lyase inhibitor) resulted in a decrease in hepatic ACC and AMPK activities, but had no effect on skeletal muscle ACC and AMPK. Our data suggest that alterations in ACC or AMPK activity in muscle do not contribute to the development of insulin resistance. However, increased liver ACC activity in the JCR:LA-cp rat appears to contribute to the development of lipid abnormalities, although this increase does not appear to occur secondary to a decrease in AMPK activity.

  13. Structural Evidence for Direct Interactions Between the BRCT Domains of Human BRCA1 and a Phospho-Peptide from Human ACC1

    SciTech Connect

    Shen,Y.; Tong, L.

    2008-01-01

    The tandem BRCA1 C-terminal (BRCT) domains are phospho-serine/threonine recognition modules essential for the function of BRCA1. Recent studies suggest that acetyl-CoA carboxylase 1 (ACC1), an enzyme with crucial roles in de novo fatty acid biosynthesis and lipogenesis and essential for cancer cell survival, may be a novel binding partner for BRCA1, through interactions with its BRCT domains. We report here the crystal structure at 3.2 Angstroms resolution of human BRCA1 BRCT domains in complex with a phospho-peptide from human ACC1 (p-ACC1 peptide, with the sequence 1258-DSPPQ-pS-PTFPEAGH-1271), which provides molecular evidence for direct interactions between BRCA1 and ACC1. The p-ACC1 peptide is bound in an extended conformation, located in a groove between the tandem BRCT domains. There are recognizable and significant structural differences to the binding modes of two other phospho-peptides to these domains, from BACH1 and CtIP, even though they share a conserved pSer-Pro-(Thr/Val)-Phe motif. Our studies establish a framework for understanding the regulation of lipid biosynthesis by BRCA1 through its inhibition of ACC1 activity, which could be a novel tumor suppressor function of BRCA1.

  14. GENERAL: The effect of ACC vehicles to mixed traffic flow consisting of manual and ACC vehicles

    NASA Astrophysics Data System (ADS)

    Xie, Dong-Fan; Gao, Zi-You; Zhao, Xiao-Mei

    2008-12-01

    This paper studies the effect of adaptive cruise control (ACC) system on traffic flow by using simulations. The multiple headway and velocity direrence (MHVD) model is used to depict the motion of ACC vehicles, and the simulation results are compared with the optimal velocity (OV) model which is used to depict the motion of manual vehicles. Compared the cases between the manual and the ACC vehicle flow, the fundamental diagram can be classified into four regions: I, II, III, IV. In low and high density the flux of the two models is the same; in region II the free flow region of the MHVD model is enlarged, and the flux of the MHVD model is larger than that of the OV model; in region III serious jams occur in the OV model while the ACC system suppresses the jams in the MHVD model and the traffic flow is in order, but the flux of the OV model is larger than that of the MHVD model. Similar phenomena also appeared in mixed traffic flow which consists of manual and ACC vehicles. The results indicate that ACC vehicles have significant effect on traffic flow. The improvement induced by ACC vehicles decreases with the increasing proportion of ACC vehicles.

  15. The human ACC2 CT-domain C-terminus is required for full functionality and has a novel twist

    PubMed Central

    Madauss, Kevin P.; Burkhart, William A.; Consler, Thomas G.; Cowan, David J.; Gottschalk, William K.; Miller, Aaron B.; Short, Steven A.; Tran, Thuy B.; Williams, Shawn P.

    2009-01-01

    Inhibition of acetyl-CoA carboxylase (ACC) may prevent lipid-induced insulin resistance and type 2 diabetes, making the enzyme an attractive pharmaceutical target. Although the enzyme is highly conserved amongst animals, only the yeast enzyme structure is available for rational drug design. The use of biophysical assays has permitted the identification of a specific C-terminal truncation of the 826-residue human ACC2 carboxyl transferase (CT) domain that is both functionally competent to bind inhibitors and crystallizes in their presence. This C-terminal truncation led to the determination of the human ACC2 CT domain–CP-640186 complex crystal structure, which revealed distinctions from the yeast-enzyme complex. The human ACC2 CT-domain C-terminus is com­prised of three intertwined α-helices that extend outwards from the enzyme on the opposite side to the ligand-binding site. Differences in the observed inhibitor conformation between the yeast and human structures are caused by differing residues in the binding pocket. PMID:19390150

  16. The human ACC2 CT-domain C-terminus is required for full functionality and has a novel twist

    SciTech Connect

    Madauss, Kevin P.; Burkhart, William A.; Consler, Thomas G.; Cowan, David J.; Gottschalk, William K.; Miller, Aaron B; Short, Steven A.; Tran, Thuy B.; Williams, Shawn P.

    2009-06-15

    Inhibition of acetyl-CoA carboxylase (ACC) may prevent lipid-induced insulin resistance and type 2 diabetes, making the enzyme an attractive pharmaceutical target. Although the enzyme is highly conserved amongst animals, only the yeast enzyme structure is available for rational drug design. The use of biophysical assays has permitted the identification of a specific C-terminal truncation of the 826-residue human ACC2 carboxyl transferase (CT) domain that is both functionally competent to bind inhibitors and crystallizes in their presence. This C-terminal truncation led to the determination of the human ACC2 CT domain-CP-640186 complex crystal structure, which revealed distinctions from the yeast-enzyme complex. The human ACC2 CT-domain C-terminus is comprised of three intertwined -helices that extend outwards from the enzyme on the opposite side to the ligand-binding site. Differences in the observed inhibitor conformation between the yeast and human structures are caused by differing residues in the binding pocket.

  17. Continuous fat oxidation in acetyl–CoA carboxylase 2 knockout mice increases total energy expenditure, reduces fat mass, and improves insulin sensitivity

    PubMed Central

    Choi, Cheol Soo; Savage, David B.; Abu-Elheiga, Lutfi; Liu, Zhen-Xiang; Kim, Sheene; Kulkarni, Ameya; Distefano, Alberto; Hwang, Yu-Jin; Reznick, Richard M.; Codella, Roberto; Zhang, Dongyan; Cline, Gary W.; Wakil, Salih J.; Shulman, Gerald I.

    2007-01-01

    Acetyl–CoA carboxylase 2 (ACC)2 is a key regulator of mitochondrial fat oxidation. To examine the impact of ACC2 deletion on whole-body energy metabolism, we measured changes in substrate oxidation and total energy expenditure in Acc2−/− and WT control mice fed either regular or high-fat diets. To determine insulin action in vivo, we also measured whole-body insulin-stimulated liver and muscle glucose metabolism during a hyperinsulinemic–euglycemic clamp in Acc2−/− and WT control mice fed a high-fat diet. Contrary to previous studies that have suggested that increased fat oxidation might result in lower glucose oxidation, both fat and carbohydrate oxidation were simultaneously increased in Acc2−/− mice. This increase in both fat and carbohydrate oxidation resulted in an increase in total energy expenditure, reductions in fat and lean body mass and prevention from diet-induced obesity. Furthermore, Acc2−/− mice were protected from fat-induced peripheral and hepatic insulin resistance. These improvements in insulin-stimulated glucose metabolism were associated with reduced diacylglycerol content in muscle and liver, decreased PKCθ activity in muscle and PKCε activity in liver, and increased insulin-stimulated Akt2 activity in these tissues. Taken together with previous work demonstrating that Acc2−/− mice have a normal lifespan, these data suggest that Acc2 inhibition is a viable therapeutic option for the treatment of obesity and type 2 diabetes. PMID:17923673

  18. ACC forum looks at 'burning' questions

    SciTech Connect

    Carter, R.

    2005-06-01

    The American Coal Council's (ACC) Spring Coal Forum had as its theme: Coal's renaissance: prospects for regenerating coal generation'. It explored US coal demand, supply, end-user technology and market trends. The article gives an overview of the conference, highlighting several presentations. 2 figs., 1 tab.

  19. Adrenocortical carcinoma (ACC): diagnosis, prognosis, and treatment

    PubMed Central

    Libé, Rossella

    2015-01-01

    Adrenocortical carticnoma (ACC) is a rare malignancy with an incidence of 0.7–2.0 cases/million habitants/year. The diagnosis of malignancy relies on careful investigations of clinical, biological, and imaging features before surgery and pathological examination after tumor removal. Most patients present with steroid hormone excess or abdominal mass effects, but 15% of patients with ACC is initially diagnosed incidentally. After the diagnosis, in order to assess the ACC prognosis and establish an adequate basis for treatment decisions different tools are proposed. The stage classification proposed by the European Network for the Study of Adrenal Tumors (ENSAT) is recommended. Pathology reports define the Weiss score, the resection status and the proliferative index, including the mitotic count and the Ki67 index. As far as the treatment is concerned, in case of tumor limited to the adrenal gland, the complete resection of the tumor is the first option. Most patients benefit from adjuvant mitotane treatment. In metastatic disease, mitotane is the cornerstone of initial treatment, and cytotoxic drugs should be added in case of progression. Recently, the First International Randomized (FIRM-ACT) Trial in metastatic ACC reported the association between mitotane and etoposide/doxorubicin/cisplatin (EDP) as the new standard in first line treatment of ACC. In last years, new targeted therapies, including the IGF-1 receptor inhibitors, have been investigated, but their efficacy remains limited. Thus, new treatment concepts are urgently needed. The ongoing “omic approaches” and next-generation sequencing will improve our understanding of the pathogenesis and hopefully will lead to better therapies. PMID:26191527

  20. Novel Insights into the Biotin Carboxylase Domain Reactions of Pyruvate Carboxylase from Rhizobium etli†

    PubMed Central

    Zeczycki, Tonya N.; Menefee, Ann L.; Adina-Zada, Abdussalam; Jitrapakdee, Sarawut; Surinya, Kathy H.; Wallace, John C.; Attwood, Paul V.; St. Maurice, Martin; Cleland, W. Wallace

    2011-01-01

    The catalytic mechanism of the MgATP-dependent carboxylation of biotin in the biotin carboxylase domain of pyruvate carboxylase from R. etli (RePC) is common to the biotin-dependent carboxylases. The current site-directed mutagenesis study has clarified the catalytic functions of several residues proposed to be pivotal in MgATP-binding and cleavage (Glu218 and Lys245), HCO3− deprotonation (Glu305 and Arg301) and biotin enolization (Arg353). The E218A mutant was inactive for any reaction involving the BC domain and the E218Q mutant exhibited a 75-fold decrease in kcat for both pyruvate carboxylation and the full reverse reaction. The E305A mutant also showed a 75- and 80-fold decrease in kcat for both pyruvate carboxylation and the full reverse reaction, respectively. While Glu305 appears to be the active site base which deprotonates HCO3−, Lys245, Glu218 and Arg301 are proposed to contribute to catalysis through substrate binding interactions. The reactions of the biotin carboxylase and carboxyl transferase domains were uncoupled in the R353M-catalyzed reactions, indicating that Arg353 may not only facilitate the formation of the biotin enolate, but also assist in coordinating catalysis between the two spatially distinct active sites. The 2.5 and 4-fold increase in kcat for the full reverse reaction with the R353K and R353M mutants, respectively, suggests that mutation of Arg353 allows carboxybiotin increased access to the biotin carboxylase domain active site. The proposed chemical mechanism is initiated by the deprotonation of HCO3− by Glu305 and concurrent nucleophilic attack on the γ-phosphate of MgATP. The trianionic carboxyphosphate intermediate formed reversibly decomposes in the active site to CO2 and PO43−. PO43− then acts as the base to deprotonate the tethered biotin at the N1-position. Stabilized by interactions between the ureido oxygen and Arg353, the biotin-enolate reacts with CO2 to give carboxybiotin. The formation of a distinct salt

  1. Molecular basis for the inhibition of the carboxyltransferase domain of acetyl-coenzyme-A carboxylase by haloxyfop and diclofop

    PubMed Central

    Zhang, Hailong; Tweel, Benjamin; Tong, Liang

    2004-01-01

    Acetyl-CoA carboxylases (ACCs) are crucial for the metabolism of fatty acids, making these enzymes important targets for the development of therapeutics against obesity, diabetes, and other diseases. The carboxyltransferase (CT) domain of ACC is the site of action of commercial herbicides, such as haloxyfop, diclofop, and sethoxydim. We have determined the crystal structures at up to 2.5-Å resolution of the CT domain of yeast ACC in complex with the herbicide haloxyfop or diclofop. The inhibitors are bound in the active site, at the interface of the dimer of the CT domain. Unexpectedly, inhibitor binding requires large conformational changes for several residues in this interface, which create a highly conserved hydrophobic pocket that extends deeply into the core of the dimer. Two residues that affect herbicide sensitivity are located in this binding site, and mutation of these residues disrupts the structure of the domain. Other residues in the binding site are strictly conserved among the CT domains. PMID:15079078

  2. DNA inhibits catalysis by the carboxyltransferase subunit of acetyl-CoA carboxylase: implications for active site communication.

    PubMed

    Benson, Brian K; Meades, Glen; Grove, Anne; Waldrop, Grover L

    2008-01-01

    Acetyl-CoA carboxylase (ACC) catalyzes the first committed step in the synthesis of long-chain fatty acids. The crystal structure of the Escherichia coli carboxyltransferase component of ACC revealed an alpha(2)beta(2) subunit composition with two active sites and, most importantly, a unique zinc domain in each alphabeta pair that is absent in the eukaryotic enzyme. We show here that carboxyltransferase binds DNA. Half-maximal saturation of different single-stranded or double-stranded DNA constructs is seen at 0.5-1.0 muM, and binding is cooperative and nonspecific. The substrates (malonyl-CoA and biocytin) inhibit DNA:carboxyltransferase complex formation. More significantly, single-stranded DNA, double-stranded DNA, and heparin inhibit the reaction catalyzed by carboxyltransferase, with single-stranded DNA and heparin acting as competitive inhibitors. However, double-inhibition experiments revealed that both DNA and heparin can bind the enzyme in the presence of a bisubstrate analog (BiSA), and the binding of BiSA has a very weak synergistic effect on the binding of the second inhibitor (DNA or heparin) and vice versa. In contrast, DNA and heparin can also bind to the enzyme simultaneously, but the binding of either molecule has a strong synergistic effect on binding of the other. An important mechanistic implication of these observations is that the dual active sites of ACC are functionally connected.

  3. Biochemical characterization of a Rhizobium etli monovalent cation-stimulated acyl-coenzyme A carboxylase with a high substrate specificity constant for propionyl-coenzyme A.

    PubMed

    Dunn, Michael F; Araíza, Gisela; Mora, Jaime

    2004-02-01

    Biotin has a profound effect on the metabolism of rhizobia. It is reported here that the activities of the biotin-dependent enzymes acetyl-coenzyme A carboxylase (ACC; EC 6.4.1.2) and propionyl-coenzyme A carboxylase (PCC; EC 6.4.1.3) are present in all species of the five genera comprising the Rhizobiaceae which were examined. Evidence is presented that the ACC and PCC activities detectable in Rhizobium etli extracts are catalysed by a single acyl-coenzyme A carboxylase. The enzyme from R. etli strain 12-53 was purified 478-fold and displayed its highest activity with propionyl-CoA as substrate, with apparent K(m) and V(max) values of 0.064 mM and 2885 nmol min(-1) (mg protein)(-1), respectively. The enzyme carboxylated acetyl-CoA and butyryl-CoA with apparent K(m) values of 0.392 and 0.144 mM, respectively, and V(max) values of 423 and 268 nmol min(-1) (mg protein)(-1), respectively. K(+), or Cs(+) markedly activated the enzyme, which was essentially inactive in their absence. Electrophoretic analysis indicated that the acyl-CoA carboxylase was composed of a 74 kDa biotin-containing alpha subunit and a 45 kDa biotin-free beta subunit, and gel chromatography indicated a total molecular mass of 620 000 Da. The strong kinetic preference of the enzyme for propionyl-CoA is consistent with its participation in an anaplerotic pathway utilizing this substrate.

  4. Molecular evolution of urea amidolyase and urea carboxylase in fungi

    PubMed Central

    2011-01-01

    Background Urea amidolyase breaks down urea into ammonia and carbon dioxide in a two-step process, while another enzyme, urease, does this in a one step-process. Urea amidolyase has been found only in some fungal species among eukaryotes. It contains two major domains: the amidase and urea carboxylase domains. A shorter form of urea amidolyase is known as urea carboxylase and has no amidase domain. Eukaryotic urea carboxylase has been found only in several fungal species and green algae. In order to elucidate the evolutionary origin of urea amidolyase and urea carboxylase, we studied the distribution of urea amidolyase, urea carboxylase, as well as other proteins including urease, across kingdoms. Results Among the 64 fungal species we examined, only those in two Ascomycota classes (Sordariomycetes and Saccharomycetes) had the urea amidolyase sequences. Urea carboxylase was found in many but not all of the species in the phylum Basidiomycota and in the subphylum Pezizomycotina (phylum Ascomycota). It was completely absent from the class Saccharomycetes (phylum Ascomycota; subphylum Saccharomycotina). Four Sordariomycetes species we examined had both the urea carboxylase and the urea amidolyase sequences. Phylogenetic analysis showed that these two enzymes appeared to have gone through independent evolution since their bacterial origin. The amidase domain and the urea carboxylase domain sequences from fungal urea amidolyases clustered strongly together with the amidase and urea carboxylase sequences, respectively, from a small number of beta- and gammaproteobacteria. On the other hand, fungal urea carboxylase proteins clustered together with another copy of urea carboxylases distributed broadly among bacteria. The urease proteins were found in all the fungal species examined except for those of the subphylum Saccharomycotina. Conclusions We conclude that the urea amidolyase genes currently found only in fungi are the results of a horizontal gene transfer event from

  5. Reduced AMPK-ACC and mTOR signaling in muscle from older men, and effect of resistance exercise

    PubMed Central

    Li, Mengyao; Verdijk, Lex B.; Sakamoto, Kei; Ely, Brian; van Loon, Luc J.C.; Musi, Nicolas

    2012-01-01

    AMP-activated protein kinase (AMPK) is a key energy-sensitive enzyme that controls numerous metabolic and cellular processes. Mammalian target of rapamycin (mTOR) is another energy/nutrient-sensitive kinase that controls protein synthesis and cell growth. In this study we determined whether older versus younger men have alterations in the AMPK and mTOR pathways in skeletal muscle, and examined the effect of a long term resistance type exercise training program on these signaling intermediaries. Older men had decreased AMPKα2 activity and lower phosphorylation of AMPK and its downstream signaling substrate acetyl-CoA carboxylase (ACC). mTOR phosphylation also was reduced in muscle from older men. Exercise training increased AMPKα1 activity in older men, however, AMPKα2 activity, and the phosphorylation of AMPK, ACC and mTOR, were not affected. In conclusion, older men have alterations in the AMPK-ACC and mTOR pathways in muscle. In addition, prolonged resistance type exercise training induces an isoform-selective up regulation of AMPK activity. PMID:23000302

  6. Ribulose 1,5-bisphosphate carboxylase and phosphoribulokinase in Prochloron

    NASA Technical Reports Server (NTRS)

    Berhow, M. A.; Mcfadden, B. A.

    1983-01-01

    Ribulose 1,5-bisphosphate (RuBP) carboxylase and phosphoribulokinase, enzymes in the reductive pentose-phosphate cycle, were measured in cell-free extracts of Prochloran didemni. The partial purification and characterization of RuBP carboxylase were described. Prochloron RuBP carboxylase, when purified by isopycnic centrifugation in reoriented linear 0.2 to 0.8 M sucrose gradients, sedimented to a position which corresponded to that of the 520,000-dalton spinach enzyme. Sodium dodecyl sulfate polyacrylamide gel electrophoresis showed that the Prochloron enzyme was composed of large and small subunits (MW = 57,500 and 18,800). Though results established that the enzymes RuBP carboxylase and phosphoribulokinase were present in levels comparable to other CO2-fixing microorganisms, it was suggested that other enzymes in the Calvin cycle limit growth or that additional enzymic insufficiencies exist.

  7. Drosophila melanogaster Acetyl-CoA-carboxylase sustains a fatty acid-dependent remote signal to waterproof the respiratory system.

    PubMed

    Parvy, Jean-Philippe; Napal, Laura; Rubin, Thomas; Poidevin, Mickael; Perrin, Laurent; Wicker-Thomas, Claude; Montagne, Jacques

    2012-01-01

    Fatty acid (FA) metabolism plays a central role in body homeostasis and related diseases. Thus, FA metabolic enzymes are attractive targets for drug therapy. Mouse studies on Acetyl-coenzymeA-carboxylase (ACC), the rate-limiting enzyme for FA synthesis, have highlighted its homeostatic role in liver and adipose tissue. We took advantage of the powerful genetics of Drosophila melanogaster to investigate the role of the unique Drosophila ACC homologue in the fat body and the oenocytes. The fat body accomplishes hepatic and storage functions, whereas the oenocytes are proposed to produce the cuticular lipids and to contribute to the hepatic function. RNA-interfering disruption of ACC in the fat body does not affect viability but does result in a dramatic reduction in triglyceride storage and a concurrent increase in glycogen accumulation. These metabolic perturbations further highlight the role of triglyceride and glycogen storage in controlling circulatory sugar levels, thereby validating Drosophila as a relevant model to explore the tissue-specific function of FA metabolic enzymes. In contrast, ACC disruption in the oenocytes through RNA-interference or tissue-targeted mutation induces lethality, as does oenocyte ablation. Surprisingly, this lethality is associated with a failure in the watertightness of the spiracles-the organs controlling the entry of air into the trachea. At the cellular level, we have observed that, in defective spiracles, lipids fail to transfer from the spiracular gland to the point of air entry. This phenotype is caused by disrupted synthesis of a putative very-long-chain-FA (VLCFA) within the oenocytes, which ultimately results in a lethal anoxic issue. Preventing liquid entry into respiratory systems is a universal issue for air-breathing animals. Here, we have shown that, in Drosophila, this process is controlled by a putative VLCFA produced within the oenocytes.

  8. Nuclear-Cytoplasmic Conflict in Pea (Pisum sativum L.) Is Associated with Nuclear and Plastidic Candidate Genes Encoding Acetyl-CoA Carboxylase Subunits

    PubMed Central

    Bogdanova, Vera S.; Zaytseva, Olga O.; Mglinets, Anatoliy V.; Shatskaya, Natalia V.; Kosterin, Oleg E.; Vasiliev, Gennadiy V.

    2015-01-01

    In crosses of wild and cultivated peas (Pisum sativum L.), nuclear-cytoplasmic incompatibility frequently occurs manifested as decreased pollen fertility, male gametophyte lethality, sporophyte lethality. High-throughput sequencing of plastid genomes of one cultivated and four wild pea accessions differing in cross-compatibility was performed. Candidate genes for involvement in the nuclear-plastid conflict were searched in the reconstructed plastid genomes. In the annotated Medicago truncatula genome, nuclear candidate genes were searched in the portion syntenic to the pea chromosome region known to harbor a locus involved in the conflict. In the plastid genomes, a substantial variability of the accD locus represented by nucleotide substitutions and indels was found to correspond to the pattern of cross-compatibility among the accessions analyzed. Amino acid substitutions in the polypeptides encoded by the alleles of a nuclear locus, designated as Bccp3, with a complementary function to accD, fitted the compatibility pattern. The accD locus in the plastid genome encoding beta subunit of the carboxyltransferase of acetyl-coA carboxylase and the nuclear locus Bccp3 encoding biotin carboxyl carrier protein of the same multi-subunit enzyme were nominated as candidate genes for main contribution to nuclear-cytoplasmic incompatibility in peas. Existence of another nuclear locus involved in the accD-mediated conflict is hypothesized. PMID:25789472

  9. Structural analysis, plastid localization, and expression of the biotin carboxylase subunit of acetyl-coenzyme A carboxylase from tobacco.

    PubMed Central

    Shorrosh, B S; Roesler, K R; Shintani, D; van de Loo, F J; Ohlrogge, J B

    1995-01-01

    Acetyl-coenzyme A carboxylase (ACCase, EC 6.4.1.2) catalyzes the synthesis of malonyl-coenzyme A, which is utilized in the plastid for de novo fatty acid synthesis and outside the plastid for a variety of reactions, including the synthesis of very long chain fatty acids and flavonoids. Recent evidence for both multifunctional and multisubunit ACCase isozymes in dicot plants has been obtained. We describe here the isolation of a tobacco (Nicotiana tabacum L. cv bright yellow 2 [NT1]) cDNA clone (E3) that encodes a 58.4-kD protein that shares 80% sequence similarity and 65% identity with the Anabaena biotin carboxylase subunit of ACCase. Similar to other biotin carboxylase subunits of acetyl-CoA carboxylase, the E3-encoded protein contains a putative ATP-binding motif but lacks a biotin-binding site (methionine-lysine-methionine or methionine-lysine-leucine). The deduced protein sequence contains a putative transit peptide whose function was confirmed by its ability to direct in vitro chloroplast uptake. The subcellular localization of this biotin carboxylase has also been confirmed to be plastidial by western blot analysis of pea (Pisum sativum), alfalfa (Medicago sativa L.), and castor (Ricinus communis L.) plastid preparations. Northern blot analysis indicates that the plastid biotin carboxylase transcripts are expressed at severalfold higher levels in castor seeds than in leaves. PMID:7610168

  10. Ethanolic Extract of Vitis thunbergii Exhibits Lipid Lowering Properties via Modulation of the AMPK-ACC Pathway in Hypercholesterolemic Rabbits

    PubMed Central

    Pan, Chun-Hsu; Tsai, Chia-Hua; Lin, Wen-Hsin; Chen, Guo-Yan; Wu, Chieh-Hsi

    2012-01-01

    Vitis thunbergii (VT) is a wild grape that has been shown to provide various cardioprotective effects. The present study was designed to examine whether a VT extract could reduce serum lipid levels and prevent atherogenesis in a hypercholesterolemic rabbit model. At the end of an 8-week study, our results showed that a VT extract supplement markedly suppressed the serum levels of cholesterol and low-density lipoprotein, reduced lipid accumulation in liver tissues, and limited aortic fatty streaks. Our findings suggest that the VT extract activated AMPK (5′-adenosine monophosphate-activated protein kinase) with subsequent inhibition of the activation of ACC (acetyl-CoA carboxylase). Our results suggest that this VT extract could be further developed as a potential lipid-lowering agent and as a natural health food to prevent atherogenesis. PMID:22536284

  11. Lattice QCD simulations using the OpenACC platform

    NASA Astrophysics Data System (ADS)

    Majumdar, Pushan

    2016-10-01

    In this article we will explore the OpenACC platform for programming Graphics Processing Units (GPUs). The OpenACC platform offers a directive based programming model for GPUs which avoids the detailed data flow control and memory management necessary in a CUDA programming environment. In the OpenACC model, programs can be written in high level languages with OpenMP like directives. We present some examples of QCD simulation codes using OpenACC and discuss their performance on the Fermi and Kepler GPUs.

  12. Improving production of malonyl coenzyme A-derived metabolites by abolishing Snf1-dependent regulation of Acc1.

    PubMed

    Shi, Shuobo; Chen, Yun; Siewers, Verena; Nielsen, Jens

    2014-05-06

    ABSTRACT Acetyl coenzyme A (acetyl-CoA) carboxylase (ACCase) plays a central role in carbon metabolism and has been the site of action for the development of therapeutics or herbicides, as its product, malonyl-CoA, is a precursor for production of fatty acids and other compounds. Control of Acc1 activity in the yeast Saccharomyces cerevisiae occurs mainly at two levels, i.e., regulation of transcription and repression by Snf1 protein kinase at the protein level. Here, we demonstrate a strategy for improving the activity of ACCase in S. cerevisiae by abolishing posttranslational regulation of Acc1 via site-directed mutagenesis. It was found that introduction of two site mutations in Acc1, Ser659 and Ser1157, resulted in an enhanced activity of Acc1 and increased total fatty acid content. As Snf1 regulation of Acc1 is particularly active under glucose-limited conditions, we evaluated the effect of the two site mutations in chemostat cultures. Finally, we showed that our modifications of Acc1 could enhance the supply of malonyl-CoA and therefore successfully increase the production of two industrially important products derived from malonyl-CoA, fatty acid ethyl esters and 3-hydroxypropionic acid. IMPORTANCE ACCase is responsible for carboxylation of acetyl-CoA to produce malonyl-CoA, which is a crucial step in the control of fatty acid metabolism. ACCase opened the door for pharmaceutical treatments of obesity and diabetes as well as the development of new herbicides. ACCase is also recognized as a promising target for developing cell factories, as its malonyl-CoA product serves as a universal precursor for a variety of high-value compounds in white biotechnology. Yeast ACCase is a good model in understanding the enzyme's catalysis, regulation, and inhibition. The present study describes the importance of protein phosphorylation in regulation of yeast ACCase and identifies potential regulation sites. This study led to the generation of a more efficient ACCase, which

  13. Inhibition of acetyl-coenzyme A carboxylase by two classes of grass-selective herbicides

    SciTech Connect

    Rendina, A.R.; Craig-Kennard, A.C.; Beaudoin, J.D.; Breen, M.K. )

    1990-05-01

    The selective grass herbicides diclofop, haloxyfop, and trifop (((aryloxy)phenoxy)propionic acids) and alloxydim, sethoxydim, and clethodim (cyclohexanediones) are potent, reversible inhibitors of acetyl-coenzyme A carboxylase (ACC) partially purified from barley, corn, and wheat. Although inhibition of the wheat enzyme by clethodim and diclofop is noncompetitive versus each of the substrates adenosine triphosphate (ATP), HCO{sub 3}{sup {minus}}, and acetyl-coenzyme A (acetyl-CoA), diclofop and clethodim are nearly competitive versus acetyl-CoA since the level of inhibition is most sensitive to the concentration of acetyl-CoA (K{sub is} < K{sub ii}). To conclusively show whether the herbicides interact at the biotin carboxylation site or the carboxyl transfer site, the inhibition of isotope exchange and partial reactions catalyzed at each site was studied with the wheat enzyme. Only the ({sup 14}C)acetyl-CoA-malonyl-CoA exchange and decarboxylation of ({sup 14}C)malonyl-CoA reactions are strongly inhibited by clethodim and diclofop, suggesting that the herbicides interfere with the carboxyl transfer site rather than the biotin carboxylation site of the enzyme. Double-inhibition studies with diclofop and clethodim suggest that the ((aryloxy)phenoxy)propionic acid and cyclohexanedione herbicides may bind to the same region of the enzyme.

  14. In silico structural and functional analysis of Mesorhizobium ACC deaminase.

    PubMed

    Pramanik, Krishnendu; Soren, Tithi; Mitra, Soumik; Maiti, Tushar Kanti

    2017-02-11

    Nodulation is one of the very important processes of legume plants as it is the initiating event of fixing nitrogen. Although ethylene has essential role in normal plant metabolism but it has also negative impact on plants particularly in nodule formation in legume plants. It is also produced due to a variety of biotic or abiotic stresses. 1-aminocyclopropane-1-carboxylic acid (ACC) deaminase is a rhizobial enzyme which cleaves ACC (immediate precursor of ethylene) into α-ketobutyrate and ammonia. As a result, the level of ethylene from the plant cells is decreased and the negative impact of ethylene on nodule formation is reduced. ACC deaminase is widely studied in several plant growth promoting rhizobacterial (PGPR) strains including many legume nodulating bacteria like Mesorhizobium sp. It is an important symbiotic nitrogen fixer belonging to the class - alphaproteobacteria under the order Rhizobiales. ACC deaminase has positive role in Legume-rhizobium symbiosis. Rhizobial ACC deaminase has the potentiality to reduce the adverse effects of ethylene, thereby triggering the nodulation process. The present study describes an in silico comparative structural (secondary structure prediction, homology modeling) and functional analysis of ACC deaminase from Mesorhizobium spp. to explore physico-chemical properties using a number of bio-computational tools. M. loti was selected as a representative species of Mesorhizobium genera for 3D modelling of ACC deaminase protein. Correlation by the phylogenetic relatedness on the basis of both ACC deaminase enzymes and respective acdS genes of different strains of Mesorhizobium has also studied.

  15. Characterization of ribulose diphosphate carboxylase and phosphoribulokinase from Thiobacillus thioparus and Thiobacillus neapolitanus.

    NASA Technical Reports Server (NTRS)

    Johnson, E. J.; Johnson, M. K.; Macelroy, R. D.

    1968-01-01

    Ribulose diphosphate carboxylase and phosphoribulokinase activity in chemosynthetic autotrophs Thiobacillus thioparus and Thiobacillus neapolitanus, noting sedimentation and gel filtration characteristics

  16. Differentiation of 1-aminocyclopropane-1-carboxylate (ACC) deaminase from its homologs is the key for identifying bacteria containing ACC deaminase.

    PubMed

    Li, Zhengyi; Chang, Siping; Ye, Shuting; Chen, Mingyue; Lin, Li; Li, Yuanyuan; Li, Shuying; An, Qianli

    2015-10-01

    1-Aminocyclopropane-1-carboxylate (ACC) deaminase-mediated reduction of ethylene generation in plants under abiotic stresses is a key mechanism by which bacteria can promote plant growth. Misidentification of ACC deaminase and the ACC deaminase structure gene (acdS) can lead to overestimation of the number of bacteria containing ACC deaminase and their function in ecosystems. Previous non-specific amplification of acdS homologs has led to an overestimation of the horizontal transfer of acdS genes. Here, we designed consensus-degenerate hybrid oligonucleotide primers (acdSf3, acdSr3 and acdSr4) based on differentiating the key residues in ACC deaminases from those of homologs for specific amplification of partial acdS genes. PCR amplification, sequencing and phylogenetic analysis identified acdS genes from a wide range of proteobacteria and actinobacteria. PCR amplification and a genomic search did not find the acdS gene in bacteria belonging to Pseudomonas stutzeri or in the genera Enterobacter, Klebsiella or Bacillus. We showed that differentiating the acdS gene and ACC deaminase from their homologs was crucial for the molecular identification of bacteria containing ACC deaminase and for understanding the evolution of the acdS gene. We provide an effective method for screening and identifying bacteria containing ACC deaminase.

  17. Susceptibility of podocytes to palmitic acid is regulated by fatty acid oxidation and inversely depends on acetyl-CoA carboxylases 1 and 2.

    PubMed

    Kampe, Kapil; Sieber, Jonas; Orellana, Jana Marina; Mundel, Peter; Jehle, Andreas Werner

    2014-02-15

    Type 2 diabetes is characterized by dyslipidemia with elevated free fatty acids (FFAs). Loss of podocytes is a hallmark of diabetic nephropathy, and podocytes are susceptible to saturated FFAs, which induce endoplasmic reticulum (ER) stress and podocyte death. Genome-wide association studies indicate that expression of acetyl-CoA carboxylase (ACC) 2, a key enzyme of fatty acid oxidation (FAO), is associated with proteinuria in type 2 diabetes. Here, we show that stimulation of FAO by aminoimidazole-4-carboxamide-1β-D-ribofuranoside (AICAR) or by adiponectin, activators of the low-energy sensor AMP-activated protein kinase (AMPK), protects from palmitic acid-induced podocyte death. Conversely, inhibition of carnitine palmitoyltransferase (CPT-1), the rate-limiting enzyme of FAO and downstream target of AMPK, augments palmitic acid toxicity and impedes the protective AICAR effect. Etomoxir blocked the AICAR-induced FAO measured with tritium-labeled palmitic acid. The beneficial effect of AICAR was associated with a reduction of ER stress, and it was markedly reduced in ACC-1/-2 double-silenced podocytes. In conclusion, the stimulation of FAO by modulating the AMPK-ACC-CPT-1 pathway may be part of a protective mechanism against saturated FFAs that drive podocyte death. Further studies are needed to investigate the potentially novel therapeutic implications of these findings.

  18. RNAi knockdown of acetyl-CoA carboxylase gene eliminates jinggangmycin-enhanced reproduction and population growth in the brown planthopper, Nilaparvata lugens

    PubMed Central

    Zhang, Yi-Xin; Ge, Lin-Quan; Jiang, Yi-Ping; Lu, Xiu-Li; Li, Xin; Stanley, David; Song, Qi-Sheng; Wu, Jin-Cai

    2015-01-01

    A major challenge in ecology lies in understanding the coexistence of intraguild species, well documented at the organismal level, but not at the molecular level. This study focused on the effects of the antibiotic, jinggangmycin (JGM), a fungicide widely used in Asian rice agroecosystems, on reproduction of insects within the planthopper guild, including the brown planthopper (BPH) Nilaparvata lugens and the white-backed planthopper (WBPH) Sogatella furcifera, both serious resurgence rice pests. JGM exposure significantly increased BPH fecundity and population growth, but suppressed both parameters in laboratory and field WBPH populations. We used digital gene expression and transcriptomic analyses to identify a panel of differentially expressed genes, including a set of up-regulated genes in JGM-treated BPH, which were down-regulated in JGM-treated WBPH. RNAi silencing of Acetyl Co-A carboxylase (ACC), highly expressed in JGM-treated BPH, reduced ACC expression (by > 60%) and eliminated JGM-induced fecundity increases in BPH. These findings support our hypothesis that differences in ACC expression separates intraguild species at the molecular level. PMID:26482193

  19. RNAi knockdown of acetyl-CoA carboxylase gene eliminates jinggangmycin-enhanced reproduction and population growth in the brown planthopper, Nilaparvata lugens.

    PubMed

    Zhang, Yi-Xin; Ge, Lin-Quan; Jiang, Yi-Ping; Lu, Xiu-Li; Li, Xin; Stanley, David; Song, Qi-Sheng; Wu, Jin-Cai

    2015-10-20

    A major challenge in ecology lies in understanding the coexistence of intraguild species, well documented at the organismal level, but not at the molecular level. This study focused on the effects of the antibiotic, jinggangmycin (JGM), a fungicide widely used in Asian rice agroecosystems, on reproduction of insects within the planthopper guild, including the brown planthopper (BPH) Nilaparvata lugens and the white-backed planthopper (WBPH) Sogatella furcifera, both serious resurgence rice pests. JGM exposure significantly increased BPH fecundity and population growth, but suppressed both parameters in laboratory and field WBPH populations. We used digital gene expression and transcriptomic analyses to identify a panel of differentially expressed genes, including a set of up-regulated genes in JGM-treated BPH, which were down-regulated in JGM-treated WBPH. RNAi silencing of Acetyl Co-A carboxylase (ACC), highly expressed in JGM-treated BPH, reduced ACC expression (by > 60%) and eliminated JGM-induced fecundity increases in BPH. These findings support our hypothesis that differences in ACC expression separates intraguild species at the molecular level.

  20. Covalent dimerization of ribulose bisphosphate carboxylase subunits by UV radiation.

    PubMed

    Ferreira, R M; Franco, E; Teixeira, A R

    1996-08-15

    The effect of UV radiation (UV-A, UV-B and UV-C) on ribulose bisphosphate carboxylase from a variety of plant species was examined. The exposition of plant leaves or the pure enzyme to UV radiation produced a UV-dependent accumulation of a +5 kDa polypeptide (P65). Different approaches were utilized to elucidate the origin and structure of P65: electrophoretic and fluorographic analyses of 35S-labelled ribulose bisphosphate carboxylase exposed to UV radiation and immunological experiments using antibodies specific for P65, for the large and small subunits of ribulose bisphosphate carboxylase and for high-molecular-mass aggregates of the enzyme. These studies revealed that P65 is a dimer, formed by the covalent, non-disulphide linkage of one small subunit with one large subunit of ribulose bisphosphate carboxylase. For short periods of time (< 1 h), the amount of P65 formed increased with the duration of the exposure to the UV radiation and with the energy of the radiation applied. Prolonged exposure to UV radiation (1-6 h) resulted in the formation of high-molecular-mass aggregates of ribulose bisphosphate carboxylase. Formation of P65 was shown to depend on the native state of the protein, was stimulated by inhibitors of enzyme activity, and was inhibited by activators of enzyme activity. A UV-independent accumulation of P65 was also achieved by the in vitro incubation of plant crude extracts. However, the UV-dependent and the UV-independent formation of P65 seemed to occur by distinct molecular mechanisms. The UV-dependent accumulation of P65 was immunologically detected in all species examined, including Lemna minor, Arum italicum, Brassica oleracea, Triticum aestivum, Zea mays, Pisum sativum and Phaseolus vulgaris, suggesting that it may constitute a universal response to UV radiation, common to all photo-synthetic tissues.

  1. 24 CFR 969.105 - Extension of ACC upon payment of operating subsidy.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... for which Operating Subsidy is paid with respect to the project. (b) Consolidated ACC. Where a single... Consolidated ACC. In any event, no Operating Subsidy payable under a Consolidated ACC or otherwise shall...

  2. Adrenocortical cancer (ACC) - literature overview and own experience.

    PubMed

    Dworakowska, Dorota; Drabarek, Agata; Wenzel, Ingrid; Babińska, Anna; Świątkowska-Stodulska, Renata; Sworczak, Krzysztof

    2014-01-01

    Adrenocortical carcinoma (ACC) is a malignant endocrine tumour. The rarity of the disease has stymied therapeutic development. Age distribution shows two peaks: the first and fifth decades of life, with children and women more frequently affected. Although 60-70% of ACCs are biochemically found to overproduce hormones, it is not clinically apparent in many cases. If present, endocrine symptoms include signs of hypercortisolaemia, virilisation or gynaecomastia. ACC carries a poor prognosis, and a cure can be achieved only by complete surgical resection. Mitotane is used both as an adjuvant treatment and also in non-operative patients. The role of radio- and chemotherapy is still controversial. The post-operative disease free survival is low and oscillates around 30% due to high tumour recurrence rate. The diagnosis is based on tumour histological assessment with the use of the Weiss score, however urinary steroid profiling (if available) can serve to differentiate between ACC and other adrenal tumours. Conventional prognostic markers in ACC include stage and grade of disease, and, as currently reported, the presence of hypercortisolaemia. Molecular analysis has had a significant impact on the understanding of the pathogenetic mechanism of ACC development and the evaluation of prognostic and predictive markers, among which alterations of the IGF system, the Wnt pathway, p53 and molecules involved in cancer cell invasion properties and angiogenesis seem to be very promising. We here summarise our own experience related to the management of ACC and present a literature overview. We have not aimed to include a detailed summary of the molecular alterations biology described in ACC, as this has already been addressed in other papers.

  3. Acetyl CoA carboxylase inactivation and meiotic maturation in mouse oocytes.

    PubMed

    Valsangkar, Deepa S; Downs, Stephen M

    2015-09-01

    In mouse oocytes, meiotic induction by pharmacological activation of PRKA (adenosine monophosphate-activated protein kinase; formerly known as AMPK) or by hormones depends on stimulation of fatty acid oxidation (FAO). PRKA stimulates FAO by phosphorylating and inactivating acetyl CoA carboxylase (ACAC; formerly ACC), leading to decreased malonyl CoA levels and augmenting fatty-acid transport into mitochondria. We investigated a role for ACAC inactivation in meiotic resumption by testing the effect of two ACAC inhibitors, CP-640186 and Soraphen A, on mouse oocytes maintained in meiotic arrest in vitro. These inhibitors significantly stimulated the resumption of meiosis in arrested cumulus cell-enclosed oocytes, denuded oocytes, and follicle-enclosed oocytes. This stimulation was accompanied by an increase in FAO. Etomoxir, a malonyl CoA analogue, prevented meiotic resumption as well as the increase in FAO induced by ACAC inhibition. Citrate, an ACAC activator, and CBM-301106, an inhibitor of malonyl CoA decarboxylase, which converts malonyl CoA to acetyl CoA, suppressed both meiotic induction and FAO induced by follicle-stimulating hormone, presumably by maintaining elevated malonyl CoA levels. Mouse oocyte-cumulus cell complexes contain both isoforms of ACAC (ACACA and ACACB); when wild-type and Acacb(-/-) oocytes characteristics were compared, we found that these single-knockout oocytes showed a significantly higher FAO level and a reduced ability to maintain meiotic arrest, resulting in higher rates of germinal vesicle breakdown. Collectively, these data support the model that ACAC inactivation contributes to the maturation-promoting activity of PRKA through stimulation of FAO.

  4. Properties of ribulose diphosphate carboxylase immobilized on porous glass

    NASA Technical Reports Server (NTRS)

    Shapira, J.; Hanson, C. L.; Lyding, J. M.; Reilly, P. J.

    1974-01-01

    Ribulose-1,5-diphosphate carboxylase from spinach has been bound to arylamine porous glass with a diazo linkage and to alklamine porous glass with glutaraldehyde. Stability at elevated temperatures and responses to changes of pH and ribulose-1,5-diphosphate, Mg(2+), and dithiothreitol concentrations were not significantly different from the soluble enzyme, though stability at 4 C was somewhat improved.

  5. Ribulose diphosphate carboxylase of the cyanobacterium Spirulina platensis

    SciTech Connect

    Terekhova, I.V.; Chernyad'ev, I.I.; Doman, N.G.

    1986-11-20

    The ribulose diphosphate (RDP) carboxylase activity of the cyanobacterium Spirulina platensis is represented by two peaks when a cell homogenate is centrifuged in a sucrose density gradient. In the case of differential centrifugation (40,000 g, 1 h), the activity of the enzyme was distributed between the supernatant liquid (soluble form) and the precipitate (carboxysomal form). From the soluble fraction, in which 80-95% of the total activity of the enzyme is concentrated, electrophoretically homogeneous RDP carboxylase was isolated by precipitation with ammonium sulfate and centrifugation in a sucrose density gradient. The purified enzyme possessed greater electrophoretic mobility in comparison with the RDP carboxylase of beans Vicia faba. The molecular weight of the enzyme, determined by gel filtration, was 450,000. The enzyme consists of monotypic subunits with a molecular weight of 53,000. The small subunits were not detected in electrophoresis in polyacrylamide gel in the presence of SDS after fixation and staining of the gels by various methods.

  6. 24 CFR 905.302 - Timely submission of the CF ACC amendment by the PHA.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... 24 Housing and Urban Development 4 2014-04-01 2014-04-01 false Timely submission of the CF ACC... Requirements § 905.302 Timely submission of the CF ACC amendment by the PHA. Upon being provided with a CF ACC Amendment from HUD, the PHA must sign and date the CF ACC Amendment and return it to HUD by the...

  7. The active site of ribulose-bisphosphate carboxylase/oxygenase

    SciTech Connect

    Hartman, F.C.

    1991-01-01

    The active site of ribulose-bisphosphate carboxylase/oxygenase requires interacting domains of adjacent, identical subunits. Most active-site residues are located within the loop regions of an eight-stranded {beta}/{alpha}-barrel which constitutes the larger C-terminal domain; additional key residues are located within a segment of the smaller N-terminal domain which partially covers the mouth of the barrel. Site-directed mutagenesis of the gene encoding the enzyme from Rhodospirillum rubrum has been used to delineate functions of active-site residues. 6 refs., 2 figs.

  8. Decreasing the Rate of Metabolic Ketone Reduction in the Discovery of a Clinical Acetyl-CoA Carboxylase Inhibitor for the Treatment of Diabetes

    PubMed Central

    2015-01-01

    Acetyl-CoA carboxylase (ACC) inhibitors offer significant potential for the treatment of type 2 diabetes mellitus (T2DM), hepatic steatosis, and cancer. However, the identification of tool compounds suitable to test the hypothesis in human trials has been challenging. An advanced series of spirocyclic ketone-containing ACC inhibitors recently reported by Pfizer were metabolized in vivo by ketone reduction, which complicated human pharmacology projections. We disclose that this metabolic reduction can be greatly attenuated through introduction of steric hindrance adjacent to the ketone carbonyl. Incorporation of weakly basic functionality improved solubility and led to the identification of 9 as a clinical candidate for the treatment of T2DM. Phase I clinical studies demonstrated dose-proportional increases in exposure, single-dose inhibition of de novo lipogenesis (DNL), and changes in indirect calorimetry consistent with increased whole-body fatty acid oxidation. This demonstration of target engagement validates the use of compound 9 to evaluate the role of DNL in human disease. PMID:25423286

  9. Decreasing the Rate of Metabolic Ketone Reduction in the Discovery of a Clinical Acetyl-CoA Carboxylase Inhibitor for the Treatment of Diabetes

    SciTech Connect

    Griffith, David A.; Kung, Daniel W.; Esler, William P.; Amor, Paul A.; Bagley, Scott W.; Beysen, Carine; Carvajal-Gonzalez, Santos; Doran, Shawn D.; Limberakis, Chris; Mathiowetz, Alan M.; McPherson, Kirk; Price, David A.; Ravussin, Eric; Sonnenberg, Gabriele E.; Southers, James A.; Sweet, Laurel J.; Turner, Scott M.; Vajdos, Felix F.

    2014-12-26

    We found that Acetyl-CoA carboxylase (ACC) inhibitors offer significant potential for the treatment of type 2 diabetes mellitus (T2DM), hepatic steatosis, and cancer. However, the identification of tool compounds suitable to test the hypothesis in human trials has been challenging. An advanced series of spirocyclic ketone-containing ACC inhibitors recently reported by Pfizer were metabolized in vivo by ketone reduction, which complicated human pharmacology projections. Here, we disclose that this metabolic reduction can be greatly attenuated through introduction of steric hindrance adjacent to the ketone carbonyl. Incorporation of weakly basic functionality improved solubility and led to the identification of 9 as a clinical candidate for the treatment of T2DM. Phase I clinical studies demonstrated dose-proportional increases in exposure, single-dose inhibition of de novo lipogenesis (DNL), and changes in indirect calorimetry consistent with increased whole-body fatty acid oxidation. This demonstration of target engagement validates the use of compound 9 to evaluate the role of DNL in human disease.

  10. Isoelectric focusing of wound-induced tomato ACC synthase

    SciTech Connect

    White, J.A.; Kende, H. )

    1990-05-01

    Several techniques of electrofocusing have been used to determine whether 1-aminocyclopropane-1-carboxylate (ACC) synthase isolated from wounded tomato pericarp tissue exists in different isoforms, each with its characteristic isoelectric point (pI). The pI of the native enzyme was found to be 6.0 {plus minus} 0.2. When radiolabeled, denatured ACC synthase was electrofocused by non-equilibrium pH gradient electrophoresis (NEpHGE), the enzyme separated into four discernible spots which, upon reaching equilibrium, ranged in pI from 6.6 to 6.9. Immunopurified ACC synthase from four tomato cultivars (Duke, Cornell, Mountain Pride and Pik Red) migrated in each case as a 50-kDa protein on sodium dodecyl sulfate polyacrylamide gels (SDS-PAGE). We propose that native ACC synthase in extracts of tomato pericarp tissue exists in one single form and that the charge heterogeneities observed upon electrofocusing of denatured enzyme result from modifications of preexisting protein.

  11. Coordinate expression of hydrogenase and ribulose bisphosphate carboxylase in Rhizobium japonicum Hupc mutants.

    PubMed Central

    Merberg, D; Maier, R J

    1984-01-01

    In contrast to the wild type, H2 uptake-constitutive mutants of Rhizobium japonicum expressed both hydrogenase and ribulose bisphosphate carboxylase activities when grown heterotrophically. However, as bacteroids from soybean root nodules, the H2 uptake-constitutive mutants, like the wild type, did not express ribulose bisphosphate carboxylase activity. PMID:6384199

  12. A distinct holoenzyme organization for two-subunit pyruvate carboxylase

    PubMed Central

    Choi, Philip H.; Jo, Jeanyoung; Lin, Yu-Cheng; Lin, Min-Han; Chou, Chi-Yuan; Dietrich, Lars E. P.; Tong, Liang

    2016-01-01

    Pyruvate carboxylase (PC) has important roles in metabolism and is crucial for virulence for some pathogenic bacteria. PC contains biotin carboxylase (BC), carboxyltransferase (CT) and biotin carboxyl carrier protein (BCCP) components. It is a single-chain enzyme in eukaryotes and most bacteria, and functions as a 500 kD homo-tetramer. In contrast, PC is a two-subunit enzyme in a collection of Gram-negative bacteria, with the α subunit containing the BC and the β subunit the CT and BCCP domains, and it is believed that the holoenzyme has α4β4 stoichiometry. We report here the crystal structures of a two-subunit PC from Methylobacillus flagellatus. Surprisingly, our structures reveal an α2β4 stoichiometry, and the overall architecture of the holoenzyme is strikingly different from that of the homo-tetrameric PCs. Biochemical and mutagenesis studies confirm the stoichiometry and other structural observations. Our functional studies in Pseudomonas aeruginosa show that its two-subunit PC is important for colony morphogenesis. PMID:27708276

  13. 1-Aminocyclopropane-1-carboxylate (ACC) deaminases from Methylobacterium radiotolerans and Methylobacterium nodulans with higher specificity for ACC.

    PubMed

    Fedorov, Dmitry N; Ekimova, Galina A; Doronina, Nina V; Trotsenko, Yuri A

    2013-06-01

    The 1-aminocyclopropane-1-carboxylate (ACC) deaminases (EC 3.4.99.7), the key enzymes of degradation of the precursor of the phytohormone ethylene, have not been well studied despite their great importance for plant-bacterial interactions. Using blast, the open reading frames encoding ACC deaminases were found in the genomes of epiphytic methylotroph Methylobacterium radiotolerans JCM2831 and nodule-forming endosymbiont Methylobacterium nodulans ORS2060. These genes were named acdS and cloned; recombinant proteins were expressed and purified from Escherichia coli. The enzyme from M. nodulans displayed the highest substrate specificity among all of the characterized ACC deaminases (Km 0.80 ± 0.04 mM), whereas the enzyme from M. radiotolerans had Km 1.8 ± 0.3 mM. The kcat values were 111.8 ± 0.2 and 65.8 ± 2.8 min(-1) for the enzymes of M. nodulans and M. radiotolerans, respectively. Both enzymes are homotetramers with a molecular mass of 144 kDa, as was demonstrated by size exclusion chromatography and native PAGE. The purified enzymes displayed the maximum activity at 45-50 °C and pH 8.0. Thus, the priority data have been obtained, extending the knowledge of biochemical properties of bacterial ACC deaminases.

  14. Biomarker analysis of the MITO2 phase III trial of first-line treatment in ovarian cancer: predictive value of DNA-PK and phosphorylated ACC

    PubMed Central

    Perrone, Francesco; Baldassarre, Gustavo; Indraccolo, Stefano; Signoriello, Simona; Chiappetta, Gennaro; Esposito, Franca; Ferrandina, Gabriella; Franco, Renato; Mezzanzanica, Delia; Sonego, Maura; Zulato, Elisabetta; Zannoni, Gian F.; Canzonieri, Vincenzo; Scambia, Giovanni; Sorio, Roberto; Savarese, Antonella; Breda, Enrico; Scollo, Paolo; Ferro, Antonella; Tamberi, Stefano; Febbraro, Antonio; Natale, Donato; Maio, Massimo Di; Califano, Daniela; Scognamiglio, Giosuè; Lorusso, Domenica; Canevari, Silvana; Losito, Simona; Gallo, Ciro; Pignata, Sandro

    2016-01-01

    Background No biomarker is available to predict prognosis of patients with advanced ovarian cancer (AOC) and guide the choice of chemotherapy. We performed a prospective-retrospective biomarker study within the MITO2 trial on the treatment of AOC. Patients and methods: MITO2 is a randomised multicentre phase 3 trial conducted with 820 AOC patients assigned carboplatin/paclitaxel (carboplatin: AUC5, paclitaxel: 175 mg/m², every 3 weeks for 6 cycles) or carboplatin/PLD-pegylated liposomal doxorubicin (carboplatin: AUC5, PLD: 30 mg/m², every 3 weeks for 6 cycles) as first line treatment. Sixteen biomarkers (pathways of adhesion/invasion, apoptosis, transcription regulation, metabolism, and DNA repair) were studied in 229 patients, in a tissue microarray. Progression-free and overall survival were analysed with multivariable Cox model. Results After 72 months median follow-up, 594 progressions and 426 deaths were reported; there was no significant difference between the two arms in the whole trial. No biomarker had significant prognostic value. Statistically significant interactions with treatment were found for DNA-dependent protein kinase (DNA-PK) and phosphorylated acetyl-coenzymeA carboxylase (pACC), both predicting worse outcome for patients receiving carboplatin/paclitaxel. Conclusion These data show that in presence of DNA-PK or pACC overexpression, carboplatin/paclitaxel might be less effective than carboplatin/PLD as first line treatment of ovarian cancer patients. Further validation of these findings is warranted. PMID:27655643

  15. Structure of the acetophenone carboxylase core complex: prototype of a new class of ATP-dependent carboxylases/hydrolases

    PubMed Central

    Weidenweber, Sina; Schühle, Karola; Demmer, Ulrike; Warkentin, Eberhard; Ermler, Ulrich; Heider, Johann

    2017-01-01

    Degradation of the aromatic ketone acetophenone is initiated by its carboxylation to benzoylacetate catalyzed by acetophenone carboxylase (Apc) in a reaction dependent on the hydrolysis of two ATP to ADP and Pi. Apc is a large protein complex which dissociates during purification into a heterooctameric Apc(αα′βγ)2 core complex of 482 kDa and Apcε of 34 kDa. In this report, we present the X-ray structure of the Apc(αα′βγ)2 core complex from Aromatoleum aromaticum at ca. 3 Å resolution which reveals a unique modular architecture and serves as model of a new enzyme family. Apcβ contains a novel domain fold composed of two β-sheets in a barrel-like arrangement running into a bundle of eight short polyproline (type II)-like helical segments. Apcα and Apcα′ possess ATP binding modules of the ASKHA superfamily integrated into their multidomain structures and presumably operate as ATP-dependent kinases for acetophenone and bicarbonate, respectively. Mechanistic aspects of the novel carboxylation reaction requiring massive structural rearrangements are discussed and criteria for specifically annotating the family members Apc, acetone carboxylase and hydantoinase are defined. PMID:28054554

  16. Mechanism of action of ribulose bisphosphate carboxylase/oxygenase.

    PubMed

    Lane, M D; Miziorko, H M

    1978-01-01

    RuBP carboxylase-oxygenase appears to catalyze carboxylation and oxygenation by homologous mechanisms. A common binding site exists on the enzyme for the acceptor substrate, RuBP. A mechanism is proposed whereby RuBP is isomerized, and a carbanion is generated at C2. Then, either CO2 or O2 is added as an electrophile at C2 to form the corresponding 3-keto-2-carboxy-RBP or 3-keto-2-hydroperoxy-RBP adduct. Hydrolytic cleavage at the C2-C3 bonds of these intermediates by the enzyme is envisioned to produce 2 molecules of 3-phosphoglycerate in the carboxylation sequence and 1 molecule of phosphoglycolate and 1 molecule of 3-phosphoglycerate in the oxygenation sequence. Further work will be necessary to establish the validity of the proposed mechanism.

  17. The formation of ACC and competition between polyamines and ethylene for SAM

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Ethylene biosynthesis involves the conversion of S-adenosylmethionine (SAM) to 1-aminocyclopropane-1-carboxylic acid (ACC) by ACC synthase (ACS). ACC is then converted to ethylene. The genes that encode enzymes in this pathway all belong to a family of genes. Differential transcriptional regulation ...

  18. In vitro synthesis and stabilization of amorphous calcium carbonate (ACC) nanoparticles within liposomes

    SciTech Connect

    Tester, Chantel C.; Brock, Ryan E.; Wu, Ching-Hsuan; Krejci, Minna R.; Weigand, Steven; Joester, Derk

    2012-02-07

    We show that amorphous calcium carbonate (ACC) can be synthesized in phospholipid bilayer vesicles (liposomes). Liposome-encapsulated ACC nanoparticles are stable against aggregation, do not crystallize for at least 20 h, and are ideally suited to investigate the influence of lipid chemistry, particle size, and soluble additives on ACC in situ.

  19. 24 CFR 905.304 - CF ACC term and covenant to operate.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... 24 Housing and Urban Development 4 2014-04-01 2014-04-01 false CF ACC term and covenant to operate... URBAN DEVELOPMENT THE PUBLIC HOUSING CAPITAL FUND PROGRAM General Program Requirements § 905.304 CF ACC... operate all public housing projects in accordance with the CF ACC, as amended, and applicable...

  20. Ribulose diphosphate carboxylase/oxygenase. III. Isolation and properties.

    PubMed

    Ryan, F J; Tolbert, N E

    1975-06-10

    Similarities in properties of ribulose diphosphate carboxylase and oxygenase activities further substantiate the hypothesis that the same protein catalyzes both reactions. The Km (ribulose diphosphate) is 0.33 mM for the ribulose diphosphate oxygenase, when assayed in air with an oxygen electrode. Maximum activity is obtained with 10 to 35 mM MgCl2. Higher MgCl2 concentrations are inhibitory, but they shift the pH optimum from 9.3 or 9.4 to 8.7 or 9.0. MnCl2 is an effective cofactor of the oxygenase and some activity is obtained with CoCl2. Both the ribulose diphosphate carboxylase and oxygenase activity of the purified protein from spinach leaves are slowly inactivated by storage at 0 degrees and reactivated in 10 min at 50 degrees, provided both 25 mM MgCl2 and 1 mM dithiothreitol are present. The sulfhydryl groups of the enzyme which react rapidly with 5,5'-dithiobis(2-nitrobenzoic acid) are approximately 4 at pH 7.8 and 11 at pH 9.4. At both pH values ribulose diphosphate prevents two of these sulfhydryl groups from reacting with this reagent. About 50% inhibition of the oxygenase activity at pH 9.0 occurs with 50 mM bicarbonate in the presence of 3 mM ribulose diphosphate, and from variations in these parameters the inhibition is attributed to the CO2 species. The purified enzyme of acrylamide gels prevented the reduction of nitroblue tetrazolium in the presence of the superoxide radical, but the enzyme in solution did not react as a superoxide dismutase.

  1. Mitochondrial storage form of acetyl CoA carboxylase in fasted and alloxan diabetic rats

    SciTech Connect

    Roman-Lopez, C.R.; Allred, J.B.

    1986-05-01

    Sodium dodecyl sulfate-denatured biotinyl proteins will bind (/sup 14/C)methyl avidin which remains bound through polyacrylamide gel electrophoresis. The method has been used to demonstrate the presence of two high molecular weight subunit forms of acetyl CoA carboxylase in rat liver cytoplasm, both of which are precipitated by antibody to purifed rat liver acetyl CoA carboxylase prepared from sheep serum. Rat liver mitochondria contained five distinct biotinyl protein subunits, the two largest of which have been identified as acetyl CoA carboxylase subunits on the basis of precipitation by anti-acetyl CoA carboxylase antibody. The small quantity of acetyl CoA carboxylase associated with rat liver microsomes could be attributed to cytoplasmic contamination. The binding of radioactive avidin is sufficiently tight to use as a measure of the quantity of acetyl CoA carboxylase. The quantity and activity of the cytoplasmic enzyme was reduced in fasted and in alloxan diabetic rats compared to that in fed controls but the quantity of the enzyme associated with isolated mitochondria was not reduced. The results indicate that there is a mitochondrial storage form of acetyl CoA carboxylase.

  2. A first insight into the occurrence and expression of functional amoA and accA genes of autotrophic and ammonia-oxidizing bathypelagic Crenarchaeota of Tyrrhenian Sea

    NASA Astrophysics Data System (ADS)

    Yakimov, Michail M.; Cono, Violetta La; Denaro, Renata

    2009-05-01

    The autotrophic and ammonia-oxidizing crenarchaeal assemblage at offshore site located in the deep Mediterranean (Tyrrhenian Sea, depth 3000 m) water was studied by PCR amplification of the key functional genes involved in energy (ammonia mono-oxygenase alpha subunit, amoA) and central metabolism (acetyl-CoA carboxylase alpha subunit, accA). Using two recently annotated genomes of marine crenarchaeons, an initial set of primers targeting archaeal accA-like genes was designed. Approximately 300 clones were analyzed, of which 100% of amoA library and almost 70% of accA library were unambiguously related to the corresponding genes from marine Crenarchaeota. Even though the acetyl-CoA carboxylase is phylogenetically not well conserved and the remaining clones were affiliated to various bacterial acetyl-CoA/propionyl-CoA carboxylase genes, the pool of archaeal sequences was applied for development of quantitative PCR analysis of accA-like distribution using TaqMan ® methodolgy. The archaeal accA gene fragments, together with alignable gene fragments from the Sargasso Sea and North Pacific Subtropical Gyre (ALOHA Station) metagenome databases, were analyzed by multiple sequence alignment. Two accA-like sequences, found in ALOHA Station at the depth of 4000 m, formed a deeply branched clade with 64% of all archaeal Tyrrhenian clones. No close relatives for residual 36% of clones, except of those recovered from Eastern Mediterranean, was found, suggesting the existence of a specific lineage of the crenarchaeal accA genes in deep Mediterranean water. Alignment of Mediterranean amoA sequences defined four cosmopolitan phylotypes of Crenarchaeota putative ammonia mono-oxygenase subunit A gene occurring in the water sample from the 3000 m depth. Without exception all phylotypes fell into Deep Marine Group I cluster that contain the vast majority of known sequences recovered from global deep-sea environment. Remarkably, three phylotypes accounted for 91% of all Mediterranean

  3. Reward salience and risk aversion underlie differential ACC activity in substance dependence

    PubMed Central

    Alexander, William H.; Fukunaga, Rena; Finn, Peter; Brown, Joshua W.

    2015-01-01

    The medial prefrontal cortex, especially the dorsal anterior cingulate cortex (ACC), has long been implicated in cognitive control and error processing. Although the association between ACC and behavior has been established, it is less clear how ACC contributes to dysfunctional behavior such as substance dependence. Evidence from neuroimaging studies investigating ACC function in substance users is mixed, with some studies showing disengagement of ACC in substance dependent individuals (SDs), while others show increased ACC activity related to substance use. In this study, we investigate ACC function in SDs and healthy individuals performing a change signal task for monetary rewards. Using a priori predictions derived from a recent computational model of ACC, we find that ACC activity differs between SDs and controls in factors related to reward salience and risk aversion between SDs and healthy individuals. Quantitative fits of a computational model to fMRI data reveal significant differences in best fit parameters for reward salience and risk preferences. Specifically, the ACC in SDs shows greater risk aversion, defined as concavity in the utility function, and greater attention to rewards relative to reward omission. Furthermore, across participants risk aversion and reward salience are positively correlated. The results clarify the role that ACC plays in both the reduced sensitivity to omitted rewards and greater reward valuation in SDs. Clinical implications of applying computational modeling in psychiatry are also discussed. PMID:26106528

  4. Chemical and Physical Characterization of the Activation of Ribulosebiphosphate Carboxylase/Oxygenase

    DOE R&D Accomplishments Database

    Donnelly, M. I.; Ramakrishnan, V.; Hartman, F. C.

    1983-08-01

    Molecular structure of ribulosebiphosphate carboxylase/oxygenase isolated from Rhodospirillium was compared with the enzyme isolated from Alcaligens eutrophus. Peptides derived from the active center of the bacterial enzyme were highly homologous with those isolated from spinach. Molecular shapes of the carboxylases were estimated using neutron scattering data. These studies suggested that the enzyme as isolated from R. rubrum is a solid prolate ellipsoid or cylinder, while the spinach enzyme resembles a hollow sphere.

  5. Crystallization and structure of a recombinant ribulose-1,5-bisphosphate carboxylase

    NASA Astrophysics Data System (ADS)

    Schneider, Gunter; Lindqvist, Ylva; Brändén, Carl-Ivar; Lorimer, George

    1988-07-01

    Ribulose-1,5-bisphosphate carboxylase/oxygenase is the key enzyme in photosynthetic carbon dioxide fixation and photorespiration. The dimeric carboxylase from the photosynthetic bacterium Rhodospirillum rubrum has been cloned and expressed in E. coli. The recombinant enzyme has been crystallized in a number of different crystal forms. The three-dimensional structure of the enzyme has been determined by X-ray crystallographic methods to 2.9Åresolution.

  6. Variations in Km(CO2) of Ribulose-1,5-bisphosphate Carboxylase among Grasses

    PubMed Central

    Yeoh, Hock-Hin; Badger, Murray R.; Watson, Leslie

    1980-01-01

    A survey of the Km(CO2) values of ribulose-1,5-bisphosphate carboxylase from 60 grass species shows that enzyme from C3 grasses consistently exhibits lower Km(CO2) than does that from C4 grasses. Systematically ordered variation in Km(CO2) of ribulose-1,5-bisphosphate carboxylases from C3 and C4 grasses is also apparent and, among C4 grasses, this shows some correlation with C4 types. PMID:16661586

  7. Chemical and physical characterization of the activation of ribulosebiphosphate carboxylase/oxygenase

    SciTech Connect

    Donnelly, M.I.; Ramakrishnan, V.; Hartman, F.C.

    1983-01-01

    Molecular structure of ribulosebiphosphate carboxylase/oxygenase isolated from Rhodospirillium was compared with the enzyme isolated from Alcaligens eutrophus. Peptides derived from the active center of the bacterial enzyme were highly homologous with those isolated from spinach. Molecular shapes of the carboxylases were estimated using neutron scattering data. These studies suggested that the enzyme as isolated from R. rubrum is a solid prolate ellipsoid or cylinder, while the spinach enzyme resembles a hollow sphere. 1 drawing.

  8. Structural Analysis of Substrate, Reaction Intermediate, and Product Binding in Haemophilus influenzae Biotin Carboxylase.

    PubMed

    Broussard, Tyler C; Pakhomova, Svetlana; Neau, David B; Bonnot, Ross; Waldrop, Grover L

    2015-06-23

    Acetyl-CoA carboxylase catalyzes the first and regulated step in fatty acid synthesis. In most Gram-negative and Gram-positive bacteria, the enzyme is composed of three proteins: biotin carboxylase, a biotin carboxyl carrier protein (BCCP), and carboxyltransferase. The reaction mechanism involves two half-reactions with biotin carboxylase catalyzing the ATP-dependent carboxylation of biotin-BCCP in the first reaction. In the second reaction, carboxyltransferase catalyzes the transfer of the carboxyl group from biotin-BCCP to acetyl-CoA to form malonyl-CoA. In this report, high-resolution crystal structures of biotin carboxylase from Haemophilus influenzae were determined with bicarbonate, the ATP analogue AMPPCP; the carboxyphosphate intermediate analogues, phosphonoacetamide and phosphonoformate; the products ADP and phosphate; and the carboxybiotin analogue N1'-methoxycarbonyl biotin methyl ester. The structures have a common theme in that bicarbonate, phosphate, and the methyl ester of the carboxyl group of N1'-methoxycarbonyl biotin methyl ester all bound in the same pocket in the active site of biotin carboxylase and as such utilize the same set of amino acids for binding. This finding suggests a catalytic mechanism for biotin carboxylase in which the binding pocket that binds tetrahedral phosphate also accommodates and stabilizes a tetrahedral dianionic transition state resulting from direct transfer of CO₂ from the carboxyphosphate intermediate to biotin.

  9. Effect of Pyruvate Carboxylase Overexpression on the Physiology of Corynebacterium glutamicum

    PubMed Central

    Koffas, Mattheos A. G.; Jung, Gyoo Yeol; Aon, Juan C.; Stephanopoulos, Gregory

    2002-01-01

    Pyruvate carboxylase was recently sequenced in Corynebacterium glutamicum and shown to play an important role of anaplerosis in the central carbon metabolism and amino acid synthesis of these bacteria. In this study we investigate the effect of the overexpression of the gene for pyruvate carboxylase (pyc) on the physiology of C. glutamicum ATCC 21253 and ATCC 21799 grown on defined media with two different carbon sources, glucose and lactate. In general, the physiological effects of pyc overexpression in Corynebacteria depend on the genetic background of the particular strain studied and are determined to a large extent by the interplay between pyruvate carboxylase and aspartate kinase activities. If the pyruvate carboxylase activity is not properly matched by the aspartate kinase activity, pyc overexpression results in growth enhancement instead of greater lysine production, despite its central role in anaplerosis and aspartic acid biosynthesis. Aspartate kinase regulation by lysine and threonine, pyruvate carboxylase inhibition by aspartate (shown in this study using permeabilized cells), as well as well-established activation of pyruvate carboxylase by lactate and acetyl coenzyme A are the key factors in determining the effect of pyc overexpression on Corynebacteria physiology. PMID:12406733

  10. Genes encoding biotin carboxylase subunit of acetyl-CoA carboxylase from Brassica napus and parental species: cloning, expression patterns, and evolution

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Comparative genomics is a useful tool to investigate gene and genome evolution. Biotin carboxylase (BC), an important subunit of heteromeric ACCase that is a rate-limiting enzyme in fatty acid biosynthesis in dicots, catalyzes ATP, biotin-carboxyl-carrier protein and CO2 to form carboxybiotin-carbo...

  11. Genes encoding plastid acetyl-CoA carboxylase and 3-phosphoglycerate kinase of the Triticum/Aegilops complex and the evolutionary history of polyploid wheat

    PubMed Central

    Huang, Shaoxing; Sirikhachornkit, Anchalee; Su, Xiujuan; Faris, Justin; Gill, Bikram; Haselkorn, Robert; Gornicki, Piotr

    2002-01-01

    The classic wheat evolutionary history is one of adaptive radiation of the diploid Triticum/Aegilops species (A, S, D), genome convergence and divergence of the tetraploid (Triticum turgidum AABB, and Triticum timopheevii AAGG) and hexaploid (Triticum aestivum, AABBDD) species. We analyzed Acc-1 (plastid acetyl-CoA carboxylase) and Pgk-1 (plastid 3-phosphoglycerate kinase) genes to determine phylogenetic relationships among Triticum and Aegilops species of the wheat lineage and to establish the timeline of wheat evolution based on gene sequence comparisons. Triticum urartu was confirmed as the A genome donor of tetraploid and hexaploid wheat. The A genome of polyploid wheat diverged from T. urartu less than half a million years ago (MYA), indicating a relatively recent origin of polyploid wheat. The D genome sequences of T. aestivum and Aegilops tauschii are identical, confirming that T. aestivum arose from hybridization of T. turgidum and Ae. tauschii only 8,000 years ago. The diploid Triticum and Aegilops progenitors of the A, B, D, G, and S genomes all radiated 2.5–4.5 MYA. Our data suggest that the Acc-1 and Pgk-1 loci have different histories in different lineages, indicating genome mosaicity and significant intraspecific differentiation. Some loci of the S genome of Aegilops speltoides and the G genome of T. timophevii are closely related, suggesting the same origin of some parts of their genomes. None of the Aegilops genomes analyzed is a close relative of the B genome, so the diploid progenitor of the B genome remains unknown. PMID:12060759

  12. Deep sea water modulates blood pressure and exhibits hypolipidemic effects via the AMPK-ACC pathway: an in vivo study.

    PubMed

    Sheu, Ming-Jyh; Chou, Pei-Yu; Lin, Wen-Hsin; Pan, Chun-Hsu; Chien, Yi-Chung; Chung, Yun-Lung; Liu, Fon-Chang; Wu, Chieh-Hsi

    2013-06-17

    Deep sea water (DSW), originally pumped from the Pacific Rim off the coast of Hualien County (Taiwan), and its mineral constituents, were concentrated by a low-temperature vacuum evaporation system to produce a hardness of approximately 400,000 mg/L of seawater mineral concentrate. The primary composition of this seawater mineral concentrate was ionic magnesium (Mg²⁺), which was approximately 96,000 mg/L. Referring to the human recommended daily allowance (RDA) of magnesium, we diluted the mineral concentrate to three different dosages: 0.1 × DSW (equivalent to 3.75 mg Mg²⁺/kg DSW); 1 × DSW (equivalent to 37.5 mg Mg²⁺/kg DSW); and 2 × DSW (equivalent to 75 mg Mg²⁺/kg DSW). Additionally, a magnesium chloride treatment was conducted for comparison with the DSW supplement. The study indicated that 0.1 × DSW, 1 × DSW and 2 × DSW decreased the systolic and diastolic pressures in spontaneous hypertensive rats in an eight-week experiment. DSW has been shown to reduce serum lipids and prevent atherogenesis in a hypercholesterolemic rabbit model. Our results demonstrated that 1 × DSW and 2 × DSW significantly suppressed the serum cholesterol levels, reduced the lipid accumulation in liver tissues, and limited aortic fatty streaks. These findings indicated that the antiatherogenic effects of DSW are associated with 5'-adenosine monophosphate-activated protein kinase (AMPK) stimulation and the consequent inhibition of phosphorylation of acetyl-CoA carboxylase (ACC) in atherosclerotic rabbits. We hypothesize that DSW could potentially be used as drinking water because it modulates blood pressure, reduces lipids, and prevents atherogenesis.

  13. Deep Sea Water Modulates Blood Pressure and Exhibits Hypolipidemic Effects via the AMPK-ACC Pathway: An in Vivo Study

    PubMed Central

    Sheu, Ming-Jyh; Chou, Pei-Yu; Lin, Wen-Hsin; Pan, Chun-Hsu; Chien, Yi-Chung; Chung, Yun-Lung; Liu, Fon-Chang; Wu, Chieh-Hsi

    2013-01-01

    Deep sea water (DSW), originally pumped from the Pacific Rim off the coast of Hualien County (Taiwan), and its mineral constituents, were concentrated by a low-temperature vacuum evaporation system to produce a hardness of approximately 400,000 mg/L of seawater mineral concentrate. The primary composition of this seawater mineral concentrate was ionic magnesium (Mg2+), which was approximately 96,000 mg/L. Referring to the human recommended daily allowance (RDA) of magnesium, we diluted the mineral concentrate to three different dosages: 0.1 × DSW (equivalent to 3.75 mg Mg2+/kg DSW); 1 × DSW (equivalent to 37.5 mg Mg2+/kg DSW); and 2 × DSW (equivalent to 75 mg Mg2+/kg DSW). Additionally, a magnesium chloride treatment was conducted for comparison with the DSW supplement. The study indicated that 0.1 × DSW, 1 × DSW and 2 × DSW decreased the systolic and diastolic pressures in spontaneous hypertensive rats in an eight-week experiment. DSW has been shown to reduce serum lipids and prevent atherogenesis in a hypercholesterolemic rabbit model. Our results demonstrated that 1 × DSW and 2 × DSW significantly suppressed the serum cholesterol levels, reduced the lipid accumulation in liver tissues, and limited aortic fatty streaks. These findings indicated that the antiatherogenic effects of DSW are associated with 5′-adenosine monophosphate-activated protein kinase (AMPK) stimulation and the consequent inhibition of phosphorylation of acetyl-CoA carboxylase (ACC) in atherosclerotic rabbits. We hypothesize that DSW could potentially be used as drinking water because it modulates blood pressure, reduces lipids, and prevents atherogenesis. PMID:23774889

  14. Resistance to acetyl-CoA carboxylase-inhibiting herbicides.

    PubMed

    Kaundun, Shiv S

    2014-09-01

    Resistance to acetyl-CoA carboxylase herbicides is documented in at least 43 grass weeds and is particularly problematic in Lolium, Alopecurus and Avena species. Genetic studies have shown that resistance generally evolves independently and can be conferred by target-site mutations at ACCase codon positions 1781, 1999, 2027, 2041, 2078, 2088 and 2096. The level of resistance depends on the herbicides, recommended field rates, weed species, plant growth stages, specific amino acid changes and the number of gene copies and mutant ACCase alleles. Non-target-site resistance, or in essence metabolic resistance, is prevalent, multigenic and favoured under low-dose selection. Metabolic resistance can be specific but also broad, affecting other modes of action. Some target-site and metabolic-resistant biotypes are characterised by a fitness penalty. However, the significance for resistance regression in the absence of ACCase herbicides is yet to be determined over a practical timeframe. More recently, a fitness benefit has been reported in some populations containing the I1781L mutation in terms of vegetative and reproductive outputs and delayed germination. Several DNA-based methods have been developed to detect known ACCase resistance mutations, unlike metabolic resistance, as the genes remain elusive to date. Therefore, confirmation of resistance is still carried out via whole-plant herbicide bioassays. A growing number of monocotyledonous crops have been engineered to resist ACCase herbicides, thus increasing the options for grass weed control. While the science of ACCase herbicide resistance has progressed significantly over the past 10 years, several avenues provided in the present review remain to be explored for a better understanding of resistance to this important mode of action.

  15. Light Modulation of Maize Leaf Phosphoenolpyruvate Carboxylase 1

    PubMed Central

    Huber, Steven C.; Sugiyama, Tatsuo; Akazawa, Takashi

    1986-01-01

    Phosphoenolpyruvate carboxylase (PEPC) was extracted from maize (Zea mays L. cv Golden Cross Bantam T51) leaves harvested in the dark or light and was partially purified by (NH4)2SO4 fractionation and gel filtration to yield preparations that were 80% homogeneous. Malate sensitivity, PEPC activity, and PEPC protein (measured immunochemically) were monitored during purification. As reported previously, PEPC from dark leaves was more sensitive to malate inhibition compared to enzyme extracted from light leaves. Extraction and purification in the presence of malate stabilized the characteristics of the two forms. During gel filtration on Sephacryl S-300, all of the PEPC activity and PEPC protein emerged in a single high molecular weight peak, indicating that no inactive dissociated forms (dimers, monomers) were present. However, there was a slight difference between the light and dark enzymes in elution volume during gel filtration. In addition, specific activity (units at pH 7/milligram PEPC protein) decreased through the peak for both enzyme samples; because the dark enzyme emerged at a slightly higher elution volume, it contained enzyme with a relatively lower specific activity. The variation in specific activity of the dark enzyme corresponded with changes in malate sensitivity. Immunoblotting of samples with different specific activity and malate sensitivity, obtained from gel filtration, revealed only a single polypeptide with a relative molecular mass of 100,000. When the enzyme was extracted and purified in the absence of malate, characteristic differences of the light and dark enzymes were lost, the enzymes eluted at the same volume during gel filtration, and specific activity was constant through the peak. We conclude that maize leaf PEPC exists in situ as a tetramer of a single polypeptide and that subtle conformation changes can affect both enzymic activity and sensitivity to malate inhibition. Images Fig. 6 PMID:16665065

  16. Further studies of auxin and ACC induced feminization in the cucumber plant using ethylene inhibitors

    NASA Technical Reports Server (NTRS)

    Takahashi, H.; Jaffe, M. J.

    1984-01-01

    The present study was designed to establish the role of an essential hormone controlling sex expression in cucumber. A potent anti-ethylene agent, AgNO3, completely inhibited pistillate flower formation caused by IAA, ACC or ethephon. Inhibitors of ethylene biosynthesis, AVG and CoCl2 also suppressed feminization due to exogenous IAA or ACC. Though AVG also suppressed ethephon-induced feminization, this may be due to the second effect of AVG rather than the effect on ACC biosynthesis. These results confirm that ethylene is a major factor regulating feminization and that exogenous auxin induces pistillate flower formation through its stimulation of ethylene production, rather than ACC production.

  17. Crystallization and characterization of ribulose 1,5-bisphosphate carboxylase/oxygenase from eight plant species.

    PubMed

    Johal, S; Bourque, D P; Smith, W W; Suh, S W; Eisenberg, D

    1980-09-25

    Ribulose bisphosphate carboxylase/oxygenase was isolated and crystallized from eight plant species. Crystals grew from either of two similar sets of crystallizing conditions: crystals of the enzyme from alfalfa, corn, cotton, potato, spinach, tobacco, and tomato grew from solutions containing phosphate and polyethylene glycol 6000 as a precipitant, and those from potato, tobacco (both Nicotiana sylvestris and Nicotiana tabacum), and tomato grew from a mixture of ammonium sulfate and phosphate. Crystals of the enzyme from potato and both species of tobacco were large enough to characterize by x-ray diffraction and were found to have the Form III structure, previously reported for crystals of ribulose bisphosphate carboxylase/oxygenase from N. tabacum. For crystalline material from several species, both carboxylase and oxygenase activites have been assayed and copper and iron contents have been determined. The possible significance of the observed general conditions of crystallization of this enzyme is discussed.

  18. Changing ribulose diphosphate carboxylase/oxygenase activity in ripening tomato fruit.

    PubMed

    Bravdo, B A; Palgi, A; Lurie, S

    1977-08-01

    Tomato fruit (Lycopersicum esculentum Mill) from green, pink, and red stages were assayed for changes in the activity of ribulose diphosphate carboxylase and oxygenase, phosphoenolpyruvate carboxylase, changes in the levels of glycolate and respiratory gas exchange. The ribulose diphosphate carboxylase activity decreased as the fruit ripened. By comparison, the ribulose diphosphate oxygenase activity increased during the transition from the green to the pink stage, and declined afterward. The changes in the endogenous glycolate levels and the respiratory gas exchange, as observed at different stages of ripening, resembled the changes in the ribulose diphosphate oxygenase activity. The utilization of glycolate in further metabolic activity may result in the formation of peroxidases required for the onset of ripening.

  19. Freeze-drying yields stable and pure amorphous calcium carbonate (ACC).

    PubMed

    Ihli, Johannes; Kulak, Alexander N; Meldrum, Fiona C

    2013-04-18

    A simple synthetic method is presented for the precipitation of high purity, dry amorphous calcium carbonate (ACC) based on freeze-drying saturated, counter ion free CaCO3 solutions, where the ACC produced shows an extended atmospheric stability. Translation of the methodology to amorphous calcium phosphate demonstrates the generality of the approach.

  20. ACCE/ACS National Educator and Leader of the Year Winners: AEC Congratulates These Outstanding Educators

    ERIC Educational Resources Information Center

    Australian Educational Computing, 2012

    2012-01-01

    This article presents the ACCE/ACS National Educator and Leader of the Year winners. Anne Mirtschin is the recipient of the ACCE/ACS 2012 Educator of the Year Award. Mirtschin is an innovative teacher at Hawkesdale P-12 College a small rural school that is isolated culturally and geographically. She uses online tools and technology to create…

  1. 24 CFR 882.805 - HA application process, ACC execution, and pre-rehabilitation activities.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    .... (4) The owner is responsible for selecting a competent contractor to undertake the rehabilitation...; (ii) Assure that the owner has selected a contractor in accordance with paragraph (c)(4) of this... ACC for an additional 10 years. (3) Section 882.403(a) (Maximum Total ACC Commitments) applies to...

  2. NP-40 reduces contamination by endogenous biotinylated carboxylases during purification of biotin tagged nuclear proteins.

    PubMed

    Papageorgiou, Dimitris N; Demmers, Jeroen; Strouboulis, John

    2013-05-01

    We describe here a simple procedure for greatly reducing contamination of nuclear extracts by naturally biotinylated cytoplasmic carboxylases, which represent a major source of non-specific background when employing BirA-mediated biotinylation tagging for the purification and characterization of nuclear protein complexes by mass spectrometry. We show that the use of 0.5% of the non-ionic detergent Nonidet-40 (NP-40) during cell lysis and nuclei isolation is sufficient to practically eliminate contamination of nuclear extracts by carboxylases and to greatly reduce background signals in downstream mass spectrometric analyses.

  3. New Insights into 1-Aminocyclopropane-1-Carboxylate (ACC) Deaminase Phylogeny, Evolution and Ecological Significance

    PubMed Central

    Nascimento, Francisco X.; Rossi, Márcio J.; Soares, Cláudio R. F. S.; McConkey, Brendan J.; Glick, Bernard R.

    2014-01-01

    The main objective of this work is the study of the phylogeny, evolution and ecological importance of the enzyme 1-aminocyclopropane-1-carboxylate (ACC) deaminase, the activity of which represents one of the most important and studied mechanisms used by plant growth–promoting microorganisms. The ACC deaminase gene and its regulatory elements presence in completely sequenced organisms was verified by multiple searches in diverse databases, and based on the data obtained a comprehensive analysis was conducted. Strain habitat, origin and ACC deaminase activity were taken into account when analyzing the results. In order to unveil ACC deaminase origin, evolution and relationships with other closely related pyridoxal phosphate (PLP) dependent enzymes a phylogenetic analysis was also performed. The data obtained show that ACC deaminase is mostly prevalent in some Bacteria, Fungi and members of Stramenopiles. Contrary to previous reports, we show that ACC deaminase genes are predominantly vertically inherited in various bacterial and fungal classes. Still, results suggest a considerable degree of horizontal gene transfer events, including interkingdom transfer events. A model for ACC deaminase origin and evolution is also proposed. This study also confirms the previous reports suggesting that the Lrp-like regulatory protein AcdR is a common mechanism regulating ACC deaminase expression in Proteobacteria, however, we also show that other regulatory mechanisms may be present in some Proteobacteria and other bacterial phyla. In this study we provide a more complete view of the role for ACC deaminase than was previously available. The results show that ACC deaminase may not only be related to plant growth promotion abilities, but may also play multiple roles in microorganism's developmental processes. Hence, exploring the origin and functioning of this enzyme may be the key in a variety of important agricultural and biotechnological applications. PMID:24905353

  4. Veratri Nigri Rhizoma et Radix (Veratrum nigrum L.) and Its Constituent Jervine Prevent Adipogenesis via Activation of the LKB1-AMPKα-ACC Axis In Vivo and In Vitro

    PubMed Central

    Park, Jinbong; Jeon, Yong-Deok; Kim, Hye-Lin; Kim, Dae-Seung; Han, Yo-Han; Jung, Yunu; Youn, Dong-Hyun; Kang, JongWook; Yoon, Daeyeon; Jeong, Mi-Young; Lee, Jong-Hyun; Hong, Seung-Heon; Lee, Junhee; Um, Jae-Young

    2016-01-01

    This study was performed in order to investigate the antiobese effects of the ethanolic extract of Veratri Nigri Rhizoma et Radix (VN), a herb with limited usage, due to its toxicology. An HPLC analysis identified jervine as a constituent of VN. By an Oil Red O assay and a Real-Time RT-PCR assay, VN showed higher antiadipogenic effects than jervine. In high-fat diet- (HFD-) induced obese C57BL/6J mice, VN administration suppressed body weight gain. The levels of peroxisome proliferator-activated receptor gamma (PPARγ), CCAAT-enhancer-binding protein alpha (C/EBPα), adipocyte fatty-acid-binding protein (aP2), adiponectin, resistin, and LIPIN1 were suppressed by VN, while SIRT1 was upregulated. Furthermore, VN activated phosphorylation of the liver kinase B1- (LKB1-) AMP-activated protein kinase alpha- (AMPKα-) acetyl CoA carboxylase (ACC) axis. Further investigation of cotreatment of VN with the AMPK agonist AICAR or AMPK inhibitor Compound C showed that VN can activate the phosphorylation of AMPKα in compensation to the inhibition of Compound C. In conclusion, VN shows antiobesity effects in HFD-induced obese C57BL/6J mice. In 3T3-L1 adipocytes, VN has antiadipogenic features, which is due to activating the LKB1-AMPKα-ACC axis. These results suggest that VN has a potential benefit in preventing obesity. PMID:27143989

  5. Veratri Nigri Rhizoma et Radix (Veratrum nigrum L.) and Its Constituent Jervine Prevent Adipogenesis via Activation of the LKB1-AMPKα-ACC Axis In Vivo and In Vitro.

    PubMed

    Park, Jinbong; Jeon, Yong-Deok; Kim, Hye-Lin; Kim, Dae-Seung; Han, Yo-Han; Jung, Yunu; Youn, Dong-Hyun; Kang, JongWook; Yoon, Daeyeon; Jeong, Mi-Young; Lee, Jong-Hyun; Hong, Seung-Heon; Lee, Junhee; Um, Jae-Young

    2016-01-01

    This study was performed in order to investigate the antiobese effects of the ethanolic extract of Veratri Nigri Rhizoma et Radix (VN), a herb with limited usage, due to its toxicology. An HPLC analysis identified jervine as a constituent of VN. By an Oil Red O assay and a Real-Time RT-PCR assay, VN showed higher antiadipogenic effects than jervine. In high-fat diet- (HFD-) induced obese C57BL/6J mice, VN administration suppressed body weight gain. The levels of peroxisome proliferator-activated receptor gamma (PPARγ), CCAAT-enhancer-binding protein alpha (C/EBPα), adipocyte fatty-acid-binding protein (aP2), adiponectin, resistin, and LIPIN1 were suppressed by VN, while SIRT1 was upregulated. Furthermore, VN activated phosphorylation of the liver kinase B1- (LKB1-) AMP-activated protein kinase alpha- (AMPKα-) acetyl CoA carboxylase (ACC) axis. Further investigation of cotreatment of VN with the AMPK agonist AICAR or AMPK inhibitor Compound C showed that VN can activate the phosphorylation of AMPKα in compensation to the inhibition of Compound C. In conclusion, VN shows antiobesity effects in HFD-induced obese C57BL/6J mice. In 3T3-L1 adipocytes, VN has antiadipogenic features, which is due to activating the LKB1-AMPKα-ACC axis. These results suggest that VN has a potential benefit in preventing obesity.

  6. Isolation of Ribulose-1,5-Bisphosphate Carboxylase/Oxygenase from Leaves

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) is a multi-functional enzyme that catalyzes the fixation of CO2 and O2 in photosynthesis and photorespiration, respectively. As the rate-limiting step in photosynthesis, improving the catalytic properties of Rubisco has long been viewed as a...

  7. 1-aminocyclopropane-1-carboxylic acid (ACC) in plants: more than just the precursor of ethylene!

    PubMed Central

    Van de Poel, Bram; Van Der Straeten, Dominique

    2014-01-01

    Ethylene is a simple two carbon atom molecule with profound effects on plants. There are quite a few review papers covering all aspects of ethylene biology in plants, including its biosynthesis, signaling and physiology. This is merely a logical consequence of the fascinating and pleiotropic nature of this gaseous plant hormone. Its biochemical precursor, 1-aminocyclopropane-1-carboxylic acid (ACC) is also a fairly simple molecule, but perhaps its role in plant biology is seriously underestimated. This triangularly shaped amino acid has many more features than just being the precursor of the lead-role player ethylene. For example, ACC can be conjugated to three different derivatives, but their biological role remains vague. ACC can also be metabolized by bacteria using ACC-deaminase, favoring plant growth and lowering stress susceptibility. ACC is also subjected to a sophisticated transport mechanism to ensure local and long-distance ethylene responses. Last but not least, there are now a few exciting studies where ACC has been reported to function as a signal itself, independently from ethylene. This review puts ACC in the spotlight, not to give it the lead-role, but to create a picture of the stunning co-production of the hormone and its precursor. PMID:25426135

  8. OpenARC: Extensible OpenACC Compiler Framework for Directive-Based Accelerator Programming Study

    SciTech Connect

    Lee, Seyong; Vetter, Jeffrey S

    2014-01-01

    Directive-based, accelerator programming models such as OpenACC have arisen as an alternative solution to program emerging Scalable Heterogeneous Computing (SHC) platforms. However, the increased complexity in the SHC systems incurs several challenges in terms of portability and productivity. This paper presents an open-sourced OpenACC compiler, called OpenARC, which serves as an extensible research framework to address those issues in the directive-based accelerator programming. This paper explains important design strategies and key compiler transformation techniques needed to implement the reference OpenACC compiler. Moreover, this paper demonstrates the efficacy of OpenARC as a research framework for directive-based programming study, by proposing and implementing OpenACC extensions in the OpenARC framework to 1) support hybrid programming of the unified memory and separate memory and 2) exploit architecture-specific features in an abstract manner. Porting thirteen standard OpenACC programs and three extended OpenACC programs to CUDA GPUs shows that OpenARC performs similarly to a commercial OpenACC compiler, while it serves as a high-level research framework.

  9. Role of pyruvate carboxylase in facilitation of synthesis of glutamate and glutamine in cultured astrocytes.

    PubMed

    Gamberino, W C; Berkich, D A; Lynch, C J; Xu, B; LaNoue, K F

    1997-12-01

    CO2 fixation was measured in cultured astrocytes isolated from neonatal rat brain to test the hypothesis that the activity of pyruvate carboxylase influences the rate of de novo glutamate and glutamine synthesis in astrocytes. Astrocytes were incubated with 14CO2 and the incorporation of 14C into medium or cell extract products was determined. After chromatographic separation of 14C-labelled products, the fractions of 14C cycled back to pyruvate, incorporated into citric acid cycle intermediates, and converted to the amino acids glutamate and glutamine were determined as a function of increasing pyruvate carboxylase flux. The consequences of increasing pyruvate, bicarbonate, and ammonia were investigated. Increasing extracellular pyruvate from 0 to 5 mM increased pyruvate carboxylase flux as observed by increases in the 14C incorporated into pyruvate and citric acid cycle intermediates, but incorporation into glutamate and glutamine, although relatively high at low pyruvate levels, did not increase as pyruvate carboxylase flux increased. Increasing added bicarbonate from 15 to 25 mM almost doubled CO2 fixation. When 25 mM bicarbonate plus 0.5 mM pyruvate increased pyruvate carboxylase flux to approximately the same extent as 15 mM bicarbonate plus 5 mM pyruvate, the rate of appearance of [14C] glutamate and glutamine was higher with the lower level of pyruvate. The conclusion was drawn that, in addition to stimulating pyruvate carboxylase, added pyruvate (but not added bicarbonate) increases alanine aminotransferase flux in the direction of glutamate utilization, thereby decreasing glutamate as pyruvate + glutamate --> alpha-ketoglutarate + alanine. In contrast to previous in vivo studies, the addition of ammonia (0.1 and 5 mM) had no effect on net 14CO2 fixation, but did alter the distribution of 14C-labelled products by decreasing glutamate and increasing glutamine. Rather unexpectedly, ammonia did not increase the sum of glutamate plus glutamine (mass amounts or

  10. Recalibration of the ACC/AHA Risk Score in Two Population-Based German Cohorts

    PubMed Central

    de las Heras Gala, Tonia; Geisel, Marie Henrike; Peters, Annette; Thorand, Barbara; Baumert, Jens; Lehmann, Nils; Jöckel, Karl-Heinz; Moebus, Susanne; Erbel, Raimund; Meisinger, Christine

    2016-01-01

    Background The 2013 ACC/AHA guidelines introduced an algorithm for risk assessment of atherosclerotic cardiovascular disease (ASCVD) within 10 years. In Germany, risk assessment with the ESC SCORE is limited to cardiovascular mortality. Applicability of the novel ACC/AHA risk score to the German population has not yet been assessed. We therefore sought to recalibrate and evaluate the ACC/AHA risk score in two German cohorts and to compare it to the ESC SCORE. Methods We studied 5,238 participants from the KORA surveys S3 (1994–1995) and S4 (1999–2001) and 4,208 subjects from the Heinz Nixdorf Recall (HNR) Study (2000–2003). There were 383 (7.3%) and 271 (6.4%) first non-fatal or fatal ASCVD events within 10 years in KORA and in HNR, respectively. Risk scores were evaluated in terms of calibration and discrimination performance. Results The original ACC/AHA risk score overestimated 10-year ASCVD rates by 37% in KORA and 66% in HNR. After recalibration, miscalibration diminished to 8% underestimation in KORA and 12% overestimation in HNR. Discrimination performance of the ACC/AHA risk score was not affected by the recalibration (KORA: C = 0.78, HNR: C = 0.74). The ESC SCORE overestimated by 5% in KORA and by 85% in HNR. The corresponding C-statistic was 0.82 in KORA and 0.76 in HNR. Conclusions The recalibrated ACC/AHA risk score showed strongly improved calibration compared to the original ACC/AHA risk score. Predicting only cardiovascular mortality, discrimination performance of the commonly used ESC SCORE remained somewhat superior to the ACC/AHA risk score. Nevertheless, the recalibrated ACC/AHA risk score may provide a meaningful tool for estimating 10-year risk of fatal and non-fatal cardiovascular disease in Germany. PMID:27732641

  11. Report of the American College of Cardiology (ACC) Scientific Sessions 2015, San Diego.

    PubMed

    Murohara, Toyoaki

    2015-01-01

    The 64th Annual Scientific Sessions and Exposition of the American College of Cardiology (ACC) were held at the San Diego Convention Center from March 14-16, 2015. The ACC Scientific Sessions are 1 of 2 major scientific cardiology meetings in the United States, with nearly 20,000 attendees, including 15,000 cardiovascular professionals. There were over 2,100 oral and poster abstracts, and more than 15 late-breaking clinical trials (LBCTs) abstructs. This report presents the highlights and several key presentations, especially the LBCTs, from the ACC Scientific Sessions 2015. I hope this review will help cardiologists update to the latest information.

  12. Report of the American College of Cardiology (ACC) Scientific Sessions 2016, Chicago.

    PubMed

    Mano, Toshiaki; Yamamoto, Kazuhiro

    2016-05-25

    The 65(th)Annual Scientific Sessions of the American College of Cardiology (ACC) were held at McCormick Place, Chicago, from April 2-4, 2016. The ACC Scientific Sessions are one of the 2 major scientific cardiology meetings in the USA and one of the major scientific meetings of cardiology in the world. It had an attendance of 18,769 and over 2,000 oral and poster abstracts, including 8 late-breaking clinical trials. This report presents the key presentations and the highlights from the ACC Scientific Sessions 2016 in Chicago. (Circ J 2016; 80: 1308-1313).

  13. Isolation, identification, and synthesis of 2-carboxyarabinitol 1-phosphate, a diurnal regulator of ribulase-bisphosphate carboxylase activity

    SciTech Connect

    Berry, J.A.; Lorimer, G.H.; Pierce, J.; Seemann, J.R.; Meek, J.; Freas, S.

    1987-02-01

    The diurnal change in activity of ribulose 1,5-bisphosphate (Rbu-1,5-P/sub 2/) carboxylase (3-phospho-D-glycerate carboxy-lyase (dimerizing); EC 4.1.1.39) of leaves of Phaseolus vulgaris is regulated (in part) by mechanisms that control the level of an endogenous inhibitor that binds tightly to the activated (carbamoylated) form of Rbu-1,5-P/sub 2/ carboxylase. This inhibitor was extracted from leaves and copurified with the Rbu-1,5-P/sub 2/ carboxylase of the leaves. Further purification by ion-exchange chromatography, adsorption to purified Rbu-1,5-P/sub 2/ carboxylase, barium precipitation, and HPLC separation yielded a phosphorylated compound that was a strong inhibitor of Rbu-1,5-P/sub 2/ carboxylase. The compound was analyzed by GC/MS, /sup 13/C NMR, and /sup 1/H NMR and shown to be 2-carboxyarabinitol 1-phosphate ((2-C-phosphohydroxymethyl)-D-ribonic acid). The structure of the isolated compound differs from the Rbu-1,5-P/sub 2/ carboxylase transition-state analogue 2-carboxyarabinitol 1,5-bisphosphate only by the lack of the C-5 phosphate group. This difference results in a higher binding constant for the monophosphate compared with the bisphosphate. The less tightly bound compound acts in a light-dependent, reversible regulation of Rbu-1,5-P/sub 2/ carboxylase activity in vivo.

  14. IMPACC: A Tightly Integrated MPI+OpenACC Framework Exploiting Shared Memory Parallelism

    SciTech Connect

    Lee, Seyong; Vetter, Jeffrey S

    2016-01-01

    We propose IMPACC, an MPI+OpenACC framework for heterogeneous accelerator clusters. IMPACC tightly integrates MPI and OpenACC, while exploiting the shared memory parallelism in the target system. IMPACC dynamically adapts the input MPI+OpenACC applications on the target heterogeneous accelerator clusters to fully exploit target system-specific features. IMPACC provides the programmers with the unified virtual address space, automatic NUMA-friendly task-device mapping, efficient integrated communication routines, seamless streamlining of asynchronous executions, and transparent memory sharing. We have implemented IMPACC and evaluated its performance using three heterogeneous accelerator systems, including Titan supercomputer. Results show that IMPACC can achieve easier programming, higher performance, and better scalability than the current MPI+OpenACC model.

  15. Technology Awareness Workshop on Active Combustion Control (ACC) in Propulsion Systems: JANNAF Combustion Subcommittee Workshop

    NASA Technical Reports Server (NTRS)

    Fry, Ronald S. (Editor); Gannaway, Mary T. (Editor)

    1997-01-01

    A JANNAF Combustion Subcommittee Technology Awareness Seminar on Active Combustion Control (ACC) in Propulsion Systems' was held 12 November 1997 at the NASA Lewis Research Center (LeRC), Cleveland, Ohio. The objectives of the seminar were: 1) Define the need and potential of ACC to meet future requirements for gas turbines and ramjets; 2) Explain general principles of ACC and discuss recent successes to suppress combustion instabilities, increase combustion efficiency, reduce emission, and extend flammability limits; 3) Identify R&D barriers/needs for practical implementation of ACC; 4) Explore potential for improving coordination of future R&D activities funded by various government agencies. Over 40 individuals representing senior management from over 20 industry and government organizations participated. This document summarizes the presentations and findings of this seminar.

  16. OpenACC programs of the Swendsen-Wang multi-cluster spin flip algorithm

    NASA Astrophysics Data System (ADS)

    Komura, Yukihiro

    2015-12-01

    We present sample OpenACC programs of the Swendsen-Wang multi-cluster spin flip algorithm. OpenACC is a directive-based programming model for accelerators without requiring modification to the underlying CPU code itself. In this paper, we deal with the classical spin models as with the sample CUDA programs (Komura and Okabe, 2014), that is, two-dimensional (2D) Ising model, three-dimensional (3D) Ising model, 2D Potts model, 3D Potts model, 2D XY model and 3D XY model. We explain the details of sample OpenACC programs and compare the performance of the present OpenACC implementations with that of the CUDA implementations for the 2D and 3D Ising models and the 2D and 3D XY models.

  17. Effect of dexamethasone and ACC on bacteria-induced mucin expression in human airway mucosa.

    PubMed

    Hauber, Hans-Peter; Goldmann, Torsten; Vollmer, Ekkehard; Wollenberg, Barbara; Zabel, Peter

    2007-11-01

    Gram-negative bacteria can stimulate mucin production, but excessive mucus supports bacterial infection and consequently leads to airway obstruction. Therefore, the effect of dexamethasone (DEX) and the antioxidant acetyl-cysteine (ACC) on bacteria-induced mucus expression was investigated. Explanted human airway mucosa and mucoepidermoid cells (Calu-3) were stimulated with lipopolysaccharide (LPS) or PAM3 (a synthetic lipoprotein). DEX or ACC were added to either LPS- or PAM3-stimulated airway mucosa or Calu-3 cells. Mucin mRNA expression (MUC5AC) and total mucus glycoconjugates (mucin protein) were quantified using real-time PCR and periodic acid Schiff staining. LPS and PAM3 significantly increased mucin expression in airway mucosa and Calu-3 cells (P < 0.05). DEX alone had no significant effect on mucin expression in airway mucosa or Calu-3 cells (P > 0.05). In contrast, DEX significantly reduced LPS- and PAM3-induced mucin expression in explanted mucosal tissue and mucin expression in Calu-3 cells (P < 0.05). In explanted human airway mucosa ACC alone significantly increased mucin expression (P < 0.05). In contrast, ACC significantly decreased LPS- and PAM3-induced mucin expression (P < 0.05). In Calu-3 cells ACC alone had no significant effect on mucin expression (P > 0.05). ACC decreased LPS- and PAM3-induced mucin expression, but this effect was not significant (P > 0.05). These data suggest that DEX can effectively reduce bacteria-induced mucin expression in the airways. ACC alone may increase mucin expression in noninfected mucosa, but it decreased bacteria-induced mucin expression. Further studies are warranted to evaluate whether the effect of DEX or ACC is clinically relevant.

  18. Computational redesign of bacterial biotin carboxylase inhibitors using structure-based virtual screening of combinatorial libraries.

    PubMed

    Brylinski, Michal; Waldrop, Grover L

    2014-04-02

    As the spread of antibiotic resistant bacteria steadily increases, there is an urgent need for new antibacterial agents. Because fatty acid synthesis is only used for membrane biogenesis in bacteria, the enzymes in this pathway are attractive targets for antibacterial agent development. Acetyl-CoA carboxylase catalyzes the committed and regulated step in fatty acid synthesis. In bacteria, the enzyme is composed of three distinct protein components: biotin carboxylase, biotin carboxyl carrier protein, and carboxyltransferase. Fragment-based screening revealed that amino-oxazole inhibits biotin carboxylase activity and also exhibits antibacterial activity against Gram-negative organisms. In this report, we redesigned previously identified lead inhibitors to expand the spectrum of bacteria sensitive to the amino-oxazole derivatives by including Gram-positive species. Using 9,411 small organic building blocks, we constructed a diverse combinatorial library of 1.2×10⁸ amino-oxazole derivatives. A subset of 9×10⁶ of these compounds were subjected to structure-based virtual screening against seven biotin carboxylase isoforms using similarity-based docking by eSimDock. Potentially broad-spectrum antibiotic candidates were selected based on the consensus ranking by several scoring functions including non-linear statistical models implemented in eSimDock and traditional molecular mechanics force fields. The analysis of binding poses of the top-ranked compounds docked to biotin carboxylase isoforms suggests that: (1) binding of the amino-oxazole anchor is stabilized by a network of hydrogen bonds to residues 201, 202 and 204; (2) halogenated aromatic moieties attached to the amino-oxazole scaffold enhance interactions with a hydrophobic pocket formed by residues 157, 169, 171 and 203; and (3) larger substituents reach deeper into the binding pocket to form additional hydrogen bonds with the side chains of residues 209 and 233. These structural insights into drug

  19. Ozone stress induces the expression of ACC synthase in potato plants

    SciTech Connect

    Schlagnhaufer, C.D.; Arteca, R.N.; Pell, E.J. )

    1993-05-01

    When potato plants (Solanum tuberosum L. cv Norland) are subjected to oxone stress ethylene is emitted. Increases in ethylene production are often the result of increased expression of the enzyme ACC synthase. We used the polymerase chain reaction (PCR) to clone a cDNA encoding an ozone-induced ACC synthase. After treating potato plants with 300 ppb ozone for 4 h, RNA was extracted using a guanidinium isothiocyanate method. Using degenerate oligonucleotides corresponding to several conserved regions of ACC synthase sequences reported from different plant tissues as primers, we were able to reverse transcribe the RNA and amplify a cDNA for ACC synthase. The clone is 1098 bp in length encoding for 386 amino acids comprising [approximately]80% of the protein. Computer analysis of the deduced amino acid sequence showed that our clone is 50-70% homologous with ACC synthase genes cloned from other plant tissues. Using the cDNA as a probe in northern analysis we found that there is little or no expression in control tissue: however there is a large increase in the expression of the ACC synthase message in response to ozone treatment.

  20. Perspective of plant growth promoting rhizobacteria (PGPR) containing ACC deaminase in stress agriculture.

    PubMed

    Saleem, Muhammad; Arshad, Muhammad; Hussain, Sarfraz; Bhatti, Ahmad Saeed

    2007-10-01

    Ethylene is a gaseous plant growth hormone produced endogenously by almost all plants. It is also produced in soil through a variety of biotic and abiotic mechanisms, and plays a key role in inducing multifarious physiological changes in plants at molecular level. Apart from being a plant growth regulator, ethylene has also been established as a stress hormone. Under stress conditions like those generated by salinity, drought, waterlogging, heavy metals and pathogenicity, the endogenous production of ethylene is accelerated substantially which adversely affects the root growth and consequently the growth of the plant as a whole. Certain plant growth promoting rhizobacteria (PGPR) contain a vital enzyme, 1-aminocyclopropane-1-carboxylate (ACC) deaminase, which regulates ethylene production by metabolizing ACC (an immediate precursor of ethylene biosynthesis in higher plants) into alpha-ketobutyrate and ammonia. Inoculation with PGPR containing ACC deaminase activity could be helpful in sustaining plant growth and development under stress conditions by reducing stress-induced ethylene production. Lately, efforts have been made to introduce ACC deaminase genes into plants to regulate ethylene level in the plants for optimum growth, particularly under stressed conditions. In this review, the primary focus is on giving account of all aspects of PGPR containing ACC deaminase regarding alleviation of impact of both biotic and abiotic stresses onto plants and of recent trends in terms of introduction of ACC deaminase genes into plant and microbial species.

  1. Ribulose-1,5-bisphosphate carboxylase/oxygenase from thermophilic cyanobacterium Thermosynechococcus elongatus.

    PubMed

    Gubernator, Beata; Bartoszewski, Rafal; Kroliczewski, Jaroslaw; Wildner, Guenter; Szczepaniak, Andrzej

    2008-01-01

    Ribulose-1,5-bisphosphate carboxylase/oxygenase (rubisco) can be divided into two branches: the "red-like type" of marine algae and the "green-like type" of cyanobacteria, green algae, and higher plants. We found that the "green-like type" rubisco from the thermophilic cyanobacterium Thermosynechococcus elongatus has an almost 2-fold higher specificity factor compared with rubiscos of mesophilic cyanobacteria, reaching the values of higher plants, and simultaneously revealing an improvement in enzyme thermostability. The difference in the activation energies at the transition stages between the oxygenase and carboxylase reactions for Thermosynechococcus elongatus rubisco is very close to that of Galdieria partita and significantly higher than that of spinach. This is the first characterization of a "green-like type" rubisco from thermophilic organism.

  2. Nonstructural 5A Protein of Hepatitis C Virus Interacts with Pyruvate Carboxylase and Modulates Viral Propagation

    PubMed Central

    Kim, Jong-Wook; Hwang, Soon B.

    2013-01-01

    Hepatitis C virus (HCV) is highly dependent on cellular factors for its own propagation. By employing tandem affinity purification method, we identified pyruvate carboxylase (PC) as a cellular partner for NS5A protein. NS5A interacted with PC through the N-terminal region of NS5A and the biotin carboxylase domain of PC. PC expression was decreased in cells expressing NS5A and HCV-infected cells. Promoter activity of PC was also decreased by NS5A protein. However, FAS expression was increased in cells expressing NS5A and cell culture grown HCV (HCVcc)-infected cells. Silencing of PC promoted fatty acid synthase (FAS) expression level. These data suggest HCV may modulate PC via NS5A protein for its own propagation. PMID:23861867

  3. Crystal Structures of Human and Staphylococcus aureus Pyruvate Carboxylase and Molecular Insights into the Carboxyltransfer Reaction

    SciTech Connect

    Xiang,S.; Tong, L.

    2008-01-01

    Pyruvate carboxylase (PC) catalyzes the biotin-dependent production of oxaloacetate and has important roles in gluconeogenesis, lipogenesis, insulin secretion and other cellular processes. PC contains the biotin carboxylase (BC), carboxyltransferase (CT) and biotin-carboxyl carrier protein (BCCP) domains. We report here the crystal structures at 2.8-Angstroms resolution of full-length PC from Staphylococcus aureus and the C-terminal region (missing only the BC domain) of human PC. A conserved tetrameric association is observed for both enzymes, and our structural and mutagenesis studies reveal a previously uncharacterized domain, the PC tetramerization (PT) domain, which is important for oligomerization. A BCCP domain is located in the active site of the CT domain, providing the first molecular insights into how biotin participates in the carboxyltransfer reaction. There are dramatic differences in domain positions in the monomer and the organization of the tetramer between these enzymes and the PC from Rhizobium etli.

  4. Simultaneous Kinetic Analysis of Ribulose 1,5-Bisphosphate Carboxylase/Oxygenase Activities 1

    PubMed Central

    Kent, Samuel S.; Young, Joseph D.

    1980-01-01

    An assay was developed for simultaneous kinetic analysis of the activities of the bifunctional plant enzyme ribulose 1,5-bisphosphate carboxylase/oxygenase [EC 4.1.1.39]. [1-14C,5-3H]Ribulose 1,5-bisphosphate (RuBP) was used as the labeled substrate. Tritium enrichment of the doubly labeled 3-phosphoglycerate (3-PGA) product, common to both enzyme activities, may be used to calculate Vc/Vo ratios from the expression A/(B-A) where A and B represent the 3H/14C isotope ratios of doubly labeled RuBP and 3-PGA, and Vc and Vo represent the activities of carboxylase and oxygenase, respectively. Doubly labeled substrate was synthesized from [2-14C]glucose and [6-3H]glucose using the enzymes of the pentose phosphate pathway coupled with phosphoribulokinase. The kinetic properties of a commercial preparation of fully activated spinach carboxylase were studied under approximated physiological conditions of 20% O2 (252 micromolar), 295 μl/l CO2 (10 micromolar), 25 C, and pH 8.19. The Vc/Vo ratio was, within experimental error, constant at 30 seconds and 1 minute. This double label assay method may be used to calculate Vc/Vo ratios for the Laing-Ogren-Hageman equation, Vc/Vo = (VcKo/VoKc) ([CO2]/[O2]) where Vc and Vo represent Vmax, and Kc and Ko represent Michaelis constants for the carboxylase and oxygenase activities, respectively. PMID:16661214

  5. Characterization of ribulose 1,5-bisphosphate carboxylase/oxygenase from Euglena gracilis Z.

    PubMed

    Yokota, A; Harada, A; Kitaoka, S

    1989-03-01

    An improved method was devised to purify ribulose 1,5-bisphosphate carboxylase/oxygenase (RuBisCO) with high specific activity (2.1 mumol of CO2 fixed/mg protein/min) from Euglena gracilis Z. The purified enzyme stored at -80 degrees C required treatment with dithiothreitol for full activity. The dithiothreitol-treated RuBisCO was activated by 12 mM NaHCO3 and 20 mM MgCl2, and the activated state was stable at least for 60 min in the presence of 4 mM ethylenediaminetetraacetate. The form of inorganic carbon fixed by the Euglena enzyme was CO2, as for the plant enzymes. The carboxylase reaction proceeded linearly with time for at least 8 min. The optimum pH for this reaction was 7.8 to 8.0. The carboxylase activity increased with increasing temperature up to 50 degrees C. The activation energy for the carboxylation reaction was 10.0 kcal/mol. The Michaelis constants of Euglena RuBisCO were 30.9 microM for CO2, 560 microM for O2, and 10.5 microM for ribulose 1,5-bisphosphate. Mathematical comparison between the photosynthesis rate predicted from these enzymatic properties and the observed rate suggested that there is no CO2-concentrating mechanism in E. gracilis.

  6. Isolation, characterization, and crystallization of ribulosebisphosphate carboxylase from autotrophically grown Rhodospirillum rubrum.

    PubMed

    Schloss, J V; Phares, E F; Long, M V; Norton, I L; Stringer, C D; Hartman, F C

    1979-01-01

    Serial culture of Rhodospirillum rubrum with 2% CO2 in H2 as the exclusive carbon source resulted in a rather large fraction of the soluble protein (greater than 40%) being comprised of ribulosebisphosphate carboxylase (about sixfold higher than the highest value previously reported). Isolation of the enzyme from these cells revealed that it has physical and kinetic properties similar to those previously described for the enzyme derived from cells grown on butyrate. Notably, the small subunit (which is a constituent of the carboxylase from eucaryotes and most procaryotes) was absent in the enzyme from autotrophically grown R. rubrum. Edman degradation of the purified enzyme revealed that the NH2 terminus is free (in contrast to the catalytic subunit of the carboxylase from eucaryotes) and that the NH2-terminal sequence is Met-Asp-Gln-Ser-Ser-Arg-Tyr-Val-Asn-Leu-Ala-Leu-Lys-Glu-Glu-Asp-Leu-Ile-Ala-Gly-Gly-Glx-His-Val-Leu-. Crystals of the enzyme were readily obtained by dialysis against distilled water.

  7. A substrate-induced biotin binding pocket in the carboxyltransferase domain of pyruvate carboxylase.

    PubMed

    Lietzan, Adam D; St Maurice, Martin

    2013-07-05

    Biotin-dependent enzymes catalyze carboxyl transfer reactions by efficiently coordinating multiple reactions between spatially distinct active sites. Pyruvate carboxylase (PC), a multifunctional biotin-dependent enzyme, catalyzes the bicarbonate- and MgATP-dependent carboxylation of pyruvate to oxaloacetate, an important anaplerotic reaction in mammalian tissues. To complete the overall reaction, the tethered biotin prosthetic group must first gain access to the biotin carboxylase domain and become carboxylated and then translocate to the carboxyltransferase domain, where the carboxyl group is transferred from biotin to pyruvate. Here, we report structural and kinetic evidence for the formation of a substrate-induced biotin binding pocket in the carboxyltransferase domain of PC from Rhizobium etli. Structures of the carboxyltransferase domain reveal that R. etli PC occupies a symmetrical conformation in the absence of the biotin carboxylase domain and that the carboxyltransferase domain active site is conformationally rearranged upon pyruvate binding. This conformational change is stabilized by the interaction of the conserved residues Asp(590) and Tyr(628) and results in the formation of the biotin binding pocket. Site-directed mutations at these residues reduce the rate of biotin-dependent reactions but have no effect on the rate of biotin-independent oxaloacetate decarboxylation. Given the conservation with carboxyltransferase domains in oxaloacetate decarboxylase and transcarboxylase, the structure-based mechanism described for PC may be applicable to the larger family of biotin-dependent enzymes.

  8. Characterisation and purification of ribulose-bisphosphate carboxylase from heterotrophically grown halophilic archaebacterium, Haloferax mediterranei.

    PubMed

    Rajagopalan, R; Altekar, W

    1994-04-15

    The CO2-fixing enzyme of Calvin cycle ribulose-1,5-bisphosphate-carboxylase/oxygenase has been isolated from a halophilic bacterium, Haloferax mediterranei grown heterotrophically. A homogeneous preparation was obtained from sonicated extract of the cells by three steps, resulting in a specific activity of 52 nmol.min-1.mg protein-1. The physicochemical and catalytic properties of the enzyme were studied. The halobacterial ribulose-bisphosphate carboxylase is an oligomer of 54-kDa and 14-kDa subunits as detected by SDS/PAGE. By sucrose-density-gradient centrifugation, the molecular mass of the enzyme was estimated as approximately 500 kDa indicating a hexadecameric nature. No evidence for an additional form of the enzyme devoid of small subunits was obtained. The enzyme required Mg2+ for activity, KCl for activity and stability, and an optimal pH of 7.8. In contrast to many halophilic proteins, ribulose-bisphosphate carboxylase from H. mediterranei is not an acidic protein. From the comparison of amino acid composition of halobacterial enzyme with its counterparts from a few eukaryotic and eubacterial sources, the S delta Q values showed that these proteins share some compositional similarities.

  9. 3-Methylcrotonyl-CoA carboxylase deficiency: phenotypic variability in a family.

    PubMed

    Eminoglu, F Tuba; Ozcelik, Aysima A; Okur, Ilyas; Tumer, Leyla; Biberoglu, Gursel; Demir, Ercan; Hasanoglu, Alev; Baumgartner, Matthias R

    2009-04-01

    A family with 3-methylcrotonyl-CoA carboxylase deficiency with different clinical features is described. A 15-month-old boy, who was the index patient, was admitted to the hospital with atonic seizure. His brother had delayed language development and their uncle had been followed with diagnosis of epilepsy for the last 5 years. Urinary organic acid analysis displayed elevated 3-hydroxyisovaleric acid and 3-methylcrotonylglycine, analysis of acylcarnitines showed elevated 3-hydroxyisovalerylcarnitine and decreased free carnitine levels in both the patients and their uncle. Methylcrotonyl-CoA carboxylase activity in cultured fibroblasts displayed a low residual activity of 2.2% of the median control value while propionyl-CoA carboxylase activity was normal in the index patient. Mutation analysis revealed a large homozygous deletion of 2264 bp (c.873+4524_6787de12264) in the MCCA gene, which has not been described to date. Adult-onset afebrile seizures have not been reported in the literature. Our cases are an example of this wide phenotypic variability within a single family.

  10. Variations in Kinetic Properties of Ribulose-1,5-bisphosphate Carboxylases among Plants

    PubMed Central

    Yeoh, Hock-Hin; Badger, Murray R.; Watson, Leslie

    1981-01-01

    Studies of ribulose-1,5-bisphosphate (RuBP) carboxylase from taxonomically diverse plants show that the enzyme from C3 and crassulacean acid metabolism pathway species exhibits lower Km(CO2) values (12-25 micromolar) than does that from C4 species (28-34 micromolar). RuBP carboxylase from aquatic angiosperms, an aquatic bryophyte, fresh water and marine algae has yielded consistently high Km(CO2) values (30-70 micromolar), similar in range to that of the enzyme from C4 terrestrial plants. This variation in Km(CO2) is discussed in relation to the correlation between the existence of CO2-concentrating mechanisms for photosynthesis and the affinity of the enzyme for CO2. The Km(RuBP) of the enzyme from various sources ranges from 10 to 136 micromolar; mean ± sd = 36 ± 20 micromolar. This variation in Km(RuBP) does not correlate with different photosynthetic pathways, but shows taxonomic patterns. Among the dicotyledons, the enzyme from crassinucellate species exhibits lower Km(RuBP) (18 ± 4 micromolar) than does that from tenuinucellate species (25 ± 7 micromolar). Among the Poaceae, RuBP carboxylase from Triticeae, chloridoids, andropogonoids, Microlaena, and Tetrarrhena has yielded lower Km(RuBP) values (29 ± 11 micromolar) than has that from other members of the grass family (46 ± 10 micromolar). PMID:16661826

  11. Positive coping styles and perigenual ACC volume: two related mechanisms for conferring resilience?

    PubMed

    Holz, Nathalie E; Boecker, Regina; Jennen-Steinmetz, Christine; Buchmann, Arlette F; Blomeyer, Dorothea; Baumeister, Sarah; Plichta, Michael M; Esser, Günter; Schmidt, Martin; Meyer-Lindenberg, Andreas; Banaschewski, Tobias; Brandeis, Daniel; Laucht, Manfred

    2016-05-01

    Stress exposure has been linked to increased rates of depression and anxiety in adults, particularly in females, and has been associated with maladaptive changes in the anterior cingulate cortex (ACC), which is an important brain structure involved in internalizing disorders. Coping styles are important mediators of the stress reaction by establishing homeostasis, and may thus confer resilience to stress-related psychopathology. Anatomical scans were acquired in 181 healthy participants at age 25 years. Positive coping styles were determined using a self-report questionnaire (German Stress Coping Questionnaire, SVF78) at age 22 years. Adult anxiety and depression symptoms were assessed at ages 22, 23 and 25 years with the Young Adult Self-Report. Information on previous internalizing diagnoses was obtained by diagnostic interview (2-19 years). Positive coping styles were associated with increased ACC volume. ACC volume and positive coping styles predicted anxiety and depression in a sex-dependent manner with increased positive coping and ACC volume being related to lower levels of psychopathology in females, but not in males. These results remained significant when controlled for previous internalizing diagnoses. These findings indicate that positive coping styles and ACC volume are two linked mechanisms, which may serve as protective factors against internalizing disorders.

  12. OpenACC to FPGA: A Framework for Directive-based High-Performance Reconfigurable Computing

    SciTech Connect

    Lee, Seyong; Kim, Jungwon; Vetter, Jeffrey S

    2016-01-01

    This paper presents a directive-based, high-level programming framework for high-performance reconfigurable computing. It takes a standard, portable OpenACC C program as input and generates a hardware configuration file for execution on FPGAs. We implemented this prototype system using our open-source OpenARC compiler; it performs source-to-source translation and optimization of the input OpenACC program into an OpenCL code, which is further compiled into a FPGA program by the backend Altera Offline OpenCL compiler. Internally, the design of OpenARC uses a high- level intermediate representation that separates concerns of program representation from underlying architectures, which facilitates portability of OpenARC. In fact, this design allowed us to create the OpenACC-to-FPGA translation framework with minimal extensions to our existing system. In addition, we show that our proposed FPGA-specific compiler optimizations and novel OpenACC pragma extensions assist the compiler in generating more efficient FPGA hardware configuration files. Our empirical evaluation on an Altera Stratix V FPGA with eight OpenACC benchmarks demonstrate the benefits of our strategy. To demonstrate the portability of OpenARC, we show results for the same benchmarks executing on other heterogeneous platforms, including NVIDIA GPUs, AMD GPUs, and Intel Xeon Phis. This initial evidence helps support the goal of using a directive-based, high-level programming strategy for performance portability across heterogeneous HPC architectures.

  13. Prebiotic Fiber Increases Hepatic Acetyl CoA Carboxylase Phosphorylation and Suppresses Glucose-Dependent Insulinotropic Polypeptide Secretion More Effectively When Used with Metformin in Obese Rats1,2

    PubMed Central

    Pyra, Kim A.; Saha, Dolan C.; Reimer, Raylene A.

    2013-01-01

    Independently, metformin (MET) and the prebiotic, oligofructose (OFS), have been shown to increase glucagon-like peptide (GLP-1) secretion. Our objective was to determine whether using OFS as an adjunct with MET augments GLP-1 secretion in obese rats. Male, diet-induced obese Sprague Dawley rats were randomized to: 1) high-fat/-sucrose diet [HFHS; control (C); 20% fat, 50% sucrose wt:wt]; 2) HFHS+10% OFS (OFS); 3) HFHS + MET [300 mg/kg/d (MET)]; 4) HFHS+10% OFS+MET (OFS +MET). Body composition, glycemia, satiety hormones, and mechanisms related to dipeptidyl peptidase 4 (DPP4) activity in plasma, hepatic AMP-activated protein kinase (AMPK; Western blots), and gut microbiota (qPCR) were examined. Direct effects of MET and SCFA were examined in human enteroendocrine cells. The interaction between OFS and MET affected fat mass, hepatic TG, secretion of glucose-dependent insulinotropic polypeptide (GIP) and leptin, and AMPKα2 mRNA and phosphorylated acetyl CoA carboxylase (pACC) levels (P < 0.05). Combined, OFS and MET reduced GIP secretion to a greater extent than either treatment alone (P < 0.05). The hepatic pACC level was increased by OFS+MET by at least 50% above all other treatments, which did not differ from each other (P < 0.05). OFS decreased plasma DPP4 activity (P < 0.001). Cecal Bifidobacteria (P < 0.001) were markedly increased and C. leptum decreased (P < 0.001) with OFS consumption. In human enteroendocrine cells, the interaction between MET and SCFA affected GLP-1 secretion (P < 0.04) but was not associated with higher GLP-1 than the highest individual doses. In conclusion, the combined actions of OFS and MET were associated with important interaction effects that have the potential to improve metabolic outcomes associated with obesity. PMID:22223580

  14. Transmembrane domain interactions and residue proline 378 are essential for proper structure, especially disulfide bond formation, in the human vitamin K-dependent gamma-glutamyl carboxylase.

    PubMed

    Tie, Jian-Ke; Zheng, Mei-Yan; Hsiao, Kuang-Ling N; Perera, Lalith; Stafford, Darrel W; Straight, David L

    2008-06-17

    We used recombinant techniques to create a two-chain form (residues 1-345 and residues 346-758) of the vitamin K-dependent gamma-glutamyl carboxylase, a glycoprotein located in the endoplasmic reticulum containing five transmembrane domains. The two-chain carboxylase had carboxylase and epoxidase activities similar to those of one-chain carboxylase. In addition, it had normal affinity for the propeptide of factor IX. We employed this molecule to investigate formation of the one disulfide bond in carboxylase, the transmembrane structure of carboxylase, and the potential interactions among the carboxylase's transmembrane domains. Our results indicate that the two peptides of the two-chain carboxylase are joined by a disulfide bond. Proline 378 is important for the structure necessary for disulfide formation. Results with the P378L carboxylase indicate that noncovalent bonds maintain the two-chain structure even when the disulfide bond is disrupted. As we had previously proposed, the fifth transmembrane domain of carboxylase is the last and only transmembrane domain in the C-terminal peptide of the two-chain carboxylase. We show that the noncovalent association between the two chains of carboxylase involves an interaction between the fifth transmembrane domain and the second transmembrane domain. Results of a homology model of transmembrane domains 2 and 5 suggest that not only do these two domains associate but that transmembrane domain 2 may interact with another transmembrane domain. This latter interaction may be mediated at least in part by a motif of glycine residues in the second transmembrane domain.

  15. An ACC Design Method for Achieving Both String Stability and Ride Comfort

    NASA Astrophysics Data System (ADS)

    Yamamura, Yoshinori; Seto, Yoji; Nishira, Hikaru; Kawabe, Taketoshi

    An investigation was made of a method for designing adaptive cruise control (ACC) so as to achieve a headway distance response that feels natural to the driver while at the same time obtaining high levels of both string stability and ride comfort. With this design method, the H∞ norm is adopted as the index of string stability. Additionally, two norms are introduced for evaluating ride comfort and natural vehicle behavior. The relationship between these three norms and headway distance response characteristics was analyzed, and an evaluation method was established for achieving high levels of the various performance characteristics required of ACC. An ACC system designed with this method was evaluated in driving tests conducted on a proving ground course, and the results confirmed that it achieved the targeted levels of string stability, ride comfort and natural vehicle behavior.

  16. Precipitation of ACC in liposomes-a model for biomineralization in confined volumes

    SciTech Connect

    Tester, Chantel C; Wu, Ching-Hsuan; Weigand, Steven; Joester, Derk

    2013-01-10

    Biomineralizing organisms frequently precipitate minerals in small phospholipid bilayer-delineated compartments. We have established an in vitro model system to investigate the effect of confinement in attoliter to femtoliter volumes on the precipitation of calcium carbonate. In particular, we analyze the growth and stabilization of liposome-encapsulated amorphous calcium carbonate (ACC) nanoparticles using a combination of in situ techniques, cryo-transmission electron microscopy (Cryo-TEM), and small angle X-ray scattering (SAXS). Herein, we discuss ACC nanoparticle growth rate as a function of liposome size, carbon dioxide flux across the liposome membrane, pH, and osmotic pressure. Based on these experiments, we argue that the stabilization of ACC nanoparticles in liposomes is a consequence of a low nucleation rate (high activation barrier) of crystalline polymorphs of calcium carbonate.

  17. 1-Aminocyclopropane-1-carboxylic acid (ACC) concentration and ACC synthase expression in soybean roots, root tips, and soybean cyst nematode (Heterodera glycines)-infected roots.

    PubMed

    Tucker, Mark L; Xue, Ping; Yang, Ronghui

    2010-01-01

    Colonization of plant roots by root knot and cyst nematodes requires a functional ethylene response pathway. However, ethylene plays many roles in root development and whether its role in nematode colonization is direct or indirect, for example lateral root initiation or root hair growth, is not known. The temporal requirement for ethylene and localized synthesis of ethylene during the life span of soybean cyst nematode (SCN) on soybean roots was further investigated. Although a significant increase in ethylene evolution was not detected from SCN-colonized roots, the concentration of the immediate precursor to ethylene, 1-aminocyclopropane-1-carboxylic acid (ACC), was higher in SCN-colonized root pieces and root tips than in other parts of the root. Moreover, expression analysis of 17 ACC synthase (ACS) genes indicated that a select set of ACS genes is expressed in SCN-colonized root pieces that is clearly different from the set of genes expressed in non-colonized roots or root tips. Semi-quantitative real-time PCR indicated that ACS transcript accumulation correlates with the high concentration of ACC in root tips. In addition, an ACS-like sequence was found in the public SCN nucleotide database. Acquisition of a full-length sequence for this mRNA (accession GQ389647) and alignment with transcripts for other well-characterized ACS proteins indicated that the nematode sequence is missing a key element required for ACS activity and therefore probably is not a functional ACS. Moreover, no significant amount of ACC was found in any growth stage of SCN that was tested.

  18. A feasibility study on porting the community land model onto accelerators using OpenACC

    SciTech Connect

    Wang, Dali; Wu, Wei; Winkler, Frank; Ding, Wei; Hernandez, Oscar R.

    2014-01-01

    As environmental models (such as Accelerated Climate Model for Energy (ACME), Parallel Reactive Flow and Transport Model (PFLOTRAN), Arctic Terrestrial Simulator (ATS), etc.) became more and more complicated, we are facing enormous challenges regarding to porting those applications onto hybrid computing architecture. OpenACC appears as a very promising technology, therefore, we have conducted a feasibility analysis on porting the Community Land Model (CLM), a terrestrial ecosystem model within the Community Earth System Models (CESM)). Specifically, we used automatic function testing platform to extract a small computing kernel out of CLM, then we apply this kernel into the actually CLM dataflow procedure, and investigate the strategy of data parallelization and the benefit of data movement provided by current implementation of OpenACC. Even it is a non-intensive kernel, on a single 16-core computing node, the performance (based on the actual computation time using one GPU) of OpenACC implementation is 2.3 time faster than that of OpenMP implementation using single OpenMP thread, but it is 2.8 times slower than the performance of OpenMP implementation using 16 threads. On multiple nodes, MPI_OpenACC implementation demonstrated very good scalability on up to 128 GPUs on 128 computing nodes. This study also provides useful information for us to look into the potential benefits of “deep copy” capability and “routine” feature of OpenACC standards. In conclusion, we believe that our experience on the environmental model, CLM, can be beneficial to many other scientific research programs who are interested to porting their large scale scientific code using OpenACC onto high-end computers, empowered by hybrid computing architecture.

  19. A feasibility study on porting the community land model onto accelerators using OpenACC

    DOE PAGES

    Wang, Dali; Wu, Wei; Winkler, Frank; ...

    2014-01-01

    As environmental models (such as Accelerated Climate Model for Energy (ACME), Parallel Reactive Flow and Transport Model (PFLOTRAN), Arctic Terrestrial Simulator (ATS), etc.) became more and more complicated, we are facing enormous challenges regarding to porting those applications onto hybrid computing architecture. OpenACC appears as a very promising technology, therefore, we have conducted a feasibility analysis on porting the Community Land Model (CLM), a terrestrial ecosystem model within the Community Earth System Models (CESM)). Specifically, we used automatic function testing platform to extract a small computing kernel out of CLM, then we apply this kernel into the actually CLM dataflowmore » procedure, and investigate the strategy of data parallelization and the benefit of data movement provided by current implementation of OpenACC. Even it is a non-intensive kernel, on a single 16-core computing node, the performance (based on the actual computation time using one GPU) of OpenACC implementation is 2.3 time faster than that of OpenMP implementation using single OpenMP thread, but it is 2.8 times slower than the performance of OpenMP implementation using 16 threads. On multiple nodes, MPI_OpenACC implementation demonstrated very good scalability on up to 128 GPUs on 128 computing nodes. This study also provides useful information for us to look into the potential benefits of “deep copy” capability and “routine” feature of OpenACC standards. In conclusion, we believe that our experience on the environmental model, CLM, can be beneficial to many other scientific research programs who are interested to porting their large scale scientific code using OpenACC onto high-end computers, empowered by hybrid computing architecture.« less

  20. Phosphoenol Pyruvate Carboxylase in Parasitic Plants: Further Characterization in Various Species and Localization at the Level of Cells and Tissues in Lathraea clandestina L.

    PubMed

    Renaudin, S; Thalouarn, P; Rey, L; Vidal, J; Larher, F

    1984-11-01

    Phosphoenolpyruvate carboxylase (PEP carboxylase, EC 4.1.1.31) activity was demonstrated in a range of holo and hemiparasitic phanerogams. Lathraea clandestina was used as a model for a more detailed study. Enzyme activity levels were determined in the various plant parts. Great changes in enzyme capacity were observed in the shoots according to the time of measurement during a 24 hr cycle. PEP carboxylase characterized at the cellular level by using an indirect immunofluorescence method was found to be mainly located in the cytosol. The possible functions of PEP carboxylase in parasitic plants are discussed.

  1. Distribution of fallover in the carboxylase reaction and fallover-inducible sites among ribulose 1,5-bisphosphate carboxylase/oxygenases of photosynthetic organisms.

    PubMed

    Uemura, K; Tokai, H; Higuchi, T; Murayama, H; Yamamoto, H; Enomoto, Y; Fujiwara, S; Hamada, J; Yokota, A

    1998-02-01

    The biphasic reaction course, fallover, of carboxylation catalysed by ribulose 1,5-bisphosphate carboxylase/oxygenase (RuBisCO) has been known as a characteristic of the enzyme from higher land plants. Fallover consists of hysteresis in the reaction seen during the initial several minutes and a very slow suicide inhibition by inhibitors formed from the substrate ribulose-1,5-bisphosphate (RuBP). This study examined the relationship between occurrence of fallover and non-catalytic RuBP-binding sites, and the putative hysteresis-inducible sites (Lys-21 and Lys-305 of the large subunit in spinach RuBisCO) amongst RuBisCOs of a wide variety of photosynthetic organisms. Fallover could be detected by following the course of the carboxylase reaction at 1 mM RuBP and the non-catalytic binding sites by alleviation of fallover at 5 mM RuBP. RuBisCO from Euglena gracilis showed the same linear reaction course at both RuBP concentrations, indicating an association between an absence of fallover and an absence of the non-catalytic binding sites. This was supported by the results of an equilibrium binding assay for this enzyme with a transition state analogue. Green macroalgae and non-green algae contained the plant-type, fallover enzyme. RuBisCOs from Conjugatae, Closterium ehrenbergii, Gonatozygon monotaenium and Netrium digitus, showed a much smaller decrease in activity at 1 mM RuBP than the spinach enzyme and the reaction courses of these enzymes at 5 mM RuBP were almost linear. RuBisCO of a primitive type Conjugatae, Mesotaenium caldariorum, showed the same linear course at both RuBP concentrations. Sequencing of rbcL of these organisms indicated that Lys-305 was changed into arginine with Lys-21 conserved.

  2. 5th International ACC Symposium: Future and Current Therapeutic Trials in Adrenocortical Carcinoma.

    PubMed

    Hoff, Ana O; Berruti, Alfredo

    2016-02-01

    Adrenocortical carcinoma (ACC) is a rare and complex disease associated with a high mortality rate. Despite intensive translational and clinical research, prognosis remains poor. Over the past decade, a significant effort has been made to develop multinational, collaborative studies to better understand the pathogenesis and clinical features of this rare disease in attempt to improve the therapeutic strategies and patient outcome. The results of both standard and newer treatments are discussed in this review as well as the recent discovery of pathways involved in ACC pathogenesis that provide the rationale to introduce new molecular target therapies. Finally, remaining issues regarding how to improve available therapies in adjuvant setting are raised and addressed.

  3. Synthesis of stable ACC using mesoporous silica gel as a support

    PubMed Central

    2014-01-01

    Stable amorphous calcium carbonate supported by mesoporous silica gel was successfully synthesized. The silica gel support is prepared through the hydrolytic polycondensation of ethyl silicate under suitable conditions. Laser scanning confocal microscopy (LSCM) observations reveal that the morphology of the products is branched with cruciform-like and flower-like structure. Raman spectroscopic analysis and scanning electron microscopy (SEM) observation of the products confirm the combination of stable amorphous calcium carbonate (ACC) nanoparticles and mesoporous silica gel. A possible growth mechanism for the branched structure has been proposed. Results indicate potential application of this work to ACC storage, crystal engineering, biomimetic synthesis, etc. PMID:25246865

  4. An OpenACC-Based Unified Programming Model for Multi-accelerator Systems

    SciTech Connect

    Kim, Jungwon; Lee, Seyong; Vetter, Jeffrey S

    2015-01-01

    This paper proposes a novel SPMD programming model of OpenACC. Our model integrates the different granularities of parallelism from vector-level parallelism to node-level parallelism into a single, unified model based on OpenACC. It allows programmers to write programs for multiple accelerators using a uniform programming model whether they are in shared or distributed memory systems. We implement a prototype of our model and evaluate its performance with a GPU-based supercomputer using three benchmark applications.

  5. Structural evidence for substrate-induced synergism and half-sites reactivity in biotin carboxylase

    SciTech Connect

    Mochalkin, Igor; Miller, J. Richard; Evdokimov, Artem; Lightle, Sandra; Yan, Chunhong; Stover, Charles Ken; Waldrop, Grover L.

    2008-10-24

    Bacterial acetyl-CoA carboxylase is a multifunctional biotin-dependent enzyme that consists of three separate proteins: biotin carboxylase (BC), biotin carboxyl carrier protein (BCCP), and carboxyltransferase (CT). Acetyl-CoA carboxylase is a potentially attractive target for novel antibiotics because it catalyzes the first committed step in fatty acid biosynthesis. In the first half-reaction, BC catalyzes the ATP-dependent carboxylation of BCCP. In the second half-reaction, the carboxyl group is transferred from carboxybiotinylated BCCP to acetyl-CoA to produce malonyl-CoA. A series of structures of BC from several bacteria crystallized in the presence of various ATP analogs is described that addresses three major questions concerning the catalytic mechanism. The structure of BC bound to AMPPNP and the two catalytically essential magnesium ions resolves inconsistencies between the kinetics of active-site BC mutants and previously reported BC structures. Another structure of AMPPNP bound to BC shows the polyphosphate chain folded back on itself, and not in the correct (i.e., extended) conformation for catalysis. This provides the first structural evidence for the hypothesis of substrate-induced synergism, which posits that ATP binds nonproductively to BC in the absence of biotin. The BC homodimer has been proposed to exhibit half-sites reactivity where the active sites alternate or 'flip-flop' their catalytic cycles. A crystal structure of BC showed the ATP analog AMPPCF{sub 2}P bound to one subunit while the other subunit was unliganded. The liganded subunit was in the closed or catalytic conformation while the unliganded subunit was in the open conformation. This provides the first structural evidence for half-sites reactivity in BC.

  6. Structural evidence for substrate-induced synergism and half-sites reactivity in biotin carboxylase

    PubMed Central

    Mochalkin, Igor; Miller, J. Richard; Evdokimov, Artem; Lightle, Sandra; Yan, Chunhong; Stover, Charles Ken; Waldrop, Grover L.

    2008-01-01

    Bacterial acetyl-CoA carboxylase is a multifunctional biotin-dependent enzyme that consists of three separate proteins: biotin carboxylase (BC), biotin carboxyl carrier protein (BCCP), and carboxyltransferase (CT). Acetyl-CoA carboxylase is a potentially attractive target for novel antibiotics because it catalyzes the first committed step in fatty acid biosynthesis. In the first half-reaction, BC catalyzes the ATP-dependent carboxylation of BCCP. In the second half-reaction, the carboxyl group is transferred from carboxybiotinylated BCCP to acetyl-CoA to produce malonyl-CoA. A series of structures of BC from several bacteria crystallized in the presence of various ATP analogs is described that addresses three major questions concerning the catalytic mechanism. The structure of BC bound to AMPPNP and the two catalytically essential magnesium ions resolves inconsistencies between the kinetics of active-site BC mutants and previously reported BC structures. Another structure of AMPPNP bound to BC shows the polyphosphate chain folded back on itself, and not in the correct (i.e., extended) conformation for catalysis. This provides the first structural evidence for the hypothesis of substrate-induced synergism, which posits that ATP binds nonproductively to BC in the absence of biotin. The BC homodimer has been proposed to exhibit half-sites reactivity where the active sites alternate or “flip-flop” their catalytic cycles. A crystal structure of BC showed the ATP analog AMPPCF2P bound to one subunit while the other subunit was unliganded. The liganded subunit was in the closed or catalytic conformation while the unliganded subunit was in the open conformation. This provides the first structural evidence for half-sites reactivity in BC. PMID:18725455

  7. Isolation of ribulose-1,5-bisphosphate carboxylase/oxygenase from leaves.

    PubMed

    Carmo-Silva, A Elizabete; Barta, Csengele; Salvucci, Michael E

    2011-01-01

    Ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) is a multifunctional enzyme that catalyzes the fixation of CO2 and O2 in photosynthesis and photorespiration, respectively. As the rate-limiting step in photosynthesis, improving the catalytic properties of Rubisco has long been viewed as a viable strategy for increasing plant productivity. Advances in biotechnology have made this goal more attainable by making it possible to modify Rubisco in planta. To properly evaluate the properties of Rubisco, it is necessary to isolate the enzyme in pure form. This chapter describes procedures for rapid and efficient purification of Rubisco from leaves of several species.

  8. GPER agonist G-1 decreases adrenocortical carcinoma (ACC) cell growth in vitro and in vivo

    PubMed Central

    Zolea, Fabiana; Rizza, Pietro; Avena, Paola; Malivindi, Rocco; De Luca, Arianna; Campana, Carmela; Martire, Emilia; Domanico, Francesco; Fallo, Francesco; Carpinelli, Giulia; Cerquetti, Lidia; Amendola, Donatella; Stigliano, Antonio; Pezzi, Vincenzo

    2015-01-01

    We have previously demonstrated that estrogen receptor (ER) alpha (ESR1) increases proliferation of adrenocortical carcinoma (ACC) through both an estrogen-dependent and -independent (induced by IGF-II/IGF1R pathways) manner. Then, the use of tamoxifen, a selective estrogen receptor modulator (SERM), appears effective in reducing ACC growth in vitro and in vivo. However, tamoxifen not only exerts antiestrogenic activity, but also acts as full agonist on the G protein-coupled estrogen receptor (GPER). Aim of this study was to investigate the effect of a non-steroidal GPER agonist G-1 in modulating ACC cell growth. We found that G-1 is able to exert a growth inhibitory effect on H295R cells both in vitro and, as xenograft model, in vivo. Treatment of H295R cells with G-1 induced cell cycle arrest, DNA damage and cell death by the activation of the intrinsic apoptotic mechanism. These events required sustained extracellular regulated kinase (ERK) 1/2 activation. Silencing of GPER by a specific shRNA partially reversed G-1-mediated cell growth inhibition without affecting ERK activation. These data suggest the existence of G-1 activated but GPER-independent effects that remain to be clarified. In conclusion, this study provides a rational to further study G-1 mechanism of action in order to include this drug as a treatment option to the limited therapy of ACC. PMID:26131713

  9. Usability of AcceSS for Web Site Accessibility. Research Report

    ERIC Educational Resources Information Center

    Hackett, Stephanie; Parmanto, Bambang

    2006-01-01

    The standard display of web pages is inadequate for users who are visually impaired. Most visually impaired people obtain information from a web page in a linear fashion via a screen reader, whereas sighted users can immediately obtain a bird's-eye view of a web page's organization and content by quickly scanning the page. AcceSS (which stands for…

  10. AccesSports: A Model for Adapting Mainstream Sports Activities for Individuals with Visual Impairments.

    ERIC Educational Resources Information Center

    Ponchilla, Paul E.

    1995-01-01

    The AccesSports Model allows professionals with basic knowledge of visual impairments and mainstream sports to analyze any sports activity and design adaptations needed for targets or goals, boundaries, and rules to enable individuals with visual impairments to participate. Suggestions for modifying baseball, table tennis, swim racing, wrestling,…

  11. A functional tomato ACC synthase expressed in Escherichia coli demonstrates suicidal inactivation by its substrate S-adenosylmethionine.

    PubMed

    Li, N; Wiesman, Z; Liu, D; Mattoo, A K

    1992-07-20

    1-Aminocyclopropane-1-carboxylate (ACC) synthase is a key enzyme in the biosynthesis of the plant hormone, ethylene. We have isolated, sequenced and expressed a functional tomato (cv Pik-Red) ACC synthase gene in Escherichia coli. ACC synthase expressed in E. coli was inactivated by incubation with S-adenosylmethionine (SAM), the half-time of which was concentration dependent. Mixing the tomato fruit protein extract with the cell-free extract from transformed E. coli did not affect SAM-dependent inactivation of ACC synthase activity. Thus, single isoforms of the ACC synthase enzyme, which demonstrate the biochemical features expected of the tomato fruit enzyme, can be expressed in E. coli and their structure-function relationships investigated.

  12. Real-time dynamic optical imaging of ACC-M tumor cells killed by HSV-tk/ACV system.

    PubMed

    Xiong, Tao; Li, Yongjin; Li, Zhiyang; Xie, Xiangmo; Lu, Lisha

    2013-01-01

    HSV-tk/ACV induced and killed human adenoid cystic carcinoma cell (ACC-M) in vivo and in vitro, which were observed through optical imaging and green fluorescence protein (GFP) tagging technique. ACC-M was transfected with TK-GFP, and the single clone cell ACC-M-TK-GFP was selected by G418. With fluorescent stereomicroscope, whole-body fluorescent imaging system and fluorescent microscope, we could observe ACV treated ACC-M-TK-GFP cells in cell level and nude mice. The therapies of tumor were visualized clearly with optical imaging. This study proves that optical imaging is a very good approach for studying the effect of HSV-tk/ACV on the ACC-M tumor cells and decreasing the amount of vessel about tumors cell. Optical imaging will become a visual groundwork for monitoring tumor growth and evaluating in vivo curative effect of antitumor drugs.

  13. Purification and characterization of the thermostable ribulose-1,5-bisphosphate carboxylase/oxygenase from the thermophilic purple bacterium Chromatium tepidum.

    PubMed

    Heda, G D; Madigan, M T

    1989-09-15

    The Calvin cycle enzyme ribulose-bisphosphate carboxylase/oxygenase has been purified and characterized from the thermophilic and obligately anaerobic purple sulfur bacterium, Chromatium tepidum. The enzyme is an L8S8 carboxylase with a molecular mass near 550 kDa. No evidence for a second form of the enzyme lacking small subunits was obtained. C. tepidum ribulose-bisphosphate carboxylase/oxygenase was stable to heating to temperatures of 60 degrees C and could be readily purified in an active form at room temperature. Both carboxylase and oxygenase activities of this enzyme were Mg2+-dependent and carboxylase activity was sensitive to the effector 6-phosphogluconic acid. The Km for ribulose bisphosphate for the carboxylase activity of the C. tepidum enzyme was substantially higher than that observed in mesophilic Calvin cycle autotrophs. Amino acid composition and immunological analyses of C. tepidum and Chromatium vinosum ribulose-bisphosphate carboxylases showed the enzymes to be highly related despite significant differences in heat stability. It is hypothesized that thermal stability of C. tepidum ribulose-bisphosphate carboxylase/oxygenase is due to differences in primary structure affecting folding patterns in both the large and small subunits and is clearly not the result of any unique quaternary structure of the thermostable enzyme.

  14. A Symmetrical Tetramer for S. aureus Pyruvate Carboxylase in Complex with Coenzyme A

    SciTech Connect

    Yu, L.; Xiang, S; Lasso, G; Gil, D; Valle, M; Tong, L

    2009-01-01

    Pyruvate carboxylase (PC) is a conserved metabolic enzyme with important cellular functions. We report crystallographic and cryo-electron microscopy (EM) studies of Staphylococcus aureus PC (SaPC) in complex with acetyl-CoA, an allosteric activator, and mutagenesis, biochemical, and structural studies of the biotin binding site of its carboxyltransferase (CT) domain. The disease-causing A610T mutation abolishes catalytic activity by blocking biotin binding to the CT active site, and Thr908 might play a catalytic role in the CT reaction. The crystal structure of SaPC in complex with CoA reveals a symmetrical tetramer, with one CoA molecule bound to each monomer, and cryo-EM studies confirm the symmetrical nature of the tetramer. These observations are in sharp contrast to the highly asymmetrical tetramer of Rhizobium etli PC in complex with ethyl-CoA. Our structural information suggests that acetyl-CoA promotes a conformation for the dimer of the biotin carboxylase domain of PC that might be catalytically more competent.

  15. Dark/Light Modulation of Ribulose Bisphosphate Carboxylase Activity in Plants from Different Photosynthetic Categories 1

    PubMed Central

    Vu, J. Cu V.; Allen, Leon H.; Bowes, George

    1984-01-01

    Ribulose bisphosphate carboxylase/oxygenase (RuBPCase) from several plants had substantially greater activity in extracts from lightexposed leaves than dark leaves, even when the extracts were incubated in vitro with saturating HCO3− and Mg2+ concentrations. This occurred in Glycine max, Lycopersicon esculentum, Nicotiana tabacum, Panicum bisulcatum, and P. hylaeicum (C3); P. maximum (C4 phosphoenolpyruvate carboxykinase); P. milioides (C3/C4); and Bromelia pinguin and Ananas comosus (Crassulacean acid metabolism). Little or no difference between light and dark leaf extracts of RuBPCase was observed in Triticum aestivum (C3); P. miliaceum (C4 NAD malic enzyme); Zea mays and Sorghum bicolor (C4 NADP malic enzyme); Moricandia arvensis (C3/C4); and Hydrilla verticillata (submersed aquatic macrophyte). It is concluded that, in many plants, especially Crassulacean acid metabolism and C3 species, a large fraction of ribulose-1,5-bisphosphate carboxylase/oxygenase in the dark is in an inactivatable state that cannot respond to CO2 and Mg2+ activation, but which can be converted to an activatable state upon exposure of the leaf to light. PMID:16663937

  16. Dark/Light modulation of ribulose bisphosphate carboxylase activity in plants from different photosynthetic categories.

    PubMed

    Vu, J C; Allen, L H; Bowes, G

    1984-11-01

    Ribulose bisphosphate carboxylase/oxygenase (RuBPCase) from several plants had substantially greater activity in extracts from lightexposed leaves than dark leaves, even when the extracts were incubated in vitro with saturating HCO(3) (-) and Mg(2+) concentrations. This occurred in Glycine max, Lycopersicon esculentum, Nicotiana tabacum, Panicum bisulcatum, and P. hylaeicum (C(3)); P. maximum (C(4) phosphoenolpyruvate carboxykinase); P. milioides (C(3)/C(4)); and Bromelia pinguin and Ananas comosus (Crassulacean acid metabolism). Little or no difference between light and dark leaf extracts of RuBPCase was observed in Triticum aestivum (C(3)); P. miliaceum (C(4) NAD malic enzyme); Zea mays and Sorghum bicolor (C(4) NADP malic enzyme); Moricandia arvensis (C(3)/C(4)); and Hydrilla verticillata (submersed aquatic macrophyte). It is concluded that, in many plants, especially Crassulacean acid metabolism and C(3) species, a large fraction of ribulose-1,5-bisphosphate carboxylase/oxygenase in the dark is in an inactivatable state that cannot respond to CO(2) and Mg(2+) activation, but which can be converted to an activatable state upon exposure of the leaf to light.

  17. Post-translational modifications in the large subunit of ribulose bisphosphate carboxylase/oxygenase.

    PubMed

    Houtz, R L; Stults, J T; Mulligan, R M; Tolbert, N E

    1989-03-01

    Two adjacent N-terminal tryptic peptides of the large subunit of ribulose bisphosphate carboxylase/oxygenase [3-phospho-D-glycerate carboxy-lyase (dimerizing), EC 4.1.1.39] from spinach, wheat, tobacco, and muskmelon were removed by limited tryptic proteolysis. Characterization by peptide sequencing, amino acid composition, and tandem mass spectrometry revealed that the N-terminal residue from the large subunit of the enzyme from each plant species was acetylated proline. The sequence of the penultimate N-terminal tryptic peptide from the large subunit of the spinach and wheat enzyme was consistent with previous primary structure determinations. However, the penultimate N-terminal peptide from the large subunit of both the tobacco and muskmelon enzymes, while identical, differed from the corresponding peptide from spinach and wheat by containing a trimethyllysyl residue at position 14. Thus, tryptic proteolysis occurred at lysine-18 rather than lysine-14 as with the spinach and wheat enzymes. A comparison of the DNA sequences for the large subunit of ribulose bisphosphate carboxylase/oxygenase indicates that the N terminus has been post-translationally processed by removal of methionine-1 and serine-2 followed by acetylation of proline-3. In addition, for the enzyme from tobacco and muskmelon a third post-translational modification occurs at lysine-14 in the form of N epsilon-trimethylation.

  18. Insight into the carboxyl transferase domain mechanism of pyruvate carboxylase from Rhizobium etli†

    PubMed Central

    Zeczycki, Tonya N.; Maurice, Martin St.; Jitrapakdee, Sarawut; Wallace, John C.; Attwood, Paul V.; Cleland, W. Wallace

    2009-01-01

    The effects of mutations in the active site of the carboxyl transferase domain of R. etli pyruvate carboxylase have been determined for the forward reaction to form oxaloacetate, the reverse reaction to form MgATP, the oxamate-induced decarboxylation of oxaloacetate, the phosphorylation of MgADP by carbamoyl phosphate and the bicarbonate-dependent ATPase reaction. Additional studies with these mutants examined the effect of pyruvate and oxamate on the reactions of the biotin carboxylase domain. From these mutagenic studies, putative roles for catalytically relevant active site residues were assigned and a more accurate description of the mechanism of the carboxyl transferase domain is presented. The T882A mutant showed no catalytic activity for reactions involving the carboxyl transferase domain, but surprisingly showed a 7- and 3.5-fold increase in activity, as compared to the wild-type enzyme, for the ADP phosphorylation and bicarbonate-dependent ATPase reactions, respectively. Furthermore, the partial inhibition of the T882A catalyzed BC domain reactions by oxamate and pyruvate further supports the critical role of Thr882 in the proton transfer between biotin and pyruvate in the carboxyl transferase domain. The catalytic mechanism appears to involve the decarboxylation of carboxybiotin and proton removal from Thr882 by the resulting biotin enolate with either a concerted or subsequent transfer of a proton from pyruvate to Thr882. The resulting enolpyruvate then reacts with CO2 to form oxaloacetate and complete the reaction. PMID:19341298

  19. The role of biotin and oxamate in the carboxyltransferase reaction of pyruvate carboxylase.

    PubMed

    Lietzan, Adam D; Lin, Yi; St Maurice, Martin

    2014-11-15

    Pyruvate carboxylase (PC) is a biotin-dependent enzyme that catalyzes the MgATP-dependent carboxylation of pyruvate to oxaloacetate, an important anaplerotic reaction in central metabolism. During catalysis, carboxybiotin is translocated to the carboxyltransferase domain where the carboxyl group is transferred to the acceptor substrate, pyruvate. Many studies on the carboxyltransferase domain of PC have demonstrated an enhanced oxaloacetate decarboxylation activity in the presence of oxamate and it has been shown that oxamate accepts a carboxyl group from carboxybiotin during oxaloacetate decarboxylation. The X-ray crystal structure of the carboxyltransferase domain from Rhizobium etli PC reveals that oxamate is positioned in the active site in an identical manner to the substrate, pyruvate, and kinetic data are consistent with the oxamate-stimulated decarboxylation of oxaloacetate proceeding through a simple ping-pong bi bi mechanism in the absence of the biotin carboxylase domain. Additionally, analysis of truncated PC enzymes indicates that the BCCP domain devoid of biotin does not contribute directly to the enzymatic reaction and conclusively demonstrates a biotin-independent oxaloacetate decarboxylation activity in PC. These findings advance the description of catalysis in PC and can be extended to the study of related biotin-dependent enzymes.

  20. Interaction of ribulose bisphosphate carboxylase/oxygenase with 2-carboxyhexitol 1,6-bisphosphates.

    PubMed

    Roach, D J; Gollnick, P D; McFadden, B A

    1983-04-01

    2-C-Carboxy-D-glucitol 1,6-bisphosphate (CGBP) and 2-C-carboxy-D-mannitol 1,6-bisphosphate (CMBP) have been synthesized, isolated, and the structures of these compounds and the derived lactones elucidated by NMR spectroscopy and periodate oxidation. Both carboxyhexitol bisphosphates, which are homologs of the transition state analog 2-C-carboxy-D-arabinitol 1,5-bisphosphate, exhibit competitive inhibiton of ribulose bisphosphate carboxylase/oxygenase (EC 4.1.1.9) isolated from spinach (Spinacia oleracea), with respect to ribulose 1,5-bisphosphate. CMBP was a more potent inhibitor (100-fold) displaying an inhibition constant (Ki at pH 8.0 and 30 degrees C) of 1-2 microM with enzymes from spinach, barley (Hordeum vulgare), and Chromatium vinosum. In contrast the Rhodospirillum rubrum enzyme was inhibited about 40-fold more weakly (Ki = 53 microM at pH 8.0 and 30 degrees C). Both CGBP and CMBP potentiated activation of RuBP carboxylase from spinach and R. rubrum.

  1. Dark/light modulation of ribulose bisphosphate carboxylase activity in plants from different photosynthetic categories

    SciTech Connect

    Vu, J.C.V.; Allen, L.H. Jr.; Bowes, G.

    1984-11-01

    Ribulose bisphosphate carboxylase/oxygenase (RuBPCase) from several plants had substantially greater activity in extracts from light-exposed leaves than dark leaves, even when the extracts were incubated in vitro with saturating HCO/sub 3//sup -/ and Mg/sup 2 +/ concentrations. This occurred in Glycine max, Lycopersicon esculentum, Nicotiana tabacum, Panicum bisulcatum, and P. hylaeicum (C/sub 3/); P. maximum (C/sub 4/ phosphoenolpyruvate carboxykinase); P. milioides (C/sub 3//C/sub 4/); and Bromelia pinguin and Ananas comosus (Crassulacean acid metabolism). Little or no difference between light and dark leaf extracts of RuBPCase was observed in Triticum aestivum (C/sub 3/); P. miliaceum (C/sub 4/ NAD malic enzyme); Zea mays and Sorghum bicolor (C/sub 4/ NADP malic enzyme); Moricandia arvensis (C/sub 3//C/sub 4/); and Hydrilla verticillata (submersed aquatic macrophyte). It is concluded that, in many plants, especially Crassulacean acid metabolism and C/sub 3/ species, a large fraction of ribulose-1,5-bisphosphate carboxylase/oxygenase in the dark is in an inactivatable state that cannot respond to CO/sub 2/ and Mg/sup 2 +/ activation, but which can be converted to an activatable state upon exposure of the leaf to light. 16 references, 2 tables.

  2. Discovery of Antibacterial Biotin Carboxylase Inhibitors by Virtual Screening and Fragment-Based Approaches

    SciTech Connect

    Mochalkin, Igor; Miller, J. Richard; Narasimhan, Lakshmi; Thanabal, Venkataraman; Erdman, Paul; Cox, Philip B.; Prasad, J.V.N. Vara; Lightle, Sandra; Huband, Michael D.; Stover, C. Kendall; Pfizer

    2009-07-24

    As part of our effort to inhibit bacterial fatty acid biosynthesis through the recently validated target biotin carboxylase, we employed a unique combination of two emergent lead discovery strategies. We used both de novo fragment-based drug discovery and virtual screening, which employs 3D shape and electrostatic property similarity searching. We screened a collection of unbiased low-molecular-weight molecules and identified a structurally diverse collection of weak-binding but ligand-efficient fragments as potential building blocks for biotin carboxylase ATP-competitive inhibitors. Through iterative cycles of structure-based drug design relying on successive fragment costructures, we improved the potency of the initial hits by up to 3000-fold while maintaining their ligand-efficiency and desirable physicochemical properties. In one example, hit-expansion efforts resulted in a series of amino-oxazoles with antibacterial activity. These results successfully demonstrate that virtual screening approaches can substantially augment fragment-based screening approaches to identify novel antibacterial agents.

  3. Translational regulation of light-induced ribulose 1,5-bisphosphate carboxylase gene expression in amaranth.

    PubMed Central

    Berry, J O; Nikolau, B J; Carr, J P; Klessig, D F

    1986-01-01

    The regulation of the genes encoding the large and small subunits of ribulose 1,5-bisphosphate carboxylase was examined in amaranth cotyledons in response to changes in illumination. When dark-grown cotyledons were transferred into light, synthesis of the large- and small-subunit polypeptides was initiated very rapidly, before any increase in the levels of their corresponding mRNAs. Similarly, when light-grown cotyledons were transferred to total darkness, synthesis of the large- and small-subunit proteins was rapidly depressed without changes in mRNA levels for either subunit. In vitro translation or in vivo pulse-chase experiments indicated that these apparent changes in protein synthesis were not due to alterations in the functionality of the mRNAs or to protein turnover, respectively. These results, in combination with our previous studies, suggest that the expression of ribulose 1,5-bisphosphate carboxylase genes can be adjusted rapidly at the translational level and over a longer period through changes in mRNA accumulation. Images PMID:3785198

  4. Light-mediated control of translational initiation of ribulose-1, 5-bisphosphate carboxylase in amaranth cotyledons.

    PubMed Central

    Berry, J O; Breiding, D E; Klessig, D F

    1990-01-01

    In cotyledons of 6-day-old amaranth seedlings, the large subunit (LSU) and the small subunit (SSU) polypeptides of ribulose-1,5-bisphosphate carboxylase are not synthesized in the absence of light. When dark-grown seedlings were transferred into light, synthesis of both polypeptides was induced within the first 3 to 5 hr of illumination without any significant changes in levels of their mRNAs. In cotyledons of light-grown seedlings and of dark-grown seedlings transferred into light for 5 hr (where ribulose-1,5-bisphosphate carboxylase synthesis was readily detected in vivo), the LSU and SSU mRNAs were associated with polysomes. In cotyledons of dark-grown seedlings, these two mRNAs were not found on polysomes. In contrast to the SSU message, mRNAs encoding the nonlight-regulated, nuclear-encoded proteins actin and ubiquitin were associated with polysomes regardless of the light conditions. Similarly, mRNA from at least one chloroplast-encoded gene (rpl2) was found on polysomes in the dark as well as in the light. These results indicate an absence of translational initiation in cotyledons of dark-grown seedlings which is specific to a subset of nuclear- and chloroplast-encoded genes including the SSU and LSU, respectively. Upon illumination, synthesis of both polypeptides, and possibly other proteins involved in light-mediated chloroplast development, was induced at the level of translational initiation. PMID:2152128

  5. Active site histidine in spinach ribulosebisphosphate carboxylase/oxygenase modified by diethyl pyrocarbonate

    SciTech Connect

    Igarashi, Y.; McFadden, B.A.; el-Gul, T.

    1985-07-16

    (TH) Diethyl pyrocarbonate was synthesized from (TH) ethanol prepared by the reduction of acetaldehyde by NaB3H4. Ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO) from spinach was inactivated with this reagent at pH 7.0 the presence of 20 mM MgS , and tryptic peptides that contained modified histidine residues were isolated by reverse-phase high-performance liquid chromatography. Labeling of the enzyme was conducted in the presence and absence of the competitive inhibitor sedoheptulose 1,7-bisphosphate. The amount of one peptide that was heavily labeled in the absence of this compound was reduced 10-fold in its presence. The labeled residue was histidine-298. This result, in combination with earlier experiments, suggests that His-298 in spinach RuBisCO is located in the active site domain and is essential to enzyme activity. This region of the primary structure is strongly conserved in seven other ribulosebisphosphate carboxylases from divergent sources.

  6. Pathway of assembly of ribulosebisphosphate carboxylase/oxygenase from Anabaena 7210 expressed in Escherichia coli

    SciTech Connect

    Gurevitz, M.; Somerville, C.R.; McIntosh, L.

    1985-10-01

    The authors have placed the genes encoding ribulosebisphosphate carboxylase/oxygenase from the Anabaena 7120 operon under transcriptional control of the lac promoter carried on the Escherichia coli plasmid pUC19. The genes encoding both the large and small subunit polypeptides (rbcL and rbcS) are transcribed and translated so that approx. = 0.6% of the soluble protein in E. coli extracts is a fully functional holoenzyme with a sedimentation coefficient of approximately 18S, which contains stoichiometric amounts of the two subunits. However, expression of the large subunit polypeptide vastly exceeds that of the small subunit because the majority of transcripts terminate in the intergenic region between the rbcL and rbcS genes. As a result, excess large subunit is synthesized and accumulates in E. coli as an insoluble and catalytically inactive form. Because small subunit is found only in the high molecular weight soluble form of ribulosebisphosphate carboxylase/oxygenase, the authors propose that the small subunit promotes assembly of the hexadecameric form of the enzyme via heterodimers of large and small subunits.

  7. The glossyhead1 Allele of ACC1 Reveals a Principal Role for multidomain acetyl-coenzyme a carboxylase in the biosynthesis of cuticular waxes by arabidopsis[c][w][oa

    Technology Transfer Automated Retrieval System (TEKTRAN)

    A novel mutant of Arabidopsis thaliana having highly glossy inflorescence stems, post-genital fusion in floral organs, and reduced fertility, was isolated from an EMS-mutagenized population and designated glossyhead1 (gsd1). The gsd1 locus was mapped to chromosome 1, and the causal gene identified a...

  8. Isolation and characterization of ACC deaminase-producing fluorescent pseudomonads, to alleviate salinity stress on canola (Brassica napus L.) growth.

    PubMed

    Jalili, Farzad; Khavazi, Kazem; Pazira, Ebrahim; Nejati, Alireza; Rahmani, Hadi Asadi; Sadaghiani, Hasan Rasuli; Miransari, Mohammad

    2009-04-01

    Salinity stress is of great importance in arid and semi-arid areas of the world due to its impact in reducing crop yield. Under salinity stress, the amount of 1-aminocyclopropane-1-carboxylate (ACC), a precursor for ethylene production in plants, increases. Here, we conducted research under the hypothesis that isolated ACC deaminase-producing Pseudomonas fluorescens and Pseudomonas putida can alleviate the stressful effects of salinity on canola (Brassica napus L.) growth. The experiments were conducted in the Soil and Water Research Institute, Tehran, Iran. Seven experimental stages were conducted to isolate and characterize ACC deaminase-producing Pseudomonas fluorescens strains and to determine factors enhancing their growth and, consequently, their effects on the germination of canola seeds. Under salinity stress, in 14% of the isolates, ACC deaminase activity was observed, indicating that they were able to utilize ACC as the sole N-source. Bacterial strains differed in their ability to synthesize auxin and hydrogen cyanide compounds, as well as in their ACC deaminase activity. Under salinity stress, the rate of germinating seeds inoculated with the strains of ACC deaminase-producing Pseudomonas fluorescens and Pseudomonas putida, and seedling growth was significantly higher. These results indicate the significance of soil biological activities, including the activities of plant growth-promoting bacteria, in the alleviation of soil stresses such as salinity on plant growth.

  9. Accumulation of wound-inducible ACC synthase transcript in tomato fruit is inhibited by salicylic acid and polyamines.

    PubMed

    Li, N; Parsons, B L; Liu, D R; Mattoo, A K

    1992-02-01

    Regulation of wound-inducible 1-aminocyclopropane-1-carboxylic acid (ACC) synthase expression was studied in tomato fruit (Lycopersicon esculentum cv. Pik-Red). A 70 base oligonucleotide probe homologous to published ACC synthase cDNA sequences was successfully used to identify and analyze regulation of a wound-inducible transcript. The 1.8 kb ACC synthase transcript increased upon wounding the fruit as well as during fruit ripening. Salicylic acid, an inhibitor of wound-responsive genes in tomato, inhibited the wound-induced accumulation of the ACC synthase transcript. Further, polyamines (putrescine, spermidine and spermine) that have anti-senescence properties and have been shown to inhibit the development of ACC synthase activity, inhibited the accumulation of the wound-inducible ACC synthase transcript. The inhibition by spermine was greater than that caused by putrescine or spermidine. The transcript level of a wound-repressible glycine-rich protein gene and that of the constitutively expressed rRNA were not affected as markedly by either salicylic acid or polyamines. These data suggest that salicylic acid and polyamines may specifically regulate ethylene biosynthesis at the level of ACC synthase transcript accumulation.

  10. Isolation and preliminary characterization of two forms of ribulose 1,5-bisphosphate carboxylase from Rhodopseudomonas capsulata.

    PubMed

    Gibson, J L; Tabita, F R

    1977-12-01

    The presence of two distinct forms of ribulose 1,5-bisphosphate carboxylase has been demonstrated in extracts of Rhodopseudomonas capsulata, similar to the form I (peak I) and form II (peak II) carboxylases previously described from R. sphaeroides (J. Gibson and F. R. Tabita, J. Biol. Chem 252:943-949, 1977). The two activities, separated by diethylaminoethyl-cellulose chromatography, were shown to be of different molecular size after assay on polyacrylamide gels. The higher-molecular-weight carboxylase from R. capsulata was designated form I-C, whereas the smaller enzyme was designated form II-C. Catalytic studies revealed significant differences between the two enzymes in response to pH and the effector 6-phosphogluconate. Immunological studies with antisera directed against the carboxylases from R. sphaeroides demonstrated antigenic differences between the two R. capsulata enzymes; cross-reactivity was observed only between R. sphaeroides anti-form II serum and the corresponding R. capsulata enzyme, form II-C.

  11. Assessing driver's mental representation of Adaptive Cruise Control (ACC) and its possible effects on behavioural adaptations.

    PubMed

    Piccinini, Giulio Francesco; Simões, Anabela; Rodrigues, Carlos Manuel; Leitão, Miguel

    2012-01-01

    The introduction of Adaptive Cruise Control (ACC) could be very helpful for making the longitudinal driving task more comfortable for the drivers and, as a consequence, it could have a global beneficial effect on road safety. However, before or during the usage of the device, due to several reasons, drivers might generate in their mind incomplete or flawed mental representations about the fundamental operation principles of ACC; hence, the resulting usage of the device might be improper, negatively affecting the human-machine interaction and cooperation and, in some cases, leading to negative behavioural adaptations to the system that might neutralise the desirable positive effects on road safety. Within this context, this paper will introduce the methodology which has been developed in order to analyse in detail the topic and foresee, in the future, adequate actions for the recovery of inaccurate mental representations of the system.

  12. Accumulation and Transport of 1-Aminocyclopropane-1-Carboxylic Acid (ACC) in Plants: Current Status, Considerations for Future Research and Agronomic Applications

    PubMed Central

    Vanderstraeten, Lisa; Van Der Straeten, Dominique

    2017-01-01

    1-aminocyclopropane-1-carboxylic acid (ACC) is a non-protein amino acid acting as the direct precursor of ethylene, a plant hormone regulating a wide variety of vegetative and developmental processes. ACC is the central molecule of ethylene biosynthesis. The rate of ACC formation differs in response to developmental, hormonal and environmental cues. ACC can be conjugated to three derivatives, metabolized in planta or by rhizobacteria using ACC deaminase, and is transported throughout the plant over short and long distances, remotely leading to ethylene responses. This review highlights some recent advances related to ACC. These include the regulation of ACC synthesis, conjugation and deamination, evidence for a role of ACC as an ethylene-independent signal, short and long range ACC transport, and the identification of a first ACC transporter. Although unraveling the complex mechanism of ACC transport is in its infancy, new questions emerge together with the identification of a first transporter. In the light of the future quest for additional ACC transporters, this review presents perspectives of the novel findings and includes considerations for future research toward applications in agronomy. PMID:28174583

  13. Biochemistry and genetics of ACC deaminase: a weapon to “stress ethylene” produced in plants

    PubMed Central

    Singh, Rajnish P.; Shelke, Ganesh M.; Kumar, Anil; Jha, Prabhat N.

    2015-01-01

    1-aminocyclopropane-1-carboxylate deaminase (ACCD), a pyridoxal phosphate-dependent enzyme, is widespread in diverse bacterial and fungal species. Owing to ACCD activity, certain plant associated bacteria help plant to grow under biotic and abiotic stresses by decreasing the level of “stress ethylene” which is inhibitory to plant growth. ACCD breaks down ACC, an immediate precursor of ethylene, to ammonia and α-ketobutyrate, which can be further metabolized by bacteria for their growth. ACC deaminase is an inducible enzyme whose synthesis is induced in the presence of its substrate ACC. This enzyme encoded by gene AcdS is under tight regulation and regulated differentially under different environmental conditions. Regulatory elements of gene AcdS are comprised of the regulatory gene encoding LRP protein and other regulatory elements which are activated differentially under aerobic and anaerobic conditions. The role of some additional regulatory genes such as AcdB or LysR may also be required for expression of AcdS. Phylogenetic analysis of AcdS has revealed that distribution of this gene among different bacteria might have resulted from vertical gene transfer with occasional horizontal gene transfer (HGT). Application of bacterial AcdS gene has been extended by developing transgenic plants with ACCD gene which showed increased tolerance to biotic and abiotic stresses in plants. Moreover, distribution of ACCD gene or its homolog's in a wide range of species belonging to all three domains indicate an alternative role of ACCD in the physiology of an organism. Therefore, this review is an attempt to explore current knowledge of bacterial ACC deaminase mediated physiological effects in plants, mode of enzyme action, genetics, distribution among different species, ecological role of ACCD and, future research avenues to develop transgenic plants expressing foreign AcdS gene to cope with biotic and abiotic stressors. Systemic identification of regulatory circuits

  14. Improved method for effective screening of ACC (1-aminocyclopropane-1-carboxylate) deaminase producing microorganisms.

    PubMed

    Patil, Chandrashekhar; Suryawanshi, Rahul; Koli, Sunil; Patil, Satish

    2016-12-01

    Aminocyclopropane-1-carboxylate deaminase (ACCD) producing microorganisms support plant growth under a variety of biotic and abiotic stress conditions such as drought, soil salinity, flooding, heavy metal pollution and phyto-pathogen attack. Available screening methods for ACCD give idea only about its primary microbial ACCD activity than the actual potential. In the present investigation, we have simply improved screening method by incorporating pH indicator dyes (phenol red and bromothymol blue) in ACC containing medium. This modification is based on the basic principle that ACCD action releases ammonia which can be detected by color change and zone around the bacterial colony. High color intensity and zone around the colony indicates most potent producer, colony showing only a color change indicates moderate potential and no change in colony color indicates least efficiency. Enzymatic bioassays as well as root elongation studies revealed that ACC-deaminase activity of Pseudomonas aeruginosa, Stenotrophomonas maltophilia and Bacillus subtilis clearly corresponds to their growth on dye incorporated ACC medium. This method could be used to complement the existing screening methods and to speed up the targeted isolation of agriculturally important microorganisms.

  15. The ACC strategy in biomineralization: the case of earthworm's amorphous spherulites

    NASA Astrophysics Data System (ADS)

    Briones, Maria J. I.; Alvarez-Otero, Rosa; Méndez, Jesús; Gago Duport, Luis

    2010-05-01

    The occurrence of amorphous calcium carbonate (ACC), an hydrated and highly soluble form of solid CaCO3, seems to be a common feature in all carbonate forming organisms such as mollusks, corals, echinoderms and crustaceans. The ubiquity of ACC in these Ca-carbonate biomineralizing systems, as a precursor of further crystalline phases, has recently open the interesting question about if the formation of an amorphous phase is a necessary step in the calcification process of all organisms and consequently, whether it would be possible to define the "amorphous precursor estategy" as a general mechanism in biomineralization. Neverthelees, although ACC appears to be widespread in these organisms very little is known about its particular role in the biomineralization scheme of the different phyla. The formation of CaCO3 spherulites in the calciferous glands of earthworms is a particular case of calcareous biomineralization involving the presence of ACC as a transient precursor phase [2]. Interestingly, the formation of crystalline carbonates via ACC in these organisms is not connected with skeleton building so it must play another functional role. In addition, the transient transformation stages can be followed by in situ spectrometric techniques and therefore, earthworms provide and adequate model to analyse the mutual interactions between ACC-solvent-and crystalline phases. In this study, we have analysed the morphological and structural transformations from the initial ACC spherulites until the formation of the crystalline phases: vaterite (and/or aragonite) and finally calcite, is accomplished. The characterization of ACC was done by performing in situ FT-IR, together with and HREM and Debye scherrer -XRD. The structural results were interpreted in the light of the histological study of the gland. The geometry of the secretory epithelium of the calciferous gland, as evidenced by TEM [2], shows the presence of irregulary shaped cells with their apical surface

  16. A type III ACC synthase, ACS7, is involved in root gravitropism in Arabidopsis thaliana.

    PubMed

    Huang, Shih-Jhe; Chang, Chia-Lun; Wang, Po-Hsun; Tsai, Min-Chieh; Hsu, Pang-Hung; Chang, Ing-Feng

    2013-11-01

    Ethylene is an important plant hormone that regulates developmental processes in plants. The ethylene biosynthesis pathway is a highly regulated process at both the transcriptional and post-translational level. The transcriptional regulation of these ethylene biosynthesis genes is well known. However, post-translational modifications of the key ethylene biosynthesis enzyme 1-aminocyclopropane-1-carboxylate (ACC) synthase (ACS) are little understood. In vitro kinase assays were conducted on the type III ACS, AtACS7, fusion protein and peptides to determine whether the AtACS7 protein can be phosphorylated by calcium-dependent protein kinase (CDPK). AtACS7 was phosphorylated at Ser216, Thr296, and Ser299 by AtCDPK16 in vitro. To investigate further the function of the ACS7 gene in Arabidopsis, an acs7-1 loss-of-function mutant was isolated. The acs7-1 mutant exhibited less sensitivity to the inhibition of root gravitropism by treatment with the calcium chelator ethylene glycol tetraacetic acid (EGTA). Seedlings were treated with gradient concentrations of ACC. The results showed that a certain concentration of ethylene enhanced the gravity response. Moreover, the acs7-1 mutant was less sensitive to inhibition of the gravity response by treatment with the auxin polar transport inhibitor 1-naphthylphthalamic acid, but exogenous ACC application recovered root gravitropism. Altogether, the results indicate that AtACS7 is involved in root gravitropism in a calcium-dependent manner in Arabidopsis.

  17. The kinetics and mechanisms of amorphous calcium carbonate (ACC) crystallization to calcite, via vaterite.

    PubMed

    Rodriguez-Blanco, Juan Diego; Shaw, Samuel; Benning, Liane G

    2011-01-01

    The kinetics and mechanisms of nanoparticulate amorphous calcium carbonate (ACC) crystallization to calcite, via vaterite, were studied at a range of environmentally relevant temperatures (7.5-25 °C) using synchrotron-based in situ time-resolved Energy Dispersive X-ray Diffraction (ED-XRD) in conjunction with high-resolution electron microscopy, ex situ X-ray diffraction and infrared spectroscopy. The crystallization process occurs in two stages; firstly, the particles of ACC rapidly dehydrate and crystallize to form individual particles of vaterite; secondly, the vaterite transforms to calcite via a dissolution and reprecipitation mechanism with the reaction rate controlled by the surface area of calcite. The second stage of the reaction is approximately 10 times slower than the first. Activation energies of calcite nucleation and crystallization are 73±10 and 66±2 kJ mol(-1), respectively. A model to calculate the degree of calcite crystallization from ACC at environmentally relevant temperatures (7.5-40 °C) is also presented.

  18. Bacteria with ACC deaminase can promote plant growth and help to feed the world.

    PubMed

    Glick, Bernard R

    2014-01-20

    To feed all of the world's people, it is necessary to sustainably increase agricultural productivity. One way to do this is through the increased use of plant growth-promoting bacteria; recently, scientists have developed a more profound understanding of the mechanisms employed by these bacteria to facilitate plant growth. Here, it is argued that the ability of plant growth-promoting bacteria that produce 1-aminocyclopropane-1-carboxylate (ACC) deaminase to lower plant ethylene levels, often a result of various stresses, is a key component in the efficacious functioning of these bacteria. The optimal functioning of these bacteria includes the synergistic interaction between ACC deaminase and both plant and bacterial auxin, indole-3-acetic acid (IAA). These bacteria not only directly promote plant growth, they also protect plants against flooding, drought, salt, flower wilting, metals, organic contaminants, and both bacterial and fungal pathogens. While a considerable amount of both basic and applied work remains to be done before ACC deaminase-producing plant growth-promoting bacteria become a mainstay of plant agriculture, the evidence indicates that with the expected shift from chemicals to soil bacteria, the world is on the verge of a major paradigm shift in plant agriculture.

  19. Species Variation in the Predawn Inhibition of Ribulose-1,5-Bisphosphate Carboxylase/Oxygenase 1

    PubMed Central

    Servaites, Jerome C.; Parry, Martin A. J.; Gutteridge, Steven; Keys, Alfred J.

    1986-01-01

    The activity of ribulose-1,5-bisphosphate carboxylase/oxygenase was measured in extracts of leaves collected before dawn (predawn activity, pa) and at midday (midday activity, ma). Twenty-three of the 37 species examined showed a pa/ma ratio (≤0.75, while only Capsicum frutescens, Cucumis sativa, Glycine max, Nicotiana tabacum, Vigna unguiculata, and 3 Solanum species showed a pa/ma ratio ≤0.5. Phaseolus vulgaris consistently showed a pa/ma ratio of ≤0.1. Activities and pa/ma ratios of the same species grown in the United States and the United Kingdom were very similar. Gel filtration of extracts before assay had no effect on the observed activities and the pa/ma ratios. These data are consistent with the hypothesis that in a number of species the enzyme is partially inhibited following the night period by the presence of a tight-binding inhibitor. PMID:16665155

  20. Purification and characterization of ribulose-1,5-bisphosphate carboxylase from triticale.

    PubMed

    Khan, M A; Dixit, A; Upadhyaya, K C

    1994-04-01

    Ribulose-1,5-bisphosphate carboxylase has been isolated from a synthetic cereal triticale and purified using a newly developed rapid procedure involving precipitation with ammonium sulphate (35-55% saturation), DEAE-cellulose (DE-52) chromatography and filtration through Sepharose CL-68. Molecular weights of the enzyme subunits are 15.5 and 52 kDa which corresponds to 540 kDa for the hexadecameric holoenzyme. Isoelectric focussing showed that the enzyme has a pI of 4.2. Various kinetic constants determined under aerobic conditions are: Km (CO2), 118 microM; Km (RuBP), 220 microM (at 20 mM NaHCO3) and Vmax, 690 nmole CO2 fixed/mg enzyme/min.

  1. Cloning and characterization of ribulose bisphosphate carboxylase gene of a carboxydobacterium, hydrogenophagea pseudoflava DSM 1084.

    PubMed

    Lee, S N; Kim, Y M

    1998-10-31

    The ribulose bisphosphate carboxylase/oxygenase rbcL and rbcS genes of a carbon monoxide-oxidizing bacterium, Hydrogenophaga pseudoflava DSM 1084, were cloned and sequenced. The cloned rbcL and rbcS genes had open reading frames of 1422 and 351 nucleotides encoding RbcL and RbcS with calculated molecular masses of 52,689 and 13,541, respectively. The known active site residues in other RbcL proteins were conserved in the H. pseudoflava proteins. The H. pseudoflava RbcS protein lacked the 12-residue internal sequence found in the plant enzymes. The 2 genes were separated by a 134 bp intergenic region and cotranscribed as a 2.0 kb rbcLS mRNA. Novel two perfect 9 bp direct repeats overlapping with two dyad symmetries were found in the rbcLS promoter region.

  2. Postimport methylation of the small subunit of ribulose-1,5-bisphosphate carboxylase in chloroplasts.

    PubMed

    Grimm, R; Grimm, M; Eckerskorn, C; Pohlmeyer, K; Röhl, T; Soll, J

    1997-05-26

    Electron impact mass spectronomy analysis of the amino-terminal amino acid of the small subunit (SSU) of ribulose-1,5-bisphosphate carboxylase (Rubisco) showed that the amino-terminal methionine residue is post-translationally modified to N-methyl-methionine. Modification of the amino-terminal methionine residue was found in mature SSU proteins from the dicotyledonous plants pea and spinach as well as the monocotyledonous plants barley and corn. SSU methyltransferase is a soluble protein in the chloroplast stroma and accepts heterologously expressed non-methylated SSU as a substrate using S-adenosylmethionine as methyl-group donor. We show that this modification occurs after post-translational uptake of the precursor form of SSU into chloroplasts and processing to its mature size. This reaction represents a new step in the import and assembly pathway of Rubisco holoenzyme.

  3. Maternal 3-methylcrotonyl-coenzyme A carboxylase deficiency with elevated 3-hydroxyisovalerylcarnitine in breast milk

    PubMed Central

    Cho, Kyung Lae; Kim, Yeo Jin; Yang, Song Hyun; Kim, Gu-Hwan

    2016-01-01

    We report here a case of maternal 3-methylcrotonyl-coenzyme A carboxylase (3-MCC) deficiency in a Korean woman. Her 2 infants had elevated 3-hydroxyisovalerylcarnitine (C5-OH) on a neonatal screening test by liquid chromatography-tandem mass spectrometry (LC-MS/MS), but normal results were found on urine organic acid analysis. The patient was subjected to serial testing and we confirmed a maternal 3-MCC deficiency by blood spot and breast milk spot test by LC-MS/MS, serum amino acid analysis, urine organic acid and molecular genetic analysis that found c.838G>T (p.Asp280Tyr) homozygous mutation within exon 9 of the MCCB gene. Especially, we confirmed marked higher levels of C5-OH on breast milk spot by LC-MS/MS, in the case of maternal 3-MCC deficiency vs. controls. PMID:28018443

  4. Acetyl-CoA carboxylase inhibitors from avocado (Persea americana Mill) fruits.

    PubMed

    Hashimura, H; Ueda, C; Kawabata, J; Kasai, T

    2001-07-01

    A methanol extract of avocado fruits showed potent inhibitory activity against acetyl-CoA carboxylase, a key enzyme in fatty acid biosynthesis. The active principles were isolated and identified as (5E,12Z,15Z)-2-hydroxy-4-oxoheneicosa-5,12,15-trienyl (1), (2R,12Z,15Z)-2-hydroxy-4-oxoheneicosa-12,15-dienyl (2), (2R*,4R*)-2,4-dihydroxyheptadec-16-enyl (3) and (2R*,4R*)-2,4-dihydroxyheptadec-16-ynyl (4) acetates by instrumental analyses. The IC50 of the compounds were 4.0 x 10(-6), 4.9 x 10(-6), 9.4 x 10(-6), and 5.1 x 10(-6) M, respectively.

  5. [Functions of plant phosphoenolpyruvate carboxylase and its applications for genetic engineering].

    PubMed

    Wei, Shaowei; Li, Yin

    2011-12-01

    Phosphoenolpyruvate carboxylase (PEPC, EC 4.1.1.31) is an important ubiquitous cytosol enzyme that fixes HCO3 together with phosphoenolpyruvate (PEP) and yields oxaloacetate that can be converted to intermediates of the citric acid cycle. In plant cells, PEPC participates in CO2 assimilation and other important metabolic pathways, and it has broad functions in different plant tissues. PEPC is also involved in the regulation of storage product synthesis and metabolism in seeds, such as affecting the metabolic fluxes from sugars/starch towards the synthesis of fatty acids or amino acids and proteins. In this review, we introduced the progress in classification, structure and regulation of PEPC in plant tissues. We discussed the potential applications of plant PEPCs in genetic engineering. The researches in functions and regulation mechanism of plant PEPCs will provide beneficial approaches to applications of plant PEPCs in high-yield crops breeding, energy crop and microbe genetic engineering.

  6. Structural and Biochemical Studies on the Regulation of Biotin Carboxylase by Substrate Inhibition and Dimerization

    SciTech Connect

    Chou, Chi-Yuan; Tong, Liang

    2012-06-19

    Biotin carboxylase (BC) activity is shared among biotin-dependent carboxylases and catalyzes the Mg-ATP-dependent carboxylation of biotin using bicarbonate as the CO{sub 2} donor. BC has been studied extensively over the years by structural, kinetic, and mutagenesis analyses. Here we report three new crystal structures of Escherichia coli BC at up to 1.9 {angstrom} resolution, complexed with different ligands. Two structures are wild-type BC in complex with two ADP molecules and two Ca{sup 2+} ions or two ADP molecules and one Mg{sup 2+} ion. One ADP molecule is in the position normally taken by the ATP substrate, whereas the other ADP molecule occupies the binding sites of bicarbonate and biotin. One Ca{sup 2+} ion and the Mg{sup 2+} ion are associated with the ADP molecule in the active site, and the other Ca{sup 2+} ion is coordinated by Glu-87, Glu-288, and Asn-290. Our kinetic studies confirm that ATP shows substrate inhibition and that this inhibition is competitive against bicarbonate. The third structure is on the R16E mutant in complex with bicarbonate and Mg-ADP. Arg-16 is located near the dimer interface. The R16E mutant has only a 2-fold loss in catalytic activity compared with the wild-type enzyme. Analytical ultracentrifugation experiments showed that the mutation significantly destabilized the dimer, although the presence of substrates can induce dimer formation. The binding modes of bicarbonate and Mg-ADP are essentially the same as those to the wild-type enzyme. However, the mutation greatly disrupted the dimer interface and caused a large re-organization of the dimer. The structures of these new complexes have implications for the catalysis by BC.

  7. Structural and Biochemical Studies on the Regulation of Biotin Carboxylase by Substrate Inhibition and Dimerization

    SciTech Connect

    C Chou; L Tong

    2011-12-31

    Biotin carboxylase (BC) activity is shared among biotin-dependent carboxylases and catalyzes the Mg-ATP-dependent carboxylation of biotin using bicarbonate as the CO{sub 2} donor. BC has been studied extensively over the years by structural, kinetic, and mutagenesis analyses. Here we report three new crystal structures of Escherichia coli BC at up to 1.9 {angstrom} resolution, complexed with different ligands. Two structures are wild-type BC in complex with two ADP molecules and two Ca{sup 2+} ions or two ADP molecules and one Mg{sup 2+} ion. One ADP molecule is in the position normally taken by the ATP substrate, whereas the other ADP molecule occupies the binding sites of bicarbonate and biotin. One Ca{sup 2+} ion and the Mg{sup 2+} ion are associated with the ADP molecule in the active site, and the other Ca{sup 2+} ion is coordinated by Glu-87, Glu-288, and Asn-290. Our kinetic studies confirm that ATP shows substrate inhibition and that this inhibition is competitive against bicarbonate. The third structure is on the R16E mutant in complex with bicarbonate and Mg-ADP. Arg-16 is located near the dimer interface. The R16E mutant has only a 2-fold loss in catalytic activity compared with the wild-type enzyme. Analytical ultracentrifugation experiments showed that the mutation significantly destabilized the dimer, although the presence of substrates can induce dimer formation. The binding modes of bicarbonate and Mg-ADP are essentially the same as those to the wild-type enzyme. However, the mutation greatly disrupted the dimer interface and caused a large re-organization of the dimer. The structures of these new complexes have implications for the catalysis by BC.

  8. Ribulose 1,5-bisphosphate carboxylase from the halophilic cyanobacterium Aphanothece halophytica.

    PubMed

    Asami, S; Takabe, T; Akazawa, T; Codd, G A

    1983-09-01

    Various structural and functional properties of ribulose 1,5-bisphosphate carboxylase/oxygenase (RuBisCO) isolated from the halophilic cyanobacterium (blue-green alga) Aphanothece halophytica were reexamined. The ready dissociation of this algal RuBisCO during sedimentation in a linear sucrose density gradient was observed. Low NaCl concentrations promote the dissociation of small subunit (B) from the original native enzyme molecule as evidenced by the sucrose density gradient centrifugation and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. It is thus possible that the intracellular osmoticum of A. halophytica might influence the structural integrity and activity of RuBisCO. The low residual carboxylase activity ascribed to the catalytic core, an oligomer form of the large subunit (A) apparently deficient in small subunit (B), was found to be markedly stimulated by a protein component which appears identical to subunit B. The purification and structural characterization of the catalytic core and subunit B were attempted by step-wise column chromatography on DEAE-cellulose, Utrogel AcA 34, Sephadex G-75, and hydroxylapatite, and at the final stage each component was purified to near homogeneity, although the catalytic core is still associated with a small quantity of subunit B. The addition of subunit B to the catalytic core does not alter the Km (HCO-3, RuBP) values, but Vmax values are markedly enhanced. Sucrose density gradient centrifugation gave a value of 16 S for the catalytic core. The molecular weights of the monomeric forms of the catalytic core (subunit A) and subunit B were 5.0 X 10(4) and 1.4 X 10(4), respectively.

  9. Crystal Structure of Biotin Carboxylase in Complex with Substrates and Implications for Its Catalytic Mechanism

    SciTech Connect

    Chou, C.; Yu, L; Tong, L

    2009-01-01

    Biotin-dependent carboxylases are widely distributed in nature and have important functions in many cellular processes. These enzymes share a conserved biotin carboxylase (BC) component, which catalyzes the ATP-dependent carboxylation of biotin using bicarbonate as the donor. Despite the availability of a large amount of biochemical and structural information on BC, the molecular basis for its catalysis is currently still poorly understood. We report here the crystal structure at 2.0 {angstrom} resolution of wild-type Escherichia coli BC in complex with its substrates biotin, bicarbonate, and Mg-ADP. The structure suggests that Glu{sup 296} is the general base that extracts the proton from bicarbonate, and Arg{sup 338} is the residue that stabilizes the enolate biotin intermediate in the carboxylation reaction. The B domain of BC is positioned closer to the active site, leading to a 2-{angstrom} shift in the bound position of the adenine nucleotide and bringing it near the bicarbonate for catalysis. One of the oxygen atoms of bicarbonate is located in the correct position to initiate the nucleophilic attack on ATP to form the carboxyphosphate intermediate. This oxygen is also located close to the N1' atom of biotin, providing strong evidence that the phosphate group, derived from decomposition of carboxyphosphate, is the general base that extracts the proton on this N1' atom. The structural observations are supported by mutagenesis and kinetic studies. Overall, this first structure of BC in complex with substrates offers unprecedented insights into the molecular mechanism for the catalysis by this family of enzymes.

  10. Ribulose 1,5-bisphosphate carboxylase/oxygenase from Pseudomonas oxalacticus.

    PubMed Central

    Lawlis, V B; Gordon, G L; McFadden, B A

    1979-01-01

    Ribulose 1,5-bisphosphate carboxylase/oxygenase was purified by a rapid, facile procedure from formate-grown Pseudomonas oxalaticus. The electrophoretically homogeneous enzyme had specific activities of 1.9 mumol of CO2 fixed per min per mg of protein and 0.15 mumol of O2 consumed per min per mg of protein. The amino acid composition was similar to that of other bacterial sources of the enzyme. The molecular weights determined by sedimentation equilibrium and by gel filtration were 421,000 and 450,000, respectively. Upon sodium dodecyl sulfate electrophoresis of enzyme purified under conditions which would limit proteolysis, two types of large (L) subunits and two types of small (S) subunits were observed with apparent molecular weights of 57,000, 55,000, 17,000 and 15,000. By densitometric scans at two different protein concentrations the stoichiometry of the total large to total small subunits was 1:1, implying an L6S6 structure. Electron micrographs of the enzyme revealed an unusual structure that was inconsistent with a cubical structure. The enzyme had an unusually high Km for ribulose 1,5-bisphosphate (220 microM) and was strongly inhibited by 6-phosphogluconate in the ribulose 1,5-bisphosphate carboxylase assay (Ki = 270 microM). One, 5, and 12 days after purification the enzyme was half-maximally activated at 0.13 microM, 0.23 mM, and 0.70 mM CO2, respectively, at saturating Mg2+. At saturating CO2, enzyme 1 day afer purification responded sigmoidally to Mg2+ and was half-maximally activated by 0.85 mM Mg2+ in the absence of 6-phosphogluconate (Hill coefficient, h = 2.0) and by 0.19 mM Mg2+ in the presence of mM 6-phosphogluconate (h = 1.7). Images PMID:457602

  11. Nitric oxide regulation of leaf phosphoenolpyruvate carboxylase-kinase activity: implication in sorghum responses to salinity.

    PubMed

    Monreal, José A; Arias-Baldrich, Cirenia; Tossi, Vanesa; Feria, Ana B; Rubio-Casal, Alfredo; García-Mata, Carlos; Lamattina, Lorenzo; García-Mauriño, Sofía

    2013-11-01

    Nitric oxide (NO) is a signaling molecule that mediates many plant responses to biotic and abiotic stresses, including salt stress. Interestingly, salinity increases NO production selectively in mesophyll cells of sorghum leaves, where photosynthetic C₄ phosphoenolpyruvate carboxylase (C₄ PEPCase) is located. PEPCase is regulated by a phosphoenolpyruvate carboxylase-kinase (PEPCase-k), which levels are greatly enhanced by salinity in sorghum. This work investigated whether NO is involved in this effect. NO donors (SNP, SNAP), the inhibitor of NO synthesis NNA, and the NO scavenger cPTIO were used for long- and short-term treatments. Long-term treatments had multifaceted consequences on both PPCK gene expression and PEPCase-k activity, and they also decreased photosynthetic gas-exchange parameters and plant growth. Nonetheless, it could be observed that SNP increased PEPCase-k activity, resembling salinity effect. Short-term treatments with NO donors, which did not change photosynthetic gas-exchange parameters and PPCK gene expression, increased PEPCase-k activity both in illuminated leaves and in leaves kept at dark. At least in part, these effects were independent on protein synthesis. PEPCase-k activity was not decreased by short-term treatment with cycloheximide in NaCl-treated plants; on the contrary, it was decreased by cPTIO. In summary, NO donors mimicked salt effect on PEPCase-k activity, and scavenging of NO abolished it. Collectively, these results indicate that NO is involved in the complex control of PEPCase-k activity, and it may mediate some of the plant responses to salinity.

  12. Methylation of γ-carboxylated Glu (Gla) allows detection by liquid chromatography-mass spectrometry and the identification of Gla residues in the γ-glutamyl carboxylase.

    PubMed

    Hallgren, K W; Zhang, D; Kinter, M; Willard, B; Berkner, K L

    2013-06-07

    γ-Carboxylated Glu (Gla) is a post-translational modification required for the activity of vitamin K-dependent (VKD) proteins that has been difficult to study by mass spectrometry due to the properties of this negatively charged residue. Gla is generated by a single enzyme, the γ-glutamyl carboxylase, which has broad biological impact because VKD proteins have diverse functions that include hemostasis, apoptosis, and growth control. The carboxylase also contains Glas, of unknown function, and is an integral membrane protein with poor sequence coverage. To locate these Glas, we first established methods that resulted in high coverage (92%) of uncarboxylated carboxylase. Subsequent analysis of carboxylated carboxylase identified a Gla peptide (729-758) and a missing region (625-647) that was detected in uncarboxylated carboxylase. We therefore developed an approach to methylate Gla, which efficiently neutralized Gla and improved mass spectrometric analysis. Methylation eliminated CO2 loss from Gla, increased the ionization of Gla-containing peptide, and appeared to facilitate trypsin digestion. Methylation of a carboxylated carboxylase tryptic digest identified Glas in the 625-647 peptide. These studies provide valuable information for testing the function of carboxylase carboxylation. The methylation approach for studying Gla by mass spectrometry is an important advance that will be broadly applicable to analyzing other VKD proteins.

  13. Methylation of gamma-carboxylated Glu (Gla) allows detection by liquid chromatography-mass spectrometry and the identification of Gla residues in the gamma-glutamyl carboxylase

    PubMed Central

    Hallgren, K. W.; Zhang, D.; Kinter, M.; Willard, B.; Berkner, K. L.

    2013-01-01

    Gamma-carboxylated Glu (Gla) is a post-translational modification required for the activity of vitamin K-dependent (VKD) proteins that has been difficult to study by mass spectrometry due to the properties of this negatively-charged residue. Gla is generated by a single enzyme, the gamma-glutamyl carboxylase, which has broad biological impact because VKD proteins have diverse functions that include hemostasis, apoptosis, and growth control. The carboxylase also contains Glas, of unknown function, and is an integral membrane protein with poor sequence coverage. To locate these Glas, we first established methods that resulted in high coverage (92%) of uncarboxylated carboxylase. Subsequent analysis of carboxylated carboxylase identified a Gla-peptide (729-758) and a missing region (625-647) that was detected in uncarboxylated carboxylase. We therefore developed an approach to methylate Gla, which efficiently neutralized Gla and improved mass spectrometric analysis. Methylation eliminated CO2 loss from Gla, increased the ionization of Gla-containing peptide, and appeared to facilitate trypsin digestion. Methylation of a carboxylated carboxylase tryptic digest identified Glas in the 625-647 peptide. These studies provide valuable information for testing the function of carboxylase carboxylation. The methylation approach for studying Gla by mass spectrometry is an important advance that will be broadly applicable to analyzing other VKD proteins. PMID:22536908

  14. Genes encoding the biotin carboxylase subunit of acetyl-CoA carboxylase from Brassica napus and parental species: cloning, expression patterns, and evolution.

    PubMed

    Li, Zhi-Guo; Yin, Wei-Bo; Song, Li-Ying; Chen, Yu-Hong; Guan, Rong-Zhan; Wang, Jing-Qiao; Wang, Richard R-C; Hu, Zan-Min

    2011-03-01

    Comparative genomics is a useful tool to investigate gene and genome evolution. Biotin carboxylase (BC), an important subunit of heteromeric acetyl-CoA carboxylase (ACCase) that is a rate-limiting enzyme in fatty acid biosynthesis in dicots, catalyzes ATP, biotin carboxyl carrier protein, and CO2 to form carboxybiotin carboxyl carrier protein. In this study, we cloned four genes encoding BC from Brassica napus L. (namely BnaC.BC.a, BnaC.BC.b, BnaA.BC.a, and BnaA.BC.b), and two were cloned from each of the two parental species Brassica rapa L. (BraA.BC.a and BraA.BC.b) and Brassica oleracea L. (BolC.BC.a and BolC.BC.b). Sequence analyses revealed that in B. napus the genes BnaC.BC.a and BnaC.BC.b were from the C genome of B. oleracea, whereas BnaA.BC.a and BnaA.BC.b were from the A genome of B. rapa. Comparative and cluster analysis indicated that these genes were divided into two major groups, BnaC.BC.a, BnaA.BC.a, BraA.BC.a, and BolC.BC.a in group-1 and BnaC.BC.b, BnaA.BC.b, BraA.BC.b, and BolC.BC.b in group-2. The divergence of group-1 and group-2 genes occurred in their common ancestor 13-17 million years ago (MYA), soon after the divergence of Arabidopsis and Brassica (15-20 MYA). This time of divergence is identical to the previously reported triplicated time of paralogous subgenomes of diploid Brassica species and the divergence date of group-1 and group-2 genes of α-carboxyltransferase, another subunit of heteromeric ACCase, in Brassica. Reverse transcription PCR revealed that the expression level of group-1 and group-2 genes varied in different organs, and the expression patterns of the two groups of genes were similar in different organs, except in flower. However, two paralogs of group-2 BC genes from B. napus could express differently in mature plants tested by generating BnaA.BC.b and BnaC.BC.b promoter-β-glucuronidase (GUS) fusions. The amino acid sequences of proteins encoded by these genes were highly conserved, except the sequence encoding

  15. Amelioration of high salinity stress damage by plant growth-promoting bacterial endophytes that contain ACC deaminase.

    PubMed

    Ali, Shimaila; Charles, Trevor C; Glick, Bernard R

    2014-07-01

    Plant growth and productivity is negatively affected by soil salinity. However, it is predicted that plant growth-promoting bacterial (PGPB) endophytes that contain 1-aminocyclopropane-1-carboxylate (ACC) deaminase (E.C. 4.1.99.4) can facilitate plant growth and development in the presence of a number of different stresses. In present study, the ability of ACC deaminase containing PGPB endophytes Pseudomonas fluorescens YsS6, Pseudomonas migulae 8R6, and their ACC deaminase deficient mutants to promote tomato plant growth in the absence of salt and under two different levels of salt stress (165 mM and 185 mM) was assessed. It was evidence that wild-type bacterial endophytes (P. fluorescens YsS6 and P. migulae 8R6) promoted tomato plant growth significantly even in the absence of stress (salinity). Plants pretreated with wild-type ACC deaminase containing endophytic strains were healthier and grew to a much larger size under high salinity stress compared to plants pretreated with the ACC deaminase deficient mutants or no bacterial treatment (control). The plants pretreated with ACC deaminase containing bacterial endophytes exhibit higher fresh and dry biomass, higher chlorophyll contents, and a greater number of flowers and buds than the other treatments. Since the only difference between wild-type and mutant bacterial endophytes was ACC deaminase activity, it is concluded that this enzyme is directly responsible for the different behavior of tomato plants in response to salt stress. The use of PGPB endophytes with ACC deaminase activity has the potential to facilitate plant growth on land that is not normally suitable for the majority of crops due to their high salt contents.

  16. Assessing the effects of heavy metals in ACC deaminase and IAA production on plant growth-promoting bacteria.

    PubMed

    Carlos, Mendoza-Hernández José; Stefani, Perea-Vélez Yazmin; Janette, Arriola-Morales; Melani, Martínez-Simón Sara; Gabriela, Pérez-Osorio

    2016-01-01

    This study poses a methodology in order to simultaneously quantify ACC deaminase and IAA levels in the same culture medium. Ten bacterial strains isolated from plant rhizosphere naturally settled in mining residues were chosen. These bacterial strains were characterized as PGPB, and all of them showed at least three characteristics (indole-3 acetic acid and siderophore production, ACC deaminase enzyme activity, and inorganic phosphate solubilization). Taxonomic identification showed that the strains belong to Enterobacter, Serratia, Klebsiella, and Escherichia genera. Similarly, both the ACC deaminase enzyme activity and the IAA synthesis in the presence of Cu, As, Pb, Ni, Cd, and Mn were measured. The results showed that both the ACC deaminase enzyme activity and the IAA synthesis were higher with the Pb, As, and Cu treatments than with the Escherichia N16, Enterobacter K131, Enterobacter N9, and Serratia K120 control treatments. On the other hand, Ni, Cd, and Mn negatively affected both the ACC deaminase enzyme activity and the IAA production on every bacterium except on the Klebsiella Mc173 strain. Serratia K120 bacterium got a positive correlation between ACC deaminase and IAA in the presence of every heavy metal, and it also promoted Helianthus annuus plant growth, showing a potential use in phytoremediation systems.

  17. Molecular cloning, expression, and stress response of the estrogen-related receptor gene (AccERR) from Apis cerana cerana

    NASA Astrophysics Data System (ADS)

    Zhang, Weixing; Zhu, Ming; Zhang, Ge; Liu, Feng; Wang, Hongfang; Guo, Xingqi; Xu, Baohua

    2016-04-01

    Estrogen-related receptor (ERR), which belongs to the nuclear receptor superfamily, has been implicated in diverse physiological processes involving the estrogen signaling pathway. However, little information is available on ERR in Apis cerana cerana. In this report, we isolated the ERR gene and investigated its involvement in antioxidant defense. Quantitative real-time polymerase chain reaction (qPCR) revealed that the highest mRNA expression occurred in eggs during different developmental stages. The expression levels of AccERR were highest in the muscle, followed by the rectum. The predicted transcription factor binding sites in the promoter of AccERR suggested that AccERR potentially functions in early development and in environmental stress responses. The expression of AccERR was induced by cold (4 °C), heat (42 °C), ultraviolet light (UV), HgCl2, and various types of pesticides (phoxim, deltamethrin, triadimefon, and cyhalothrin). Western blot was used to measure the expression levels of AccERR protein. These data suggested that AccERR might play a vital role in abiotic stress responses.

  18. Capteurs monopodes pour mesures accélérométriques

    NASA Astrophysics Data System (ADS)

    Delaite, R.; Valentin, J.-P.

    1993-08-01

    A new design for accelerometric measurements sensors is described. It uses a plate vibrating in thickness shear mode, maintained by the means of a single holder located at the crystal edge. This mounting does cancel the mechanical and thermal stresses which generally modify the sensor output signal. So the ratio signal/noise of a thickness shear accelerometer is improved and the intrinsic sensitivity is multiplied by a factor 40, by comparison with the sensitivity of a thickness shear plate bonded by the means of two opposite holders. Un nouveau dispositif destiné aux mesures d'accélération est présenté. Il met en œuvre une lame vibrant en cisaillement d'épaisseur, fixée à sa structure de maintien par l'intermédiaire d'une unique liaison. Ce montage permet d'éliminer les contraintes mécaniques et thermiques qui perturbent habituellement le signal de mesure, et qui sont liées soit au montage des éléments du capteur, soit aux variations rapides de température qui interviennent lors de la mise en fonctionnement du capteur. Le rapport signal/bruit d'un accéléromètre à lame vibrant en cisaillement d'épaisseur s'en trouve amélioré et la sensibilité à l'accélération est multipliée par un facteur 40, comparée à celle d'un capteur qui serait constitué d'une lame vibrant en cisaillement d'épaisseur, fixée par deux liaisons diamétralement opposées.

  19. Characterization of three members of the ACC synthase gene family in Solanum tuberosum L.

    PubMed

    Destéfano-Beltrán, L J; van Caeneghem, W; Gielen, J; Richard, L; van Montagu, M; van der Straeten, D

    1995-02-20

    Two genomic clones corresponding to three members of the 1-aminocyclopropane-1-carboxylic acid (ACC) synthase gene family in potato (Solanum tuberosum L.) have been isolated and sequenced. Two highly homologous genes, ST-ACS1A and ST-ACS1B, transcribed in opposite directions were found in an 8.9 kb region. Their coding sequences are interrupted by two introns at identical positions. Their closest relative in tomato is the LE-ACS3 gene. The third gene in potato, ST-ACS2, was found in a 4 kb region and shows a gene structure similar to that of the tomato LE-ACS4 gene and to the mung bean VR-ACS4 and VR-ACS5 genes. Based on its lack of significant homology to the tomato gene family and its closeness to the VR-ACS4 and VR-ACS5 genes, we propose that LE-ACS7 represents an additional isoform in the tomato genome. Moreover, in a phylogenetic comparison of known ACC synthases, the ST-ACS2 isoform was grouped in a separate lineage together with the mung bean VR-ACS4 and VR-ACS5, and the moth orchid DS-ACS1A and DS-ACS1B gene products. Expression of the three potato genes was studied by reverse transcription-polymerase chain reaction on total RNA. The twin genes are positively regulated by indole-3-acetic acid in hypocotyls and expression is modulated by wounding in the leaves. The third gene is responsive to ethylene and wounding mainly in tubers. The roles of these three genes and of other members of the ACC synthase gene family in vegetative processes of potato such as tuberization, dormancy, and sprouting have yet to be determined.

  20. Dissociating contributions of ACC and vmPFC in reward prediction, outcome, and choice.

    PubMed

    Vassena, Eliana; Krebs, Ruth M; Silvetti, Massimo; Fias, Wim; Verguts, Tom

    2014-07-01

    Acting in an uncertain environment requires estimating the probability and the value of potential outcomes. These computations are typically ascribed to various parts of the medial prefrontal cortex (mPFC), but the functional architecture of this region remains debated. The anterior cingulate cortex (ACC) encodes reward prediction and outcome (i.e. win vs lose, Silvetti, Seurinck, & Verguts, 2013. Cortex, 49(6), 1627-35. doi:10.1016/j.cortex.2012.05.008). An outcome-related value signal has also been reported in the ventromedial Prefrontal Cortex (vmPFC, Rangel & Hare, 2010. Current Opinion in Neurobiology, 20(2), 262-70. doi:10.1016/j.conb.2010.03.001). Whether a functional dissociation can be traced in these regions with respect to reward prediction and outcome has been suggested but not rigorously tested. Hence an fMRI study was designed to systematically examine the contribution of ACC and vmPFC to reward prediction and outcome. A striking dissociation was identified, with ACC coding for positive prediction errors and vmPFC responding to outcome, irrespective of probability. Moreover, ACC has been assigned a crucial role in the selection of intentional actions (decision-making) and computing the value associated to these actions (action-based value). Conversely, vmPFC seems to implement stimulus-based value processing (Rudebeck et al., 2008. Journal of Neuroscience, 28(51), 13775-85. doi:10.1523/JNEUROSCI.3541-08.2008; Rushworth, Behrens, Rudebeck, & Walton, 2007. Trends in Cognitive Sciences, 11(4), 168-76. doi:10.1016/j.tics.2007.01.004). Therefore, a decision-making factor (choice vs. no choice condition) was also implemented in the present paradigm to distinguish stimulus-based versus action-based value coding in the mPFC during both decision and outcome phase. We found that vmPFC was more activated during the outcome phase in the no-choice than in the choice condition, potentially confirming the role of this area in stimulus-based (more than action

  1. Effects of heterologous expression of phosphoenolpyruvate carboxykinase and phosphoenolpyruvate carboxylase on organic acid production in Aspergillus carbonarius.

    PubMed

    Yang, Lei; Lübeck, Mette; Lübeck, Peter S

    2015-11-01

    Aspergillus carbonarius has a potential as a cell factory for production of various organic acids. In this study, the organic acid profile of A. carbonarius was investigated under different cultivation conditions. Moreover, two heterologous genes, pepck and ppc, which encode phosphoenolpyruvate carboxykinase in Actinobacillus succinogenes and phosphoenolpyruvate carboxylase in Escherichia coli, were inserted individually and in combination in A. carbonarius to enhance the carbon flux toward the reductive TCA branch. Results of transcription analysis and measurement of enzyme activities of phosphoenolpyruvate carboxykinase and phosphoenolpyruvate carboxylase in the corresponding single and double transformants demonstrated that the two heterologous genes were successfully expressed in A. carbonarius. The production of citric acid increased in all the transformants in both glucose- and xylose-based media at pH higher than 3 but did not increase in the pH non-buffered cultivation compared with the wild type.

  2. Nitrate-Dependent Degradation of Acetone by Alicycliphilus and Paracoccus Strains and Comparison of Acetone Carboxylase Enzymes ▿

    PubMed Central

    Dullius, Carlos Henrique; Chen, Ching-Yuan; Schink, Bernhard

    2011-01-01

    A novel acetone-degrading, nitrate-reducing bacterium, strain KN Bun08, was isolated from an enrichment culture with butanone and nitrate as the sole sources of carbon and energy. The cells were motile short rods, 0.5 to 1 by 1 to 2 μm in size, which gave Gram-positive staining results in the exponential growth phase and Gram-negative staining results in the stationary-growth phase. Based on 16S rRNA gene sequence analysis, the isolate was assigned to the genus Alicycliphilus. Besides butanone and acetone, the strain used numerous fatty acids as substrates. An ATP-dependent acetone-carboxylating enzyme was enriched from cell extracts of this bacterium and of Alicycliphilus denitrificans K601T by two subsequent DEAE Sepharose column procedures. For comparison, acetone carboxylases were enriched from two additional nitrate-reducing bacterial species, Paracoccus denitrificans and P. pantotrophus. The products of the carboxylase reaction were acetoacetate and AMP rather than ADP. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis of cell extracts and of the various enzyme preparations revealed bands corresponding to molecular masses of 85, 78, and 20 kDa, suggesting similarities to the acetone carboxylase enzymes described in detail for the aerobic bacterium Xanthobacter autotrophicus strain Py2 (85.3, 78.3, and 19.6 kDa) and the phototrophic bacterium Rhodobacter capsulatus. Protein bands were excised and compared by mass spectrometry with those of acetone carboxylases of aerobic bacteria. The results document the finding that the nitrate-reducing bacteria studied here use acetone-carboxylating enzymes similar to those of aerobic and phototrophic bacteria. PMID:21841031

  3. GroE heat shock protein is required for in vivo assembly of recombinant Anabaena ribulose bisphosphate (Ru-P sub 2 ) carboxylase/oxygenase

    SciTech Connect

    Larimer, F.W.; Soper, T.S. )

    1991-03-11

    As a prerequisite for site-directed mutagenesis of a L{sub 8}S{sub 8} form of Ru-P{sub 2} carboxylase, the rbc operon from Anabaena 7120 was placed under control of the tac promoter (tac-rbcLrbcS, bla, ori(pMB1), from pFL260) in E. coli MV1190 (recA). Substantial amounts of insoluble large subunit were produced, but not active enzyme, suggesting that the carboxylase was not being correctly assembled in vivo. Coexpression of rbcLrbcS and the operon encoding the GroESL (HSP10, HSP60) complex from a compatible plasmid (tac-groESgroEL, cat, ori(p15A), from pFL261) resulted in high levels of active, soluble enzyme. Supplementation of rich medium with potassium ions, required for GroE complex function in vitro enhanced recovery of active enzyme. Under optimal expression conditions, active Ru-P{sub 2} carboxylase comprised 7-10% of soluble protein. The recombinant carboxylase, purified to homogeneity, was similar to the enzyme purified from the authentic cyanobacterium. Chaperonins are required for assembly of many complex proteins. The stringent requirement of Anabaena carboxylase for elevated levels of E. coli GroE chaperonin for proper assembly suggests that the GroE complex differs from the Anabaena chaperonin complex that is normally involved in the assembly of this L{sub 8}S{sub 8} carboxylase.

  4. The opportunity for and significance of alteration of ribulose 1,5-bisphosphate carboxylase activities in crop production.

    PubMed

    Hardy, R W; Havelka, U D; Quebedeaux, B

    1978-01-01

    Increased population and the dietary changes accompanying increased affluence are creating a need for a suggested doubling of world cereal grain production (a 3% per year compounding rate) and quadrupling of grain legume production (a 6% per year compounding rate) during this quarter century (1). CO2 enrichment of field-grown crops has demonstrated the possibility of enhancing RuBP carboxylase activity to achieve improved crop production; it increases the production of grain legumes by 50 to 100% and that of cereal grains, for which the studies are less complete, by perhaps 10 to 50%. Results of O2 alteration of growth-room legumes and cereal grains are consistent with the results of CO2 enrichment except for a second role of O2 in assimilate partitioning. It may be necessary to include other components of the system, e.g., additional soil fertility, especially for non-N2-fixing plants, to enable an improved RuBP carboxylase to increase production. No practical method--chemical, genetic, or physical--of improving RuBP carboxylase activity has been reported.

  5. Isolation and partial characterization of Rhodopseudomonas sphaeroides mutants defective in the regulation of ribulose bisphosphate carboxylase/oxygenase.

    PubMed

    Weaver, K E; Tabita, F R

    1983-11-01

    Several mutants of Rhodopseudomonas sphaeroides defective in the derepression of the enzyme ribulose 1,5-bisphosphate carboxylase have been isolated by using the unstable Tn5 vectors pJB4JI and pRK340. Transpositional insertion mutants obtained with pJB4JI were demonstrated to be incapable of increasing ribulose 1,5-bisphosphate carboxylase/oxygenase levels when grown on butyrate-bicarbonate medium or under conditions of carbon starvation, whereas the wild-type strain increased activity four- to eightfold. When the wild-type strain was starved for carbon in the presence of chloramphenicol, no derepression was observed. Crude extracts from mutant and wild-type strains had distinct and consistent differences in protein content as observed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Chromatographic evidence indicated that mutants were defective in the regulation of only one of the two forms of ribulose 1,5-bisphosphate carboxylase/oxygenase synthesized by R. sphaeroides.

  6. Acetyl Coenzyme A Carboxylase Activity in Developing Seedlings and Chloroplasts of Barley and Its Virescens Mutant 1

    PubMed Central

    Thomson, Lawrence W.; Zalik, Saul

    1981-01-01

    Acetyl coenzyme A (CoA) carboxylase activity of whole tissue homogenates and chloroplast preparations was analyzed as the acetyl-CoA-dependent incorporation of [14C]bicarbonate into an acid-stable product. The absolute requirement for ATP and MgCl2, the complete inhibition with avidin, and end-product analysis were consistent with the presence of acetyl-CoA carboxylase activity. Little difference was found between the mutant and normal tissue homogenates from the 1- to 3-day growth stages, during which period both showed a 3-fold increase. However, by 4 days, the activity of the mutant exceeded that of the normal. Fractionation studies showed that the enzyme was a soluble protein present in the stromal fraction of chloroplasts. The biotin content was also highest in the stroma, although it was found in the lamellar fraction as well. For both the mutant and the normal, the highest acetyl-CoA carboxylase activities were obtained in the stromal preparations from 4-day seedlings (54 and 31 nmoles per milligram protein per minute for the mutant and the normal, respectively) with a progressive decline by 6 and 8 days. The difference between the mutant and the normal was not due to the accumulation of an inhibitor in the normal. PMID:16661731

  7. Evaluation of 3-methylcrotonyl-CoA carboxylase deficiency detected by tandem mass spectrometry newborn screening.

    PubMed

    Koeberl, D D; Millington, D S; Smith, W E; Weavil, S D; Muenzer, J; McCandless, S E; Kishnani, P S; McDonald, M T; Chaing, S; Boney, A; Moore, E; Frazier, D M

    2003-01-01

    Since the addition of tandem mass spectrometry (MS/MS) to the North Carolina Newborn Screening Program, 20 infants with two consecutive elevated 3-hydroxyisovalerylcarnitine (C5OH) levels have been evaluated for evidence of inborn errors of metabolism associated with this metabolite. Ten of these 20 infants had significant concentrations of both 3-hydroxyisovaleric acid and 3-methylcrotonylglycine in their urine, suggestive of 3-methylcrotonyl-CoA carboxylase (3-MCC) deficiency. Four of these 10 were infants whose abnormal metabolites were found to be of maternal origin. Of 8 patients with probable 3-MCC deficiency, 7 have been tested and found to have the enzyme deficiency confirmed in lymphoblasts or cultured fibroblasts; one of these 7 infants had only marginally decreased 3-MCC activity in lymphocytes but deficient 3-MCC in fibroblasts. We estimate the incidence of 3-MCC deficiency at 1:64000 live births in North Carolina. We conclude that MS/MS newborn screening will detect additional inborn errors of metabolism, such as 3-MCC deficiency, not traditionally associated with newborn screening. The evaluation of newborns with two abnormally elevated C5OH levels on MS/MS newborn screening should include, at least, urine organic acid analysis by capillary GC-MS and a plasma acylcarnitine profile by MS/MS. Long-term follow-up is needed to determine the outcome of presymptomatically diagnosed patients with 3-MCC deficiency by MS/MS newborn screening.

  8. Pyruvate carboxylase from Rhizobium etli: mutant characterization, nucleotide sequence, and physiological role.

    PubMed Central

    Dunn, M F; Encarnación, S; Araíza, G; Vargas, M C; Dávalos, A; Peralta, H; Mora, Y; Mora, J

    1996-01-01

    Pyruvate carboxylase (PYC), a biotin-dependent enzyme which catalyzes the conversion of pyruvate to oxaloacetate, was hypothesized to play an important anaplerotic role in the growth of Rhizobium etli during serial subcultivation in minimal media containing succinate (S. Encarnación, M. Dunn, K. Willms, and J. Mora, J. Bacteriol. 177:3058-3066, 1995). R. etli and R. tropici pyc::Tn5-mob mutants were selected for their inability to grow in minimal medium with pyruvate as a sole carbon source. During serial subcultivation in minimal medium containing 30 mM succinate, the R. etli parent and pyc mutant strains exhibited similar decreases in growth rate with each subculture. Supplementation of the medium with biotin prevented the growth decrease of the parent but not the mutant strain, indicating that PYC was necessary for the growth of R. etli under these conditions. The R. tropici pyc mutant grew normally in subcultures regardless of biotin supplementation. The symbiotic phenotypes of the pyc mutants from both species were similar to those of the parent strains. The R. etli pyc was cloned, sequenced, and found to encode a 126-kDa protein of 1,154 amino acids. The deduced amino acid sequence is highly homologous to other PYC sequences, and the catalytic domains involved in carboxylation, pyruvate binding, and biotinylation are conserved. The sequence and biochemical data show that the R. etli PYC is a member of the alpha4, homotetrameric, acetyl coenzyme A-activated class of PYCs. PMID:8830693

  9. Insulin stimulates the dephosphorylation and activation of acetyl-CoA carboxylase.

    PubMed Central

    Witters, L A; Watts, T D; Daniels, D L; Evans, J L

    1988-01-01

    The mechanism underlying the ability of insulin to acutely activate acetyl-CoA carboxylase [acetyl-CoA: carbon-dioxide ligase (ADP-forming), EC 6.4.1.2; AcCoA-Case] has been examined in Fao Reuber hepatoma cells. Insulin promotes the rapid activation of AcCoACase, as measured in cell lysates, and this stimulation persists to the same degree after isolation of AcCoACase by avidin-Sepharose chromatography. The insulin-stimulated enzyme, as compared with control enzyme, exhibits an increase in both citrate-independent and -dependent activity and a decrease in the Ka for citrate. Direct examination of the phosphorylation state of isolated 32P-labeled AcCoACase after insulin exposure reveals a marked decrease in total enzyme phosphorylation coincident with activation. The dephosphorylation due to insulin appears to be restricted to the phosphorylation sites previously shown to regulate AcCoACase activity. All of these effects of insulin are mimicked by a low molecular weight autocrine factor, tentatively identified as an oligosaccharide, present in conditioned medium of hepatoma cells. These data suggest that insulin may activate AcCoACase by inhibiting the activity of protein kinase(s) or stimulating the activity of protein phosphatase(s) that control the phosphorylation state of the enzyme. Images PMID:2899891

  10. Activating Phosphoenolpyruvate Carboxylase and Phosphoenolpyruvate Carboxykinase in Combination for Improvement of Succinate Production

    PubMed Central

    Tan, Zaigao; Zhu, Xinna; Chen, Jing; Li, Qingyan

    2013-01-01

    Phosphoenolpyruvate (PEP) carboxylation is an important step in the production of succinate by Escherichia coli. Two enzymes, PEP carboxylase (PPC) and PEP carboxykinase (PCK), are responsible for PEP carboxylation. PPC has high substrate affinity and catalytic velocity but wastes the high energy of PEP. PCK has low substrate affinity and catalytic velocity but can conserve the high energy of PEP for ATP formation. In this work, the expression of both the ppc and pck genes was modulated, with multiple regulatory parts of different strengths, in order to investigate the relationship between PPC or PCK activity and succinate production. There was a positive correlation between PCK activity and succinate production. In contrast, there was a positive correlation between PPC activity and succinate production only when PPC activity was within a certain range; excessive PPC activity decreased the rates of both cell growth and succinate formation. These two enzymes were also activated in combination in order to recruit the advantages of each for the improvement of succinate production. It was demonstrated that PPC and PCK had a synergistic effect in improving succinate production. PMID:23747698

  11. Soybean ribulose bisphosphate carboxylase small subunit: Mechanisms and determinants of RNA turnover. Annual progress report

    SciTech Connect

    Meagher, R.B.

    1993-12-31

    An in vitro degradation system has been developed from petunia and soybean polysomes in order to investigate the mechanisms and determinants controlling RNA turnover in higher plants. This system faithfully degrades soybean ribulose-1,5-bisphosphate carboxylase small subunit (rbcS) mRNA into the same products observed in total RNA preparations. In previous years it was shown that the most stable products represent a nested constellation of fragments, which are shortened from their 3{prime} ends, and have intact 5{prime} ends. Exogenous rbcS RNA tagged with novel 5{prime} sequence 15 or 56 bp long were synthesized in vitro as Sp6 and T7 runoff transcripts, respectively. When added to the system they were degraded faithfully into constellation of products which were 15 or 56 bp longer than the endogenous products, respectively. Detailed kinetics on the appearance of these exogenous products confirmed degradation proceeds in an overall 3{prime} to 5{prime} direction but suggested that there are multiple pathways through which the RNA may be degraded. To further demonstrate a precursor product relationships, in vitro synthesized transcripts truncated at their 3{prime} ends were shown to degrade into the expected smaller fragments previously mapped in the 5{prime} portion of the rbcS RNA.

  12. Slow inactivation of ribulosebisphosphate carboxylase during catalysis is not due to decarbamylation of the catalytic site

    SciTech Connect

    Edmondson, D.L.; Badger, M.R.; Andrews, T.J. )

    1990-08-01

    An investigation was made of the proposal that the slow inactivation of ribulosebisphosphate carboxylase (Rubisco) activity, which occurs during in vitro assays, is due to decarbamylation of the enzyme. The level of carbamylation was compared with catalytic activity during assay conditions in which activity was both increasing and decreasing. A dual isotope procedure was used in which ({sup 3}H)carboxyarabinitol-P{sub 2} measured total active sites and {sup 14}CO{sub 2} reported the level of carbamylation. The efficacy of the procedure was verified both in the presence and in the absence of the substrate D-ribulose-1,5-bisphosphate (ribulose-P{sub 2}). These measurements showed that changes in activity during assays were not correlated with carbamylation status. Inactivation during assays initiated with both fully and partially carbamylated enzyme was not associated with any change in carbamylation level. This implies that the loss of activity during assays is not due to ribulose-P{sub 2} binding and sequestering the E form of the enzyme. Ribulose-P{sub 2} did not appear to alter the equilibrium between carbamylated and uncarbamylated enzyme, but it did slow the rate at which enzyme was both decarbamylated and carbamylated. The most likely explanation for the loss of activity during assays appears to be the sequestration of carbamylated, Mg{sup 2+}-bound active sites by an inhibitor.

  13. Crystalline ribulose bisphosphate carboxylase/oxygenase of high integrity and catalytic activity from Nicotiana tabacum.

    PubMed

    Servaites, J C

    1985-04-01

    Crystalline tobacco (Nicotiana tabacum L.) ribulose-1,5-bisphosphate carboxylase/oxygenase (EC 4.1.1.39) was prepared using a procedure which protected the enzyme from hydrolysis by endogenous proteases. Leaves were extracted in a buffered medium containing casein, leupeptin, and high concentrations of MgSO4 and NaHCO3. After filtration through ion-exchange resin to remove contaminants, the enzyme was concentrated by precipitation with polyethylene glycol and crystal formation was induced by low-salt dialysis. The crystalline enzyme had a measured specific activity of 1.7 mumol CO2 mg protein-1 min-1, and about 93% of the enzyme could be activated with Mg2+ and CO2. Crystalline enzyme prepared in the absence of casein exhibited an activity which was only one-third of this rate and only about 70% of the enzyme could be activated with Mg2+ and CO2. Casein-extracted enzyme was resolved into distinct bands corresponding to the large (55,000) and small (14,000) subunits by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The large subunit of enzyme prepared according to the latter procedure was found to be composed of five different polypeptides of slightly decreasing molecular weight. Only about one-third of the large subunits were of the 55,000 molecular weight type. No differences between the two preparations were observed in the Km (CO2) and apparent Km (ribulose bisphosphate).

  14. [Efficacy of thiamine pyrophosphate or carboxylase in the salvage of diabetic foot].

    PubMed

    Carmona-Cervantes, J

    2014-01-01

    Diabetic foot represents one of the most common complications in patients with a long standing disease. The etiology is neuropathy, infections and ischemia that together contribute to the sequence of tissue necrosis, ulceration and gangrene. Since treatment is very difficult, we must look for several options to solve these problems caused by chronic hyperglycemia. Thiamine pyrophosphate or carboxylase perform multiple metabolic and non-metabolic activities that are considered important in the resolution of diabetic impairments, therefore, this work shows the results when using it in patients with diabetic foot. 29 patients with diabetic foot were treated between January 1998 and July 2012: 19 Wagner type III and 12 Wagner type IV. Management was the administration of antibiotics, partial surgical procedures and thiamine pyrophosphate. The infectious process was controlled, the appearance of granulation tissue and scarring of the lesion in a period of 2 to 6 months depending on the severity of the problem. Given the clinical data and evolution of the patients, we conclude that the administration of thiamine pyrophosphate was able to control metabolic and non-metabolic dysfunctions that lead to complications in diabetic patients, therefore we must consider it a tool in the treatment of diabetic patients in general and for diabetic foot salvage in particular.

  15. Catalytic by-product formation and ligand binding by ribulose bisphosphate carboxylases from different phylogenies.

    PubMed

    Pearce, F Grant

    2006-11-01

    During catalysis, all Rubisco (D-ribulose-1,5-bisphosphate carboxylase/oxygenase) enzymes produce traces of several by-products. Some of these by-products are released slowly from the active site of Rubisco from higher plants, thus progressively inhibiting turnover. Prompted by observations that Form I Rubisco enzymes from cyanobacteria and red algae, and the Form II Rubisco enzyme from bacteria, do not show inhibition over time, the production and binding of catalytic by-products was measured to ascertain the underlying differences. In the present study we show that the Form IB Rubisco from the cyanobacterium Synechococcus PCC6301, the Form ID enzyme from the red alga Galdieria sulfuraria and the low-specificity Form II type from the bacterium Rhodospirillum rubrum all catalyse formation of by-products to varying degrees; however, the by-products are not inhibitory under substrate-saturated conditions. Study of the binding and release of phosphorylated analogues of the substrate or reaction intermediates revealed diverse strategies for avoiding inhibition. Rubisco from Synechococcus and R. rubrum have an increased rate of inhibitor release. G. sulfuraria Rubisco releases inhibitors very slowly, but has an increased binding constant and maintains the enzyme in an activated state. These strategies may provide information about enzyme dynamics, and the degree of enzyme flexibility. Our observations also illustrate the phylogenetic diversity of mechanisms for regulating Rubisco and raise questions about whether an activase-like mechanism should be expected outside the green-algal/higher-plant lineage.

  16. Acetyl-CoA carboxylase alpha is essential to breast cancer cell survival.

    PubMed

    Chajès, Véronique; Cambot, Marie; Moreau, Karen; Lenoir, Gilbert M; Joulin, Virginie

    2006-05-15

    Activation of de novo fatty acid synthesis is a characteristic feature of cancer cells. We have recently described an interaction between acetyl-CoA carboxylase alpha (ACCalpha), a key enzyme in fatty acid synthesis, and BRCA1, which indicates a possible connection between lipid synthesis and genetic factors involved in susceptibility to breast and ovarian cancers. For this reason, we explored the role of ACCalpha in breast cancer cell survival using an RNA interference (RNAi) approach. We show that specific silencing of either the ACCalpha or the fatty acid synthase (FAS) genes in cancer cells results in a major decrease in palmitic acid synthesis. Depletion of the cellular pool of palmitic acid is associated with induction of apoptosis concomitant with the formation of reactive oxygen species (ROS) and mitochondrial impairment. Expression of a small interfering RNA (siRNA)-resistant form of ACCalpha mRNA prevented the effect of ACCalpha-RNAi but failed to prevent the effect of FAS gene silencing. Furthermore, supplementation of the culture medium with palmitate or with the antioxidant vitamin E resulted in the complete rescue of cells from both ACCalpha and FAS siRNA-induced apoptosis. Finally, human mammary epithelial cells are resistant to RNAi against either ACCalpha or FAS. These data confirm the importance of lipogenesis in cancer cell survival and indicate that this pathway represents a key target for antineoplastic therapy that, however, might require specific dietary recommendation for full efficacy.

  17. 3-methylcrotonyl-CoA carboxylase deficiency: Clinical, biochemical, enzymatic and molecular studies in 88 individuals

    PubMed Central

    2012-01-01

    Background Isolated 3-methylcrotonyl-CoA carboxylase (MCC) deficiency is an autosomal recessive disorder of leucine metabolism caused by mutations in MCCC1 or MCCC2 encoding the α and β subunit of MCC, respectively. The phenotype is highly variable ranging from acute neonatal onset with fatal outcome to asymptomatic adults. Methods We report clinical, biochemical, enzymatic and mutation data of 88 MCC deficient individuals, 53 identified by newborn screening, 26 diagnosed due to clinical symptoms or positive family history and 9 mothers, identified following the positive newborn screening result of their baby. Results Fifty-seven percent of patients were asymptomatic while 43% showed clinical symptoms, many of which were probably not related to MCC deficiency but due to ascertainment bias. However, 12 patients (5 of 53 identified by newborn screening) presented with acute metabolic decompensations. We identified 15 novel MCCC1 and 16 novel MCCC2 mutant alleles. Additionally, we report expression studies on 3 MCCC1 and 8 MCCC2 mutations and show an overview of all 132 MCCC1 and MCCC2 variants known to date. Conclusions Our data confirm that MCC deficiency, despite low penetrance, may lead to a severe clinical phenotype resembling classical organic acidurias. However, neither the genotype nor the biochemical phenotype is helpful in predicting the clinical course. PMID:22642865

  18. Improvement of the phosphoenolpyruvate carboxylase activity of Phaeodactylum tricornutum PEPCase 1 through protein engineering.

    PubMed

    Chang, Kwang Suk; Jeon, Hancheol; Seo, Seungbeom; Lee, Yew; Jin, EonSeon

    2014-06-10

    In order to mitigate CO2 accumulation and decrease the rate of global warming and climate change, we previously presented a strategy for the development of an efficient CO2 capture and utilization system. The system employs two recombinant enzymes, carbonic anhydrase and phosphoenolpyruvate carboxylase, which were originated from microalgae. Although utilization of this integrated system would require a large quantity of high quality PEPCase protein, such quantities could be produced by increasing the solubility of the Phaeodactylum tricornutum PEPCase 1 (PtPEPCase 1) protein in the Escherichia coli heterologous expression system. We first expressed the putative mitochondria targeting peptide- and chloroplast transit peptide-truncated proteins of PtPEPCase 1, mPtPEPCase 1 and cPtPEPCase 1, respectively, in E. coli. After affinity chromatography, the amount of purified PEPCase protein from 500mL of E. coli culture was greatest for cPtPEPCase 1 (1.99mg), followed by mPtPEPCase 1 (0.82mg) and PtPEPCase 1 (0.61mg). Furthermore, the enzymatic activity of mPtPEPCase 1 and cPtPEPCase 1 showed approximately 1.6-fold (32.19 units/mg) and 3-fold (59.48 units/mg) increases, respectively. Therefore, cPtPEPCase 1 purified using the E. coli heterogeneous expression system could be a strong candidate for a platform technology to capture CO2 and produce value-added four-carbon platform chemicals.

  19. Characterization of lysine acetylation of a phosphoenolpyruvate carboxylase involved in glutamate overproduction in Corynebacterium glutamicum.

    PubMed

    Nagano-Shoji, Megumi; Hamamoto, Yuma; Mizuno, Yuta; Yamada, Ayuka; Kikuchi, Masaki; Shirouzu, Mikako; Umehara, Takashi; Yoshida, Minoru; Nishiyama, Makoto; Kosono, Saori

    2017-03-03

    Protein Nε-acylation is emerging as a ubiquitous post-translational modification. In Corynebacterium glutamicum, which is utilized for industrial production of L-glutamate, the levels of protein acetylation and succinylation change drastically under the conditions that induce glutamate overproduction. Here, we characterized the acylation of phosphoenolpyruvate carboxylase (PEPC), an anaplerotic enzyme that supplies oxaloacetate for glutamate overproduction. We showed that acetylation of PEPC at lysine 653 decreased enzymatic activity, leading to reduced glutamate production. An acetylation-mimic (KQ) mutant of K653 showed severely reduced glutamate production, while the corresponding KR mutant showed normal production levels. Using an acetyllysine-incorporated PEPC protein, we verified that K653-acetylation negatively regulates PEPC activity. In addition, NCgl0616, a sirtuin-type deacetylase, deacetylated K653-acetylated PEPC in vitro. Interestingly, the specific activity of PEPC was increased during glutamate overproduction, which was blocked by the K653R mutation or deletion of sirtuin-type deacetylase homologues. These findings suggested that deacetylation of K653 by NCgl0616 likely plays a role in the activation of PEPC, which maintains carbon flux under glutamate-producing conditions. PEPC deletion increased protein acetylation levels in cells under glutamate-producing conditions, supporting our hypothesis that PEPC is responsible for a large carbon flux change under glutamate-producing conditions. This article is protected by copyright. All rights reserved.

  20. Cloning and characterization of the pyruvate carboxylase from Sinorhizobium meliloti Rm1021.

    PubMed

    Dunn, M F; Araíza, G; Finan, T M

    2001-11-01

    The gene encoding pyruvate carboxylase (pyc) was isolated from a Sinorhizobium meliloti Rm1021 cosmid bank by complementation of a Rhizobium tropici pyc mutant. PYC-negative mutants of S. meliloti Rm1021 were isolated by transposon mutagenesis and were unable to grow with glucose or pyruvate as sole carbon sources, but were symbiotically competent in combination with alfalfa plants. PYC activity assays, pyc::lacZ gene fusion studies and an in vivo biotinylation assay showed that PYC activity in S. meliloti was dependent mainly on biotin availability and not on changes in gene transcription. The subunit and holo-enzyme molecular masses of the S. meliloti PYC indicated that the enzyme was an alpha4 homotetramer. The S. meliloti PYC had a high apparent Ka (0.23 mM) for the allosteric activator acetyl-CoA and was product-inhibited by sub-millimolar concentrations of oxaloacetate. In contrast to other bacterial alpha4-PYCs which have been characterized, the S. meliloti enzyme was not strongly inhibited by L-aspartate.

  1. Allosteric Inhibition of Phosphoenolpyruvate Carboxylases is Determined by a Single Amino Acid Residue in Cyanobacteria

    PubMed Central

    Takeya, Masahiro; Hirai, Masami Yokota; Osanai, Takashi

    2017-01-01

    Phosphoenolpyruvate carboxylase (PEPC) is an important enzyme for CO2 fixation and primary metabolism in photosynthetic organisms including cyanobacteria. The kinetics and allosteric regulation of PEPCs have been studied in many organisms, but the biochemical properties of PEPC in the unicellular, non-nitrogen-fixing cyanobacterium Synechocystis sp. PCC 6803 have not been clarified. In this study, biochemical analysis revealed that the optimum pH and temperature of Synechocystis 6803 PEPC proteins were 7.3 and 30 °C, respectively. Synechocystis 6803 PEPC was found to be tolerant to allosteric inhibition by several metabolic effectors such as malate, aspartate, and fumarate compared with other cyanobacterial PEPCs. Comparative sequence and biochemical analysis showed that substitution of the glutamate residue at position 954 with lysine altered the enzyme so that it was inhibited by malate, aspartate, and fumarate. PEPC of the nitrogen-fixing cyanobacterium Anabaena sp. PCC 7120 was purified, and its activity was inhibited in the presence of malate. Substitution of the lysine at position 946 (equivalent to position 954 in Synechocystis 6803) with glutamate made Anabaena 7120 PEPC tolerant to malate. These results demonstrate that the allosteric regulation of PEPC in cyanobacteria is determined by a single amino acid residue, a characteristic that is conserved in different orders. PMID:28117365

  2. Phosphoenolpyruvate carboxylase in mistletoe leaves: Regulation of gene expression, protein content, and covalent modification.

    PubMed

    Li, Xuemei; Wanek, Wolfgang; Nehls, U.; Popp, Marianne; Hampp, Rüdiger; Rennenberg, Heinz; Einig, Werner

    2001-07-01

    Seasonal changes in the activity of phosphoenolpyruvate carboxylase (PEPCase, EC 4.1.1.31), a key enzyme in the interaction of carbohydrate and nitrogen metabolism, were studied in leaves of the C3 semiparasitic mistletoe, Viscum album, growing on different host trees. Maximum extractable PEPCase activities were higher in leaves of mistletoes growing on Betula pendula and Alnus glutinosa hosts compared with those on the conifers, Abies alba and Larix decidua. Independent of host, maximum extractable PEPCase activities were high in spring and autumn while low in summer. Samples with higher PEPCase activities showed higher amounts of PEPCase protein and higher PEPCase mRNA levels. A curvilinear correlation between leaf total nitrogen content and the maximum extractable PEPCase activity as well as PEPCase mRNA level suggested that nitrogen might affect the activity of PEPCase of mistletoe by up-regulating gene expression. In addition to extractable activity, seasonal changes of the PEPCase activation state, the ratio of activities resulting from limited:non-limited assays, were found, which was correlated to the variation of malate content in leaves of mistletoe. ATP-dependent activation of PEPCase was characterized by an increase in I0.5(L-malate), indicating that PEPCase of leaves of mistletoes is probably regulated via phosphorylation.

  3. A curving ACC system with coordination control of longitudinal car-following and lateral stability

    NASA Astrophysics Data System (ADS)

    Zhang, Dezhao; Li, Keqiang; Wang, Jianqiang

    2012-07-01

    The paper presents a curving adaptive cruise control (ACC) system that is coordinated with a direct yaw-moment control (DYC) system and gives consideration to both longitudinal car-following capability and lateral stability on curved roads. A model including vehicle longitudinal and lateral dynamics is built first, which is as discrete as the predictive model of the system controller. Then, a cost function is determined to reflect the contradictions between vehicle longitudinal and lateral dynamics. Meanwhile, some I/O constraints are formulated with a driver permissible longitudinal car-following range and the road adhesion condition. After that, desired longitudinal acceleration and desired yaw moment are obtained by a linear matrix inequality based robust constrained state feedback method. Finally, driver-in-the-loop tests on a driving simulator are conducted and the results show that the developed control system provides significant benefits in weakening the impact of DYC on ACC longitudinal car-following capability while also improving lateral stability.

  4. Preparation of ethylene gas and comparison of ethylene responses induced by ethylene, ACC, and ethephon.

    PubMed

    Zhang, Wei; Wen, Chi-Kuang

    2010-01-01

    Ethylene is a gaseous plant hormone used in many physiological studies examining its role in plant growth and development. However, ethylene gas may not be conveniently available to many laboratories for occasional use, and therefore several chemicals can be used as replacements. Here we report that the kinetics of the ethylene response induced by ethylene and two widely-used ethylene replacements are different. ACC failed to efficiently replace prolonged ethylene treatments, while the decomposition products of ethephon may cause non-specific responses and the efficiency of ethephon conversion to ethylene was relatively low. A cost-effective method to prepare ethylene gas was developed. Analyzed by gas chromatography, the chemically produced ethylene exhibited an identical chromatogram to that from the commercial source. Our synthetic ethylene gave the same dose-response curve in Arabidopsis as gaseous ethylene. Our study shows that the use of the ethylene gas is essential to experiments that are sensitive to treatment duration and dosage. When ACC and ethephon are used as replacements, caution should be taken in the experimental design. For laboratories that do not have an ethylene tank, ethylene gas can be easily prepared by a chemical approach without further purification.

  5. Various effects of fluorescent bacteria of the genus Pseudomonas containing ACC deaminase on wheat seedling growth.

    PubMed

    Magnucka, Elżbieta G; Pietr, Stanisław J

    2015-12-01

    The study evaluates the effect of rhizobacteria having 1-aminocyclopropane-1-carboxylate deaminase (ACCd) on the development of wheat seedlings. This enzyme has been proposed to play a key role in microbe-plant association. Three fluorescent pseudomonads containing this deaminase were selected from 70 strains of pseudomonads isolated from rhizosphere of wheat (Triticum aestivum L.) and rape (Brassica napus L.). These bacteria, varied significantly in the ability to both biosynthesize auxins and hydrolyze ACC. Among them, Pseudomonas brassicacearum subsp. brassicacearum strain RZ310 presented the highest activities of ACC deaminase during 96h of growth in liquid Dworkin and Foster (DF) salt medium. Additionally, this rape rhizosphere strain did not produce indoles. Two other isolates, Pseudomonas sp. PO283 and Pseudomonas sp. PO366, secreted auxins only in the presence of their precursor. Phylogenetic analysis of the 16S rRNA gene and four other protein-encoding genes indicated that these wheat rhizosphere isolates belonged to the fluorescent Pseudomonas group. Moreover, the effects of these strains on wheat seedling growth under in vitro conditions were markedly dependent on both their cell suspensions used to grain inoculation and nutrient conditions. Strains tested had beneficial influence on wheat seedlings mainly at low cell densities. In addition, access to nutrients markedly changed bacteria action on cereal growth. Their presence generally favored the positive effects of pseudomonads on length and the estimated biomasses of wheat coleoptiles. Despite these general rules, impacts of each isolate on the growth parameters of cereal seedlings were unique.

  6. 5th International ACC Symposium: Classification of Adrenocortical Cancers from Pathology to Integrated Genomics: Real Advances or Lost in Translation?

    PubMed

    de Krijger, Ronald E; Bertherat, Jérôme

    2016-02-01

    For the clinician, despite its rarity, adrenocortical cancer is a heterogeneous tumor both in term of steroid excess and tumor evolution. For patient management, it is crucial to have an accurate vision of this heterogeneity, in order to use a correct tumor classification. Pathology is the best way to classify operated adrenocortical tumors: to recognize their adrenocortical nature and to differentiate benign from malignant tumors. Among malignant tumors pathology also aims at prognosis assessment. Although progress has being made for prognosis assessment, there is still a need for improvement. Recent studies have established the value of Ki67 for adrenocortical cancer (ACC) prognostication, aiming also at standardization to reduce variability. The use of genomics to study adrenocortical tumors gives a very new insight in their pathogenesis and molecular classification. Genomics studies of ACC give now a clear description of the mRNA (transcriptome) and miRNA expression profile, as well as chromosomal and methylation alterations. Exome sequencing also established firmly the list of the main ACC driver genes. Interestingly, genomics study of ACC also revealed subtypes of malignant tumors with different pattern of molecular alterations, associated with different outcome. This leads to a new vision of adrenocortical tumors classification based on molecular analysis. Interestingly, these molecular classifications meet also the results of pathological analysis. This opens new perspectives on the development and use of various molecular tools to classify, along with pathological analysis, ACC, and guides patient management at the area of precision medicine.

  7. Monitoring apoptosis of TK-GFP-expressing ACC-M cells induced by ACV using FRET technique

    NASA Astrophysics Data System (ADS)

    Xiong, Tao; Zhang, Zhihong; Lin, Juqiang; Yang, Jie; Zeng, Shaoqun; Luo, Qingming

    2006-05-01

    Apoptosis is an evolutionary conserved cellular process that plays an important role during development, but it is also involved in tissue homeostasis and in many diseases. To study the characteristics of suicide gene system of the herpes simplex virus thymidine kinase (HSV-tk) gene in tumor cells and explore the apoptosis phenomena in this system and its effect on the human adenoid cystic carcinoma line ACC-M cell, we detected apoptosis of CD3- (ECFP-CRS-DsRed) and TK-GFP-expressing ACC-M (ACC-M-TK-GFP-CD3) cells induced by acyclovir (ACV) using fluorescence resonance energy transfer (FRET) technique. CD3 is a FRET-based indicator for activity of caspase-3, which is composed of an enhanced cyan fluorescent protein, a caspase-3 sensitive linker, and a red fluorescent protein from Discosoma with efficient maturation property. FRET from ECFP to DsRed could be detected in normal ACC-M-TK-GFP-CD3 cells, and the FRET efficient was remarkably decreased and then disappeared during the cells apoptosis induced by ACV. It was due to the activated caspase-3 cleaved the CD3 fusion protein. In this study, the results suggested that the ACV-induced apoptosis of ACC-M-TK-GFP-CD3 cells was through caspase-3 pathway.

  8. Monitoring apoptosis of TK-GFP-expressing ACC-M cells induced by ACV using FRET technique

    NASA Astrophysics Data System (ADS)

    Xiong, Tao; Zhang, Zhihong; Lin, Juqiang; Yang, Jie; Zeng, Shaoqun; Luo, Qingming

    2006-09-01

    Apoptosis is an evolutionary conserved cellular process that plays an important role during development, but it is also involved in tissue homeostasis and in many diseases. To study the characteristics of suicide gene system of the herpes simplex virus thymidine kinase (HSV-tk) gene in tumor cells and explore the apoptosis phenomena in this system and its effect on the human adenoid cystic carcinoma line ACC-M cell, we detected apoptosis of CD3- (ECFP-CRS-DsRed) and TK-GFP-expressing ACC-M (ACC-M-TK-GFP-CD3) cells induced by acyclovir (ACV) using fluorescence resonance energy transfer (FRET) technique. CD3 is a FRET-based indicator for activity of caspase-3, which is composed of an enhanced cyan fluorescent protein, a caspase-3 sensitive linker, and a red fluorescent protein from Discosoma with efficient maturation property. FRET from ECFP to DsRed could be detected in normal ACC-M-TK-GFP-CD3 cells, and the FRET efficient was remarkably decreased and then disappeared during the cells apoptosis induced by ACV. It was due to the activated caspase-3 cleaved the CD3 fusion protein. In this study, the results suggested that the AVC-induced apoptosis of ACC-M-TK-GFP-CD3 cells was through caspase-3 pathway.

  9. Traffic flow characteristics in a mixed traffic system consisting of ACC vehicles and manual vehicles: A hybrid modelling approach

    NASA Astrophysics Data System (ADS)

    Yuan, Yao-Ming; Jiang, Rui; Hu, Mao-Bin; Wu, Qing-Song; Wang, Ruili

    2009-06-01

    In this paper, we have investigated traffic flow characteristics in a traffic system consisting of a mixture of adaptive cruise control (ACC) vehicles and manual-controlled (manual) vehicles, by using a hybrid modelling approach. In the hybrid approach, (i) the manual vehicles are described by a cellular automaton (CA) model, which can reproduce different traffic states (i.e., free flow, synchronised flow, and jam) as well as probabilistic traffic breakdown phenomena; (ii) the ACC vehicles are simulated by using a car-following model, which removes artificial velocity fluctuations due to intrinsic randomisation in the CA model. We have studied the traffic breakdown probability from free flow to congested flow, the phase transition probability from synchronised flow to jam in the mixed traffic system. The results are compared with that, where both ACC vehicles and manual vehicles are simulated by CA models. The qualitative and quantitative differences are indicated.

  10. Coexistence of Two Forms of LTP in ACC Provides a Synaptic Mechanism for the Interactions between Anxiety and Chronic Pain

    PubMed Central

    Koga, Kohei; Descalzi, Giannina; Chen, Tao; Ko, Hyoung-Gon; Lu, Jinshan; Li, Shermaine; Son, Junehee; Kim, TaeHyun; Kwak, Chuljung; Huganir, Richard L.; Zhao, Ming-gao; Kaang, Bong-Kiun; Collingridge, Graham L.; Zhuo, Min

    2015-01-01

    SUMMARY Chronic pain can lead to anxiety and anxiety can enhance the sensation of pain. Unfortunately, little is known about the synaptic mechanisms that mediate these re-enforcing interactions. Here we characterized two forms of long-term potentiation (LTP) in the anterior cingulate cortex (ACC); a presynaptic form (pre-LTP) that requires kainate receptors and a postsynaptic form (post-LTP) that requires N-methyl-D-aspartate receptors. Pre-LTP also involves adenylyl cyclase and protein kinase A and is expressed via a mechanism involving hyperpolarization-activated cyclic nucleotide-gated (HCN) channels. Interestingly, chronic pain and anxiety both result in selective occlusion of pre-LTP. Significantly, microinjection of the HCN blocker ZD7288 into the ACC in vivo produces both anxiolytic and analgesic effects. Our results provide a mechanism by which two forms of LTP in the ACC may converge to mediate the interaction between anxiety and chronic pain. PMID:25556835

  11. Acceleration of a Particle-in-Cell Code for Space Plasma Simulations with OpenACC

    NASA Astrophysics Data System (ADS)

    Peng, Ivy Bo; Markidis, Stefano; Vaivads, Andris; Vencels, Juris; Deca, Jan; Lapenta, Giovanni; Hart, Alistair; Laure, Erwin

    2015-04-01

    We simulate space plasmas with the Particle-in-cell (PIC) method that uses computational particles to mimic electrons and protons in solar wind and in Earth magnetosphere. The magnetic and electric fields are computed by solving the Maxwell's equations on a computational grid. In each PIC simulation step, there are four major phases: interpolation of fields to particles, updating the location and velocity of each particle, interpolation of particles to grids and solving the Maxwell's equations on the grid. We use the iPIC3D code, which was implemented in C++, using both MPI and OpenMP, for our case study. By November 2014, heterogeneous systems using hardware accelerators such as Graphics Processing Unit (GPUs) and the Many Integrated Core (MIC) coprocessors for high performance computing continue growth in the top 500 most powerful supercomputers world wide. Scientific applications for numerical simulations need to adapt to using accelerators to achieve portability and scalability in the coming exascale systems. In our work, we conduct a case study of using OpenACC to offload the computation intensive parts: particle mover and interpolation of particles to grids, in a massively parallel Particle-in-Cell simulation code, iPIC3D, to multi-GPU systems. We use MPI for inter-node communication for halo exchange and communicating particles. We identify the most promising parts suitable for GPUs accelerator by profiling using CrayPAT. We implemented manual deep copy to address the challenges of porting C++ classes to GPU. We document the necessary changes in the exiting algorithms to adapt for GPU computation. We present the challenges and findings as well as our methodology for porting a Particle-in-Cell code to multi-GPU systems using OpenACC. In this work, we will present the challenges, findings and our methodology of porting a Particle-in-Cell code for space applications as follows: We profile the iPIC3D code by Cray Performance Analysis Tool (CrayPAT) and identify

  12. Identification and characterization of an Apis cerana cerana Delta class glutathione S-transferase gene (AccGSTD) in response to thermal stress.

    PubMed

    Yan, Huiru; Jia, Haihong; Wang, Xiuling; Gao, Hongru; Guo, Xingqi; Xu, Baohua

    2013-02-01

    Glutathione S-transferases (GSTs) are members of a multifunctional enzyme super family that plays a pivotal role in both insecticide resistance and protection against oxidative stress. In this study, we identified a single-copy gene, AccGSTD, as being a Delta class GST in the Chinese honey bee (Apis cerana cerana). A predicted antioxidant response element, CREB, was found in the 1,492-bp 5'-flanking region, suggesting that AccGSTD may be involved in oxidative stress response pathways. Real-time PCR and immunolocalization studies demonstrated that AccGSTD exhibited both developmental- and tissue-specific expression patterns. During development, AccGSTD transcript was increased in adults. The AccGSTD expression level was the highest in the honey bee brain. Thermal stress experiments demonstrated that AccGSTD could be significantly upregulated by temperature changes in a time-dependent manner. It is hypothesized that high expression levels might be due to the increased levels of oxidative stress caused by the temperature challenges. Additionally, functional assays of the recombinant AccGSTD protein revealed that AccGSTD has the capability to protect DNA from oxidative damage. Taken together, these data suggest that AccGSTD may be responsible for antioxidant defense in adult honey bees.

  13. Identification and characterization of an Apis cerana cerana Delta class glutathione S-transferase gene ( AccGSTD) in response to thermal stress

    NASA Astrophysics Data System (ADS)

    Yan, Huiru; Jia, Haihong; Wang, Xiuling; Gao, Hongru; Guo, Xingqi; Xu, Baohua

    2013-02-01

    Glutathione S-transferases (GSTs) are members of a multifunctional enzyme super family that plays a pivotal role in both insecticide resistance and protection against oxidative stress. In this study, we identified a single-copy gene, AccGSTD, as being a Delta class GST in the Chinese honey bee ( Apis cerana cerana). A predicted antioxidant response element, CREB, was found in the 1,492-bp 5'-flanking region, suggesting that AccGSTD may be involved in oxidative stress response pathways. Real-time PCR and immunolocalization studies demonstrated that AccGSTD exhibited both developmental- and tissue-specific expression patterns. During development, AccGSTD transcript was increased in adults. The AccGSTD expression level was the highest in the honey bee brain. Thermal stress experiments demonstrated that AccGSTD could be significantly upregulated by temperature changes in a time-dependent manner. It is hypothesized that high expression levels might be due to the increased levels of oxidative stress caused by the temperature challenges. Additionally, functional assays of the recombinant AccGSTD protein revealed that AccGSTD has the capability to protect DNA from oxidative damage. Taken together, these data suggest that AccGSTD may be responsible for antioxidant defense in adult honey bees.

  14. Epigenetic regulation of pyruvate carboxylase gene expression in the postpartum liver.

    PubMed

    Walker, C G; Crookenden, M A; Henty, K M; Handley, R R; Kuhn-Sherlock, B; White, H M; Donkin, S S; Snell, R G; Meier, S; Heiser, A; Loor, J J; Mitchell, M D; Roche, J R

    2016-07-01

    Hepatic gluconeogenesis is essential for maintenance of whole body glucose homeostasis and glucose supply for mammary lactose synthesis in the dairy cow. Upregulation of the gluconeogenic enzyme pyruvate carboxylase (PC) during the transition period is vital in the adaptation to the greater glucose demands associated with peripartum lactogenesis. The objective of this study was to determine if PC transcription in hepatocytes is regulated by DNA methylation and if treatment with a nonsteroidal anti-inflammatory drug (NSAID) alters methylation of an upstream DNA sequence defined as promoter 1. Dairy cows were left untreated (n=20), or treated with a NSAID during the first 5 d postcalving (n=20). Liver was biopsied at d 7 precalving and d 7, 14, and 28 postcalving. Total PC and transcript specific gene expression was quantified using quantitative PCR and DNA methylation of promoter 1 was quantified using bisulfite Sanger sequencing. Expression of PC changed over the transition period, with increased expression postcalving occurring concurrently with increased circulating concentration of nonesterified fatty acids. The DNA methylation percentage was variable at all sites quantified and ranged from 21 to 54% across the 15 CpG dinucleotides within promoter 1. The DNA methylation at wk 1 postcalving, however, was not correlated with gene expression of promoter 1-regulated transcripts and we did not detect an effect of NSAID treatment on DNA methylation or PC gene expression. Our results do not support a role for DNA methylation in regulating promoter 1-driven gene expression of PC at wk 1 postcalving. Further research is required to determine the mechanisms regulating increased PC expression over the transition period.

  15. Unusual ribulose 1,5-bisphosphate carboxylase/oxygenase of anoxic Archaea.

    PubMed

    Watson, G M; Yu, J P; Tabita, F R

    1999-03-01

    The predominant pool of organic matter on earth is derived from the biological reduction and assimilation of carbon dioxide gas, catalyzed primarily by the enzyme ribulose 1,5-bisphosphate carboxylase/oxygenase (RubisCO). By virtue of its capacity to use molecular oxygen as an alternative and competing gaseous substrate, the catalytic efficiency of RubisCO and the enzyme's ability to assimilate CO2 may be severely limited, with consequent environmental and agricultural effects. Recent genomic sequencing projects, however, have identified putative RubisCO genes from anoxic Archaea. In the present study, these potential RubisCO sequences, from Methanococcus jannaschii and Archaeoglobus fulgidus, were analyzed in order to ascertain whether such sequences might encode functional proteins. We also report the isolation and properties of recombinant RubisCO using sequences obtained from the obligately anaerobic hyperthermophilic methanogen M. jannaschii. This is the first description of an archaeal RubisCO sequence; this study also represents the initial characterization of a RubisCO molecule that has evolved in the absence of molecular oxygen. The enzyme was shown to be a homodimer whose deduced sequence, along with other recently obtained archaeal RubisCO sequences, differs substantially from those of known RubisCO molecules. The recombinant M. jannaschii enzyme has a somewhat low, but reasonable kcat, however, unlike previously isolated RubisCO molecules, this enzyme is very oxygen sensitive yet it is stable to hyperthermal temperatures and catalyzes the formation of the expected carboxylation product. Despite inhibition by oxygen, this unusual RubisCO still catalyzes a weak yet demonstrable oxygenase activity, with perhaps the lowest capacity for CO2/O2 discrimination ever encountered for any RubisCO.

  16. Effects of phosphoenolpyruvate carboxylase desensitization on glutamic acid production in Corynebacterium glutamicum ATCC 13032.

    PubMed

    Wada, Masaru; Sawada, Kazunori; Ogura, Kotaro; Shimono, Yuta; Hagiwara, Takuya; Sugimoto, Masakazu; Onuki, Akiko; Yokota, Atsushi

    2016-02-01

    Phosphoenolpyruvate carboxylase (PEPC) in Corynebacterium glutamicum ATCC13032, a glutamic-acid producing actinobacterium, is subject to feedback inhibition by metabolic intermediates such as aspartic acid and 2-oxoglutaric acid, which implies the importance of PEPC in replenishing oxaloacetic acid into the TCA cycle. Here, we investigated the effects of feedback-insensitive PEPC on glutamic acid production. A single amino-acid substitution in PEPC, D299N, was found to relieve the feedback control by aspartic acid, but not by 2-oxoglutaric acid. A simple mutant, strain R1, having the D299N substitution in PEPC was constructed from ATCC 13032 using the double-crossover chromosome replacement technique. Strain R1 produced glutamic acid at a concentration of 31.0 g/L from 100 g/L glucose in a jar fermentor culture under biotin-limited conditions, which was significantly higher than that of the parent, 26.0 g/L (1.19-fold), indicative of the positive effect of desensitized PEPC on glutamic acid production. Another mutant, strain DR1, having both desensitized PEPC and PYK-gene deleted mutations, was constructed in a similar manner using strain D1 with a PYK-gene deleted mutation as the parent. This mutation had been shown to enhance glutamic acid production in our previous study. Although marginal, strain D1 produced higher glutamic acid, 28.8 g/L, than ATCC13032 (1.11-fold). In contrast, glutamic acid production by strain DR-1 was elevated up to 36.9 g/L, which was 1.42-fold higher than ATCC13032 and significantly higher than the other three strains. The results showed a synergistic effect of these two mutations on glutamic acid production in C. glutamicum.

  17. Synthesis of catalytically active form III ribulose 1,5-bisphosphate carboxylase/oxygenase in archaea.

    PubMed

    Finn, Michael W; Tabita, F Robert

    2003-05-01

    Ribulose 1,5 bisphosphate carboxylase/oxygenase (RubisCO) catalyzes the biological reduction and assimilation of carbon dioxide gas to organic carbon; it is the key enzyme responsible for the bulk of organic matter found on earth. Until recently it was believed that there are only two forms of RubisCO, form I and form II. However, the recent completion of several genome-sequencing projects uncovered open reading frames resembling RubisCO in the third domain of life, the archaea. Previous work and homology comparisons suggest that these enzymes represent a third form of RubisCO, form III. While earlier work indicated that two structurally distinct recombinant archaeal RubisCO proteins catalyzed bona fide RubisCO reactions, it was not established that the rbcL genes of anaerobic archaea can be transcribed and translated to an active enzyme in the native organisms. In this report, it is shown not only that Methanococcus jannaschii, Archaeoglobus fulgidus, Methanosarcina acetivorans, and Methanosarcina barkeri possess open reading frames with the residues required for catalysis but also that the RubisCO protein from these archaea accumulates in an active form under normal growth conditions. In addition, the form III RubisCO gene (rbcL) from M. acetivorans was shown to complement RubisCO deletion strains of Rhodobacter capsulatus and Rhodobacter sphaeroides under both photoheterotrophic and photoautotrophic growth conditions. These studies thus indicate for the first time that archaeal form III RubisCO functions in a physiologically significant fashion to fix CO(2). Furthermore, recombinant M. jannaschii, M. acetivorans, and A. fulgidus RubisCO possess unique properties with respect to quaternary structure, temperature optima, and activity in the presence of molecular oxygen compared to the previously described Thermococcus kodakaraensis and halophile proteins.

  18. In vivo monoubiquitination of anaplerotic phosphoenolpyruvate carboxylase occurs at Lys624 in germinating sorghum seeds

    PubMed Central

    Echevarría, Cristina

    2014-01-01

    Phosphoenolpyruvate carboxylase (PEPC; EC 4.1.1.31) is an important cytosolic regulatory enzyme that plays a pivotal role in numerous physiological processes in plants, including seed development and germination. Previous studies demonstrated the occurrence of immunoreactive PEPC polypeptides of ~110kDa and 107kDa (p110 and p107, respectively) on immunoblots of clarified extracts of germinating sorghum (Sorghum bicolor) seeds. In order to establish the biochemical basis for this observation, a 460kDa PEPC heterotetramer composed of an equivalent ratio of p110 and p107 subunits was purified to near homogeneity from the germinated seeds. Mass spectrometry established that p110 and p107 are both encoded by the same plant-type PEPC gene (CP21), but that p107 was in vivo monoubiquitinated at Lys624 to form p110. This residue is absolutely conserved in vascular plant PEPCs and is proximal to a PEP-binding/catalytic domain. Anti-ubiquitin IgG immunodetected p110 but not p107, whereas incubation with a deubiquitinating enzyme (USP-2 core) efficiently converted p110 into p107, while relieving the enzyme’s feedback inhibition by l-malate. Partial PEPC monoubiquitination was also detected during sorghum seed development. It is apparent that monoubiquitination at Lys624 is opposed to phosphorylation at Ser7 in terms of regulating the catalytic activity of sorghum seed PEPC. PEPC monoubiquitination is hypothesized to fine-tune anaplerotic carbon flux according to the cell’s immediate physiological requirements for tricarboxylic acid cycle intermediates needed in support of biosynthesis and carbon–nitrogen interactions. PMID:24288181

  19. Multiple Inter-Kingdom Horizontal Gene Transfers in the Evolution of the Phosphoenolpyruvate Carboxylase Gene Family

    PubMed Central

    Wang, Wen; Su, Bing

    2012-01-01

    Pepcase is a gene encoding phosphoenolpyruvate carboxylase that exists in bacteria, archaea and plants,playing an important role in plant metabolism and development. Most plants have two or more pepcase genes belonging to two gene sub-families, while only one gene exists in other organisms. Previous research categorized one plant pepcase gene as plant-type pepcase (PTPC) while the other as bacteria-type pepcase (BTPC) because of its similarity with the pepcase gene found in bacteria. Phylogenetic reconstruction showed that PTPC is the ancestral lineage of plant pepcase, and that all bacteria, protistpepcase and BTPC in plants are derived from a lineage of pepcase closely related with PTPC in algae. However, their phylogeny contradicts the species tree and traditional chronology of organism evolution. Because the diversification of bacteria occurred much earlier than the origin of plants, presumably all bacterialpepcase derived from the ancestral PTPC of algal plants after divergingfrom the ancestor of vascular plant PTPC. To solve this contradiction, we reconstructed the phylogeny of pepcase gene family. Our result showed that both PTPC and BTPC are derived from an ancestral lineage of gamma-proteobacteriapepcases, possibly via an ancient inter-kingdom horizontal gene transfer (HGT) from bacteria to the eukaryotic common ancestor of plants, protists and cellular slime mold. Our phylogenetic analysis also found 48other pepcase genes originated from inter-kingdom HGTs. These results imply that inter-kingdom HGTs played important roles in the evolution of the pepcase gene family and furthermore that HGTsare a more frequent evolutionary event than previouslythought. PMID:23251445

  20. Identification of Interactions between Abscisic Acid and Ribulose-1,5-Bisphosphate Carboxylase/Oxygenase

    PubMed Central

    Galka, Marek M.; Rajagopalan, Nandhakishore; Buhrow, Leann M.; Nelson, Ken M.; Switala, Jacek; Cutler, Adrian J.; Palmer, David R. J.; Loewen, Peter C.; Abrams, Suzanne R.; Loewen, Michele C.

    2015-01-01

    Abscisic acid ((+)-ABA) is a phytohormone involved in the modulation of developmental processes and stress responses in plants. A chemical proteomics approach using an ABA mimetic probe was combined with in vitro assays, isothermal titration calorimetry (ITC), x-ray crystallography and in silico modelling to identify putative (+)-ABA binding-proteins in crude extracts of Arabidopsis thaliana. Ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) was identified as a putative ABA-binding protein. Radiolabelled-binding assays yielded a Kd of 47 nM for (+)-ABA binding to spinach Rubisco, which was validated by ITC, and found to be similar to reported and experimentally derived values for the native ribulose-1,5-bisphosphate (RuBP) substrate. Functionally, (+)-ABA caused only weak inhibition of Rubisco catalytic activity (Ki of 2.1 mM), but more potent inhibition of Rubisco activation (Ki of ~ 130 μM). Comparative structural analysis of Rubisco in the presence of (+)-ABA with RuBP in the active site revealed only a putative low occupancy (+)-ABA binding site on the surface of the large subunit at a location distal from the active site. However, subtle distortions in electron density in the binding pocket and in silico docking support the possibility of a higher affinity (+)-ABA binding site in the RuBP binding pocket. Overall we conclude that (+)-ABA interacts with Rubisco. While the low occupancy (+)-ABA binding site and weak non-competitive inhibition of catalysis may not be relevant, the high affinity site may allow ABA to act as a negative effector of Rubisco activation. PMID:26197050

  1. Towards efficient photosynthesis: overexpression of Zea mays phosphoenolpyruvate carboxylase in Arabidopsis thaliana.

    PubMed

    Kandoi, Deepika; Mohanty, Sasmita; Govindjee; Tripathy, Baishnab C

    2016-12-01

    Plants with C4 photosynthesis are efficient in carbon assimilation and have an advantage over C3 photosynthesis. In C4 photosynthesis, the primary CO2 fixation is catalyzed by phosphoenolpyruvate carboxylase (PEPC). Here, we show that overexpression of Zea mays PEPC cDNA, under the control of (35)S promoter, in Arabidopsis thaliana resulted in ~7-10 fold higher protein abundance and ~7-10 fold increase in PEPC activity in the transgenic lines than that in the vector control. We suggest that overexpression of PEPC played an anaplerotic role to increase the supply of 4-carbon carboxylic acids, which provided carbon skeletons for increased amino acid and protein synthesis. Higher protein content must have been responsible for increased metabolic processes including chlorophyll biosynthesis, photosynthesis, and respiration. Consequently, the PEPC-overexpressed transgenic plants had higher chlorophyll content, enhanced electron transport rate (ETR), lower non-photochemical quenching (NPQ) of chlorophyll a fluorescence, and a higher performance index (PI) than the vector control. Consistent with these observations, the rate of CO2 assimilation, the starch content, and the dry weight of PEPC-overexpressed plants increased by 14-18 %, 10-18 %, and 6.5-16 %, respectively. Significantly, transgenics were tolerant to salt stress as they had increased ability to synthesize amino acids, including the osmolyte proline. NaCl (150 mM)-treated transgenic plants had higher variable to maximum Chl a fluorescence (F v/F m) ratio, higher PI, higher ETR, and lower NPQ than the salt-treated vector controls. These results suggest that expression of C4 photosynthesis enzyme(s) in a C3 plant can improve its photosynthetic capacity with enhanced tolerance to salinity stress.

  2. Coordinating Role of His216 in MgATP Binding and Cleavage in Pyruvate Carboxylase

    PubMed Central

    2015-01-01

    His216 is a well-conserved residue in pyruvate carboxylases and, on the basis of structures of the enzyme, appears to have a role in the binding of MgATP, forming an interaction with the 3′-hydroxyl group of the ribose ring. Mutation of this residue to asparagine results in a 9-fold increase in the Km for MgATP in its steady-state cleavage in the absence of pyruvate and a 3-fold increase in the Km for MgADP in its steady-state phosphorylation by carbamoyl phosphate. However, from single-turnover experiments of MgATP cleavage, the Kd of the enzyme·MgATP complex is essentially the same in the wild-type enzyme and H216N. Direct stopped-flow measurements of nucleotide binding and release using the fluorescent analogue FTP support these observations. However, the first-order rate constant for MgATP cleavage in the single-turnover experiments in H216N is only 0.75% of that for the wild-type enzyme, and thus, the MgATP cleavage step is rate-limiting in the steady state for H216N but not for the wild-type enzyme. Close examination of the structure of the enzyme suggested that His216 may also interact with Glu218, which in turn interacts with Glu305 to form a proton relay system involved in the deprotonation of bicarbonate. Single-turnover MgATP cleavage experiments with mutations of these two residues resulted in kinetic parameters similar to those observed in H216N. We suggest that the primary role of His216 is to coordinate the binding of MgATP and the deprotonation of bicarbonate in the reaction to form the putative carboxyphosphate intermediate by participation in a proton relay system involving Glu218 and Glu305. PMID:24460480

  3. Molecular Evolution of the Small Subunit of Ribulose Bisphosphate Carboxylase: Nucleotide Substitution and Gene Conversion

    PubMed Central

    Meagher, R. B.; Berry-Lowe, S.; Rice, K.

    1989-01-01

    The nucleotide sequences encoding the mature portion of 31 ribulose 1,5-bisphosphate carboxylase small subunit (SSU) genes from 17 genera of plants, green algae and cyanobacteria were examined. Among the 465 pairwise sequence comparisons, SSU multigene family members within the same species were more similar to each other in nonsynonymous or replacement nucleotide substitutions (RNS) than they were to SSU sequences in any other organism. The concerted evolution of independent SSU gene lineages within closely related plant species suggests that homogenization of RNS positions has occurred at least once in the life of each genus. The rate of expected RNS among mature SSU sequences was calculated to be 1.25 X 10(-9)/site/yr for the first 70 million years (MY) of divergence with a significant slowing to 0.13 X 10(-9)/site/yr for the next 1,400 MY. The data suggest that mature SSU sequences do not accumulate more than 20% differences in the RNS positions without compensatory changes in other components of this enzyme system. During the first 70 MY of divergence between species, the rate of expected synonymous or silent nucleotide substitutions (SNS) is ~6.6 X 10(-9)/site/yr. This is five times the RNS rate and is similar to the silent rate observed in animals. In striking contrast, SNS and RNS do not show this correlation among SSU gene family members within a species. A mechanism involving gene conversion within the exons followed by selection for biased gene conversion products with conservation of RNS positions and divergence of SNS positions is discussed. A SSU gene tree based on corrected RNS for 31 SSU sequences is presented and agrees well with a species tree based on morphological and cytogenetic traits for the 17 genera examined. SSU gene comparisons may be useful in predicting phylogenetic relationships and in some cases divergence times of various plant, algal and cyanobacterial species. PMID:2515110

  4. Passive monitoring of VOC in air using ACC. Final report, revised edition

    SciTech Connect

    Gesser, H.D.

    1997-12-31

    The report describes a project that developed a method of using activated carbon cloth (ACC) as a sampler for measuring volatile organic compounds (VOCs) in air. Strips of carbon cloth mounted in slide holders were tested as diffusive samplers exposed to known concentrations of standard chemicals in test chambers. Adsorbed chemicals were extracted with solvents and analyzed, and comparison was made between the results obtained with the cloths and with passive samplers. Preliminary tests were carried out to determine the effects on carbon cloth performance of relative humidity, methods of extraction, materials for storing the cloth strips, and the type of cloth. Appendices include tables of experimental data and a report on a novel method of solventless extraction based on a combination of solid phase micro-extraction and purge/trap methods.

  5. Overview of the arthritis Cost Consequence Evaluation System (ACCES): a pharmacoeconomic model for celecoxib.

    PubMed

    Pettitt, D; Goldstein, J L; McGuire, A; Schwartz, J S; Burke, T; Maniadakis, N

    2000-12-01

    Pharmacoeconomic analyses have become useful and essential tools for health care decision makers who increasingly require such analyses prior to placing a drug on a national, regional or hospital formulary. Previous health economic models of non-steroidal anti-inflammatory drugs (NSAIDs) have been restricted to evaluating a narrow range of agents within specific health care delivery systems using medical information derived from homogeneous clinical trial data. This paper summarizes the Arthritis Cost Consequence Evaluation System (ACCES)--a pharmacoeconomic model that has been developed to predict and evaluate the costs and consequences associated with the use of celecoxib in patients with arthritis, compared with other NSAIDs and NSAIDs plus gastroprotective agents. The advantage of this model is that it can be customized to reflect local practice patterns, resource utilization and costs, as well as provide context-specific health economic information to a variety of providers and/or decision makers.

  6. Sequencing of allotetraploid cotton (Gossypium hirsutum L. acc. TM-1) provides a resource for fiber improvement.

    PubMed

    Zhang, Tianzhen; Hu, Yan; Jiang, Wenkai; Fang, Lei; Guan, Xueying; Chen, Jiedan; Zhang, Jinbo; Saski, Christopher A; Scheffler, Brian E; Stelly, David M; Hulse-Kemp, Amanda M; Wan, Qun; Liu, Bingliang; Liu, Chunxiao; Wang, Sen; Pan, Mengqiao; Wang, Yangkun; Wang, Dawei; Ye, Wenxue; Chang, Lijing; Zhang, Wenpan; Song, Qingxin; Kirkbride, Ryan C; Chen, Xiaoya; Dennis, Elizabeth; Llewellyn, Danny J; Peterson, Daniel G; Thaxton, Peggy; Jones, Don C; Wang, Qiong; Xu, Xiaoyang; Zhang, Hua; Wu, Huaitong; Zhou, Lei; Mei, Gaofu; Chen, Shuqi; Tian, Yue; Xiang, Dan; Li, Xinghe; Ding, Jian; Zuo, Qiyang; Tao, Linna; Liu, Yunchao; Li, Ji; Lin, Yu; Hui, Yuanyuan; Cao, Zhisheng; Cai, Caiping; Zhu, Xiefei; Jiang, Zhi; Zhou, Baoliang; Guo, Wangzhen; Li, Ruiqiang; Chen, Z Jeffrey

    2015-05-01

    Upland cotton is a model for polyploid crop domestication and transgenic improvement. Here we sequenced the allotetraploid Gossypium hirsutum L. acc. TM-1 genome by integrating whole-genome shotgun reads, bacterial artificial chromosome (BAC)-end sequences and genotype-by-sequencing genetic maps. We assembled and annotated 32,032 A-subgenome genes and 34,402 D-subgenome genes. Structural rearrangements, gene loss, disrupted genes and sequence divergence were more common in the A subgenome than in the D subgenome, suggesting asymmetric evolution. However, no genome-wide expression dominance was found between the subgenomes. Genomic signatures of selection and domestication are associated with positively selected genes (PSGs) for fiber improvement in the A subgenome and for stress tolerance in the D subgenome. This draft genome sequence provides a resource for engineering superior cotton lines.

  7. Reward sensitivity modulates brain activity in the prefrontal cortex, ACC and striatum during task switching.

    PubMed

    Fuentes-Claramonte, Paola; Ávila, César; Rodríguez-Pujadas, Aina; Ventura-Campos, Noelia; Bustamante, Juan C; Costumero, Víctor; Rosell-Negre, Patricia; Barrós-Loscertales, Alfonso

    2015-01-01

    Current perspectives on cognitive control acknowledge that individual differences in motivational dispositions may modulate cognitive processes in the absence of reward contingencies. This work aimed to study the relationship between individual differences in Behavioral Activation System (BAS) sensitivity and the neural underpinnings involved in processing a switching cue in a task-switching paradigm. BAS sensitivity was hypothesized to modulate brain activity in frontal regions, ACC and the striatum. Twenty-eight healthy participants underwent fMRI while performing a switching task, which elicited activity in fronto-striatal regions during the processing of the switch cue. BAS sensitivity was negatively associated with activity in the lateral prefrontal cortex, anterior cingulate cortex and the ventral striatum. Combined with previous results, our data indicate that BAS sensitivity modulates the neurocognitive processes involved in task switching in a complex manner depending on task demands. Therefore, individual differences in motivational dispositions may influence cognitive processing in the absence of reward contingencies.

  8. Reward Sensitivity Modulates Brain Activity in the Prefrontal Cortex, ACC and Striatum during Task Switching

    PubMed Central

    Fuentes-Claramonte, Paola; Ávila, César; Rodríguez-Pujadas, Aina; Ventura-Campos, Noelia; Bustamante, Juan C.; Costumero, Víctor; Rosell-Negre, Patricia; Barrós-Loscertales, Alfonso

    2015-01-01

    Current perspectives on cognitive control acknowledge that individual differences in motivational dispositions may modulate cognitive processes in the absence of reward contingencies. This work aimed to study the relationship between individual differences in Behavioral Activation System (BAS) sensitivity and the neural underpinnings involved in processing a switching cue in a task-switching paradigm. BAS sensitivity was hypothesized to modulate brain activity in frontal regions, ACC and the striatum. Twenty-eight healthy participants underwent fMRI while performing a switching task, which elicited activity in fronto-striatal regions during the processing of the switch cue. BAS sensitivity was negatively associated with activity in the lateral prefrontal cortex, anterior cingulate cortex and the ventral striatum. Combined with previous results, our data indicate that BAS sensitivity modulates the neurocognitive processes involved in task switching in a complex manner depending on task demands. Therefore, individual differences in motivational dispositions may influence cognitive processing in the absence of reward contingencies. PMID:25875640

  9. Isolated spinach ribulose-1,5-bisphosphate carboxylase/oxygenase large subunit .sup..epsilon. N-methyltransferase and method of inactivating ribulose-1,5-bisphosphatase carboxylase/oxygenase large subunit .sup..epsilon. N-methyltransferase activity

    DOEpatents

    Houtz, Robert L.

    1999-01-01

    The gene sequence for ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) large subunit (LS) .sup..epsilon. N-methyltransferase (protein methylase III or Rubisco LSMT) from a plant which has a des(methyl) lysyl residue in the LS is disclosed. In addition, the full-length cDNA clones for Rubisco LSMT are disclosed. Transgenic plants and methods of producing same which have the Rubisco LSMT gene inserted into the DNA are also provided. Further, methods of inactivating the enzymatic activity of Rubisco LSMT are also disclosed.

  10. Leucine 332 influences the CO2/O2 specificity factor of ribulose-1,5-bisphosphate carboxylase/oxygenase from Anacystis nidulans.

    PubMed Central

    Lee, G. J.; McDonald, K. A.; McFadden, B. A.

    1993-01-01

    The role of Leu 332 in ribulose-1,5-bisphosphate carboxylase/oxygenase from the cyanobacterium Anacystis nidulans was investigated by site-directed mutagenesis. Substitutions of this residue with Met, Ile, Val, Thr, or Ala decreased the CO2/O2 specificity factor by as much as 67% and 96% for the Ile mutant in the presence of Mg2+ and Mn2+, respectively. For the Met, Ile, and Ala mutants in the presence of Mg2+, no loss of oxygenase activity was observed despite the loss of greater than 65% of the carboxylase activity relative to the wild-type enzyme. In the presence of Mn2+, carboxylase activities for mutant enzymes were reduced to approximately the same degree as was observed in the presence of Mg2+, although oxygenase activities were also reduced to similar extents as carboxylase activities. Only minor changes in Km(RuBP) were observed for all mutants in the presence of Mg2+ relative to the wild-type enzyme, indicating that Leu 332 does not function in RuBP binding. These results suggest that in the presence of Mg2+, Leu 332 contributes to the stabilization of the transition state for the carboxylase reaction, and demonstrate that it is possible to affect only one of the activities of this bifunctional enzyme. PMID:8358297

  11. Unequal synthesis and differential degradation of propionyl CoA carboxylase subunits in cells from normal and propionic acidemia patients.

    PubMed Central

    Ohura, T; Kraus, J P; Rosenberg, L E

    1989-01-01

    We have characterized further the molecular basis of human inherited propionyl CoA carboxylase deficiency by measuring steady state levels of the mRNAs coding for the enzyme's two protein subunits (alpha and beta) and by estimating initial synthesis and steady state levels of the protein subunits in skin fibroblasts from controls and affected patients. We studied cell lines from both major complementation groups (pccA and pccBC) corresponding, respectively, to defects in the carboxylase's alpha and beta subunits. Analysis of pccA lines revealed the absence of alpha chain mRNA in three and an abnormally small alpha-mRNA in a fourth. Despite the presence of normal beta-mRNA in each of these pccA lines, there was complete absence of both alpha and beta protein subunits under steady state conditions, even though new synthesis and mitochondrial import of beta precursors was normal. Results in nine pccBC lines revealed normal alpha mRNA in each, while the amounts of beta-mRNA were distinctly reduced in every case. Correspondingly, alpha protein subunits were present in normal amounts at steady-state, but beta subunits were uniformly decreased. In addition, in six of the nine beta deficient cell lines, partially degraded beta-subunits were observed. To help interpret these results, synthesis and stability of carboxylase subunits were studied in intact HeLa cells using a pulse-chase protocol. Whereas alpha chains were stable over the four hour interval studied, beta chains--initially synthesized in large excess over alpha chains--were degraded rapidly reaching equivalence with alpha chains after two hours.(ABSTRACT TRUNCATED AT 250 WORDS) Images Figure 1 Figure 2 Figure 3 Figure 4 PMID:2741949

  12. Immobilization of D-ribulose-1,5-bisphosphate carboxylase/oxygenase: a step toward carbon dioxide fixation bioprocess.

    PubMed

    Chakrabarti, Subhra; Bhattacharya, Sumana; Bhattacharya, Sanjoy K

    2003-03-20

    Immobilization of D-ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) from spinach leaves is described. This enzyme enables the fixation of carbon dioxide on a five-carbon sugar D-ribulose-1,5-bisphosphate (RuBP). Two different immobilization methods were employed: dicyclohexylcarbodiimide coupling on nylon membrane matrix and dimethylpimelimidate immobilization on protein A agarose. The reusability of immobilized enzymes, coupling efficiency, and temperature-activity relationship of soluble and immobilized Rubisco are presented. The immobilization imparted greater thermal and storage stability. The thermal deactivation rates of the immobilized enzymes were considerably lower than those of the soluble enzyme.

  13. Improved Performance of an Air Cooled Condenser (ACC) Using SPX Wind Guide Technology at Coal-Based Thermoelectric Power Plants

    SciTech Connect

    Ken Mortensen

    2010-12-31

    This project added a new airflow enhancement technology to an existing ACC cooling process at a selected coal power plant. Airflow parameters and efficiency improvement for the main plant cooling process using the applied technology were determined and compared with the capabilities of existing systems. The project required significant planning and pre-test execution in order to reach the required Air Cooled Condenser system configuration for evaluation. A host Power Plant ACC system had to be identified, agreement finalized, and addition of the SPX ACC Wind Guide Technology completed on that site. Design of the modification, along with procurement, fabrication, instrumentation, and installation of the new airflow enhancement technology were executed. Baseline and post-modification cooling system data was collected and evaluated. The improvement of ACC thermal performance after SPX wind guide installation was clear. Testing of the improvement indicates there is a 5% improvement in heat transfer coefficient in high wind conditions and 1% improvement at low wind speed. The benefit increased with increasing wind speed. This project was completed on schedule and within budget.

  14. 24 CFR 884.105 - Maximum total ACC commitment and project account (private-owner/PHA projects).

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... 24 Housing and Urban Development 4 2010-04-01 2010-04-01 false Maximum total ACC commitment and project account (private-owner/PHA projects). 884.105 Section 884.105 Housing and Urban Development Regulations Relating to Housing and Urban Development (Continued) OFFICE OF THE ASSISTANT SECRETARY...

  15. Draft Genome Sequence of Edwardsiellapiscicida Strain ACC35.1 Isolated from Diseased Turbot (Scophthalmus maximus) in Europe.

    PubMed

    Buján, Noemí; Toranzo, Alicia E; Magariños, Beatriz

    2017-02-16

    Edwardsiella piscicida is a bacterial fish pathogen with a high degree of virulence. The strain ACC35.1 was isolated from diseased turbot in Europe. The draft genome sequence comprises 3.84 Mb with a G+C content of 59.8% and >3,450 protein-coding genes.

  16. Characterization of ACC deaminase gene in Pseudomonas entomophila strain PS-PJH isolated from the rhizosphere soil.

    PubMed

    Kamala-Kannan, Seralathan; Lee, Kui-Jae; Park, Seung-Moon; Chae, Jong-Chan; Yun, Bong-Sik; Lee, Yong Hoon; Park, Yool-Jin; Oh, Byung-Taek

    2010-04-01

    The enzyme 1-aminocyclopropane-1-carboxylate (ACC) deaminase cleaves the ethylene precursor ACC into alpha-ketobutyrate and ammonia. The decreased level of ethylene allows the plant to be more resistant to a wide environmental stress including plant pathogens. In the present study, we characterized the ACC deaminase activity of a Pseudomonas entomophila strain PS-PJH isolated from the red pepper rhizosphere region of red pepper grown at Jinan, Korea. The isolate produced 23.8 +/- 0.4 micromol of alpha-ketobutyrate/mg of protein/h during ACC deamination under in vitro conditions. Polymerase chain reaction for acdS gene showed that the isolated P. entomophila strain PS-PJH carry sequences similar to the known acdS genes. Results of the multiple sequence alignment revealed >99% identity (nucleotide and amino acid) with acdS gene of Pseudomonas putida strains AM15 and UW4. The isolated bacteria promoted 43.3 and 34.1% of growth in Raphanus sativus and Lactuca sativa plants, respectively. Based on the 16S-23S internal transcribed spacer region sequences, the isolate was identified as P. entomophila. To the best of our knowledge this is the first study to report the acdS gene in P. entomophila.

  17. Tuning the light in senior care: Evaluating a trial LED lighting system at the ACC Care Center in Sacramento, CA

    SciTech Connect

    Davis, Robert G.; Wilkerson, Andrea M.

    2016-08-31

    This report summarizes the results from a trial installation of light-emitting diode (LED) lighting systems in several spaces within the ACC Care Center in Sacramento, CA. The Sacramento Municipal Utility District (SMUD) coordinated the project and invited the U.S. Department of Energy (DOE) to document the performance of the LED lighting systems as part of a GATEWAY evaluation. DOE tasked the Pacific Northwest National Laboratory (PNNL) to conduct the investigation. SMUD and ACC staff coordinated and completed the design and installation of the LED systems, while PNNL and SMUD staff evaluated the photometric performance of the systems. ACC staff also track behavioral and health measures of the residents; some of those results are reported here, although PNNL staff were not directly involved in collecting or interpreting those data. The trial installation took place in a double resident room and a single resident room, and the corridor that connects those (and other) rooms to the central nurse station. Other spaces in the trial included the nurse station, a common room called the family room located near the nurse station, and the ACC administrator’s private office.

  18. Implementation and efficiency analysis of parallel computation using OpenACC: a case study using flow field simulations

    NASA Astrophysics Data System (ADS)

    Zhang, Shanghong; Yuan, Rui; Wu, Yu; Yi, Yujun

    2016-01-01

    The Open Accelerator (OpenACC) application programming interface is a relatively new parallel computing standard. In this paper, particle-based flow field simulations are examined as a case study of OpenACC parallel computation. The parallel conversion process of the OpenACC standard is explained, and further, the performance of the flow field parallel model is analysed using different directive configurations and grid schemes. With careful implementation and optimisation of the data transportation in the parallel algorithm, a speedup factor of 18.26× is possible. In contrast, a speedup factor of just 11.77× was achieved with the conventional Open Multi-Processing (OpenMP) parallel mode on a 20-kernel computer. These results demonstrate that optimised feature settings greatly influence the degree of speedup, and models involving larger numbers of calculations exhibit greater efficiency and higher speedup factors. In addition, the OpenACC parallel mode is found to have good portability, making it easy to implement parallel computation from the original serial model.

  19. Draft Genome Sequence of Edwardsiella piscicida Strain ACC35.1 Isolated from Diseased Turbot (Scophthalmus maximus) in Europe

    PubMed Central

    Toranzo, Alicia E.; Magariños, Beatriz

    2017-01-01

    ABSTRACT   Edwardsiella piscicida is a bacterial fish pathogen with a high degree of virulence. The strain ACC35.1 was isolated from diseased turbot in Europe. The draft genome sequence comprises 3.84 Mb with a G+C content of 59.8% and >3,450 protein-coding genes. PMID:28209828

  20. NATO Network Enabled Capability (NNEC) challenges: Why NATO Air Command and Control System (ACCS) might be a good case?

    DTIC Science & Technology

    2011-06-01

    Picture ( RAP ), Air Tasking Order (ATO), Air Coordination order (ACO), etc. 2. Non Functional: providing all other functionalities, e.g. IA/security...with their respective characteristics in the project management behavior and environmental parameters. It should be noted that SOA implementation... influence its transformation. The roles and perspectives of the main ACCS transformation actors are described in this section. Several stakeholders

  1. Expression of recombinant AccMRJP1 protein from royal jelly of Chinese honeybee in Pichia pastoris and its proliferation activity in an insect cell line

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Main royal jelly protein 1 (MRJP1) is the most abundant member of the main royal jelly protein (MRJP) family among honeybees. Mature MRJP1 cDNA of the Chinese honeybee (Apis cerana cerana MRJP1, or AccMRJP1) was expressed in Pichia pastoris. SDS-PAGE showed that recombinant AccMRJP1 was identical in...

  2. The cloned 1-aminocyclopropane-1-carboxylate (ACC) deaminase gene from Sinorhizobium sp. strain BL3 in Rhizobium sp. strain TAL1145 promotes nodulation and growth of Leucaena leucocephala.

    PubMed

    Tittabutr, Panlada; Awaya, Jonathan D; Li, Qing X; Borthakur, Dulal

    2008-06-01

    The objective of this study was to determine the role of 1-aminocyclopropane-1-carboxylate (ACC) deaminase of symbionts in nodulation and growth of Leucaena leucocephala. The acdS genes encoding ACC deaminase were cloned from Rhizobium sp. strain TAL1145 and Sinorhizobium sp. BL3 in multicopy plasmids, and transferred to TAL1145. The BL3-acdS gene greatly enhanced ACC deaminase activity in TAL1145 compared to the native acdS gene. The transconjugants of TAL1145 containing the native or BL3 acdS gene could grow in minimal media containing 1.5mM ACC, whereas BL3 could tolerate up to 3mM ACC. The TAL1145 acdS gene was inducible by mimosine and not by ACC, while the BL3 acdS gene was highly inducible by ACC and not by mimosine. The transconjugants of TAL1145 containing the native- and BL3-acdS genes formed nodules with greater number and sizes, and produced higher root mass on L. leucocephala than by TAL1145. This study shows that the introduction of multiple copies of the acdS gene increased ACC deaminase activities of TAL1145 and enhanced its symbiotic efficiency on L. leucocephala.

  3. Inactivation of ribulosebisphosphate carboxylase/oxygenase from Rhodospirillum rubrum and spinach with the new affinity label 2-bromo-1,5-dihydroxy-3-pentanone 1,5-bisphosphate

    SciTech Connect

    Donnelly, M.I.; Hartman, F.C.

    1981-11-16

    In an attempt to identify the active-site base believed to initiate catalysis by ribulosebisphosphate carboxylase, we have synthesized 2-bromo-1, 5-dihydroxy-3-pentanone 1,5-bisphosphate, a reactive analogue of a postulated intermediate of carboxylation. Although highly unstable, this compound can be shown to inactivate the carboxylases from both Rhodospirillum rubrum and spinach rapidly and irreversibly. Inactivation follows pseudo first-order kinetics, shows rate saturation and is greatly reduced by saturating amounts of the competitive inhibitor, 2-carboxyribitol 1,5-bisphosphate. The incorporation of reagent, quantified by reducing the modified carboxylases with (/sup 3/H)NaBH/sub 4/, shows that inactivation results from the modification of approximately one residue per catalytic subunit of the Rhodospirillum rubrum enzyme and less than one residue per protomeric unit of the spinach enzyme.

  4. Effect of Light and NO3− on Wheat Leaf Phosphoenolpyruvate Carboxylase Activity

    PubMed Central

    Le Van Quy; Foyer, Christine; Champigny, Marie-Louise

    1991-01-01

    Phosphoenolpyruvate carboxylase (PEPcase) activity was studied in excised leaves of wheat (Triticum aestivum L.) in the dark and in the light, in presence of either N-free (low-NO3− leaves) or 40 millimolar KNO3 (high-NO3− leaves) nutrient solutions. PEPcase activity increased to 2.7-fold higher than that measured in dark-adapted tissue (control) during the first 60 minutes and continued to increase more slowly to 3.8-fold that of the control. This level was reached after 200 minutes exposure of the leaves to light and high NO3−. In contrast, the lower rate of increase recorded for low-NO3− leaves ceased after 60 minutes of exposure to light at 2.3-fold the control level. The short-term NO3− effect increased linearly with the level of NO3− uptake. In immunoprecipitation experiments, the antibody concentration for PEPcase precipitation increased with the protein extracts from the different treatments in the order: control, illuminated low-NO3− leaves, illuminated high-NO3− leaves. This order also applied with regard to a decreasing sensitivity to malate and an increasing stimulation by okadaic acid (an inhibitor of P-protein phosphatases). Following these studies, 32P labeling experiments were carried out in vivo. These showed that the light-induced change in the properties of the PEPcase was due to an alteration in the phosphorylation state of the protein and that this effect was enhanced in high-NO3− conditions. Based on the responses of PEPcase and sucrose phosphate synthase in wheat leaves to light and NO3−, an interpretation of the role of NO3− as either an inhibitor of P-protein phosphatase(s) or activator of protein kinase(s) is inferred. In the presence of NO3−, the phosphorylation state of both PEPcase and sucrose phosphate synthase is increased. This causes activation of the former enzyme and inhibition of the latter. We suggest that NO3− modulates the relative protein kinase/protein phosphatase ratio to favor increased

  5. Regulation of bovine pyruvate carboxylase mRNA and promoter expression by thermal stress.

    PubMed

    White, H M; Koser, S L; Donkin, S S

    2012-09-01

    Pyruvate carboxylase (PC) catalyzes the rate-limiting step in gluconeogenesis from lactate and is a determinant of tricarboxylic acid cycle carbon flux. Bovine PC 5' untranslated region (UTR) mRNA variants are the products of a single PC gene containing 3 promoter regions (P3, P2, and P1, 5' to 3') that are responsive to physiological and nutritional stressors. The objective of this study was to determine the direct effects of thermal stress on PC mRNA and gene expression in bovine hepatocyte monolayer cultures, rat hepatoma (H4IIE) cells, and Madin-Darby bovine kidney epithelial (MDBK) cells. Hepatocytes were isolated from 3 Holstein bull calves and used to prepare monolayer cultures. Rat hepatoma cells and MDBK cells were obtained from American Type Culture Collection, Manassas, VA. Beginning 24 h after initial seeding, cells were subjected to either 37°C (control) or 42°C (thermal stress) for 24 h. Treatments were applied in triplicate in a minimum of 3 independent cell preparations. For bovine primary hepatocytes, endogenous expression of bovine PC mRNA increased (P < 0.1) with 24 h of thermal stress (1.31 vs. 2.79 ± 0.49, arbitrary units, control vs. thermal stress, respectively), but there was no change (P ≥ 0.1) in cytosolic phosphoenolpyruvate carboxykinase (PEPCK-C) mRNA expression. Similarly, exposure of MDBK cells to thermal stress increased (P < 0.1) expression of bovine PC mRNA without altering (P ≥ 0.1) PEPCK-C mRNA expression. Conversely, there was no effect (P ≥ 0.1) of thermal stress on endogenous rat PC (0.47 vs. 0.30 ± 0.08, control vs. thermal stress) or PEPCK-C (1.61 vs. 1.20 ± 0.48, arbitrary units, control vs. thermal stress, respectively) mRNA expressions in H4IIE cells. To further investigate the regulation of PC, H4IIE cells were transiently transfected with bovine promoter-luciferase constructs containing either P1, P2, or P3, and exposed to thermal stress for 23 h. Activity of P1 was suppressed (P < 0.1) 5-fold, activity of P2

  6. Brønsted analysis reveals Lys218 as the carboxylase active site base that deprotonates vitamin K hydroquinone to initiate vitamin K-dependent protein carboxylation.

    PubMed

    Rishavy, Mark A; Hallgren, Kevin W; Yakubenko, Anna V; Shtofman, Rebecca L; Runge, Kurt W; Berkner, Kathleen L

    2006-11-07

    The vitamin K-dependent (VKD) carboxylase converts Glu's to carboxylated Glu's in VKD proteins to render them functional in a broad range of physiologies. The carboxylase uses vitamin K hydroquinone (KH(2)) epoxidation to drive Glu carboxylation, and one of its critical roles is to provide a catalytic base that deprotonates KH(2) to allow epoxidation. A long-standing model invoked Cys as the catalytic base but was ruled out by activity retention in a mutant where every Cys is substituted by Ala. Inhibitor analysis of the cysteine-less mutant suggested that the base is an activated amine [Rishavy et al. (2004) Proc. Natl. Acad. Sci. U.S.A. 101, 13732-13737], and in the present study, we used an evolutionary approach to identify candidate amines, which revealed His160, His287, His381, and Lys218. When mutational analysis was performed using an expression system lacking endogenous carboxylase, the His to Ala mutants all showed full epoxidase activity but K218A activity was not detectable. The addition of exogenous amines restored K218A activity while having little effect on wild type carboxylase, and pH studies indicated that rescue was dependent upon the basic form of the amine. Importantly, Brønsted analysis that measured the effect of amines with different pK(a) values showed that K218A activity rescue depended upon the basicity of the amine. The combined results provide strong evidence that Lys218 is the essential base that deprotonates KH(2) to initiate the reaction. The identification of this base is an important advance in defining the carboxylase active site and has implications regarding carboxylase membrane topology and the feedback mechanism by which the Glu substrate regulates KH(2) oxygenation.

  7. Kinetic Variance of Ribulose-1,5-bisphosphate Carboxylase/Oxygenase Isolated from Diverse Taxonomic Sources 1

    PubMed Central

    Kent, Samuel Sherrill; Tomany, Michael John

    1984-01-01

    Two dual label methods were used to investigate kinetic variability of ribulose 1,5-bisphosphate (RuBP) carboxylase/oxygenase (EC 4.1.1.39). In addition to using [1-14C,5-3H]RuBP (method 1), we describe here the detailed assay with 14CO2 and [5-3H]RuBP (method 2), which generates [3H,14C]3-phosphoglyceric acid and unlabeled (noncontaminating) phosphoglycolate; the carboxylase/oxygenase activity ratio (vc/vo) is calculated from 3H/14C ratios of substrates and products. vc/vo was found to be a linear function of [CO2]/[O2], constant over a 4-minute assay interval, and invariant of the degree of enzyme activity. Accurately measurable vc/vo ratios range from approximately 0.3 to 6. The Km and Vmax of both enzymes may be determined as a composite constant, VcKo/VoKc. By method 2, the directly compared, relative values at 40 micromolar CO2 and 1240 micromolar O2 were: Spinacia oleracea (74), Chlorella pyrenoidosa (31), Plectonema boryanum (32), and Rhodospirillum rubrum (8). With method 1, the values for S. oleracea and R. rubrum were 75, and 9, respectively. Under tight experimental controls, the absolute value for S. oleracea was 69 ± 3. PMID:16663680

  8. Varying Photoperiod, Ribulose 1,5-Bisphosphate Carboxylase/Oxygenase and CO2 Uptake in Thalassiosira fluviatilis (Bacillariophyceae) 1

    PubMed Central

    Hobson, Louis A.; Morris, W. James.; Guest, Kathryn P.

    1985-01-01

    The purpose of this research was to test the hypothesis that acclimation of the unicellular marine alga, Thalassiosira fluviatilis Hustedt, to short photoperiods results in decreased cellular concentrations of ribulose 1,5-bisphosphate carboxylase/oxygenase and decreased rates of light-saturated CO2 uptake. Cells were acclimated to photoperiods of 6:18, 12:12, and 18:6 h:h light:dark, and concentrations of the large subunit of the enzyme and responses of CO2 uptake to varying irradiance were measured. Concentrations of the large subunit, which weighed approximately 50 kilodaltons, were conserved while rates of CO2 uptake under light saturation and limitation, and cellular contents of chlorophyll a increased as photoperiod decreased. Apparently, these cells acclimate to short photoperiods by increasing rates of CO2 uptake under saturating irradiances by increasing in vivo activation of ribulose 1,5-bisphosphate carboxylase/oxygenase. Also, chlorophyll-specific concentrations and specific activities of the enzyme appear to be lower and higher, respectively, in diatomaceous algae than in higher plants. Images Fig. 2 PMID:16664500

  9. Improving derivatization efficiency of BMAA utilizing AccQ-Tag in a complex cyanobacterial matrix.

    PubMed

    Eriksson, Johan; Jonasson, Sara; Papaefthimiou, Dimitra; Rasmussen, Ulla; Bergman, Birgitta

    2009-01-01

    Two different assays have been developed and used in order to investigate the optimal conditions for derivatization and detection of acid beta-N-methyl-amino-L-alanine (BMAA) in a cyanobacterial sample. BMAA was extracted from cyanobacterial cultures both from the cytosolic ("free") fraction and in the precipitated ("protein") fraction using a newly developed extraction scheme and the sample matrix was standardized according to protein concentration to ensure the highest possible derivative yield. A rapid and sensitive HPLC method for fluorescence detection of the non-protein amino acid BMAA in cyanobacteria, utilizing the Waters AccQ-Tag chemistry and Chromolith Performance RP-18e columns was developed. Using this new method and utilizing a different buffer system and column than that recommended by Waters, we decreased the time between injections by 75%. The limit of quantification was determined to be 12 nmol and limit of detection as 120 fmol. The linear range was in the range of 8.5 nmol-84 pmol. Accuracy and precision were well within FDA guidelines for bioanalysis.

  10. Anatomically shaped cranial collimation (ACC) for lateral cephalometric radiography: a technical report.

    PubMed

    Hoogeveen, R C; van der Stelt, P F; Berkhout, W E R

    2014-01-01

    Lateral cephalograms in orthodontic practice display an area cranial of the base of the skull that is not required for diagnostic evaluation. Attempts have been made to reduce the radiation dose to the patient using collimators combining the shielding of the areas above the base of the skull and below the mandible. These so-called "wedge-shaped" collimators have not become standard equipment in orthodontic offices, possibly because these collimators were not designed for today's combination panoramic-cephalometric imaging systems. It also may be that the anatomical variability of the area below the mandible makes this area unsuitable for standardized collimation. In addition, a wedge-shaped collimator shields the cervical vertebrae; therefore, assessment of skeletal maturation, which is based on the stage of development of the cervical vertebrae, cannot be performed. In this report, we describe our investigations into constructing a collimator to be attached to the cephalostat and shield the cranial area of the skull, while allowing the visualization of diagnostically relevant structures and markedly reducing the size of the irradiated area. The shape of the area shielded by this "anatomically shaped cranial collimator" (ACC) was based on mean measurements of cephalometric landmarks of 100 orthodontic patients. It appeared that this collimator reduced the area of irradiation by almost one-third without interfering with the imaging system or affecting the quality of the image. Further research is needed to validate the clinical efficacy of the collimator.

  11. A portable platform for accelerated PIC codes and its application to GPUs using OpenACC

    NASA Astrophysics Data System (ADS)

    Hariri, F.; Tran, T. M.; Jocksch, A.; Lanti, E.; Progsch, J.; Messmer, P.; Brunner, S.; Gheller, C.; Villard, L.

    2016-10-01

    We present a portable platform, called PIC_ENGINE, for accelerating Particle-In-Cell (PIC) codes on heterogeneous many-core architectures such as Graphic Processing Units (GPUs). The aim of this development is efficient simulations on future exascale systems by allowing different parallelization strategies depending on the application problem and the specific architecture. To this end, this platform contains the basic steps of the PIC algorithm and has been designed as a test bed for different algorithmic options and data structures. Among the architectures that this engine can explore, particular attention is given here to systems equipped with GPUs. The study demonstrates that our portable PIC implementation based on the OpenACC programming model can achieve performance closely matching theoretical predictions. Using the Cray XC30 system, Piz Daint, at the Swiss National Supercomputing Centre (CSCS), we show that PIC_ENGINE running on an NVIDIA Kepler K20X GPU can outperform the one on an Intel Sandy bridge 8-core CPU by a factor of 3.4.

  12. AccERK2, a map kinase gene from Apis cerana cerana, plays roles in stress responses, developmental processes, and the nervous system.

    PubMed

    Li, Yuzhen; Zhang, Liang; Kang, Mingjiang; Guo, Xingqi; Baohua Xu

    2012-03-01

    Extracellular signal-regulated kinase (ERK), a mitogen-activated protein kinase (MAPK), plays roles in a variety of cellular responses. However, limited information is available on the relationship between ERKs and environmental stresses. In this report, an ERK gene, AccERK2, was cloned and characterized from Apis cerana cerana. Polypeptide sequence alignment revealed that the single-copied AccERK2 shares high identity with other known ERKs and contains the typical conserved Thr-Glu-Tyr (TEY) motif in its activation loop. Genomic sequence analysis revealed that the seven exons of AccERK2 are interrupted by six introns, and the seventh intron is located in the 3' untranslated region. Semi-quantitative reverse transcription (RT-PCR) indicated that AccERK2 was expressed at higher levels in the larval and pupal stages than in the adult stage. AccERK2 was also most highly expressed in the brain. The expression of AccERK2 was induced by abiotic stresses, including heat, ion irradiation, oxidative stress, and heavy metal ions. Based on these results, it appears that AccERK2 in A. cerana cerana participates in developmental processes, the nervous system, and responses to environmental stressors.

  13. Bringing measurement and management science to the cath laboratory: the National Cardiovascular Data Registry (ACC-NCDR) and the Cardiac Catheterization Laboratory Continuous Quality Improvement Toolkit (ACC-CathKIT).

    PubMed

    Dehmer, Gregory J; Elma, MaryAnne; Hewitt, Kathleen; Brindis, Ralph G

    2004-01-01

    Diagnostic cardiac catheterization and percutaneous coronary interventions are widely performed for the evaluation and treatment of patients with cardiac disease. Because of high utilization, cost, and complication rates, invasive cardiac procedures are closely monitored and frequently measured using national benchmark databases and public reports. Before decision makers can accept these data and reports as accurate, it is necessary that the measurement process be performed correctly. However, collecting and measuring data is only the first step and does not automatically lead to improvements in quality. For an improvement to occur, a continuous quality improvement effort must exist to transform data into improved outcomes for patients. Recognizing the need to supply healthcare providers with methods and standards for measurement reporting and tools to assist facilities in the development of effective continuous quality improvement efforts, the American College of Cardiology developed the National Cardiovascular Data Registry (ACC-NCDR). Subsequently, the American College of Cardiology Foundation, in cooperation with the Society for Cardiovascular Angiography and Interventions, the American College of Cardiovascular Administrators, and several other professional organizations, developed the ACC-Cardiac Catheterization Laboratory Continuous Quality Improvement Toolkit (ACC-CathKIT). The development and usefulness of these products is described in this paper.

  14. Characterization of a Decapentapletic Gene (AccDpp) from Apis cerana cerana and Its Possible Involvement in Development and Response to Oxidative Stress

    PubMed Central

    Wang, Hongfang; Guo, Xulei; Guo, Xingqi; Sun, Qinghua; Xu, Baohua

    2016-01-01

    To tolerate many acute and chronic oxidative stress-producing agents that exist in the environment, organisms have evolved many classes of signal transduction pathways, including the transforming growth factor β (TGFβ) signal pathway. Decapentapletic gene (Dpp) belongs to the TGFβ superfamily, and studies on Dpp have mainly focused on its role in the regulation of development. No study has investigated the response of Dpp to oxidative pressure in any organism, including Apis cerana cerana (A. cerana cerana). In this study, we identified a Dpp gene from A. cerana cerana named AccDpp. The 5΄ flanking region of AccDpp had many transcription factor binding sites that relevant to development and stress response. AccDpp was expressed at all stages of A. cerana cerana, with its highest expression in 15-day worker bees. The mRNA level of AccDpp was higher in the poison gland and midgut than other tissues. Furthermore, the transcription of AccDpp could be repressed by 4°C and UV, but induced by other treatments, according to our qRT-PCR analysis. It is worth noting that the expression level of AccDpp protein was increased after a certain time when A. cerana cerana was subjected to all simulative oxidative stresses, a finding that was not completely consistent with the result from qRT-PCR. It is interesting that recombinant AccDpp restrained the growth of Escherichia coli, a function that might account for the role of the antimicrobial peptides of AccDpp. In conclusion, these results provide evidence that AccDpp might be implicated in the regulation of development and the response of oxidative pressure. The findings may lay a theoretical foundation for further genetic studies of Dpp. PMID:26881804

  15. Enzymic and physicochemical characterization of ribulose 1,5-bisphosphate carboxylase/oxygenase from diploid and tetraploid cultivars of perennial ryegrass

    SciTech Connect

    Rejda, J.M.; Johal, S.; Chollet, R.

    1981-09-01

    Homogeneous preparations of ribulose 1,5-bisphosphate carboxylase/oxygenase were isolated from several diploid and tetraploid cultivars of perennial ryegrass by three different purification protocols. The apparent K/sub m/ values for substrate CO/sub 2/ were essentially identical for the fully CO/sub 2//Mg/sup 2 +/-activated diploid and tetraploid enzymes, as were the kinetics for deactivation and activation of the CO/sub 2//Mg/sup 2 +/-activated and -depleted carboxylases, respectively. Similarly, virtually indistinguishable electrophoretic properties were observed for both the native and dissociated diploid and tetraploid ryegrass proteins, including native and subunit molecular weights and the isoelectric points of the native proteins and the large and small subunit component polypeptides. The quantity of carboxylase protein or total soluble leaf protein did not differ significantly between the diploid and tetraploid cultivars. Contrary to a previous report, these results indicate that increased ploidy level has had essentially no effect on the quantity or enzymic and physicochemical properties of ribulosebisphosphate carboxylase/oxygenase in perennial ryegrass.

  16. Assessment of Anterior Cingulate Cortex (ACC) and Left Cerebellar Metabolism in Asperger's Syndrome with Proton Magnetic Resonance Spectroscopy (MRS)

    PubMed Central

    Goji, Aya; Ito, Hiromichi; Mori, Kenji; Harada, Masafumi; Hisaoka, Sonoka; Toda, Yoshihiro; Mori, Tatsuo; Abe, Yoko; Miyazaki, Masahito; Kagami, Shoji

    2017-01-01

    Purpose Proton magnetic resonance spectroscopy (1H MRS) is a noninvasive neuroimaging method to quantify biochemical metabolites in vivo and it can serve as a powerful tool to monitor neurobiochemical profiles in the brain. Asperger’s syndrome (AS) is a type of autism spectrum disorder, which is characterized by impaired social skills and restrictive, repetitive patterns of interest and activities, while intellectual levels and language skills are relatively preserved. Despite clinical aspects have been well-characterized, neurometabolic profiling in the brain of AS remains to be clear. The present study used proton magnetic resonance spectroscopy (1H MRS) to investigate whether pediatric AS is associated with measurable neurometabolic abnormalities that can contribute new information on the neurobiological underpinnings of the disorder. Methods Study participants consisted of 34 children with AS (2–12 years old; mean age 5.2 (±2.0); 28 boys) and 19 typically developed children (2–11 years old; mean age 5.6 (±2.6); 12 boys) who served as the normal control group. The 1H MRS data were obtained from two regions of interest: the anterior cingulate cortex (ACC) and left cerebellum. Results In the ACC, levels of N-acetylaspartate (NAA), total creatine (tCr), total choline-containing compounds (tCho) and myo-Inositol (mI) were significantly decreased in children with AS compared to controls. On the other hand, no significant group differences in any of the metabolites were found in the left cerebellum. Neither age nor sex accounted for the metabolic findings in the regions. Conclusion The finding of decreased levels of NAA, tCr, tCho, and mI in the ACC but not in left cerebellar voxels in the AS, suggests a lower ACC neuronal density in the present AS cohort compared to controls. PMID:28060873

  17. Southern blight disease of tomato control by 1-aminocyclopropane-1-carboxylate (ACC) deaminase producing Paenibacillus lentimorbus B-30488.

    PubMed

    Dixit, Ritu; Agrawal, Lalit; Gupta, Swati; Kumar, Manoj; Yadav, Sumit; Chauhan, Puneet Singh; Nautiyal, Chandra Shekhar

    2016-01-01

    Tomato cultivation is highly susceptible for soil born diseases and among them southern blight disease caused by Scelerotium rolfsii is very common. For its management use of chemical fungicides is not very successful as their spores are able to survive for many years in the soil. As an alternative eco-friendly approach to control the disease antagonistic microbes are being characterized.Among them plant growth promoting rhizobacteria Paenibacillus lentimorbus B-30488 (B-30488) with antagonistic properties, multiple PGP attributes stress tolerance and ACC deaminase enzyme activity is characterized to decipher its mode of action against S. rolfsii under in vitro and in vivo conditions. In vitro results obtained from this study clearly demonstrate that B-30488 has ability to show antagonistic properties under different abiotic stresses against S. rolfsii. Similar results were also obtained from in vivo experiments where B-30488 inoculation has efficiently controlled the disease caused by S. rolfsii and improve the plant growth. Deleterious enhanced ethylene level in S. rolfsii infected plants was also ameliorated by inoculation of ACC deaminase producing B-30488. The ACC accumulation, ACO and ACS activities were also modulated in S. rolfsii infected plants. Results from defense enzymes and other biochemical attributes were also support the role of B-30488 inoculation in ameliorating the biotic stress caused by S. rolfsii in tomato plants. These results were further validated by pathogen related gene expression analysis by real time PCR. Overall results from the present study may be concluded that ACC deaminase producing B-30488 has ability to control the southern blight disease caused by S. rolfsii and commercial bioinoculant package may be developed.

  18. Southern blight disease of tomato control by 1-aminocyclopropane-1-carboxylate (ACC) deaminase producing Paenibacillus lentimorbus B-30488

    PubMed Central

    Dixit, Ritu; Agrawal, Lalit; Gupta, Swati; Kumar, Manoj; Yadav, Sumit; Chauhan, Puneet Singh; Nautiyal, Chandra Shekhar

    2016-01-01

    abstract Tomato cultivation is highly susceptible for soil born diseases and among them southern blight disease caused by Scelerotium rolfsii is very common. For its management use of chemical fungicides is not very successful as their spores are able to survive for many years in the soil. As an alternative eco-friendly approach to control the disease antagonistic microbes are being characterized.Among them plant growth promoting rhizobacteria Paenibacillus lentimorbus B-30488 (B-30488) with antagonistic properties, multiple PGP attributes stress tolerance and ACC deaminase enzyme activity is characterized to decipher its mode of action against S. rolfsii under in vitro and in vivo conditions. In vitro results obtained from this study clearly demonstrate that B-30488 has ability to show antagonistic properties under different abiotic stresses against S. rolfsii. Similar results were also obtained from in vivo experiments where B-30488 inoculation has efficiently controlled the disease caused by S. rolfsii and improve the plant growth. Deleterious enhanced ethylene level in S. rolfsii infected plants was also ameliorated by inoculation of ACC deaminase producing B-30488. The ACC accumulation, ACO and ACS activities were also modulated in S. rolfsii infected plants. Results from defense enzymes and other biochemical attributes were also support the role of B-30488 inoculation in ameliorating the biotic stress caused by S. rolfsii in tomato plants. These results were further validated by pathogen related gene expression analysis by real time PCR. Overall results from the present study may be concluded that ACC deaminase producing B-30488 has ability to control the southern blight disease caused by S. rolfsii and commercial bioinoculant package may be developed. PMID:26825539

  19. A comparison of native GPU computing versus OpenACC for implementing flow-routing algorithms in hydrological applications

    NASA Astrophysics Data System (ADS)

    Rueda, Antonio J.; Noguera, José M.; Luque, Adrián

    2016-02-01

    In recent years GPU computing has gained wide acceptance as a simple low-cost solution for speeding up computationally expensive processing in many scientific and engineering applications. However, in most cases accelerating a traditional CPU implementation for a GPU is a non-trivial task that requires a thorough refactorization of the code and specific optimizations that depend on the architecture of the device. OpenACC is a promising technology that aims at reducing the effort required to accelerate C/C++/Fortran code on an attached multicore device. Virtually with this technology the CPU code only has to be augmented with a few compiler directives to identify the areas to be accelerated and the way in which data has to be moved between the CPU and GPU. Its potential benefits are multiple: better code readability, less development time, lower risk of errors and less dependency on the underlying architecture and future evolution of the GPU technology. Our aim with this work is to evaluate the pros and cons of using OpenACC against native GPU implementations in computationally expensive hydrological applications, using the classic D8 algorithm of O'Callaghan and Mark for river network extraction as case-study. We implemented the flow accumulation step of this algorithm in CPU, using OpenACC and two different CUDA versions, comparing the length and complexity of the code and its performance with different datasets. We advance that although OpenACC can not match the performance of a CUDA optimized implementation (×3.5 slower in average), it provides a significant performance improvement against a CPU implementation (×2-6) with by far a simpler code and less implementation effort.

  20. Global Transcriptomic Analysis of Targeted Silencing of Two Paralogous ACC Oxidase Genes in Banana

    PubMed Central

    Xia, Yan; Kuan, Chi; Chiu, Chien-Hsiang; Chen, Xiao-Jing; Do, Yi-Yin; Huang, Pung-Ling

    2016-01-01

    Among 18 1-aminocyclopropane-1-carboxylic acid (ACC) oxidase homologous genes existing in the banana genome there are two genes, Mh-ACO1 and Mh-ACO2, that participate in banana fruit ripening. To better understand the physiological functions of Mh-ACO1 and Mh-ACO2, two hairpin-type siRNA expression vectors targeting both the Mh-ACO1 and Mh-ACO2 were constructed and incorporated into the banana genome by Agrobacterium-mediated transformation. The generation of Mh-ACO1 and Mh-ACO2 RNAi transgenic banana plants was confirmed by Southern blot analysis. To gain insights into the functional diversity and complexity between Mh-ACO1 and Mh-ACO2, transcriptome sequencing of banana fruits using the Illumina next-generation sequencer was performed. A total of 32,093,976 reads, assembled into 88,031 unigenes for 123,617 transcripts were obtained. Significantly enriched Gene Oncology (GO) terms and the number of differentially expressed genes (DEGs) with GO annotation were ‘catalytic activity’ (1327, 56.4%), ‘heme binding’ (65, 2.76%), ‘tetrapyrrole binding’ (66, 2.81%), and ‘oxidoreductase activity’ (287, 12.21%). Real-time RT-PCR was further performed with mRNAs from both peel and pulp of banana fruits in Mh-ACO1 and Mh-ACO2 RNAi transgenic plants. The results showed that expression levels of genes related to ethylene signaling in ripening banana fruits were strongly influenced by the expression of genes associated with ethylene biosynthesis. PMID:27681726

  1. Circumpolar Estimates of Isopycnal Mixing in the ACC from Argo Floats

    NASA Astrophysics Data System (ADS)

    Roach, C. J.; Balwada, D.; Speer, K. G.

    2015-12-01

    There are few direct observations of cross-stream isopycnal mixing in the interior of the Southern Ocean, yet such measurements are needed to determine the role of eddies transporting properties across the ACC, and key to progress toward testing theories of meridional overturning. In light of this we examine if it is possible to obtain estimates of mixing from Argo float trajectories. We divided the Southern Ocean into overlapping 15ο longitude bins before estimating mixing. Resulting diffusivities ranged from 300 to 3000 m2s-1, with peaks corresponding to the Scotia Sea; Kerguelen and Campbell Plateaus. Comparison of our diffusivities with previous regional studies demonstrated good agreement. Tests of the methodology in the DIMES region found that mixing from Argo floats agreed closely with mixing from RAFOS floats. To further test the method we used the Southern Ocean State Estimate velocity fields to advect particles with Argo and RAFOS float like behaviours. Stirring estimates from the particles agreed well with each other in the Kerguelen Island region, South Pacific and Scotia Sea, despite the differences in the imposed behaviour. Finally, these estimates were compared to mixing length suppression theory presented in Ferrari and Nikurashin 2010. This mixing length suppression theory quantifies horizontal diffusivity similar to Prandtl (1925), but the mixing length is suppressed in the presence of mean flows and eddy phase speeds. Our results suggest that the theory can explain both the structure and magnitude of mixing using mean flow data. An exception is near the Kerguelen and Campbell Plateaus where theory under-estimates mixing relative to our results.

  2. Global Transcriptomic Analysis of Targeted Silencing of Two Paralogous ACC Oxidase Genes in Banana.

    PubMed

    Xia, Yan; Kuan, Chi; Chiu, Chien-Hsiang; Chen, Xiao-Jing; Do, Yi-Yin; Huang, Pung-Ling

    2016-09-26

    Among 18 1-aminocyclopropane-1-carboxylic acid (ACC) oxidase homologous genes existing in the banana genome there are two genes, Mh-ACO1 and Mh-ACO2, that participate in banana fruit ripening. To better understand the physiological functions of Mh-ACO1 and Mh-ACO2, two hairpin-type siRNA expression vectors targeting both the Mh-ACO1 and Mh-ACO2 were constructed and incorporated into the banana genome by Agrobacterium-mediated transformation. The generation of Mh-ACO1 and Mh-ACO2 RNAi transgenic banana plants was confirmed by Southern blot analysis. To gain insights into the functional diversity and complexity between Mh-ACO1 and Mh-ACO2, transcriptome sequencing of banana fruits using the Illumina next-generation sequencer was performed. A total of 32,093,976 reads, assembled into 88,031 unigenes for 123,617 transcripts were obtained. Significantly enriched Gene Oncology (GO) terms and the number of differentially expressed genes (DEGs) with GO annotation were 'catalytic activity' (1327, 56.4%), 'heme binding' (65, 2.76%), 'tetrapyrrole binding' (66, 2.81%), and 'oxidoreductase activity' (287, 12.21%). Real-time RT-PCR was further performed with mRNAs from both peel and pulp of banana fruits in Mh-ACO1 and Mh-ACO2 RNAi transgenic plants. The results showed that expression levels of genes related to ethylene signaling in ripening banana fruits were strongly influenced by the expression of genes associated with ethylene biosynthesis.

  3. [Prolonging the vase life of carnation "Mabel" through integrating repeated ACC oxidase genes into its genome].

    PubMed

    Yu, Yi-Xun; Bao, Man-Zhu

    2004-10-01

    Carnation (Dianthus caryophyllus L.) is one of the most important cut flowers. The cultivar "Mabel" of carnation was transformed with direct repeat gene of ACC oxidase, the key enzyme in ethylene synthesis, driven by the CaMV35S promoter mediated by Agrobacterium tumefacien. Hygromycin phosphotransferase (HPT) gene was used as selection marker. Leaf explants were pre-cultured on shoot-inducing medium for 2 d, then immersed in Agrobacterium suspension for 8-12 min. Co-cultivation was carried out on the medium (MS+BA 1.0 mg/L+NAA 0.3 mg/L +Acetosyringone 100 micromol/L, pH 5.8-6.0) for 3 d. After that transformants were obtained by transferring explants to selection medium supplemented with 5 mg/L hygromycin (Hyg) and 400 mg/L cefotaxime (Cef). Southern blotting detection showed that a foreign gene was integrated into the carnation genome and 3 transgenic lines (T257, T299 and T273 line) obtained. Addition of acetosyringone and the time of co-culture were the main factors that influenced transformation frequency. After being transplanted to soil, transgenic plants were grew normally in greenhouse. Ethylene production of cut flower of transgenic T257 line was 95% lower than that of the control, and that of T299 line was reduced by 90% than that of the control, while that of transgenic T273 line has no of significantly different from control. Vase life of transgenic T257 line was 5 d longer than that of the control line at 25 degrees C.

  4. Control of light saturated photosynthesis: Concentration and activity of ribulose bisphosphate carboxylase. Final report, September 1, 1993--February 28, 1997

    SciTech Connect

    Geider, R.J. |

    1997-05-01

    Ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) is one of the most abundant enzymes on the planet and is responsible for catalysing the net fixation of CO{sub 2} into organic matter. It is central, therefore, to primary productivity in marine and terrestrial ecosystems. Rubisco is a large enzyme with low substrate affinity and low catalytic efficiency and is considered to limit the rate of light-saturated photosynthesis. This report summarizes research into the molecular basis of the regulation of phytoplankton photosynthesis. It describes experimental and theoretical studies of the role of Rubisco in regulating the photosynthetic rate of phytoplankton. It also describes the integration of a mechanistically based phytoplankton growth model into a description of primary productivity in the sea. This work was conducted as part of the Ocean Margins Program.

  5. Isolation of the catalytically competent small subunit of ribulose bisphosphate carboxylase/oxygenase from spinach under an extremely alkaline condition.

    PubMed

    Incharoensakdi, A; Takabe, T; Takabe, T; Akazawa, T

    1986-07-16

    A method for isolating the small subunit (B) of ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO) from spinach leaf using an alkaline buffer (pH 11.2) in combination with sucrose gradient centrifugation is described. Although the yield of isolated subunit B (ca. 20%) was comparable to that previously described (ca. 25%) using the acid precipitation method [Andrews, T.J. and Lorimer, G.H. (1985) J. Biol. Chem. 260: 4632-4636], the isolated subunit B in this report suffered less denaturation (ca. 30%) as estimated from kinetic analysis of its reassembly with large subunit (A) derived from Aphanothece halophytica. Studies on the kinetic properties of the reassembled enzyme molecules suggested that spinach subunit B does not influence the affinity of the enzyme for substrate CO2. The catalytic core (A8) of spinach RuBisCO could not be isolated in the native form.

  6. The nature of L8 and L8S8 forms of ribulose bisphosphate carboxylase/oxygenase from Chromatium vinosum.

    PubMed

    Torres-Ruiz, J; McFadden, B A

    1987-04-01

    L8 and L8S8 forms of ribulose bisphosphate carboxylase/oxygenase (RubisCO) have been prepared from Chromatium vinosum by the extremely mild method of centrifugal fractionation. Only the L8S8 form is detectable in crude extracts of this organism. Both forms show immunological identify in double diffusion studies using antibody to L subunits of the L8S8 form. L subunits from both L8 and L8S8 enzymes are identical by the criteria of peptides observed after limited proteolysis and N-terminal sequence analysis. In addition, these subunits show regions of homology with L subunits from Rhodospirillum rubrum, Anacystis nidulans, and spinach. S subunits of the C. vinosum enzyme are completely homologous to those from A. nidulans and higher plants from the 18th through 25th residue, a stretch preceded in all cases by two basic amino acids.

  7. Phosphorylation of chloroplast ribulose bisphosphate carboxylase/oxygenase small subunit by an envelope-bound protein kinase in situ.

    PubMed

    Soll, J; Buchanan, B B

    1983-06-10

    A new protein kinase of the cAMP independent type was found to be bound to the outer envelope membrane of spinach chloroplasts. While stimulated by Mg2+ and inhibited by ADP, the enzyme showed no response to conventional protein substrates and was essentially independent of pH in the physiological (pH 7 to 8) range. The new protein kinase phosphorylated the mature form of the small subunit of ribulose 1,5-bisphosphate carboxylase/oxygenase and, to a lesser extent, an unidentified 24-kDa polypeptide, both of which were bound to the outer envelope membrane. The results suggest that phosphorylation of cytoplasmically synthesized protein constituents of chloroplasts is involved in their transport through the chloroplast envelope membrane barrier.

  8. Activity ratios of ribulose-1,5-bisphosphate carboxylase accurately reflect carbamylation ratios. [Phaseolus vulgaris, Spinacla oleracea

    SciTech Connect

    Butz, N.D.; Sharkey, T.D. )

    1989-03-01

    Activity ratios and carbamylation ratios of ribulose-1,5-bisphosphate carboxylase (RuBPCase) were determined for leaves of Phaseolus vulgaris and Spinacia oleracea exposed to a variety of partial pressures of CO{sub 2} and O{sub 2} and photon flux densities (PFD). It was found that activity ratios accurately predicted carbamylation ratios except in extracts from leaves held in low PFD. In particular, it was confirmed that the loss of FuBPCase activity in low partial pressure of O{sub 2} and high PFD results from reduced carbamylation. Activity ratios of RuBPCase were lower than carbamylation ratios for Phaseolus leaves sampled in low PFD, presumably because of the presence of 2-carboxyarabinitol 1-phosphate. Spinacia leaves sampled in darkness also exhibited lower activity ratios than carbamylation ratios indicating that this species may also have an RuBPCase inhibitor even though carboxyarabinitol 1-phosphate has not been detected in this species in the past.

  9. In vitro reassembly of tobacco ribulose-1,5-bisphosphate carboxylase/oxygenase from fully denatured subunits.

    PubMed

    Yong, Zhen-Hua; Chen, Gen-Yun; Shi, Jiao-Nai; Xu, Da-Quan

    2006-10-01

    It has been generally proved impossible to reassemble ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) from fully denatured subunits in vitro in higher plant, because large subunit of fully denatured Rubisco is liable to precipitate when the denaturant is removed by common methods of direct dilution and one-step dialysis. In our experiment, the problem of precipitation was resolved by an improved gradual dialysis method, which gradually decreased the concentration of denaturant. However, fully denatured Rubisco subunits still could not be reassembled into holoenzyme using gradual dialysis unless chaperonin 60 was added. The restored activity of reassembled Rubisco was approximately 8% of natural enzyme. The quantity of reassembled Rubisco increased greatly when heat shock protein 70 was present in the reassembly process. ATP and Mg2+ were unnecessary for in vitro reassembly of Rubisco, and Mg2+ inhibited the reassembly process. The reassembly was weakened when ATP, Mg2+ and K+ existed together in the reassembly process.

  10. Cloning and developmental expression of pea ribulose-1,5-bisphosphate carboxylase/oxygenase large subunit epsilon N-methyltransferase

    DOEpatents

    Houtz, R.L.

    1999-02-02

    The gene sequence for ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) large subunit (LS){sup {epsilon}}N-methyltransferase (protein methylase III or Rubisco LSMT) is disclosed. This enzyme catalyzes methylation of the {epsilon}-amine of lysine-14 in the large subunit of Rubisco. In addition, a full-length cDNA clone for Rubisco LSMT is disclosed. Transgenic plants and methods of producing same which (1) have the Rubisco LSMT gene inserted into the DNA, and (2) have the Rubisco LSMT gene product or the action of the gene product deleted from the DNA are also provided. Further, methods of using the gene to selectively deliver desired agents to a plant are also disclosed. 8 figs.

  11. Cloning and developmental expression of pea ribulose-1,5-bisphosphate carboxylase/oxygenase large subunit N-methyltransferase

    DOEpatents

    Houtz, R.L.

    1998-03-03

    The gene sequence for ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) large subunit (LS) {epsilon}N-methyltransferase (protein methylase III or Rubisco LSMT) is disclosed. This enzyme catalyzes methylation of the {epsilon}-amine of lysine-14 in the large subunit of Rubisco. In addition, a full-length cDNA clone for Rubisco LSMT is disclosed. Transgenic plants and methods of producing same which (1) have the Rubisco LSMT gene inserted into the DNA, and (2) have the Rubisco LSMT gene product or the action of the gene product deleted from the DNA are also provided. Further, methods of using the gene to selectively deliver desired agents to a plant are also disclosed. 5 figs.

  12. Cloning and developmental expression of pea ribulose-1,5-bisphosphate carboxylase/oxygenase large subunit epsilon N-methyltransferase

    DOEpatents

    Houtz, Robert L.

    1999-01-01

    The gene sequence for ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) large subunit (LS) .sup..epsilon. N-methyltransferase (protein methylase III or Rubisco LSMT) is disclosed. This enzyme catalyzes methylation of the .epsilon.-amine of lysine-14 in the large subunit of Rubisco. In addition, a full-length cDNA clone for Rubisco LSMT is disclosed. Transgenic plants and methods of producing same which (1) have the Rubisco LSMT gene inserted into the DNA, and (2) have the Rubisco LSMT gene product or the action of the gene product deleted from the DNA are also provided. Further, methods of using the gene to selectively deliver desired agents to a plant are also disclosed.

  13. Cloning and developmental expression of pea ribulose-1,5-bisphosphate carboxylase/oxygenase large subunit N-methyltransferase

    DOEpatents

    Houtz, Robert L.

    1998-01-01

    The gene sequence for ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) large subunit (LS) .epsilon.N-methyltransferase (protein methylase III or Rubisco LSMT) is disclosed. This enzyme catalyzes methylation of the .epsilon.-amine of lysine-14 in the large subunit of Rubisco. In addition, a full-length cDNA clone for Rubisco LSMT is disclosed. Transgenic plants and methods of producing same which (1) have the Rubisco LSMT gene inserted into the DNA, and (2) have the Rubisco LSMT gene product or the action of the gene product deleted from the DNA are also provided. Further, methods of using the gene to selectively deliver desired agents to a plant are also disclosed.

  14. Rubisco and PEP carboxylase responses to changing irradiance in a Brazilian Cerrado tree species, Qualea grandiflora Mart. (Vochysiaceae).

    PubMed

    Paulilo, M T; Besford, R T; Wilkins, D

    1994-02-01

    The activities of ribulose-1,5-bisphosphate carboxylase-oxygenase, Rubisco (E.C. 4.1.1.39) and phosphoenolpyruvate carboxylase, PEPc (E.C. 4.1.1.31), and concentrations of protein and chlorophyll were measured in extracts from cotyledons and first leaves of Qualea grandiflora Mart. (Vochysiaceae) seedlings after transfer from high-light (20 days at 320 micro mol m(-2) s(-1), PAR) to low-light (35 days at 120 micro mol m(-2) s(-1), PAR) conditions. When Tween 20 and glycerol were added to the extraction medium, Rubisco activities obtained for Qualea grandiflora were comparable to published values for several coniferous species and the broad-leaved species, Prunus avium L. Stella, grown in a similar light environment. Rubisco activity in cotyledons of Q. grandiflora grown in high light for 20 days and then transferred to low light for a further 35 days was similar to the activity in cotyledons of plants grown continuously in high light. However, the first leaf above the cotyledons showed a greater response to the change in irradiance; in high light, Rubisco activity of the first leaf was 1.8 times higher on a fresh weight basis and 2.7 times higher on an area basis than that of leaves transferred from high to low light. Fresh weight and chlorophyll concentration expressed on a unit leaf area basis were also higher in the high-light treatment. These responses to irradiance are indicative of a species adapted to growth in an unshaded habitat. The PEPc activity in leaves was 15% of Rubisco activity, which is typical of species with a C(3) photosynthetic pathway. The relatively slow growth rate of Q. grandiflora observed in these experiments could not be attributed to a low carboxylation capacity per unit leaf area.

  15. Evolution of the Phosphoenolpyruvate Carboxylase Protein Kinase Family in C3 and C4 Flaveria spp.1[W][OPEN

    PubMed Central

    Aldous, Sophia H.; Weise, Sean E.; Sharkey, Thomas D.; Waldera-Lupa, Daniel M.; Stühler, Kai; Mallmann, Julia; Groth, Georg; Gowik, Udo; Westhoff, Peter; Arsova, Borjana

    2014-01-01

    The key enzyme for C4 photosynthesis, Phosphoenolpyruvate Carboxylase (PEPC), evolved from nonphotosynthetic PEPC found in C3 ancestors. In all plants, PEPC is phosphorylated by Phosphoenolpyruvate Carboxylase Protein Kinase (PPCK). However, differences in the phosphorylation pattern exist among plants with these photosynthetic types, and it is still not clear if they are due to interspecies differences or depend on photosynthetic type. The genus Flaveria contains closely related C3, C3-C4 intermediate, and C4 species, which are evolutionarily young and thus well suited for comparative analysis. To characterize the evolutionary differences in PPCK between plants with C3 and C4 photosynthesis, transcriptome libraries from nine Flaveria spp. were used, and a two-member PPCK family (PPCKA and PPCKB) was identified. Sequence analysis identified a number of C3- and C4-specific residues with various occurrences in the intermediates. Quantitative analysis of transcriptome data revealed that PPCKA and PPCKB exhibit inverse diel expression patterns and that C3 and C4 Flaveria spp. differ in the expression levels of these genes. PPCKA has maximal expression levels during the day, whereas PPCKB has maximal expression during the night. Phosphorylation patterns of PEPC varied among C3 and C4 Flaveria spp. too, with PEPC from the C4 species being predominantly phosphorylated throughout the day, while in the C3 species the phosphorylation level was maintained during the entire 24 h. Since C4 Flaveria spp. evolved from C3 ancestors, this work links the evolutionary changes in sequence, PPCK expression, and phosphorylation pattern to an evolutionary phase shift of kinase activity from a C3 to a C4 mode. PMID:24850859

  16. Phosphoenolpyruvate carboxylase regulation in C4-PEPC-expressing transgenic rice during early responses to drought stress.

    PubMed

    Liu, Xiaolong; Li, Xia; Zhang, Chen; Dai, Chuanchao; Zhou, Jiayu; Ren, Chenggang; Zhang, Jinfei

    2017-02-01

    Phosphoenolpyruvate carboxylase (PEPC; EC 4.1.1.31) has important functions in C4 photosynthesis and biosynthesis of intermediate metabolites. In this study, the drought resistance of C4-PEPC-expressing transgenic rice (Oryza sativa, line PC) plants was assessed using simulated drought conditions [i.e. polyethylene glycol (PEG)-6000 treatment]. The dry weight of PC plants was higher than that of wild-type (WT) plants following treatment with 15% PEG-6000 for 16 days. Furthermore, the water use efficiency, relative water content and proline content in PC plants were higher than those of WT plants, as were C4-PEPC activity and transcript levels following treatment with 5% PEG-6000 for 2 h. The protein kinase activities and transcript levels of sucrose non-fermenting-1-related protein kinases (SnRKs) genes, such as SnRK1a, OsK24 and OsK35 were also higher in PC plants than in WT plants following treatment with 5% PEG-6000 for 2 h. Additionally, phosphoenolpyruvate carboxylase kinase (PPCK, EC 4.1.1.32) activities and transcript levels (e.g. PPCK1 and PPCK2) increased following drought treatment. These changes were regulated by signaling molecules, such as calcium, nitric oxide and hydrogen peroxide. Furthermore, the -1095 to -416 region of the C4-PEPC promoter in PC plants was demethylated following exposure to drought conditions for 1 h. The demethylation coincided with an increase in C4-PEPC expression. Our data suggest that the demethylation of the C4-PEPC promoter and the phosphorylation catalyzed by PPCK have key roles in conferring drought tolerance to the transgenic rice plants.

  17. Cloning and characterization of pyruvate carboxylase gene responsible for calcium malate overproduction in Penicillium viticola 152 and its expression analysis.

    PubMed

    Khan, Ibrar; Qayyum, Sadia; Ahmed, Shehzad; Maqbool, Farhana; Tauseef, Isfahan; Haleem, Kashif Syed; Chi, Zhen-Ming

    2017-03-20

    In this study, a pyruvate carboxylase gene (PYC) from a marine fungus Penicillium viticola 152 isolated from marine algae was cloned and characterized by using Genome Walking method. An open reading frame (ORF) of The PYC gene (accession number: KM593097) had 3582bp encoding 1193 amino acid protein (isoelectric point: 5.01) with a calculated molecular weight of 131.2757kDa. A putative promoter (intronless) of the gene was located at -666bp and contained a TATA box, several CAAT boxes, the 5'-SYGGRG-3' and a 5'-HGATAR-3' sequences. A consensus polyadenylation site (AATAAA) was also observed at +10bp downstream of the ORF. The protein deduced from the PYC gene had no signal peptide, was a homotetramer (4), and had the four functional domains. Furthermore, PYC protein also had three potential N-linked glycosylation sites, among them, -N-S-T-I- at 36 amino acid, -N-G-T-V- at 237 amino acid, and -N-G-S-S- at 517 amino acid were the most possible N-glycosylation sites. After expression of the PYC gene of P. viticola 152 in medium supplemented with CSL and biotin, it was found that the specific pyruvate carboxylase activity in MA production medium supplemented with CSL was much higher (0.5U/mg) than in MA medium supplemented with biotin (0.3U/mg), suggesting that optimal concentration of CSL is required for increased expression of the PYC gene, which is responsible for high level production of malic acid in P. viticola 152 strain.

  18. Demonstration of a functional requirement for the carbamate nitrogen of ribulosebisphosphate carboxylase/oxygenase by chemical rescue

    SciTech Connect

    Smith, H.B.; Hartman, F.C. )

    1991-05-28

    Ribulosebisphosphate carboxylase/oxygenase is reversibly activated by the reaction of Co{sub 2} with a specific lysyl residue to form a carbamate that coordinates an essential Mg{sup 2+} cation. Surprisingly, the Lys191{yields}Cys mutant protein, in the presence of Co{sub 2} and Mg{sup 2+} exhibits tight binding of the reaction intermediate analogue 2-carboxyarabinitol bisphosphate a property normally equated with effective coordination of the Mg{sup 2+} by the carbamate. Catalytic ineptness of the Cys191 mutant protein, despite its ability to coordinate Mg{sup 2+} properly, might be due to the absence of the carbamate nitrogen. To investigate this possibility, the authors have evaluated the ability of exogenous amines to restore catalytic activity to the mutant protein. Significantly, the Cys191 protein manifests ribulose bisphosphate dependent fixation of {sup 14}CO{sub 2} when incubated with aminomethanewsulfonate but not ethanesulfonate. This novel activity reflects a K{sub m} value for ribulose bisphosphate which is not markedly perturbed relative to wild-type enzyme, a K{sub m} for Mg{sup 2+} which is in fact decreased 10-fold, and rate saturation with respect to aminomethanesulfonate. Chromatographic and spectrophotometric analyses reveal the product of CO{sub 2} fixation to be D-3-phosphoglycerate while turnover of (1-{sup 3}H)ribulose bisphosphate into ({sup 3}H)phosphoglycolate confirms oxygenase activity. The authors conclude that aminomethanesulfonate restored ribulosebisphosphate carboxylase/oxygenase activities to the Cys191 mutant protein by providing a nitrogenous function which satisfies a catalytic demand normally met by the carbamate nitrogen of Lys191.

  19. ACC GABA levels are associated with functional activation and connectivity in the fronto-striatal network during interference inhibition in patients with borderline personality disorder.

    PubMed

    Wang, Guo-Ying; van Eijk, Julia; Demirakca, Traute; Sack, Markus; Krause-Utz, Annegret; Cackowski, Sylvia; Schmahl, Christian; Ende, Gabriele

    2017-02-15

    Impulsivity often develops from disturbed inhibitory control, a function mainly regulated by γ-Aminobutyric acid (GABA) levels in the anterior cingulate cortex (ACC) and the fronto-striatal system. In this study, we combined MRS GABA measurements and fMRI to investigate neurochemical and neurofunctional correlates of interference inhibition, further emphasizing the direct relationship between those two systems, as well as their relations to impulsivity in patients with BPD. In addition to BOLD activation, task-dependent functional connectivity was assessed by a generalized psychophysiological interactions approach. Full factorial analyses were performed via SPM to examine the main effect (within-group associations) as well as the interaction term (group differences in the association slope). The UPPS scales were used to evaluate impulsivity traits. Compared to healthy controls (HCs), BPD patients exhibited significantly less ACC-caudate functional connectivity during interference inhibition. ACC GABA levels in BPD patients but not in HCs were positively related to the magnitude of activation in several fronto-striatal regions (e.g. ACC, frontal regions, putamen, caudate,) and the strength of ACC-caudate functional connectivity during interference inhibition. The strength of the correlations of GABA with connectivity significantly differs between the two groups. Moreover, among all the UPPS impulsivity subscales, UPPS sensation seeking in the BPD group was related to GABA and was also negatively related to the task-dependent BOLD activation and functional connectivity in the fronto-striatal network. Finally, mediation analyses revealed that the magnitude of activation in the caudate and the strength of ACC-caudate functional connectivity mediated the relationship between ACC GABA levels and UPPS sensation seeking in patients with BPD. Our findings suggest a disconnectivity of the fronto-striatal network in BPD patients during interference inhibition, particularly

  20. Silencing the NR2B gene in rat ACC neurons by lentivirus-delivered shRNA alleviates pain-related aversion.

    PubMed

    Guo, Shou-Gang; Lv, Xiu-Hua; Guan, Shan-Hui; Li, Hui-Lu; Qiao, Yong; Feng, Hao; Cong, Lin; Wang, Gong-Ming

    2015-01-01

    The N-methyl-D-aspartate (NMDA) receptor NR2B subunit on neurons in the anterior cingulate cortex (ACC) is implicated in the affective response to noxious stimuli. Selectively silencing this NR2B subunit in ACC neurons could therefore alleviate pain-related aversion. However, to date, there is no optimal approach to selectively silence the NR2B gene in ACC neurons. In the present study, we constructed lentiviral vectors and delivered shRNA (NR2B-RNAi-LV) to effectively silence the NR2B gene in ACC neurons. The use of lentivirus resulted in 95% transfection efficiency and 83% silencing of the NR2B gene in ACC neurons. Electrophysiological experiments showed that the total INMDA was similarly reduced by 48% in lentivirus-transfected ACC neurons. The biochemical and functional data demonstrated that lentiviral shRNA delivery produced a high transfection and silencing efficiency in the ACC neurons. SNI rats weighting 220-250 g were randomly divided into three groups: normal saline group (NS), lenti-siRNA/NC (LV-NC) group, and lenti-siRNA/NR2B (LV-NR2B) group, and conditioned place avoidance was conducted. The results indicated that NR2B-RNAi-LV decreased greatly the conditioning scores of F-CPA while NC-GFP-LV has no effects. NR2B mRNA expression in the NR2B-RNAi-LV group was significantly lower than that in the control group and NC-GFP-LV group. This novel approach of silencing the NR2B gene in ACC neuron could potentially be used to alleviate pain-related aversion.

  1. Austrian Carbon Calculator (ACC) - modelling soil carbon dynamics in Austrian soils

    NASA Astrophysics Data System (ADS)

    Sedy, Katrin; Freudenschuss, Alexandra; Zethner, Gehard; Spiegel, Heide; Franko, Uwe; Gründling, Ralf; Xaver Hölzl, Franz; Preinstorfer, Claudia; Haslmayr, Hans Peter; Formayer, Herbert

    2014-05-01

    Austrian Carbon Calculator (ACC) - modelling soil carbon dynamics in Austrian soils. The project funded by the Klima- und Energiefonds, Austrian Climate Research Programme, 4th call Authors: Katrin Sedy, Alexandra Freudenschuss, Gerhard Zethner (Environment Agency Austria), Heide Spiegel (Austrian Agency for Health and Food Safety), Uwe Franko, Ralf Gründling (Helmholtz Centre for Environmental Research) Climate change will affect plant productivity due to weather extremes. However, adverse effects could be diminished and satisfying production levels may be maintained with proper soil conditions. To sustain and optimize the potential of agricultural land for plant productivity it will be necessary to focus on preserving and increasing soil organic carbon (SOC). Carbon sequestration in agricultural soils is strongly influenced by management practice. The present management is affected by management practices that tend to speed up carbon loss. Crop rotation, soil cultivation and the management of crop residues are very important measures to influence carbon dynamics and soil fertility. For the future it will be crucial to focus on practical measures to optimize SOC and to improve soil structure. To predict SOC turnover the existing humus balance model the application of the "Carbon Candy Balance" was verified by results from Austrian long term field experiments and field data of selected farms. Thus the main aim of the project is to generate a carbon balancing tool box that can be applied in different agricultural production regions to assess humus dynamics due to agricultural management practices. The toolbox will allow the selection of specific regional input parameters for calculating the C-balance at field level. However farmers or other interested user can also apply their own field data to receive the result of C-dynamics under certain management practises within the next 100 years. At regional level the impact of predefined changes in agricultural management

  2. Assessment of adherence to ACC/AHA guidelines in primary management of patients with NSTEMI in a referral cardiology hospital.

    PubMed

    Farahzadi, Mohammadreza; Shafiee, Akbar; Bozorgi, Ali; Mahmoudian, Mehran; Sadeghian, Saeed

    2015-03-01

    Acute coronary syndromes are considered as a global major health-care problem, and Iran as a developing country is of no exception. We aimed to investigate the degree of adherence to American College of Cardiology and American Heart Association (ACC/AHA) guideline for the management of non-ST-segment elevation myocardial infarction (NSTEMI) in patients who presented to the emergency department at Tehran Heart Center. Data of the patients who presented with acute chest pain to the emergency department of Tehran Heart Center within 1 year and were diagnosed as NSTEMI by the cardiologist in charge were included. The details of the initial managements based on the ACC/AHA guideline for NSTEMI of the patients were recorded from the patients' files in the emergency department for this study. Then, the frequency of guideline-related management in the study population was calculated and reported. A total of 684 patients [mean age = 62.95 ± 12.19 years; male gender = 460 (67.3%)] were diagnosed as NSTEMI at the emergency department of our center. Initial management based on the current guideline including administration of aspirin and clopidogrel was performed in 98.4% and 95.0%, respectively. Intravenous heparin was administered in 67.0% of the patients, whereas 30.8% of patients received enoxaparin. Following the initial management, coronary angiography was performed in 563 (82.3%) patients within 48 hours from the admission. Adherence to ACC/AHA guideline for the management of NSTEMI in patients who presented to a tertiary health-care center was in a high degree.

  3. Assessment of Coronary Artery Calcium Scoring for Statin Treatment Strategy according to ACC/AHA Guidelines in Asymptomatic Korean Adults

    PubMed Central

    Han, Donghee; Ó Hartaigh, Bríain; Lee, Ji Hyun; Rizvi, Asim; Park, Hyo Eun; Choi, Su-Yeon; Sung, Jidong

    2017-01-01

    Purpose The 2013 American College of Cardiology (ACC)/American Heart Association (AHA) cholesterol management guidelines advocate the use of statin treatment for prevention of cardiovascular disease. We aimed to assess the usefulness of coronary artery calcium (CAC) for stratifying potential candidates of statin use among asymptomatic Korean individuals. Materials and Methods A total of 31375 subjects who underwent CAC scoring as part of a general health examination were enrolled in the current study. Statin eligibility was categorized as statin recommended (SR), considered (SC), and not recommended (SN) according to ACC/AHA guidelines. Cox regression analysis was employed to estimate hazard ratios (HR) with 95% confidential intervals (CI) after stratifying the subjects according to CAC scores of 0, 1–100, and >100. Number needed to treat (NNT) to prevent one mortality event during study follow up was calculated for each group. Results Mean age was 54.4±7.5 years, and 76.3% were male. During a 5-year median follow-up (interquartile range; 3–7), there were 251 (0.8%) deaths from all-causes. A CAC >100 was independently associated with mortality across each statin group after adjusting for cardiac risk factors (e.g., SR: HR, 1.60; 95% CI, 1.07–2.38; SC: HR, 2.98; 95% CI, 1.09–8.13, and SN: HR, 3.14; 95% CI, 1.08–9.17). Notably, patients with CAC >100 displayed a lower NNT in comparison to the absence of CAC or CAC 1–100 in SC and SN groups. Conclusion In Korean asymptomatic individuals, CAC scoring might prove useful for reclassifying patient eligibility for receiving statin therapy based on updated 2013 ACC/AHA guidelines. PMID:27873499

  4. Impact of the New ACC/AHA Guidelines on the Treatment of High Blood Cholesterol in a Managed Care Setting

    PubMed Central

    Tran, Josephine N.; Caglar, Toros; Stockl, Karen M.; Lew, Heidi C.; Solow, Brian K.; Chan, Paul S.

    2014-01-01

    Background In November 2013, the American College of Cardiology (ACC) and the American Heart Association (AHA) together issued new guidelines for the treatment of patients with high cholesterol, providing a new paradigm for the management of cholesterol in the primary and secondary prevention of coronary artery disease. Objective To examine the impact of the 2013 ACC/AHA cholesterol treatment guidelines on pharmacy utilization of cholesterol-lowering drugs in a real-world managed care setting. Methods Pharmacy claims from OptumRx, a national pharmacy benefit management provider, for the period between January 1, 2013, and December 31, 2013 (baseline period), were used to identify candidates for cholesterol-lowering therapy and to estimate the number of potential patients who will be starting or intensifying statin therapy based on the updated cholesterol treatment guidelines. Potential candidates for cholesterol-lowering treatments included patients with diabetes or hypertension aged 40 to 75 years who were not already receiving a cholesterol-lowering medication, as well as patients receiving cholesterol-lowering therapies during the baseline period. The baseline cholesterol-lowering medication market share was used to project changes in pharmacy utilization over the next 3 years. Results Based on the 2013 ACC/AHA cholesterol treatment guidelines, there will be a 25% increase in the proportion of the overall population that is treated with statins over the next 3 years, increasing from 3,909,407 (27.7%) patients to 4,892,668 (34.7%) patients. The largest proportion of the increase in statin utilization is projected to be for primary prevention in patients aged 40 to 75 years who were not receiving any cholesterol-lowering treatment at baseline. These projected changes will increase the overall number of statin prescriptions by 25% and will decrease the number of nonstatin cholesterol-lowering medication prescriptions by 68% during the next 3 years. Conclusion The

  5. High substrate specificity factor ribulose bisphosphate carboxylase/oxygenase from eukaryotic marine algae and properties of recombinant cyanobacterial RubiSCO containing "algal" residue modifications.

    PubMed

    Read, B A; Tabita, F R

    1994-07-01

    Marine algae play an important role in removing carbon dioxide from the atmosphere. In this investigation, we have determined the substrate specificity factor of ribulose 1,5-bisphosphate carboxylase/oxygenase from several marine chromophytic and rhodophytic algae. The enzymes were purified to homogeneity and all possessed significantly higher substrate specificity factors than the enzymes from terrestrial plants, green algae, or bacteria. There are substantial differences in the sequence in a helix 6 of the large subunit of these enzymes, which is intriguing since residues of this region had been previously shown to influence the ability of ribulose bisphosphate carboxylase to discriminate between CO2 and O2, presumably by influencing the adjacent flexible loop 6 region. Sequence divergence at this and other key regions might contribute to the substantial differences in the substrate specificity factor of the chromophyte/rhodophyte enzyme. Initial studies on probing the basis for the high substrate specificity factor employed single amino acid substitutions in the recombinant cyanobacterial ribulose bisphosphate carboxylase. Residues in the vicinity of loop 6 were changed to reflect the corresponding residues in the chromophyte/rhodophyte large subunit. Some changes in the substrate specificity factor were noted, as were alterations in other important kinetic parameters. Since marine algae show little evidence of photorespiratory metabolism, the high substrate specificity of ribulose bisphosphate carboxylase is consistent with the physiology of these organisms. The results of this study provide further evidence that the properties of this enzyme may evolve or change according to the environment in which the host organism is found.

  6. An 'in situ' perfusion system suitable for investigating mammary-tissue metabolism in the lactating rat. Hormonal regulation of acetyl-CoA carboxylase.

    PubMed Central

    Clegg, R A; Calvert, D T

    1988-01-01

    A technique is described for the non-recirculating perfusion of inguinal/abdominal mammary tissue in situ in anaesthetized lactating rats. Tissue viability was maintained, without resort to infusion of vasoactive chemicals which may also be effectors of cellular metabolism, for at least 90 min. Total tissue adenine nucleotides (per mg of DNA) were somewhat decreased in perfused relative to non-perfused mammary tissue. DNA content (per g wet wt. of tissue) was diminished after 90 min of perfusion to approx. 65% of its value in control tissue. Adenylate energy-charge ratios were lower in perfused tissue in the absence of hormones than in control tissue. They were increased to control values by the presence of either insulin or isoprenaline in the perfusate. No changes occurred in flow rate of the perfusate that might account for these increases. In mammary tissue perfused without addition of hormones, acetyl-CoA carboxylase activities were similar to those measured in control tissue samples, although activity-ratio measurements implied some increase in the phosphorylation of this enzyme. Insulin or isoprenaline increased the activity of acetyl-CoA carboxylase, especially when this was measured at low concentrations of citrate. Confirming conclusions from previous experiments with mammary acini and explant preparations, insulin activated acetyl-CoA carboxylase in mammary tissue, but inhibition of its activity was not mediated by cyclic AMP. PMID:2895636

  7. Site-specific mutations in a loop region of the C-terminal domain of the large subunit of ribulose bisphosphate carboxylase/oxygenase that influence substrate partitioning.

    PubMed

    Gutteridge, S; Rhoades, D F; Herrmann, C

    1993-04-15

    Amino acids composing a flexible loop (loop 6) of the eight-stranded barrel domain of the L-subunit of Synechococcus ribulose bisphosphate carboxylase/oxygenase (EC 4.1.1.39) involved in reaction intermediate stabilization have been modified by site-specific mutagenesis. Changes at positions both distant and within the active site affect overall catalysis and substrate partitioning. Most significantly, replacement of the active site Lys (Lys-334) with Arg at the apex of the loop almost completely suppressed the carboxylase activity of the enzyme relative to oxygenation, with only a modest reduction in overall catalysis. Val-331 and Thr-342, more distant from the active site but with interacting side chains, were changed to larger and smaller residues with differential effects on both turnover and substrate partitioning. Substitution of the loop with the sequence found in more efficient carboxylases only increased partitioning marginally when accompanied by alterations in the C-terminal tail of the L-subunit that interacts with the loop. Generally, modifications to the loop composition also affected enediol formation, the first step of catalysis, suggesting that the geometry and hence flexibility of this segment affect more than just stabilization of the intermediates immediately following reaction with CO2 or O2.

  8. Resistance to herbicides caused by single amino acid mutations in acetyl-CoA carboxylase in resistant populations of grassy weeds.

    PubMed

    Jang, SoRi; Marjanovic, Jasmina; Gornicki, Piotr

    2013-03-01

    Eleven spontaneous mutations of acetyl-CoA carboxylase have been identified in many herbicide-resistant populations of 42 species of grassy weeds, hampering application of aryloxyphenoxypropionate, cyclohexadione and phenylpyrazoline herbicides in agriculture. IC(50) shifts (resistance indices) caused by herbicide-resistant mutations were determined using a recombinant yeast system that allows comparison of the effects of single amino acid mutations in the same biochemical background, avoiding the complexity inherent in the in planta experiments. The effect of six mutations on the sensitivity of acetyl-CoA carboxylase to nine herbicides representing the three chemical classes was studied. A combination of partially overlapping binding sites of the three classes of herbicides and the structure of their variable parts explains cross-resistance among and between the three classes of inhibitors, as well as differences in their specificity. Some degree of resistance was detected for 51 of 54 herbicide/mutation combinations. Introduction of new herbicides targeting acetyl-CoA carboxylase will depend on their ability to overcome the high degree of cross-resistance already existing in weed populations.

  9. Susceptibilities of Different Test Systems from Maize (Zea mays), Poa annua, and Festuca rubra to Herbicides That Inhibit the Enzyme Acetyl-Coenzyme A Carboxylase

    PubMed

    Herbert; Cole; Pallett; Harwood

    1996-06-01

    The susceptibilities of maize (Zea mays cv. Champ) and two graminicide-resistant grass species, Poa annua (annual meadow grass) and Festuca rubra (red fescue), to two aryloxyphenoxypropionates (quizalofop and fluazifop) and a cyclohexanedione (sethoxydim) graminicide were evaluated in leaf blades and isolated chloroplasts, and by assaying acetyl-coenzyme A carboxylase (ACCase) in desalted leaf homogenates. The graminicide resistance of P. annua and F. rubra appeared to be at the level of ACCase. Festuca rubra ACCase was highly insensitive and P. annua ACCase was partially insensitive to the graminicides that were tested. Fatty acid synthesis in isolated maize chloroplasts was more susceptible to inhibition than was ACCase activity from whole leaves. There was a smaller difference in graminicide sensitivity between these two test systems in P. annua. The developmental pattern of ACCase specific activity and its inhibition by quizalofop was measured in maize and P. annua leaf blades. There was an age-dependent increase in the sensitivity of maize leaf ACCase activity to inhibition by quizalofop. Together with the greater susceptibility of chloroplasts compared with leaf homogenates this could imply that a graminicide-insensitive (extrachloroplastic) ACCase isoform is less highly expressed in older leaves. Poa annua ACCase did not significantly alter in sensitivity as leaves aged, consistent with the smaller difference in the level of inhibition between chloroplasts and leaf homogenates in this species. A small pyruvate carboxylase activity was detected in maize leaves after 9 days. By 38 days, when leaves were senescing, pyruvate carboxylase activity predominated over ACCase.

  10. Dose reduction in orthodontic lateral cephalography: dosimetric evaluation of a novel cephalographic thyroid protector (CTP) and anatomical cranial collimation (ACC)

    PubMed Central

    Rottke, D; van der Stelt, P F; Berkhout, W E R

    2015-01-01

    Objectives: To test the dose-reducing capabilities of a novel thyroid protection device and a recently introduced cranial collimator to be used in orthodontic lateral cephalography. Methods: Cephalographic thyroid protector (CTP) was designed to shield the thyroid while leaving the cervical vertebrae depicted. Using a RANDO® head phantom (The Phantom Laboratory, Salem, NY) equipped with dosemeters and a Proline XC (Planmeca, Helsinki, Finland) cephalograph, lateral cephalograms were taken, and the effective dose (ED) was calculated for four protocols: (1) without shielding; (2) with CTP; (3) with CTP and anatomical cranial collimator (ACC); and (4) with a thyroid collar (TC). Results: The ED for the respective protocols was (1) 8.51; (2) 5.39; (3) 3.50; and (4) 4.97 µSv. The organ dose for the thyroid was reduced from 30.17 to 4.50 µSv in Protocols 2 and 3 and to 3.33 µSv in Protocol 4. Conclusions: The use of just the CTP (Protocol 2) resulted in a 36.8% reduction of the ED of a lateral cephalogram. This was comparable to the classical TC (Protocol 4). A 58.8% reduction of the ED was obtained when combining CTP and ACC (Protocol 3). The dose to the radiosensitive thyroid gland was reduced by 85% in Protocols 2 and 3 and by 89% in Protocol 4. PMID:25564885

  11. OpenACC acceleration of an unstructured CFD solver based on a reconstructed discontinuous Galerkin method for compressible flows

    DOE PAGES

    Xia, Yidong; Lou, Jialin; Luo, Hong; ...

    2015-02-09

    Here, an OpenACC directive-based graphics processing unit (GPU) parallel scheme is presented for solving the compressible Navier–Stokes equations on 3D hybrid unstructured grids with a third-order reconstructed discontinuous Galerkin method. The developed scheme requires the minimum code intrusion and algorithm alteration for upgrading a legacy solver with the GPU computing capability at very little extra effort in programming, which leads to a unified and portable code development strategy. A face coloring algorithm is adopted to eliminate the memory contention because of the threading of internal and boundary face integrals. A number of flow problems are presented to verify the implementationmore » of the developed scheme. Timing measurements were obtained by running the resulting GPU code on one Nvidia Tesla K20c GPU card (Nvidia Corporation, Santa Clara, CA, USA) and compared with those obtained by running the equivalent Message Passing Interface (MPI) parallel CPU code on a compute node (consisting of two AMD Opteron 6128 eight-core CPUs (Advanced Micro Devices, Inc., Sunnyvale, CA, USA)). Speedup factors of up to 24× and 1.6× for the GPU code were achieved with respect to one and 16 CPU cores, respectively. The numerical results indicate that this OpenACC-based parallel scheme is an effective and extensible approach to port unstructured high-order CFD solvers to GPU computing.« less

  12. OpenACC acceleration of an unstructured CFD solver based on a reconstructed discontinuous Galerkin method for compressible flows

    SciTech Connect

    Xia, Yidong; Lou, Jialin; Luo, Hong; Edwards, Jack; Mueller, Frank

    2015-02-09

    Here, an OpenACC directive-based graphics processing unit (GPU) parallel scheme is presented for solving the compressible Navier–Stokes equations on 3D hybrid unstructured grids with a third-order reconstructed discontinuous Galerkin method. The developed scheme requires the minimum code intrusion and algorithm alteration for upgrading a legacy solver with the GPU computing capability at very little extra effort in programming, which leads to a unified and portable code development strategy. A face coloring algorithm is adopted to eliminate the memory contention because of the threading of internal and boundary face integrals. A number of flow problems are presented to verify the implementation of the developed scheme. Timing measurements were obtained by running the resulting GPU code on one Nvidia Tesla K20c GPU card (Nvidia Corporation, Santa Clara, CA, USA) and compared with those obtained by running the equivalent Message Passing Interface (MPI) parallel CPU code on a compute node (consisting of two AMD Opteron 6128 eight-core CPUs (Advanced Micro Devices, Inc., Sunnyvale, CA, USA)). Speedup factors of up to 24× and 1.6× for the GPU code were achieved with respect to one and 16 CPU cores, respectively. The numerical results indicate that this OpenACC-based parallel scheme is an effective and extensible approach to port unstructured high-order CFD solvers to GPU computing.

  13. Dominant mutations causing alterations in acetyl-coenzyme A carboxylase confer tolerance to cyclohexanedione and aryloxyphenoxypropionate herbicides in maize.

    PubMed Central

    Parker, W B; Marshall, L C; Burton, J D; Somers, D A; Wyse, D L; Gronwald, J W; Gengenbach, B G

    1990-01-01

    A partially dominant mutation exhibiting increased tolerance to cyclohexanedione and aryloxyphenoxypropionate herbicides was isolated by exposing susceptible maize (Zea mays) tissue cultures to increasingly inhibitory concentrations of sethoxydim (a cyclohexanedione). The selected tissue culture (S2) was greater than 40-fold more tolerant to sethoxydim and 20-fold more tolerant to haloxyfop (an aryloxyphenoxypropionate) than the nonselected wild-type tissue culture. Regenerated S2 plants were heterozygous for the mutant allele and exhibited a high-level, but not complete, tolerance to both herbicides. Homozygous mutant families derived by self-pollinating the regenerated S2 plants exhibited no injury after treatment with 0.8 kg of sethoxydim per ha, which was greater than 16-fold the rate lethal to wild-type plants. Acetyl-coenzyme A carboxylase (ACCase; EC 6.4.1.2) is the target enzyme of cyclohexanedione and aryloxyphenoxypropionate herbicides. ACCase activities of the nonselected wild-type and homozygous mutant seedlings were similar in the absence of herbicide. ACCase activity from homozygous tolerant plants required greater than 100-fold more sethoxydim and 16-fold more haloxyfop for 50% inhibition than ACCase from wild-type plants. These results indicate that tolerance to sethoxydim and haloxyfop is controlled by a partially dominant nuclear mutation encoding a herbicide-insensitive alteration in maize ACCase. Images PMID:1976254

  14. Multiple isoforms of phosphoenolpyruvate carboxylase in the Orchidaceae (subtribe Oncidiinae): implications for the evolution of crassulacean acid metabolism.

    PubMed

    Silvera, Katia; Winter, Klaus; Rodriguez, B Leticia; Albion, Rebecca L; Cushman, John C

    2014-07-01

    Phosphoenolpyruvate carboxylase (PEPC) catalyses the initial fixation of atmospheric CO2 into oxaloacetate and subsequently malate. Nocturnal accumulation of malic acid within the vacuole of photosynthetic cells is a typical feature of plants that perform crassulacean acid metabolism (CAM). PEPC is a ubiquitous plant enzyme encoded by a small gene family, and each member encodes an isoform with specialized function. CAM-specific PEPC isoforms probably evolved from ancestral non-photosynthetic isoforms by gene duplication events and subsequent acquisition of transcriptional control elements that mediate increased leaf-specific or photosynthetic-tissue-specific mRNA expression. To understand the patterns of functional diversification related to the expression of CAM, ppc gene families and photosynthetic patterns were characterized in 11 closely related orchid species from the subtribe Oncidiinae with a range of photosynthetic pathways from C3 photosynthesis (Oncidium cheirophorum, Oncidium maduroi, Rossioglossum krameri, and Oncidium sotoanum) to weak CAM (Oncidium panamense, Oncidium sphacelatum, Gomesa flexuosa and Rossioglossum insleayi) and strong CAM (Rossioglossum ampliatum, Trichocentrum nanum, and Trichocentrum carthagenense). Phylogenetic analysis revealed the existence of two main ppc lineages in flowering plants, two main ppc lineages within the eudicots, and three ppc lineages within the Orchidaceae. Our results indicate that ppc gene family expansion within the Orchidaceae is likely to be the result of gene duplication events followed by adaptive sequence divergence. CAM-associated PEPC isoforms in the Orchidaceae probably evolved from several independent origins.

  15. Temperature Responses of C4 Photosynthesis: Biochemical Analysis of Rubisco, Phosphoenolpyruvate Carboxylase, and Carbonic Anhydrase in Setaria viridis1[OPEN

    PubMed Central

    Boyd, Ryan A.; Gandin, Anthony; Cousins, Asaph B.

    2015-01-01

    The photosynthetic assimilation of CO2 in C4 plants is potentially limited by the enzymatic rates of Rubisco, phosphoenolpyruvate carboxylase (PEPc), and carbonic anhydrase (CA). Therefore, the activity and kinetic properties of these enzymes are needed to accurately parameterize C4 biochemical models of leaf CO2 exchange in response to changes in CO2 availability and temperature. There are currently no published temperature responses of both Rubisco carboxylation and oxygenation kinetics from a C4 plant, nor are there known measurements of the temperature dependency of the PEPc Michaelis-Menten constant for its substrate HCO3−, and there is little information on the temperature response of plant CA activity. Here, we used membrane inlet mass spectrometry to measure the temperature responses of Rubisco carboxylation and oxygenation kinetics, PEPc carboxylation kinetics, and the activity and first-order rate constant for the CA hydration reaction from 10°C to 40°C using crude leaf extracts from the C4 plant Setaria viridis. The temperature dependencies of Rubisco, PEPc, and CA kinetic parameters are provided. These findings describe a new method for the investigation of PEPc kinetics, suggest an HCO3− limitation imposed by CA, and show similarities between the Rubisco temperature responses of previously measured C3 species and the C4 plant S. viridis. PMID:26373659

  16. Reversible inactivation and characterization of purified inactivated form I ribulose 1,5-bisphosphate carboxylase/oxygenase of Rhodobacter sphaeroides.

    PubMed

    Wang, X; Tabita, F R

    1992-06-01

    Form I ribulose 1,5-bisphosphate carboxylase/oxygenase (RubisCO) from Rhodobacter sphaeroides is inactivated upon the addition of organic acids to photolithoautotrophically grown cultures. Activity recovers after the dissipation of the organic acid from the culture. The inactivation process depends on both the concentration of the organic compound and the nitrogen status of the cells. The inactivated RubisCO has been purified and was shown to exhibit mobility on both nondenaturing and sodium dodecyl sulfate gels different from that of the active enzyme prepared from cells not treated with organic acids. However, the Michaelis constants for ribulose 1,5-bisphosphate and CO2 or O2 were not dramatically altered. Purified inactivated RubisCO could be activated in vitro by increasing the temperature or the levels of Mg(II), and this activation was accompanied by changes in the electrophoretic mobility of the protein. When foreign bacterial RubisCO genes were expressed in an R. sphaeroides host strain lacking the ability to synthesize endogenous RubisCO, only slight inactivation of RubisCO activity was attained.

  17. Closely related form I ribulose bisphosphate carboxylase/oxygenase molecules that possess different CO2/O2 substrate specificities.

    PubMed

    Horken, K M; Tabita, F R

    1999-01-15

    The deduced primary sequence (cbbL and cbbS) of form I ribulose 1, 5-bisphosphate carboxylase/oxygenase (rubisco) from Bradyrhizobium japonicum places this enzyme within the Type IC subgroup of red-like rubisco enzymes. In addition, B. japonicum appears to organize most of the structural genes of the Calvin-Benson-Bassham (CBB) pathway in at least one major operon. Functional expression and characterization of the B. japonicum and Xanthobacter flavus enzymes from this group revealed that these molecules exhibit diverse kinetic properties despite their relatively high degree of sequence relatedness. Of prime importance was the fact that these closely related enzymes exhibited CO2 and O2 substrate specificities that varied from relatively low values [tau = (VcKo)/(VoKc) = 45] to values that approximated those obtained for higher plants (tau = 75). These results, combined with the metabolic and genetic versatility of the organisms from which these enzymes were derived, suggest a potential rich resource for future biological selection and structure-function studies aimed at elucidating structural features that govern key enzymological properties of rubisco.

  18. Effect of mutation of lysine-128 of the large subunit of ribulose bisphosphate carboxylase/oxygenase from Anacystis nidulans.

    PubMed

    Bainbridge, G; Anralojc, P J; Madgwick, P J; Pitts, J E; Parry, M A

    1998-12-01

    The contribution of lysine-128 within the active site of Anacystis nidulans d-ribulose 1,5-bisphosphate carboxylase/oxygenase (Rubisco; EC 4.1.1.39) was investigated by the characterization of mutants in which lysine-128 was replaced with arginine, glycine, glutamine, histidine or aspartic acid. Mutated genes encoding the Rubisco large subunit were expressed in Escherichia coli and the resultant polypeptides assembled into active complexes. All of the mutant enzymes had a lower affinity for ribulose 1,5-bisphosphate (RuBP) and lower rates of carboxylation. Substitution of lysine-128 with glutamine, histidine or aspartic acid decreased the specificity factor and led to the production of an additional monophosphate reaction product. We show that this product results from the loss of the phosphate from C-1 of RuBP, most probably by beta-elimination from the 2,3-enediolate derivative of RuBP. The results confirm that lysine-128 is important in determining the position of the essential epsilon-amino group of lysine-334 within the active site and in loop dynamics. This further demonstrates that residues remote from the active site can be manipulated to modify catalytic function.

  19. Constitutive and dark-induced expression of Solanum tuberosum phosphoenolpyruvate carboxylase enhances stomatal opening and photosynthetic performance of Arabidopsis thaliana.

    PubMed

    Kebeish, Rashad; Niessen, Markus; Oksaksin, Mehtap; Blume, Christian; Peterhaensel, Christoph

    2012-02-01

    The effect of constitutive and dark-induced expression of Solanum tuberosum phosphoenolpyruvate carboxylase (PEPC) on the opening state of stomata and photosynthetic performance in Arabidopsis thaliana plants was studied. Transcript accumulation analyses of the A. thaliana dark-induced (Din10 and Din6) and the Pisum sativum asparagine synthetase 2 promoters (Asn2) in transiently transformed tobacco leaves showed that Din10 promoter induced more DsRed accumulation in the dark compared to the other din genes. Overexpression of PEPC under the control of the constitutive enhanced CaMV 35S (p35SS) and dark-induced Din10 promoter in stably transformed A. thaliana plants increased the number of opened stomata in dark adapted leaves. Gas exchange measurements using A. thaliana plants transgenic for p35SS-PEPC and Din10-PEPC revealed a marked increase in stomatal conductance, transpiration, and dark respiration rates measured in the dark compared to wild-type plants. Moreover, measurement of CO(2) assimilation rates at different external CO(2) concentrations (C(a) ) and different light intensities shows an increase in the CO(2) assimilation rates in transgenic Arabidopsis lines compared to wild-type plants. This is considered as first step towards transferring the aspects of Crassulacean acid metabolism-like photosynthetic mechanism into C3 plants.

  20. Pyruvate Carboxylase Activates the RIG-I-like Receptor-Mediated Antiviral Immune Response by Targeting the MAVS signalosome

    PubMed Central

    Cao, Zhongying; Zhou, Yaqin; Zhu, Shengli; Feng, Jian; Chen, Xueyuan; Liu, Shi; Peng, Nanfang; Yang, Xiaodan; Xu, Gang; Zhu, Ying

    2016-01-01

    When retinoic acid-inducible gene 1 protein (RIG-I)-like receptors sense viral dsRNA in the cytosol, RIG-I and melanoma differentiation-associated gene 5 (MDA5) are recruited to the mitochondria to interact with mitochondrial antiviral signaling protein (MAVS) and initiate antiviral immune responses. In this study, we demonstrate that the biotin-containing enzyme pyruvate carboxylase (PC) plays an essential role in the virus-triggered activation of nuclear factor kappa B (NF-κB) signaling mediated by MAVS. PC contributes to the enhanced production of type I interferons (IFNs) and pro-inflammatory cytokines, and PC knockdown inhibits the virus-triggered innate immune response. In addition, PC shows extensive antiviral activity against RNA viruses, including influenza A virus (IAV), human enterovirus 71 (EV71), and vesicular stomatitis virus (VSV). Furthermore, PC mediates antiviral action by targeting the MAVS signalosome and induces IFNs and pro-inflammatory cytokines by promoting phosphorylation of NF-κB inhibitor-α (IκBα) and the IκB kinase (IKK) complex, as well as NF-κB nuclear translocation, which leads to activation of interferon-stimulated genes (ISGs), including double-stranded RNA-dependent protein kinase (PKR) and myxovirus resistance protein 1 (Mx1). Our findings suggest that PC is an important player in host antiviral signaling. PMID:26906558

  1. Diversity of the ribulose bisphosphate carboxylase/oxygenase form I gene (rbcL) in natural phytoplankton communities.

    PubMed Central

    Pichard, S L; Campbell, L; Paul, J H

    1997-01-01

    The phytoplankton of the world's oceans play an integral part in global carbon cycling and food webs by conversion of carbon dioxide into organic carbon. They accomplish this task through the action of the Calvin cycle enzyme ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO). Here we have investigated the phylogenetic diversity in the form I rbcL locus in natural phytoplankton communities of the open ocean and representative clones of marine autotrophic picoplankton by mRNA or DNA amplification and sequencing of a 480 to 483 bp internal fragment of this gene. Five gene sequences were recovered from nucleic acids of natural phytoplankton communities of the Gulf of Mexico. The rbcL genes of two Prochlorococcus isolates and one Synechococcus strain (WH8007) were also sequenced. Sequences were aligned with the database of rbcL genes and subjected to both neighbor-joining and parsimony analyses. The five sequences from the natural phytoplankton community spanned nearly the entire diversity of characterized form I rbcL genes, with some sequences closely related to isolates such as Synechococcus and Prochlorococcus (forms IA and I) and prymnesiophyte algae (form ID), while other sequences were deeply rooted. Unexpectedly, the deep euphotic zone contained an organism that possesses a transcriptionally active rbcL gene closely related to that of a recently characterized manganese-oxidizing bacterium, suggesting that such chemoautotrophs may contribute to the diversity of carbon-fixing organisms in the marine euphotic zone. PMID:9293012

  2. Photosynthetic and Other Phosphoenolpyruvate Carboxylase Isoforms in the Single-Cell, Facultative C4 System of Hydrilla verticillata1

    PubMed Central

    Rao, Srinath K.; Magnin, Noël C.; Reiskind, Julia B.; Bowes, George

    2002-01-01

    The submersed monocot Hydrilla verticillata (L.f.) Royle is a facultative C4 plant. It typically exhibits C3 photosynthetic characteristics, but exposure to low [CO2] induces a C4 system in which the C4 and Calvin cycles co-exist in the same cell and the initial fixation in the light is catalyzed by phosphoenolpyruvate carboxylase (PEPC). Three full-length cDNAs encoding PEPC were isolated from H. verticillata, two from leaves and one from root. The sequences were 95% to 99% identical and shared a 75% to 85% similarity with other plant PEPCs. Transcript studies revealed that one isoform, Hvpepc4, was exclusively expressed in leaves during C4 induction. This and enzyme kinetic data were consistent with it being the C4 photosynthesis isoform. However, the C4 signature serine of terrestrial plant C4 isoforms was absent in this and the other H. verticillata sequences. Instead, alanine, typical of C3 sequences, was present. Western analyses of C3 and C4 leaf extracts after anion-exchange chromatography showed similar dominant PEPC-specific bands at 110 kD. In phylogenetic analyses, the sequences grouped with C3, non-graminaceous C4, and Crassulacean acid metabolism PEPCs but not with the graminaceous C4, and formed a clade with a gymnosperm, which is consistent with H. verticillata PEPC predating that of other C4 angiosperms. PMID:12376652

  3. The source and characteristics of chemiluminescence associated with the oxygenase reaction catalyzed by Mn(2+)-ribulosebisphosphate carboxylase.

    PubMed

    Lilley, R M; Riesen, H; Andrews, T J

    1993-07-05

    We confirm the observation of Mogel and McFadden (Mogel, S.N., and McFadden, B. A. (1990) Biochemistry 29, 8333-8337) that ribulosebisphosphate carboxylase/oxygenase (rubisco) exhibits chemiluminescence while catalyzing its oxygenase reaction in the presence of Mn2+. However, our results with the spinach and Rhodospirillum rubrum enzymes differ markedly in the following respects. 1) Chemiluminescence intensity was directly proportional to enzyme concentration and behaved as if representing the rate of oxygenase catalysis. 2) The wavelength spectrum peaked at about 770 nm and extended beyond 810 nm. This seems inconsistent with chemiluminescence generated by simultaneous decay of pairs of singlet O2 molecules. It is consistent with manganese(II) luminescence and we discuss its possible sources. The time course of chemiluminescence (resolution, 0.25 s) was distinctively different for spinach and R. rubrum enzymes during the initial 5 s of catalysis, with the bacterial enzyme exhibiting a pronounced initial "burst." Chemiluminescence by the spinach enzyme responded to substrate concentrations in a manner consistent with known oxygenase properties, exhibiting Michaelis-Menten kinetics with ribulose-1,5-bisphosphate (Km 400 nM). Chemiluminescence required carbamylated enzyme with Mn2+ bound at the active site (activation energy, -57.1 KJ.mol-1). As an indicator of oxygenase activity, chemiluminescence represents an improvement over oxygen electrode measurements in response time and sensitivity by factors of at least 100.

  4. Mechanism of metamifop inhibition of the carboxyltransferase domain of acetyl-coenzyme A carboxylase in Echinochloa crus-galli

    PubMed Central

    Xia, Xiangdong; Tang, Wenjie; He, Shun; Kang, Jing; Ma, Hongju; Li, Jianhong

    2016-01-01

    Acetyl-coenzyme A carboxylase (ACCase) plays crucial roles in fatty acid metabolism and is an attractive target for herbicide discovery. Metamifop is a novel ACCase-inhibiting herbicide that can be applied to control sensitive weeds in paddy fields. In this study, the effects of metamifop on the chloroplasts, ACCase activity and carboxyltransferase (CT) domain gene expression in Echinochloa crus-galli were investigated. The results showed that metamifop interacted with the CT domain of ACCase in E. crus-galli. The three-dimensional structure of the CT domain of E. crus-galli ACCase in complex with metamifop was examined by homology modelling, molecular docking and molecular dynamics (MD) simulations. Metamifop has a different mechanism of inhibiting the CT domain compared with other ACCase inhibitors as it interacted with a different region in the active site of the CT domain. The protonation of nitrogen in the oxazole ring of metamifop plays a crucial role in the interaction between metamifop and the CT domain. The binding mode of metamifop provides a foundation for elucidating the molecular mechanism of target resistance and cross-resistance among ACCase herbicides, and for designing and optimizing ACCase inhibitors. PMID:27666674

  5. Promotion of photosynthesis in transgenic rice over-expressing of maize C4 phosphoenolpyruvate carboxylase gene by nitric oxide donors.

    PubMed

    Chen, Pingbo; Li, Xia; Huo, Kai; Wei, Xiaodong; Dai, Chuanchao; Lv, Chuangen

    2014-03-15

    We determined the effects of exogenous nitric oxide on photosynthesis and gene expression in transgenic rice plants (PC) over-expressing the maize C4pepc gene, which encodes phosphoenolpyruvate carboxylase (PEPC). Seedlings were subjected to treatments with NO donors, an NO scavenger, phospholipase inhibitors, a Ca(2+) chelator, a Ca(2+) channel inhibitor, and a hydrogen peroxide (H2O2) inhibitor, individually and in various combinations. The NO donors significantly increased the net photosynthetic rate (PN) of PC and wild-type (WT), especially that of PC. Treatment with an NO scavenger did inhibit the PN of rice plants. The treatments with phospholipase inhibitors and a Ca(2+) chelator decreased the PN of WT and PC, and photosynthesis was more strongly inhibited in WT than in PC. Further analyses showed that the NO donors increased endogenous levels of NO and PLD activity, but decreased endogenous levels of Ca(2+) both WT and PC. However, there was a greater increase in NO in WT and a greater increase in PLD activity and Ca(2+) level in PC. The NO donors also increased both PEPC activity and pepc gene expression in PC. PEPC activity can be increased by SNP alone. But the expression of its encoding gene in PC might be regulated by SNP, together with PA and Ca(2+).

  6. Mechanism of metamifop inhibition of the carboxyltransferase domain of acetyl-coenzyme A carboxylase in Echinochloa crus-galli

    NASA Astrophysics Data System (ADS)

    Xia, Xiangdong; Tang, Wenjie; He, Shun; Kang, Jing; Ma, Hongju; Li, Jianhong

    2016-09-01

    Acetyl-coenzyme A carboxylase (ACCase) plays crucial roles in fatty acid metabolism and is an attractive target for herbicide discovery. Metamifop is a novel ACCase-inhibiting herbicide that can be applied to control sensitive weeds in paddy fields. In this study, the effects of metamifop on the chloroplasts, ACCase activity and carboxyltransferase (CT) domain gene expression in Echinochloa crus-galli were investigated. The results showed that metamifop interacted with the CT domain of ACCase in E. crus-galli. The three-dimensional structure of the CT domain of E. crus-galli ACCase in complex with metamifop was examined by homology modelling, molecular docking and molecular dynamics (MD) simulations. Metamifop has a different mechanism of inhibiting the CT domain compared with other ACCase inhibitors as it interacted with a different region in the active site of the CT domain. The protonation of nitrogen in the oxazole ring of metamifop plays a crucial role in the interaction between metamifop and the CT domain. The binding mode of metamifop provides a foundation for elucidating the molecular mechanism of target resistance and cross-resistance among ACCase herbicides, and for designing and optimizing ACCase inhibitors.

  7. Genome-wide Analysis of Phosphoenolpyruvate Carboxylase Gene Family and Their Response to Abiotic Stresses in Soybean

    PubMed Central

    Wang, Ning; Zhong, Xiujuan; Cong, Yahui; Wang, Tingting; Yang, Songnan; Li, Yan; Gai, Junyi

    2016-01-01

    Phosphoenolpyruvate carboxylase (PEPC) plays an important role in assimilating atmospheric CO2 during C4 and crassulacean acid metabolism photosynthesis, and also participates in various non-photosynthetic processes, including fruit ripening, stomatal opening, supporting carbon–nitrogen interactions, seed formation and germination, and regulation of plant tolerance to stresses. However, a comprehensive analysis of PEPC family in Glycine max has not been reported. Here, a total of ten PEPC genes were identified in soybean and denominated as GmPEPC1-GmPEPC10. Based on the phylogenetic analysis of the PEPC proteins from 13 higher plant species including soybean, PEPC family could be classified into two subfamilies, which was further supported by analyses of their conserved motifs and gene structures. Nineteen cis-regulatory elements related to phytohormones, abiotic and biotic stresses were identified in the promoter regions of GmPEPC genes, indicating their roles in soybean development and stress responses. GmPEPC genes were expressed in various soybean tissues and most of them responded to the exogenously applied phytohormones. GmPEPC6, GmPEPC8 and GmPEPC9 were significantly induced by aluminum toxicity, cold, osmotic and salt stresses. In addition, the enzyme activities of soybean PEPCs were also up-regulated by these treatments, suggesting their potential roles in soybean response to abiotic stresses. PMID:27924923

  8. ppc, the gene for phosphoenolpyruvate carboxylase from an extremely thermophilic bacterium, Rhodothermus obamensis: cloning, sequencing and overexpression in Escherichia coli.

    PubMed

    Takai, K; Sako, Y; Uchida, A

    1998-05-01

    The ppc gene, which encodes phosphoenolpyruvate carboxylase (PEPC) of an extremely thermophilic bacterium, Rhodothermus obamensis, was directly sequenced by the thermal asymmetric interlaced (TAIL) PCR method. An ORF for a 937 amino acid polypeptide was found in the gene. The ppc gene had a high G+C content (66.2 mol%) and the third position of the codon exhibited strong preference for G or C usage (85.0 mol%). The calculated molecular mass was 107,848 Da, which was consistent with the molecular mass of the enzyme as determined by SDS-PAGE (100 kDa). The amino acid sequence of R. obamensis PEPC was closely related to that of PEPC from another thermophile, a Thermus sp., and from a mesophile, Corynebacterium glutamicum, exhibiting 45.3% or 37.7% identity and 61.5% or 56.5% similarity, respectively. By Southern analysis, the ppc gene was found to be present in a single copy in the genomic DNA of this organism. The cloned gene was expressed in Escherichia coli using a pET expression vector system and a thermostable recombinant PEPC was obtained. Comparison of the deduced amino acid sequences of the thermophilic and mesophilic PEPCs revealed distinct or common preferences for specific amino acid composition and substitutions in the two thermophilic enzymes.

  9. Algal evolution in relation to atmospheric CO2: carboxylases, carbon-concentrating mechanisms and carbon oxidation cycles

    PubMed Central

    Raven, John A.; Giordano, Mario; Beardall, John; Maberly, Stephen C.

    2012-01-01

    Oxygenic photosynthesis evolved at least 2.4 Ga; all oxygenic organisms use the ribulose bisphosphate carboxylase-oxygenase (Rubisco)–photosynthetic carbon reduction cycle (PCRC) rather than one of the five other known pathways of autotrophic CO2 assimilation. The high CO2 and (initially) O2-free conditions permitted the use of a Rubisco with a high maximum specific reaction rate. As CO2 decreased and O2 increased, Rubisco oxygenase activity increased and 2-phosphoglycolate was produced, with the evolution of pathways recycling this inhibitory product to sugar phosphates. Changed atmospheric composition also selected for Rubiscos with higher CO2 affinity and CO2/O2 selectivity correlated with decreased CO2-saturated catalytic capacity and/or for CO2-concentrating mechanisms (CCMs). These changes increase the energy, nitrogen, phosphorus, iron, zinc and manganese cost of producing and operating Rubisco–PCRC, while biosphere oxygenation decreased the availability of nitrogen, phosphorus and iron. The majority of algae today have CCMs; the timing of their origins is unclear. If CCMs evolved in a low-CO2 episode followed by one or more lengthy high-CO2 episodes, CCM retention could involve a combination of environmental factors known to favour CCM retention in extant organisms that also occur in a warmer high-CO2 ocean. More investigations, including studies of genetic adaptation, are needed. PMID:22232762

  10. Interaction between potyvirus P3 and ribulose-1,5-bisphosphate carboxylase/oxygenase (RubisCO) of host plants.

    PubMed

    Lin, Lin; Luo, Zhaopeng; Yan, Fei; Lu, Yuwen; Zheng, Hongying; Chen, Jianping

    2011-08-01

    The P3 protein encoded by Shallot yellow stripe virus onion isolate (SYSV-O) interacted in the Yeast Two-hybrid (Y2H) system and in co-immunoprecipitation (Co-IP) assays with the large subunit of the ribulose-1,5-bisphosphate carboxylase/oxygenase (RubisCO) protein that is encoded by the rbcL gene of its onion host. Dissection analysis by Y2H showed that the main part of SYSV P3 (amino acids 1-390) and onion RbcL (amino acids 1-137) were responsible for the interaction. The P3 proteins encoded by Onion yellow dwarf virus (OYDV), Soybean mosaic virus Pinellia isolate (SMV-P), and Turnip mosaic virus (TuMV) also interacted with RbcL, suggesting that a P3/RbcL interaction might exist generally for potyviruses. An interaction between P3 of these potyviruses and the small subunit of RubisCO (RbcS) was also demonstrated. Moreover, the P3N-PIPO protein encoded by a newly identified open reading frame embedded within the P3 cistron also interacted with both RbcL and RbcS. It is possible that the potyvirus P3 protein affects the normal functions of RubisCO which thus contributes to symptom development.

  11. Multiple isoforms of phosphoenolpyruvate carboxylase in the Orchidaceae (subtribe Oncidiinae): implications for the evolution of crassulacean acid metabolism

    PubMed Central

    Silvera, Katia; Winter, Klaus; Rodriguez, B. Leticia; Albion, Rebecca L.; Cushman, John C.

    2014-01-01

    Phosphoenolpyruvate carboxylase (PEPC) catalyses the initial fixation of atmospheric CO2 into oxaloacetate and subsequently malate. Nocturnal accumulation of malic acid within the vacuole of photosynthetic cells is a typical feature of plants that perform crassulacean acid metabolism (CAM). PEPC is a ubiquitous plant enzyme encoded by a small gene family, and each member encodes an isoform with specialized function. CAM-specific PEPC isoforms probably evolved from ancestral non-photosynthetic isoforms by gene duplication events and subsequent acquisition of transcriptional control elements that mediate increased leaf-specific or photosynthetic-tissue-specific mRNA expression. To understand the patterns of functional diversification related to the expression of CAM, ppc gene families and photosynthetic patterns were characterized in 11 closely related orchid species from the subtribe Oncidiinae with a range of photosynthetic pathways from C3 photosynthesis (Oncidium cheirophorum, Oncidium maduroi, Rossioglossum krameri, and Oncidium sotoanum) to weak CAM (Oncidium panamense, Oncidium sphacelatum, Gomesa flexuosa and Rossioglossum insleayi) and strong CAM (Rossioglossum ampliatum, Trichocentrum nanum, and Trichocentrum carthagenense). Phylogenetic analysis revealed the existence of two main ppc lineages in flowering plants, two main ppc lineages within the eudicots, and three ppc lineages within the Orchidaceae. Our results indicate that ppc gene family expansion within the Orchidaceae is likely to be the result of gene duplication events followed by adaptive sequence divergence. CAM-associated PEPC isoforms in the Orchidaceae probably evolved from several independent origins. PMID:24913627

  12. 1-Aminocyclopropane-1-carboxylic acid (ACC) deaminase-containing rhizobacteria protect Ocimum sanctum plants during waterlogging stress via reduced ethylene generation.

    PubMed

    Barnawal, Deepti; Bharti, Nidhi; Maji, Deepamala; Chanotiya, Chandan Singh; Kalra, Alok

    2012-09-01

    Ocimum sanctum grown as rain-fed crop, is known to be poorly adapted to waterlogged conditions. Many a times the crop suffers extreme damages because of anoxia and excessive ethylene generation due to waterlogging conditions present under heavy rain. The usefulness of 1-aminocyclopropane-1-carboxylic acid (ACC) deaminase-containing plant growth promoting rhizobacteria was investigated under waterlogging stress. The comparison of herb yield and stress induced biochemical changes of waterlogged and non-waterlogged plants with and without ACC deaminase-containing microbiological treatments were monitored in this study. Ten plant growth promoting rhizobacteria strains containing ACC-deaminase were isolated and characterized. Four selected isolates Fd2 (Achromobacter xylosoxidans), Bac5 (Serratia ureilytica), Oci9 (Herbaspirillum seropedicae) and Oci13 (Ochrobactrum rhizosphaerae) had the potential to protect Ocimum plants from flood induced damage under waterlogged glass house conditions. Pot experiments were conducted to evaluate the potential of these ACC deaminase-containing selected strains for reducing the yield losses caused by waterlogging conditions. Bacterial treatments protected plants from waterlogging induced detrimental changes like stress ethylene production, reduced chlorophyll concentration, higher lipid peroxidation, proline concentration and reduced foliar nutrient uptake. Fd2 (A. xylosoxidans) induced maximum waterlogging tolerance as treated waterlogged plants recorded maximum growth and herb yield (46.5% higher than uninoculated waterlogged plants) with minimum stress ethylene levels (53% lower ACC concentration as compared to waterlogged plants without bacterial inoculation) whereas under normal non-waterlogged conditions O. rhizosphaerae was most effective in plant growth promotion.

  13. pACC1 peptide loaded chitosan nanoparticles induces apoptosis via reduced fatty acid synthesis in MDA-MB-231 cells

    NASA Astrophysics Data System (ADS)

    Kaliaperumal, Jagatheesh; Hari, Natarajan; Pavankumar, Padarthi; Elangovan, Namasivayam

    2016-06-01

    The development of formulations with therapeutic peptides has been restricted to poor cell penetration and in this attempt; we developed pACC1 peptide loaded chitosan nanoparticles. The prepared nanoparticles were characterized with FT-IR, XRD, SEM and TEM. In addition, the suitable formulation was evaluated for hemocompatibility, plasma stability and embryo toxicity using Danio rerio embryo model. The results showed that pACC1 peptide loaded chitosan nanoparticles were compatible with plasma. They possess sustained release pattern and also found to be safe up to 300 mg/L in embryo toxicity tests. Cytotoxicity assays with MDA-MB-231 cell lines suggested that, pACC1 peptide loaded chitosan nanoparticles were capable of enhanced cellular penetration and reduced palmitic acid content, which was confirmed by H1 NMR. Hence, these nanoparticles could be employed as excellent adjuvant therapeutics while treating solid tumors with multi-drug resistance.

  14. Studies on Plant Growth Promoting Properties of Fruit-Associated Bacteria from Elettaria cardamomum and Molecular Analysis of ACC Deaminase Gene.

    PubMed

    Jasim, B; Anish, Mathew Chacko; Shimil, Vellakudiyan; Jyothis, Mathew; Radhakrishnan, E K

    2015-09-01

    Endophytic microorganisms have been reported to have diverse plant growth promoting mechanisms including phosphate solubilization, N2 fixation, production of phyto-hormones and ACC (1-aminocyclopropane-1-carboxylate) deaminase and antiphyto-pathogenic properties. Among these, ACC deaminase production is very important because of its regulatory effect on ethylene which is a stress hormone with precise role in the control of fruit development and ripening. However, distribution of these properties among various endophytic bacteria associated with fruit tissue and its genetic basis is least investigated. In the current study, 11 endophytic bacteria were isolated and identified from the fruit tissue of Elettaria cardamomum and were studied in detail for various plant growth promoting properties especially ACC deaminase activity using both culture-based and PCR-based methods. PCR-based screening identified the isolates EcB 2 (Pantoea sp.), EcB 7 (Polaromonas sp.), EcB 9 (Pseudomonas sp.), EcB 10 (Pseudomonas sp.) and EcB 11 (Ralstonia sp.) as positive for ACC deaminase. The PCR products were further subjected to sequence analysis which proved the similarity of the sequences identified in the study with ACC deaminase sequences reported from other sources. The detailed bioinformatic analysis of the sequence including homology-based modelling and molecular docking confirmed the sequences to have ACC deaminase activity. The docking of the modelled proteins was done using patch dock, and the detailed scrutiny of the protein ligand interaction revealed conservation of key amino acids like Lys51, Ser78, Tyr268 and Tyr294 which play important role in the enzyme activity. These suggest the possible regulatory effect of these isolates on fruit physiology.

  15. ACC oxidase genes expressed in the wood-forming tissues of loblolly pine (Pinus taeda L.) include a pair of nearly identical paralogs (NIPs).

    PubMed

    Yuan, S; Wang, Y; Dean, J F D

    2010-03-15

    1-Aminocyclopropane-1-carboxylate (ACC) oxidase catalyzes the final reaction of the ethylene biosynthetic pathway, converting the unusual cyclic amino acid, ACC, into ethylene. Past studies have shown a possible link between ethylene and compression wood formation in conifers, but the relationship has received no more than modest study at the gene expression level. In this study, a cDNA clone encoding a putative ACC oxidase, PtACO1, was isolated from a cDNA library produced using mRNA from lignifying xylem of loblolly pine (Pinus taeda) trunk wood. The cDNA clone comprised an open reading frame of 1461 bp encoding a protein of 333 amino acids. Using PCR amplification techniques, a genomic clone corresponding to PtACO1 was isolated and shown to contain three introns with typical GT/AG boundaries defining the splice junctions. The PtACO1 gene product shared 70% identity with an ACC oxidase from European white birch (Betula pendula), and phylogenetic analyses clearly placed the gene product in the ACC oxidase cluster of the Arabidopsis thaliana 2-oxoglutarate-dependent dioxygenase superfamily tree. The PtACO1 sequence was used to identify additional ACC oxidase clones from loblolly pine root cDNA libraries characterized as part of an expressed sequence tag (EST) discovery project. The PtACO1 sequence was also used to recover additional paralogous sequences from genomic DNA, one of which (PtACO2) turned out to be >98% identical to PtACO1 in the nucleotide coding sequence, leading to its classification as a "nearly identical paralog" (NIP). Quantitative PCR analyses showed that the expression level of PtACO1-like transcripts varied in different tissues, as well as in response to hormonal treatments and bending. Possible roles for PtACO1 in compression wood formation in loblolly pine and the discovery of its NIP are discussed in light of these results.

  16. Potentiation of motor sub-networks for motor control but not working memory: Interaction of dACC and SMA revealed by resting-state directed functional connectivity.

    PubMed

    Diwadkar, Vaibhav A; Asemi, Avisa; Burgess, Ashley; Chowdury, Asadur; Bressler, Steven L

    2017-01-01

    The dorsal Anterior Cingulate Cortex (dACC) and the Supplementary Motor Area (SMA) are known to interact during motor coordination behavior. We previously discovered that the directional influences underlying this interaction in a visuo-motor coordination task are asymmetric, with the dACC→SMA influence being significantly greater than that in the reverse direction. To assess the specificity of this effect, here we undertook an analysis of the interaction between dACC and SMA in two distinct contexts. In addition to the motor coordination task, we also assessed these effects during a (n-back) working memory task. We applied directed functional connectivity analysis to these two task paradigms, and also to the rest condition of each paradigm, in which rest blocks were interspersed with task blocks. We report here that the previously known asymmetric interaction between dACC and SMA, with dACC→SMA dominating, was significantly larger in the motor coordination task than the memory task. Moreover the asymmetry between dACC and SMA was reversed during the rest condition of the motor coordination task, but not of the working memory task. In sum, the dACC→SMA influence was significantly greater in the motor task than the memory task condition, and the SMA→dACC influence was significantly greater in the motor rest than the memory rest condition. We interpret these results as suggesting that the potentiation of motor sub-networks during the motor rest condition supports the motor control of SMA by dACC during the active motor task condition.

  17. Potentiation of motor sub-networks for motor control but not working memory: Interaction of dACC and SMA revealed by resting-state directed functional connectivity

    PubMed Central

    Diwadkar, Vaibhav A.; Asemi, Avisa; Burgess, Ashley; Chowdury, Asadur; Bressler, Steven L.

    2017-01-01

    The dorsal Anterior Cingulate Cortex (dACC) and the Supplementary Motor Area (SMA) are known to interact during motor coordination behavior. We previously discovered that the directional influences underlying this interaction in a visuo-motor coordination task are asymmetric, with the dACC→SMA influence being significantly greater than that in the reverse direction. To assess the specificity of this effect, here we undertook an analysis of the interaction between dACC and SMA in two distinct contexts. In addition to the motor coordination task, we also assessed these effects during a (n-back) working memory task. We applied directed functional connectivity analysis to these two task paradigms, and also to the rest condition of each paradigm, in which rest blocks were interspersed with task blocks. We report here that the previously known asymmetric interaction between dACC and SMA, with dACC→SMA dominating, was significantly larger in the motor coordination task than the memory task. Moreover the asymmetry between dACC and SMA was reversed during the rest condition of the motor coordination task, but not of the working memory task. In sum, the dACC→SMA influence was significantly greater in the motor task than the memory task condition, and the SMA→dACC influence was significantly greater in the motor rest than the memory rest condition. We interpret these results as suggesting that the potentiation of motor sub-networks during the motor rest condition supports the motor control of SMA by dACC during the active motor task condition. PMID:28278267

  18. Removal of Asbestos-Containing Coatings (ACC) from gas transmission pipelines. Final report, January 1991-October 1993

    SciTech Connect

    Petersen, L.E.; Blackburn, M.L.

    1994-01-01

    Corrosion control coatings on transmission pipelines may contain asbestos as a secondary component of the coating. Current environmental and health regulations require a wet removal process for asbestos materials that provides close control of airborne emissions and asbestos fibers in effluent water. Modification of current line-traveling, water jet equipment was successfully completed in developing an economic removal process for asbestos-containing coatings (ACC). Materials handling components were added in yard experiments that permitted water jet removal, slurry filtration, and residue containerization meeting emission control levels, while providing pipe cleanliness suitable for recoating. Field evaluations under in-the-ditch and over-the-ditch conditions on 16-, 26- and 30-inch pipelines verified the achievement of design coating removal rates and asbestos emission control that meets current regulations.

  19. SCAI/AATS/ACC/STS operator and institutional requirements for transcatheter valve repair and replacement, Part III: Pulmonic valve.

    PubMed

    Hijazi, Ziyad M; Ruiz, Carlos E; Zahn, Evan; Ringel, Richard; Aldea, Gabriel S; Bacha, Emile A; Bavaria, Joseph; Bolman, R Morton; Cameron, Duke E; Dean, Larry S; Feldman, Ted; Fullerton, David; Horlick, Eric; Mack, Michael J; Miller, D Craig; Moon, Marc R; Mukherjee, Debabrata; Trento, Alfredo; Tommaso, Carl L

    2015-07-01

    With the evolution of transcatheter valve replacement, an important opportunity has arisen for cardiologists and surgeons to collaborate in identifying the criteria for performing these procedures. Therefore, The Society for Cardiovascular Angiography and Interventions (SCAI), American Association for Thoracic Surgery (AATS), American College of Cardiology (ACC), and The Society of Thoracic Surgeons (STS) have partnered to provide recommendations for institutions to assess their potential for instituting and/or maintaining a transcatheter valve program. This article concerns transcatheter pulmonic valve replacement (tPVR). tPVR procedures are in their infancy with few reports available on which to base an expert consensus statement. Therefore, many of these recommendations are based on expert consensus and the few reports available. As the procedures evolve, technology advances, experience grows, and more data accumulate, there will certainly be a need to update this consensus statement. The writing committee and participating societies believe that the recommendations in this report serve as appropriate requisites. In some ways, these recommendations apply to institutions more than to individuals. There is a strong consensus that these new valve therapies are best performed using a Heart Team approach; thus, these credentialing criteria should be applied at the institutional level. Partnering societies used the ACC's policy on relationships with industry (RWI) and other entities to author this document (http://www.acc.org/guidelines/about-guidelines-and-clinical-documents). To avoid actual, potential, or perceived conflicts of interest due to industry relationships or personal interests, all members of the writing committee, as well as peer reviewers of the document, were asked to disclose all current healthcare-related relationships including those existing 12 months before the initiation of the writing effort. A committee of interventional cardiologists and

  20. Purification and characterization of large and small subunits of ribulose 1,5-bisphosphate carboxylase expressed separately in Escherichia coli.

    PubMed

    Smrcka, A V; Ramage, R T; Bohnert, H J; Jensen, R G

    1991-04-01

    Procedures were developed for 95 and 80% purification to homogeneity of the large subunit (L) and small subunit (S) of ribulose 1,5-bisphosphate carboxylase/oxygenase (L8S8) from Synechococcus PCC 6301, each expressed separately in Escherichia coli. Purified L had a low specific activity in the absence of S (0.075 mumol CO2 fixed/mg holoenzyme/min). Following elution on a Pharmacia Superose 6 or 12 gel filtration column, 50% of the purified L appeared as the octamer, L8. The rest was in equilibrium with lower polymeric species and/or was retained on the column. Large and small subunits assembled rapidly into the L8S8 holoenzyme that had high specific activities, 6.2 and 3.1 mumol CO2 fixed/mg holoenzyme/min for the homologous Synechococcus L8S8 and the hybrid Synechococcus L-pea S L8S8, respectively. The CO2 dependence for carbamylation of L8 was compared to that of L8S8 as a function of pH and CO2 concentration. The pH dependence indicated an apparent pKa for L8 of 8.28 and for L8S8 of 8.15, suggesting that S may influence the pKa of the lysine involved in carbamylation. The Kact for CO2 at pH 8.4 were similar for L8 (13.5 microM) and L8S8 (15.5 microM). L8 bound 2-[14C]carboxy-D-arabinitol 1,5-bisphosphate (CABP) tightly so that most of the bound [14C]CABP survived gel filtration. A major amount of the L8-[14C]CABP complex appeared as larger polymeric aggregates when eluted in the presence of E. coli protein.

  1. Characterization of the duplicate ribulose-1,5-bisphosphate carboxylase genes and cbb promoters of Alcaligenes eutrophus.

    PubMed

    Kusian, B; Bednarski, R; Husemann, M; Bowien, B

    1995-08-01

    Autotrophic CO2 fixation via the Calvin carbon reduction cycle in Alcaligenes eutrophus H16 is genetically determined by two highly homologous cbb operons, one of which is located on the chromosome and the other on megaplasmid pHG1 of the organism. An activator gene, cbbR, lies in divergent orientation only 167 bp upstream of the chromosomal operon and controls the expression of both cbb operons. The two 5'-terminal genes of the operons, cbbLS, coding for ribulose-1,5-bisphosphate carboxylase/oxygenase, were sequenced. Mapping of the 5' termini of the 2.1-kb cbbLS transcripts by primer extension and by nuclease S1 treatment revealed a single transcriptional start point at the same relative position for the chromosomal and plasmid-borne cbb operons. The derived cbb operon promoter showed similarity to sigma 70-dependent promoters of Escherichia coli. For the 1.4-kb transcripts of cbbR, the transcriptional start points were different in autotrophic and heterotrophic cells. The two corresponding cbbR promoters overlapped the cbb operon promoter and also displayed similarities to sigma 70-dependent promoters. The deficient cbbR gene located on pHG1 was transcribed as well. A newly constructed double operon fusion vector was used to determine the activities of the cbb promoters. Fusions with fragments carrying the cbb intergenic control regions demonstrated that the cbb operon promoters were strongly regulated in response to autotrophic versus heterotrophic growth conditions. In contrast, the cbbR promoters displayed low constitutive activities. The data suggest that the chromosomal and plasmid-borne cbb promoters of A. eutrophus H16 are functionally equivalent despite minor structural differences.

  2. Covalent modification of a highly reactive and essential lysine residue of ribulose-1,5-bisphosphate carboxylase/oxygenase activase.

    PubMed

    Salvucci, M E

    1993-10-01

    Chemical modification of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) activase with water-soluble N-hydroxysuccinimide esters was used to identify a reactive lysyl residue that is essential for activity. Incubation of Rubisco activase with sulfosuccinimidyl-7-amino-4-methylcoumarin-3-acetate (AMCA-sulfo-NHS) or sulfosuccinimidyl-acetate (sulfo-NHS-acetate) caused progressive inactivation of ATPase activity and concomitant loss of the ability to activate Rubisco. AMCA-sulfo-NHS was the more potent inactivator of Rubisco activase, exhibiting a second-order rate constant for inactivation of 239 M-1 s-1 compared to 21 M-1 s-1 for sulfo-NHS-acetate. Inactivation of enzyme activity by AMCA-sulfo-NHS correlated with the incorporation of 1.9 mol of AMCA per mol of 42-kD Rubisco activase monomer. ADP, a competitive inhibitor of Rubisco activase, afforded considerable protection against inactivation of Rubisco activase and decreased the amount of AMCA incorporated into the Rubisco activase monomer. Sequence analysis of the major labeled peptide from AMCA-sulfo-NHS-modified enzyme showed that the primary site of modification was lysine-247 (K247) in the tetrapeptide methionine-glutamic acid-lysine-phenylalanine. Upon complete inactivation of ATPase activity, modification of K247 accounted for 1 mol of AMCA incorporated per mol of Rubisco activase monomer. Photoaffinity labeling of AMCA-sulfo-NHS- and sulfo-NHS-acetate-modified Rubisco activase with ATP analogs derivatized on either the adenine base or on the gamma-phosphate showed that K247 is not essential for the binding of adenine nucleotides per se. Instead, the data indicated that the essentiality of K247 is probably due to an involvement of this highly reactive, species-invariant residue in an obligatory interaction that occurs between the protein and the nucleotide phosphate during catalysis.

  3. Changes in the net charge and subunit properties of ribulose bisphosphate carboxylase--oxygenase during cold hardening of Puma rye.

    PubMed

    Huner, N P; Macdowall, F D

    1979-02-01

    Ribulose bisphosphate carboxylase--oxygenase (RUBPCase) from leaves of cold-hardened and unhardened Puma rye was purified by gel filtration and ion exchange chromatography. The specific activity of the hardened form was twice that of the unhardened form. A difference in charge between the two forms of this enzyme was proved by gel electrofocussing. The estimated isoelectric point (pI) values were 6.4 and 6.3 for the enzyme from the hardened and unhardened source respectively. The large subunit (55,000 molecular weight) of the enzyme from only the unhardened source formed at apparent dimer during sodium dodecyl sulfate (SDS) gel electrophoresis. At pH 6,8 it was also the source of an anomalous polypeptide with an apparent molecular weight of 47,000. This anomalous polypeptide appeared in both hardened and unhardened preparations after irreversible inactivation of RUBPCase activity by NaCl. It also appeared after preparation of the purified enzymes for SDS--PAGE in the absence of beta-mercaptoethanol, but this was reversible. The enzyme from the hardened source was less affected in the absence of reducing agent. Structural evidence was obtained for the previously reported cold hardening of the enzyme against freeze inactivation. A freeze-thaw cycle applied to the enzyme in vitro caused some polymerization of the large subunit and its anomalous polypeptide, in the absence of reducing agent, especially in the unhardened case. This increased with repeated cycles until the fifth cycle when the large subunit monomer and its satellite were abolished only in preparations from the unhardened source. These data indicate that the large subunit is a probable site of change that occurred in this enzyme during cold hardening.

  4. Altered intersubunit interactions in crystal structures of catalytically compromised ribulose-1,5-bisphosphate carboxylase/oxygenase.

    PubMed

    Karkehabadi, Saeid; Taylor, Thomas C; Spreitzer, Robert J; Andersson, Inger

    2005-01-11

    Substitution of Leu290 by Phe (L290F) in the large subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase from the unicellular green alga Chlamydomonas reinhardtii causes a 13% decrease in CO(2)/O(2) specificity and reduced thermal stability. Genetic selection for restored photosynthesis at the restrictive temperature identified an Ala222 to Thr (A222T) substitution that suppresses the deleterious effects of the original mutant substitution to produce a revertant enzyme with improved thermal stability and kinetic properties virtually indistinguishable from that of the wild-type enzyme. Because the mutated residues are situated approximately 19 A away from the active site, they must affect the relative rates of carboxylation and oxygenation in an indirect way. As a means for elucidating the role of such distant interactions in Rubisco catalysis and stability, we have determined the crystal structures of the L290F mutant and L290F/A222T revertant enzymes to 2.30 and 2.05 A resolution, respectively. Inspection of the structures reveals that the mutant residues interact via van der Waals contacts within the same large subunit (intrasubunit path, 15.2 A Calpha-Calpha) and also via a path involving a neighboring small subunit (intersubunit path, 18.7 A Calpha-Calpha). Structural analysis of the mutant enzymes identified regions (residues 50-72 of the small subunit and residues 161-164 and 259-264 of the large subunit) that show significant and systematically increased atomic temperature factors in the L290F mutant enzyme compared to wild type. These regions coincide with residues on the interaction paths between the L290F mutant and A222T suppressor sites and could explain the temperature-conditional phenotype of the L290F mutant strain. This suggests that alterations in subunit interactions will influence protein dynamics and, thereby, affect catalysis.

  5. Presence of a structurally novel type ribulose-bisphosphate carboxylase/oxygenase in the hyperthermophilic archaeon, Pyrococcus kodakaraensis KOD1.

    PubMed

    Ezaki, S; Maeda, N; Kishimoto, T; Atomi, H; Imanaka, T

    1999-02-19

    We have characterized the gene encoding ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) of the hyperthermophilic archaeon, Pyrococcus kodakaraensis KOD1. The gene encoded a protein consisting of 444 amino acid residues, corresponding in size to the large subunit of previously reported Rubiscos. Rubisco of P. kodakaraensis KOD1 (Pk-Rubisco) showed only 51.4% similarity with the large subunit of type I Rubisco from spinach and 47.3% with that of type II Rubisco from Rhodospirillum rubrum, suggesting that the enzyme was not a member of either type. Active site residues identified from type I and type II Rubiscos were conserved. We expressed the gene in Escherichia coli, and we obtained a soluble protein with the expected molecular mass and N-terminal amino acid sequence. Purification of the recombinant protein revealed that Pk-Rubisco was an L8 type homo-octamer. Pk-Rubisco showed highest specific activity of 19.8 x 10(3) nmol of CO2 fixed per min/mg, and a tau value of 310 at 90 degreesC, both higher than any previously characterized Rubisco. The optimum pH was 8.3, and the enzyme possessed extreme thermostability, with a half-life of 15 h at 80 degreesC. Northern blot analysis demonstrated that the gene was transcribed in P. kodakaraensis KOD1. Furthermore, Western blot analysis with cell-free extract of P. kodakaraensis KOD1 clearly indicated the presence of Pk-Rubisco in the native host cells.

  6. Ribulose-1,5-bisphosphate carboxylase/oxygenase gene expression and diversity of Lake Erie planktonic microorganisms

    SciTech Connect

    Xu, H.H.; Tabita, F.R.

    1996-06-01

    Carbon dioxide fixation is carried out primarily through the Calvin-Benson-Bassham reductive pentose phosphate cycle, in which rubulose-1,5-bisphosphate carboxylase/oxygenase (RubisCO) is the key enzyme. The primary structure of the large subunit of form I RubisCO is well conserved; however, four distinct types, A, B, C, and D, may be distinguished. To better understand the environmental regulation of RubisCO in Lake Erie phytoplanktonic microorganisms, we have isolated total RNA and DNA from four Lake Erie sampling sites. Probes prepared from RubisCO large-subunit genes (rbcL) of the freshwater cyanobacterium Synechococcus sp. strain PCC6301 (representative of type IB) and the diatom Cylindrotheca sp. strain N1 (representative of type ID) was determined. It appeared that type ID (diatom) rbcL gene expression per gene dose decreased as the sampling sites shifted toward open water. By contrast, a similar trend was not observed for cyanobacterial (type IB) rbcL gene expression per gene dose. Thus far, a total of 21 clones of rbcL genes derived from mRNA have been obtained and completely sequenced from the Ballast Island site. For surface water samples, deduced amino acid sequences of five of six clones appeared to be representative of green algae. In contrast, six of nine sequenced rbcL clones from 10-m-deep samples were a chromophytic and rhodophytic lineages. At 5 m deep, the active CO{sub 2}-fixing planktonic organisms represented a diverse group, including organisms related to Chlorella ellipsoidea, Cylindrotheca sp. strain N1, and Olisthodiscus luteus. Although many more samplings at diverse sites must be accomplished, the discovery of distinctly different sequences of rbcL mRNA at different water depths suggests that there is a stratification of active CO{sub 2}-fixing organisms in western Lake Erie. 54 refs., 7 figs.

  7. Graminicide insensitivity correlates with herbicide-binding co-operativity on acetyl-CoA carboxylase isoforms.

    PubMed

    Price, Lindsey J; Herbert, Derek; Moss, Stephen R; Cole, David J; Harwood, John L

    2003-10-15

    The sensitivity of grass species to important classes of graminicide herbicides inhibiting ACCase (acetyl-CoA carboxylase) is associated with a specific inhibition of the multifunctional ACCase located in the plastids of grasses. In contrast, the multisubunit form of ACCase found in the chloroplasts of dicotyledonous plants is insensitive and the minor cytosolic multifunctional isoforms of the enzyme in both types of plants are also less sensitive to inhibition. We have isolated, separated and characterized the multifunctional ACCase isoforms found in exceptional examples of grasses that are either inherently insensitive to these graminicides, or from biotypes showing acquired resistance to their use. Major and minor multifunctional enzymes were isolated from cell suspension cultures of Festuca rubra and the 'Notts A1'-resistant biotype of Alopecurus myosuroides, and their properties compared with those isolated from cells of wild-type sensitive A. myosuroides or from sensitive maize. Purifications of up to 300-fold were necessary to separate the two isoforms. The molecular masses (200-230 kDa) and K(m) values for all three substrates (ATP, bicarbonate and acetyl-CoA) were similar for the different ACCases, irrespective of their graminicide sensitivity. Moreover, we found no correlation between the ability of isoforms to carboxylate propionyl-CoA and their sensitivity to graminicides. However, insensitive purified forms of ACCase were characterized by herbicide-binding co-operativity, whereas, in contrast, sensitive forms of the enzymes were not. Our studies on isolated individual isoforms of ACCase from grasses support and extend previous indications that herbicide binding co-operativity is the only kinetic property that differentiates naturally or selected insensitive enzymes from the typical sensitive forms usually found in grasses.

  8. Positive selection of Kranz and non-Kranz C4 phosphoenolpyruvate carboxylase amino acids in Suaedoideae (Chenopodiaceae).

    PubMed

    Rosnow, Josh J; Edwards, Gerald E; Roalson, Eric H

    2014-07-01

    In subfamily Suaedoideae, four independent gains of C4 photosynthesis are proposed, which includes two parallel origins of Kranz anatomy (sections Salsina and Schoberia) and two independent origins of single-cell C4 anatomy (Bienertia and Suaeda aralocaspica). Additional phylogenetic support for this hypothesis was generated from sequence data of the C-terminal portion of the phosphoenolpyruvate carboxylase (PEPC) gene used in C4 photosynthesis (ppc-1) in combination with previous sequence data. ppc-1 sequence was generated for 20 species in Suaedoideae and two outgroup Salsola species that included all types of C4 anatomies as well as two types of C3 anatomies. A branch-site test for positively selected codons was performed using the software package PAML. From labelling of the four branches where C4 is hypothesized to have developed (foreground branches), residue 733 (maize numbering) was identified to be under positive selection with a posterior probability >0.99 and residue 868 at the >0.95 interval using Bayes empirical Bayes (BEB). When labelling all the branches within C4 clades, the branch-site test identified 13 codons to be under selection with a posterior probability >0.95 by BEB; this is discussed considering current information on functional residues. The signature C4 substitution of an alanine for a serine at position 780 in the C-terminal end (which is considered a major determinant of affinity for PEP) was only found in four of the C4 species sampled, while eight of the C4 species and all the C3 species have an alanine residue; indicating that this substitution is not a requirement for C4 function.

  9. Coordinate, Organ-Specific and Developmental Regulation of Ribulose 1,5-Bisphosphate Carboxylase Gene Expression in Amaranthus hypochondriacus1

    PubMed Central

    Nikolau, Basil J.; Klessig, Daniel F.

    1987-01-01

    The expression of the genes encoding the large subunit (LSU) and small subunit (SSU) of ribulose 1,5-bisphosphate carboxylase (RuBPCase) was examined in roots, stems, cotyledons, and leaves of amaranth during the development of these tissues. The highest accumulation of LSU and SSU polypeptides occurred in cotyledons and leaves. Their steady state levels were approximately 20-fold lower in stems, while in roots neither LSU and SSU polypeptides nor their respective mRNAs could be detected. In cotyledons and leaves accumulation of these two polypeptides reached peak levels during the expansion stage of each tissue and then declined, reflecting changes in the synthesis, not turnover, of these proteins. In cotyledons and stems, the rates of synthesis of LSU and SSU polypeptides correlated with the levels of their respective mRNA, suggesting regulation primarily at the transcriptional level. In contrast, the dramatic and specific decrease in the synthesis of these two proteins during the last stages of development of the leaves could only partially be accounted for by the modest reduction in their mRNAs. Neither the translatability of these mRNAs, as assayed in cell-free systems, nor the stability of LSU and SSU polypeptides were altered, thus implying that control was being exerted at the translational level. During the development of these different organs, the expression of the LSU and SSU genes were generally coordinately regulated both at the levels of protein synthesis and mRNA accumulation. Images Fig. 2 Fig. 3 Fig. 4 Fig. 5 Fig. 6 Fig. 7 PMID:16665651

  10. Amino-terminal truncations of the ribulose-bisphosphate carboxylase small subunit influence catalysis and subunit interactions.

    PubMed Central

    Paul, K; Morell, M K; Andrews, T J

    1993-01-01

    The first 20 residues at the amino terminus of the small subunit of spinach ribulose-1,5-bisphosphate carboxylase form an irregular arm that makes extensive contacts with the large subunit and also with another small subunit (S. Knight, I. Andersson, and C.-I. Brändén [1990] J Mol Biol 215: 113-160). The influence of these contacts on subunit binding and, indirectly, on catalysis was investigated by constructing truncations from the amino terminus of the small subunit of the highly homologous enzyme from Synechococcus PCC 6301 expressed in Escherichia coli. Removal of the first six residues (and thus the region of contact with a neighboring small subunit) affected neither the affinity with which the small subunits bound to the large subunits nor the catalytic properties of the assembled holoenzyme. Extending the truncation to include the first 12 residues (which encroaches into a highly conserved region that interacts with the large subunit) also did not weaken intersubunit binding appreciably, but it reduced the catalytic activity of the holoenzyme nearly 5-fold. Removal of an additional single residue (i.e. removal of a total of 13 residues) weakened intersubunit binding approximately 80-fold. Paradoxically, this partially restored catalytic activity to approximately 40% of that of the wild-type holoenzyme. None of these truncations materially affected the Km values for ribulose-1,5-bisphosphate or CO2. Removal of all 20 residues of the irregular arm (thereby deleting the conserved region of contact with large subunits) totally abolished the small subunit's ability to bind to large subunits to form a stable holoenzyme. However, this truncated small subunit was still synthesized by the E. coli cells. These data are interpreted in terms of the role of the amino-terminal arm of the small subunit in maintaining the structure of the holoenzyme. PMID:8278544

  11. Methylcrotonoyl-CoA carboxylase 1 potentiates RLR-induced NF-κB signaling by targeting MAVS complex

    PubMed Central

    Cao, Zhongying; Xia, Zhangchuan; Zhou, Yaqin; Yang, Xiaodan; Hao, Hua; Peng, Nanfang; Liu, Shi; Zhu, Ying

    2016-01-01

    RNA virus infections are detected by the RIG-I family of receptors, which signal through the adaptor molecule mitochondrial antiviral signaling (MAVS). MAVS then recruits the adaptor’s tumor necrosis factor receptor-associated factor (TRAF) 3 and TRAF6, which in turn activate IRF3 and NF-κB, respectively, to induce interferons (IFNs) and inflammatory responses. Here we show that the biotin-containing enzyme methylcrotonoyl-CoA carboxylase 1 (MCCC1) enhances virus-induced, MAVS-mediated IFN and inflammatory cytokine expression through the NF-κB signaling pathway. MCCC1 knockdown strongly inhibits induction of IFNs and inflammatory cytokines. Furthermore, MCCC1 shows extensive antiviral activity toward RNA viruses, including influenza A virus, human enterovirus 71, and vesicular stomatitis virus. Here, we have elucidated the mechanism underlying MCCC1-mediated inhibition of viral replication. MCCC1 interacts with MAVS and components of the MAVS signalosome and contributes to enhanced production of type I IFNs and pro-inflammatory cytokines by promoting phosphorylation of the IκB kinase (IKK) complex and NF-κB inhibitor-α (IκBα), as well as NF-κB nuclear translocation. This process leads to activation of IFNs and cytokine expression and subsequent activation of IFN-stimulated genes, including double-stranded RNA-dependent protein kinase PKR and myxovirus resistance protein 1. These findings demonstrate that MCCC1 plays an essential role in virus-triggered, MAVS-mediated activation of NF-κB signaling. PMID:27629939

  12. Characterization of spinach ribulose-1,5-bisphosphate carboxylase/oxygenase activase isoforms reveals hexameric assemblies with increased thermal stability.

    PubMed

    Keown, Jeremy R; Pearce, Frederick Grant

    2014-12-15

    Most plants contain two isoforms of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) activase (Rca), a chloroplast protein that maintains the activity of Rubisco during photosynthesis. The longer (α-) Rca isoform has previously been shown to regulate the activity of Rubisco in response to both the ADP:ATP ratio and redox potential via thioredoxin-f. We have characterized the arrangement of the different spinach (Spinacia oleracea) isoforms in solution, and show how the presence of nucleotides changes the oligomeric state. Although the shorter (β-) isoform from both tobacco (Nicotiana tabacum) and spinach tend to form a range of oligomers in solution, the size of which are relatively unaffected by the addition of nucleotide, the spinach α-isoform assembles as a hexamer in the presence of adenosine 5'-[γ-thio]triphosphate (ATPγS). These hexamers have significantly higher heat stability, and may play a role in optimizing photosynthesis at higher temperatures. Hexamers were also observed for mixtures of the two isoforms, suggesting that the α-isoform can act as a structural scaffold for hexamer formation by the β-isoform. Additionally, it is shown that a variant of the tobacco β-isoform acts in a similar fashion to the α-isoform of spinach, forming thermally stable hexamers in the presence of ATPγS. Both isoforms had similar rates of ATP hydrolysis, suggesting that a propensity for hexamer formation may not necessarily be correlated with activity. Modelling of the hexameric structures suggests that although the N-terminus of Rca forms a highly dynamic, extended structure, the C-terminus is located adjacent to the intersubunit interface.

  13. Phosphoenolpyruvate carboxylase protein kinase from developing castor oil seeds: partial purification, characterization, and reversible control by photosynthate supply.

    PubMed

    Murmu, Jhadeswar; Plaxton, William C

    2007-10-01

    Phosphoenolpyruvate carboxylase (PEPC, EC 4.1.1.31) protein kinase (PPCK) was purified approximately 1,500-fold from developing castor oil seeds (COS). Gel filtration and immunoblotting with anti-(rice PPCK2)-immune serum indicated that this Ca2+-insensitive PPCK exists as a 31-kDa monomer. COS PPCK-mediated rephosphorylation of the 107-kDa subunit (p107) of COS PEPC1 (Km = 2.2 microM) activated PEPC1 by approximately 80% when assayed under suboptimal conditions (pH 7.3, 0.2 mM PEP, and 0.125 mM malate). COS PPCK displayed remarkable selectivity for phosphorylating COS PEPC1 (relative to tobacco, sorghum, or maize PEPCs), exhibited a broad pH-activity optima of approximately pH 8.5, and at pH 7.3 was activated 40-65% by 1 mM PEP, or 10 mM Gln or Asn, but inhibited 65% by 10 mM L-malate. The possible control of COS PPCK by disulfide-dithiol interconversion was suggested by its rapid inactivation and subsequent reactivation when incubated with oxidized glutathione and then dithiothreitol. In vitro PPCK activity correlated with in vivo p107 phosphorylation status, with both peaking in mid-cotyledon to full-cotyledon developing COS. Notably, PPCK activity and p107 phosphorylation of developing COS were eliminated following pod excision or prolonged darkness of intact plants. Both effects were fully reversed 12 h following reillumination of darkened plants. These results implicate a direct relationship between the up-regulation of COS PPCK and p107 phosphorylation during the recommencement of photosynthate delivery from illuminated leaves to the non-photosynthetic COS. Overall, the results support the hypothesis that PEPC and PPCK participate in the control of photosynthate partitioning into C-skeletons needed as precursors for key biosynthetic pathways of developing COS.

  14. 2015 ACC Health Policy Statement on Cardiovascular Team-Based Care and the Role of Advanced Practice Providers.

    PubMed

    Brush, John E; Handberg, Eileen M; Biga, Cathleen; Birtcher, Kim K; Bove, Alfred A; Casale, Paul N; Clark, Michael G; Garson, Arthur; Hines, Jerome L; Linderbaum, Jane A; Rodgers, George P; Shor, Robert A; Thourani, Vinod H; Wyman, Janet F

    2015-05-19

    The mission of the American College of Cardiology is "to transform cardiovascular care and improve heart health." Cardiovascular team-based care is a paradigm for practice that can transform care, improve heart health, and help meet the demands of the future. One strategic goal of the College is to help members successfully transition their clinical practices to the future, with all its complexity, challenges, and opportunities. The ACC's strategic plan is aligned with the triple aim of improved care, improved population health, and lower costs per capita. The traditional understanding of quality, access, and cost is that you cannot improve one component without diminishing the others. With cardiovascular team-based care, it is possible to achieve the triple aim of improving quality, access, and cost simultaneously to also improve cardiovascular health. Striving to serve the best interests of patients is the true north of our guiding principles. Cardiovascular team-based care is a model that can improve care coordination and communication and allow each team member to focus more on the quality of care. In addition, the cardiovascular team-based care model increases access to cardiovascular care and allows expansion of services to populations and geographic areas that are currently underserved. This document will increase awareness of the important components of cardiovascular team-based care and create an opportunity for more discussion about the most creative and effective means of implementing it. We hope that this document will stimulate further discussions and activities within the ACC and beyond about team-based care. We have identified areas that need improvement, specifically in APP education and state regulation. The document encourages the exploration of collaborative care models that should enable team members to optimize their education, training, experience, and talent. Improved team leadership, coordination, collaboration, engagement, and efficiency

  15. Measurement of acylcarnitine substrate to product ratios specific to biotin-dependent carboxylases offers a combination of indicators of biotin status in humans.

    PubMed

    Bogusiewicz, Anna; Horvath, Thomas D; Stratton, Shawna L; Mock, Donald M; Boysen, Gunnar

    2012-09-01

    This work describes a novel liquid chromatography tandem MS (LC-MS/MS) method for the determination of ratios of acylcarnitines arising from acyl-CoA substrates and products that reflect metabolic disturbances caused by marginal biotin deficiency. The urinary ratios reflecting reduced activities of biotin-dependent enzymes include the following: 1) the ratio of 3-hydroxyisovalerylcarnitine : 3-methylglutarylcarnitine (3HIAc : MGc) for methylcrotonyl-CoA carboxylase; 2) the ratio of propionylcarnitine:methylmalonylcarnitine (Pc : MMc) for propionyl-CoA carboxylase (PCC); and 3) the ratio of acetylcarnitine : malonylcarnitine (Ac : Mc) for acetyl-CoA carboxylase. To demonstrate the suitability of the LC-MS/MS method for biomonitoring, we measured the 3 ratios for 7 healthy adults at various time points (d 0, 14, and 28) during the induction of marginal biotin through the consumption of egg white. The mean change in the Pc : MMc ratio relative to d 0 was 5.3-fold by d 14 (P = 0.0049) and 8.5-fold by d 28 (P = 0.0042). The mean change in the 3HIAc : MGc ratio was 2.8-fold by d 14 (P = 0.0022) and 3.8-fold by d 28 (P = 0.0001). The mean change in the Ac : Mc ratio was 2.9-fold by d 14 (P = 0.03) and 4.7-fold by d 28 (P = 0.02). The results suggest that simultaneous assessment of ratios of multiple biotin-dependent pathways offers insight into the complex metabolic disturbances caused by marginal biotin deficiency. We hypothesize that one or a combination of the ratios might be more sensitive or robust with respect to other nutrient deficiencies or confounding metabolic processes.

  16. The rhizobacterium Variovorax paradoxus 5C-2, containing ACC deaminase, promotes growth and development of Arabidopsis thaliana via an ethylene-dependent pathway.

    PubMed

    Chen, Lin; Dodd, Ian C; Theobald, Julian C; Belimov, Andrey A; Davies, William J

    2013-04-01

    Many plant-growth-promoting rhizobacteria (PGPR) associated with plant roots contain the enzyme 1-aminocyclopropane-1-carboxylate (ACC) deaminase and can metabolize ACC, the immediate precursor of the plant hormone ethylene, thereby decreasing plant ethylene production and increasing plant growth. However, relatively few studies have explicitly linked ethylene emission and/or action to growth promotion in these plant-microbe interactions. This study examined effects of the PGPR Variovorax paradoxus 5C-2 containing ACC deaminase on the growth and development of Arabidopsis thaliana using wild-type (WT) plants and several ethylene-related mutants (etr1-1, ein2-1, and eto1-1). Soil inoculation with V. paradoxus 5C-2 promoted growth (leaf area and shoot biomass) of WT plants and the ethylene-overproducing mutant eto1-1, and also enhanced floral initiation of WT plants by 2.5 days. However, these effects were not seen in ethylene-insensitive mutants (etr1-1 and ein2-1) even though bacterial colonization of the root system was similar. Furthermore, V. paradoxus 5C-2 decreased ACC concentrations of rosette leaves of WT plants by 59% and foliar ethylene emission of both WT plants and eto1-1 mutants by 42 and 37%, respectively. Taken together, these results demonstrate that a fully functional ethylene signal transduction pathway is required for V. paradoxus 5C-2 to stimulate leaf growth and flowering of A. thaliana.

  17. Possible Role of 1-Aminocyclopropane-1-Carboxylate (ACC) Deaminase Activity of Sinorhizobium sp. BL3 on Symbiosis with Mung Bean and Determinate Nodule Senescence

    PubMed Central

    Tittabutr, Panlada; Sripakdi, Sudarat; Boonkerd, Nantakorn; Tanthanuch, Waraporn; Minamisawa, Kiwamu; Teaumroong, Neung

    2015-01-01

    Sinorhizobium sp. BL3 forms symbiotic interactions with mung bean (Vigna radiata) and contains lrpL-acdS genes, which encode the 1-aminocyclopropane-1-carboxylate (ACC) deaminase enzyme that cleaves ACC, a precursor of plant ethylene synthesis. Since ethylene interferes with nodule formation in some legumes and plays a role in senescence in plant cells, BL3-enhancing ACC deaminase activity (BL3+) and defective mutant (BL3−) strains were constructed in order to investigate the effects of this enzyme on symbiosis and nodule senescence. Nodulation competitiveness was weaker in BL3− than in the wild-type, but was stronger in BL3+. The inoculation of BL3− into mung bean resulted in less plant growth, a lower nodule dry weight, and smaller nodule number than those in the wild-type, whereas the inoculation of BL3+ had no marked effects. However, similar nitrogenase activity was observed with all treatments; it was strongly detected 3 weeks after the inoculation and gradually declined with time, indicating senescence. The rate of plant nodulation by BL3+ increased in a time-dependent manner. Nodules occupied by BL3− formed smaller symbiosomes, and bacteroid degradation was more prominent than that in the wild-type 7 weeks after the inoculation. Changes in biochemical molecules during nodulation were tracked by Fourier Transform Infrared (FT-IR) microspectroscopy, and the results obtained confirmed that aging processes differed in nodules occupied by BL3 and BL3−. This is the first study to show the possible role of ACC deaminase activity in senescence in determinate nodules. Our results suggest that an increase in ACC deaminase activity in this strain does not extend the lifespan of nodules, whereas the lack of this activity may accelerate nodule senescence. PMID:26657304

  18. Characterization of ACC deaminase-producing endophytic bacteria isolated from copper-tolerant plants and their potential in promoting the growth and copper accumulation of Brassica napus.

    PubMed

    Zhang, Yan-Feng; He, Lin-Yan; Chen, Zhao-Jin; Wang, Qing-Ya; Qian, Meng; Sheng, Xia-Fang

    2011-03-01

    One hundred Cu-resistant-endophytic bacteria were isolated from Cu-tolerant plants grown on Cu mine wasteland, of which, eight Cu-resistant and 1-aminocyclopropane-1-carboxylate (ACC) deaminase-producing endophytic bacteria were obtained based on the ACC deaminase activity of the bacteria and characterized with respect to metal resistance, production of ACC deaminase, indole-3-acetic acid (IAA) as well as siderophores and mineral phosphate solubilization. Ralstonia sp. J1-22-2, Pantoea agglomerans Jp3-3, and Pseudomonas thivervalensis Y1-3-9 with higher ACC deaminase activity (ranging from 213 to 370 μM α-ketobutyrate mg(-1)h(-1)) were evaluated for promoting plant growth and Cu uptake of rape grown in quartz sand containing 0, 2.5, and 5 mg kg(-1) of Cu in pot experiments. The eight bacteria were found to exhibit different multiple heavy metal resistance characteristics, to show different levels of ACC deaminase activity and to produce indole acetic acid. Seven bacteria produced siderophores and solubilized inorganic phosphate. Pot experiments showed that inoculation with the strains (J1-22-2, Jp3-3, and Y1-3-9) was found to increase the biomass of rape. Increases in above-ground tissue Cu contents of rape cultivated in 2.5 and 5 mg kg(-1) of Cu-contaminated substrates varied from 9% to 31% and from 3 to 4-fold respectively in inoculated-rape plants compared to the uninoculated control. The maximum Cu uptake of rape was observed after inoculation with P. agglomerans Jp3-3. The results show that metal-resistant and plant growth promoting endophytic bacteria play an important role in plant growth and Cu uptake which may provide a new endophytic bacterial-assisted phytoremediation of Cu-contaminated environment.

  19. 2013 ACC/AHA versus 2004 NECP ATP III Guidelines in the Assignment of Statin Treatment in a Korean Population with Subclinical Coronary Atherosclerosis

    PubMed Central

    Kang, Yu Mi; Yang, Dong Hyun; Kang, Joon-Won; Kim, Eun Hee; Park, Duk-Woo; Park, Joong-Yeol; Kim, Hong-Kyu; Lee, Woo Je

    2015-01-01

    Background The usefulness of the 2013 ACC/AHA guidelines for the management of blood cholesterol in the Asian population remains controversial. In this study, we investigated whether eligibility for statin therapy determined by the 2013 ACC/AHA guidelines is better aligned with the presence of subclinical coronary atherosclerosis detected by CCTA (coronary computed tomography angiography) compared to the previously recommended 2004 NCEP ATP III guidelines. Methods We collected the data from 5,837 asymptomatic subjects who underwent CCTA using MDCT during routine health examinations. Based on risk factor assessment and lipid data, we determined guideline-based eligibility for statin therapy according to the 2013 ACC/AHA and 2004 NCEP ATP III guidelines. We defined the presence and severity of subclinical coronary atherosclerosis detected in CCTA according to the presence of significant coronary artery stenosis (defined as >50% stenosis), plaques, and the degree of coronary calcification. Results As compared to the 2004 ATP III guidelines, a significantly higher proportion of subjects with significant coronary stenosis (61.8% vs. 33.8%), plaques (52.3% vs. 24.7%), and higher CACS (CACS >100, 63.6% vs. 26.5%) was assigned to statin therapy using the 2013 ACC/AHA guidelines (P < .001 for all variables). The area under the curves of the pooled cohort equation of the new guidelines in detecting significant stenosis, plaques, and higher CACS were significantly higher than those of the Framingham risk calculator. Conclusions Compared to the previous ATP III guidelines, the 2013 ACC/AHA guidelines were more sensitive in identifying subjects with subclinical coronary atherosclerosis detected by CCTA in an Asian population. PMID:26372638

  20. A kinetic study of the effects of phosphate and organic phosphates on the activity of phosphoenolpyruvate carboxylase from Crassula argentea.

    PubMed

    Meyer, C R; Rustin, P; Wedding, R T

    1989-05-15

    The effects of phosphate and several phosphate-containing compounds on the activity of purified phosphoenolpyruvate carboxylase (PEPC) from the crassulacean acid metabolism plant, Crassula argentea, were investigated. When assayed at subsaturating phosphoenolpyruvate (PEP) concentrations, low concentrations of most of the compounds tested were found to stimulate PEPC activity. This activation, variable in extent, was found in all cases to be competitive with glucose 6-phosphate (Glc-6-P) stimulation, suggesting that these effectors bind to the Glc-6-P site. At higher concentrations, depending upon the effector molecule studied, deactivation, inhibition, or no response was observed. More detailed studies were performed with Glc-6-P, AMP, phosphoglycolate, and phosphate. AMP had previously been shown to be a specific ligand for the Glc-6-P site. The main effect of Glc-6-P and AMP on the kinetic parameters was to decrease the apparent Km and increase Vmax/Km. AMP also caused a decrease in the Vmax of the reaction. In contrast, phosphoglycolate acted essentially as a competitive inhibitor increasing the apparent Km for PEP and decreasing Vmax/Km. Inorganic phosphate had a biphasic effect on the kinetic parameters, resulting in a transient decrease in Km followed by an increase of the apparent Km for PEP with increasing concentration of phosphate. The Vmax also was decreased with increasing phosphate concentrations. Further, the enzyme appeared to respond to the complex of phosphate with magnesium. In the presence of a saturating concentration of AMP, no activation but rather inhibition was observed with increasing phosphate concentration. This is consistent with the binding of phosphate to two separate sites--the Glc-6-P activation site and an inhibitory site, a phenomenon that may be occurring with other phosphate containing compounds. High concentrations of phosphate with magnesium were found to protect enzyme activity when PEPC, previously shown to contain an

  1. Role and Regulation of ACC Deaminase Gene in Sinorhizobium meliloti: Is It a Symbiotic, Rhizospheric or Endophytic Gene?

    PubMed Central

    Checcucci, Alice; Azzarello, Elisa; Bazzicalupo, Marco; De Carlo, Anna; Emiliani, Giovanni; Mancuso, Stefano; Spini, Giulia; Viti, Carlo; Mengoni, Alessio

    2017-01-01

    Plant-associated bacteria exhibit a number of different strategies and specific genes allow bacteria to communicate and metabolically interact with plant tissues. Among the genes found in the genomes of plant-associated bacteria, the gene encoding the enzyme 1-aminocyclopropane-1-carboxylate (ACC) deaminase (acdS) is one of the most diffused. This gene is supposed to be involved in the cleaving of plant-produced ACC, the precursor of the plant stress-hormone ethylene toning down the plant response to infection. However, few reports are present on the actual role in rhizobia, one of the most investigated groups of plant-associated bacteria. In particular, still unclear is the origin and the role of acdS in symbiotic competitiveness and on the selective benefit it may confer to plant symbiotic rhizobia. Here we present a phylogenetic and functional analysis of acdS orthologs in the rhizobium model-species Sinorhizobium meliloti. Results showed that acdS orthologs present in S. meliloti pangenome have polyphyletic origin and likely spread through horizontal gene transfer, mediated by mobile genetic elements. When acdS ortholog from AK83 strain was cloned and assayed in S. meliloti 1021 (lacking acdS), no modulation of plant ethylene levels was detected, as well as no increase in fitness for nodule occupancy was found in the acdS-derivative strain compared to the parental one. Surprisingly, AcdS was shown to confer the ability to utilize formamide and some dipeptides as sole nitrogen source. Finally, acdS was shown to be negatively regulated by a putative leucine-responsive regulator (LrpL) located upstream to acdS sequence (acdR). acdS expression was induced by root exudates of both legumes and non-leguminous plants. We conclude that acdS in S. meliloti is not directly related to symbiotic interaction, but it could likely be involved in the rhizospheric colonization or in the endophytic behavior. PMID:28194158

  2. Role and Regulation of ACC Deaminase Gene in Sinorhizobium meliloti: Is It a Symbiotic, Rhizospheric or Endophytic Gene?

    PubMed

    Checcucci, Alice; Azzarello, Elisa; Bazzicalupo, Marco; De Carlo, Anna; Emiliani, Giovanni; Mancuso, Stefano; Spini, Giulia; Viti, Carlo; Mengoni, Alessio

    2017-01-01

    Plant-associated bacteria exhibit a number of different strategies and specific genes allow bacteria to communicate and metabolically interact with plant tissues. Among the genes found in the genomes of plant-associated bacteria, the gene encoding the enzyme 1-aminocyclopropane-1-carboxylate (ACC) deaminase (acdS) is one of the most diffused. This gene is supposed to be involved in the cleaving of plant-produced ACC, the precursor of the plant stress-hormone ethylene toning down the plant response to infection. However, few reports are present on the actual role in rhizobia, one of the most investigated groups of plant-associated bacteria. In particular, still unclear is the origin and the role of acdS in symbiotic competitiveness and on the selective benefit it may confer to plant symbiotic rhizobia. Here we present a phylogenetic and functional analysis of acdS orthologs in the rhizobium model-species Sinorhizobium meliloti. Results showed that acdS orthologs present in S. meliloti pangenome have polyphyletic origin and likely spread through horizontal gene transfer, mediated by mobile genetic elements. When acdS ortholog from AK83 strain was cloned and assayed in S. meliloti 1021 (lacking acdS), no modulation of plant ethylene levels was detected, as well as no increase in fitness for nodule occupancy was found in the acdS-derivative strain compared to the parental one. Surprisingly, AcdS was shown to confer the ability to utilize formamide and some dipeptides as sole nitrogen source. Finally, acdS was shown to be negatively regulated by a putative leucine-responsive regulator (LrpL) located upstream to acdS sequence (acdR). acdS expression was induced by root exudates of both legumes and non-leguminous plants. We conclude that acdS in S. meliloti is not directly related to symbiotic interaction, but it could likely be involved in the rhizospheric colonization or in the endophytic behavior.

  3. Moringa oleifera leaf extract ameliorates alloxan-induced diabetes in rats by regeneration of β cells and reduction of pyruvate carboxylase expression.

    PubMed

    Abd El Latif, Amira; El Bialy, Badr El Said; Mahboub, Hamada Dahi; Abd Eldaim, Mabrouk Attia

    2014-10-01

    Moringa oleifera Lam. contains many active ingredients with nutritional and medicinal values. It is commonly used in folk medicine as an antidiabetic agent. The present study was designed to investigate how an aqueous extract from the leaves of M. oleifera reveals hypoglycemia in diabetic rats. M. oleifera leaf extract counteracted the alloxan-induced diabetic effects in rats as it normalized the elevated serum levels of glucose, triglycerides, cholesterol, and malondialdehyde, and normalized mRNA expression of the gluconeogenic enzyme pyruvate carboxylase in hepatic tissues. It also increased live body weight gain and normalized the reduced mRNA expression of fatty acid synthase in the liver of diabetic rats. Moreover, it restored the normal histological structure of the liver and pancreas damaged by alloxan in diabetic rats. This study revealed that the aqueous extract of M. oleifera leaves possesses potent hypoglycemic effects through the normalization of elevated hepatic pyruvate carboxylase enzyme and regeneration of damaged hepatocytes and pancreatic β cells via its antioxidant properties.

  4. Mild water stress effects on carbon-reduction-cycle intermediates, ribulose bisphosphate carboxylase activity, and spatial homogeneity of photosynthesis in intact leaves

    SciTech Connect

    Sharkey, T.D.; Seemann, J.R. Univ. of Nevada, Reno )

    1989-04-01

    We have examined the effect of mild water stress on photosynthetic chloroplast reactions of intact Phaseolus vulgaris leaves by measuring two parameters of ribulose bisphosphate (RuBP) carboxylase activity and the pool sizes of RuBP, 3-phosphoglycerate (PGA), triose phosphates, hexose monophosphates, and ATP. We also tested for patchy stomatal closure by feeding {sup 14}CO{sub 2}. The k{sub cat} of RuBP carboxylase (moles CO{sub 2} fixed per mole enzyme per second) which could be measured after incubating the enzyme with CO{sub 2} and Mg{sup 2+} was unchanged by water stress. The ratio of activity before and after incubation with CO{sub 2} and Mg{sup 2+} (the carbamylation state) was slightly reduced by severe stress but not by mild stress. Likewise, the concentration of RuBP was slightly reduced by severe stress but not by mild stress. The concentration of PGA was markedly reduced by both mild and severe water stress. The concentration of triose phosphates did not decline as much as PGA. We found that photosynthesis in water stressed leaves occurred in patches. The patchiness of photosynthesis during water stress may lead to an underestimation of the effect of stomatal closure. We conclude that reductions in whole leaf photosynthesis caused by mild water stress are primarily the result of stomatal closure and that there is no indication of damage to chloroplast reactions.

  5. A crotonyl-CoA reductase-carboxylase independent pathway for assembly of unusual alkylmalonyl-CoA polyketide synthase extender units

    PubMed Central

    Ray, Lauren; Valentic, Timothy R.; Miyazawa, Takeshi; Withall, David M.; Song, Lijiang; Milligan, Jacob C.; Osada, Hiroyuki; Takahashi, Shunji; Tsai, Shiou-Chuan; Challis, Gregory L.

    2016-01-01

    Type I modular polyketide synthases assemble diverse bioactive natural products. Such multienzymes typically use malonyl and methylmalonyl-CoA building blocks for polyketide chain assembly. However, in several cases more exotic alkylmalonyl-CoA extender units are also known to be incorporated. In all examples studied to date, such unusual extender units are biosynthesized via reductive carboxylation of α, β-unsaturated thioesters catalysed by crotonyl-CoA reductase/carboxylase (CCRC) homologues. Here we show using a chemically-synthesized deuterium-labelled mechanistic probe, and heterologous gene expression experiments that the unusual alkylmalonyl-CoA extender units incorporated into the stambomycin family of polyketide antibiotics are assembled by direct carboxylation of medium chain acyl-CoA thioesters. X-ray crystal structures of the unusual β-subunit of the acyl-CoA carboxylase (YCC) responsible for this reaction, alone and in complex with hexanoyl-CoA, reveal the molecular basis for substrate recognition, inspiring the development of methodology for polyketide bio-orthogonal tagging via incorporation of 6-azidohexanoic acid and 8-nonynoic acid into novel stambomycin analogues. PMID:28000660

  6. A crotonyl-CoA reductase-carboxylase independent pathway for assembly of unusual alkylmalonyl-CoA polyketide synthase extender units

    NASA Astrophysics Data System (ADS)

    Ray, Lauren; Valentic, Timothy R.; Miyazawa, Takeshi; Withall, David M.; Song, Lijiang; Milligan, Jacob C.; Osada, Hiroyuki; Takahashi, Shunji; Tsai, Shiou-Chuan; Challis, Gregory L.

    2016-12-01

    Type I modular polyketide synthases assemble diverse bioactive natural products. Such multienzymes typically use malonyl and methylmalonyl-CoA building blocks for polyketide chain assembly. However, in several cases more exotic alkylmalonyl-CoA extender units are also known to be incorporated. In all examples studied to date, such unusual extender units are biosynthesized via reductive carboxylation of α, β-unsaturated thioesters catalysed by crotonyl-CoA reductase/carboxylase (CCRC) homologues. Here we show using a chemically-synthesized deuterium-labelled mechanistic probe, and heterologous gene expression experiments that the unusual alkylmalonyl-CoA extender units incorporated into the stambomycin family of polyketide antibiotics are assembled by direct carboxylation of medium chain acyl-CoA thioesters. X-ray crystal structures of the unusual β-subunit of the acyl-CoA carboxylase (YCC) responsible for this reaction, alone and in complex with hexanoyl-CoA, reveal the molecular basis for substrate recognition, inspiring the development of methodology for polyketide bio-orthogonal tagging via incorporation of 6-azidohexanoic acid and 8-nonynoic acid into novel stambomycin analogues.

  7. ACC Neuro-over-Connectivity Is Associated with Mathematically Modeled Additional Encoding Operations of Schizophrenia Stroop-Task Performance

    PubMed Central

    Taylor, Reggie; Théberge, Jean; Williamson, Peter C.; Densmore, Maria; Neufeld, Richard W. J.

    2016-01-01

    Functional magnetic resonance imaging at 7.0 Tesla was undertaken among Schizophrenia participants (Sz), and clinical (major mood disorder; MDD) and healthy controls (HC), during performance of the Stoop task. Stroop conditions included congruent and incongruent word color items, color-only items, and word-only items. Previous modeling results extended to this most widely used selective-attention task. All groups executed item-encoding operations (subprocesses of the item encoding process) at the same rate (performance accuracy being similarly high throughout), thus displaying like processing capacity; Sz participants, however, employed more subprocesses for item completions than did the MDD participants, who in turn used more subprocesses than the HC group. The reduced efficiency in deploying cognitive-workload capacity among the Sz participants was paralleled by more diffuse neuroconnectivity (Blood-Oxygen-Level-Dependent co-activation) with the anterior cingulate cortex (ACC) (Broadman Area 32), spreading away from this encoding-intensive region; and by less evidence of network dissociation across Stroop conditions. Estimates of cognitive work done to accomplish item completion were greater for the Sz participants, as were estimates of entropy in both the modeled trial-latency distribution, and its associated neuro-circuitry. Findings are held to be symptom and assessment significant, and to have potential implications for clinical intervention. PMID:27695425

  8. Silencing of the ACC synthase gene ACACS2 causes delayed flowering in pineapple [Ananas comosus (L.) Merr.].

    PubMed

    Trusov, Yuri; Botella, José Ramón

    2006-01-01

    Flowering is a crucial developmental stage in the plant life cycle. A number of different factors, from environmental to chemical, can trigger flowering. In pineapple, and other bromeliads, it has been proposed that flowering is triggered by a small burst of ethylene production in the meristem in response to environmental cues. A 1-amino-cyclopropane-1-carboxylate synthase (ACC synthase) gene has been cloned from pineapple (ACACS2), which is induced in the meristem under the same environmental conditions that induce flowering. Two transgenic pineapple lines have been produced containing co-suppression constructs designed to down-regulate the expression of the ACACS2 gene. Northern analysis revealed that the ACACS2 gene was silenced in a number of transgenic plants in both lines. Southern hybridization revealed clear differences in the methylation status of silenced versus non-silenced plants by the inability of a methylation-sensitive enzyme to digest within the ACACS2 DNA extracted from silenced plants, indicating that methylation is the cause of the observed co-suppression of the ACACS2 gene. Flowering characteristics of the transgenic plants were studied under field conditions in South East Queensland, Australia. Flowering dynamics studies revealed significant differences in flowering behaviour, with transgenic plants exhibiting silencing showing a marked delay in flowering when compared with non-silenced transgenic plants and control non-transformed plants. It is argued that the ACACS2 gene is one of the key contributors towards triggering 'natural flowering' in mature pineapples under commercial field conditions.

  9. Anti-tumorigenic effect of nano formulated peptide pACC1 by diminishing de novo lipogenisis in DMBA induced mammary carcinoma rat model.

    PubMed

    Kaliaperumal, Jagatheesh; Padarthi, Pavankumar; Elangovan, Namasivayam; Hari, Natarajan

    2014-07-01

    At present, the majority of established treatments for breast cancer are based on clinical manifestations, some fundamental of molecular and cellular biology of cancer. In recent times, the therapy is moving towards personalized medicines. Nevertheless, both the methodologies have own demerits. In the present study, we proposed a novel idea of targeted therapy with twin pharmacological potential by a peptide pACC1. The peptide was formulated with chitosan and evaluated with DMBA induced mammary carcinoma. Results suggest that the peptide holds great control on tumor cell multiplication, fatty acid synthesis and lactate levels. In addition, peptide also brings normal metabolic signs in glycolytic and glycogenic pathways. Histological studies confirm the dual pharmacological actions. Further, it is also proven that the peptide controls membrane receptor levels of HER2 and EGFR. In conclusion, that the peptide pACC1 could be employed as greater therapeutic adjuvant with currently established drugs without considering the stage of the cancer.

  10. Comparative effectiveness of ACC-deaminase and/or nitrogen-fixing rhizobacteria in promotion of maize (Zea mays L.) growth under lead pollution.

    PubMed

    Hassan, Waseem; Bano, Rizwana; Bashir, Farhat; David, Julie

    2014-09-01

    Lead (Pb) pollution is appearing as an alarming threat nowadays. Excessive Pb concentrations in agricultural soils result in minimizing the soil fertility and health which affects the plant growth and leads to decrease in crop production. Plant growth promoting rhizobacteria (PGPR) are beneficial bacteria which can protect the plants against many abiotic stresses, and enhance the growth. The study aimed to identify important rhizobacterial strains by using the 1-aminocyclopropane-1-carboxylate (ACC) enrichment technique and examine their inoculation effects in the growth promotion of maize, under Pb pollution. A pot experiment was conducted and six rhizobacterial isolates were used. Pb was added to 2 kg soil in each pot (with 4 seeds/pot) using Pb(NO3)2 at the rate of 0, 100, 200, 300, and 400 mg kg(-1) Pb with three replications in completely randomized design. Rhizobacterial isolates performed significantly better under all Pb levels, i.e., 100 to 400 Pb mg kg(-1) soil, compared to control. Comparing the efficacy of the rhizobacterial isolates under different Pb levels, rhizobacterial isolates having both ACC-deaminase and nitrogen-fixing activities (AN8 and AN12) showed highest increase in terms of the physical, chemical and enzymatic growth parameters of maize, followed by the rhizobacterial isolates having ACC-deaminase activity only (ACC5 and ACC8), and then the nitrogen-fixing rhizobia (Azotobacter and RN5). However, the AN8 isolate showed maximum efficiency, and highest shoot and root length (14.2 and 6.1 cm), seedling fresh and dry weights (1.91 and 0.14 g), chlorophyll a, b, and carotenoids (24.1, 30.2 and 77.7 μg/l), protein (0.82 mg/g), proline (3.42 μmol/g), glutathione S-transferase, peroxidase and catalase (12.3, 4.2 and 7.2 units/mg protein), while the lowest Pb uptake in the shoot and root (0.83 and 0.48 mg/kg) were observed under this rhizobial isolate at the highest Pb level (i.e., 400 Pb mg kg(-1) soil). The results revealed that PGPR

  11. Real time expression of ACC oxidase and PR-protein genes mediated by Methylobacterium spp. in tomato plants challenged with Xanthomonas campestris pv. vesicatoria.

    PubMed

    Yim, W J; Kim, K Y; Lee, Y W; Sundaram, S P; Lee, Y; Sa, T M

    2014-07-15

    Biotic stress like pathogenic infection increases ethylene biosynthesis in plants and ethylene inhibitors are known to alleviate the severity of plant disease incidence. This study aimed to reduce the bacterial spot disease incidence in tomato plants caused by Xanthomonas campestris pv. vesicatoria (XCV) by modulating stress ethylene with 1-aminocyclopropane-1-carboxylate (ACC) deaminase activity of Methylobacterium strains. Under greenhouse condition, Methylobacterium strains inoculated and pathogen challenged tomato plants had low ethylene emission compared to pathogen infected ones. ACC accumulation and ACC oxidase (ACO) activity with ACO related gene expression increased in XCV infected tomato plants over Methylobacterium strains inoculated plants. Among the Methylobacterium spp., CBMB12 resulted lowest ACO related gene expression (1.46 Normalized Fold Expression), whereas CBMB20 had high gene expression (3.42 Normalized Fold Expression) in pathogen challenged tomato. But a significant increase in ACO gene expression (7.09 Normalized Fold Expression) was observed in the bacterial pathogen infected plants. In contrast, Methylobacterium strains enhanced β-1,3-glucanase and phenylalanine ammonia-lyase (PAL) enzyme activities in pathogen challenged tomato plants. The respective increase in β-1,3-glucanase related gene expressions due to CBMB12, CBMB15, and CBMB20 strains were 66.3, 25.5 and 10.4% higher over pathogen infected plants. Similarly, PAL gene expression was high with 0.67 and 0.30 Normalized Fold Expression, in pathogen challenged tomato plants inoculated with CBMB12 and CBMB15 strains. The results suggest that ethylene is a crucial factor in bacterial spot disease incidence and that methylobacteria with ACC deaminase activity can reduce the disease severity with ultimate pathogenesis-related protein increase in tomato.

  12. Cloning of a chicken liver cDNA encoding 5-aminoimidazole ribonucleotide carboxylase and 5-aminoimidazole-4-N-succinocarboxamide ribonucleotide synthetase by functional complementation of Escherichia coli pur mutants.

    PubMed Central

    Chen, Z D; Dixon, J E; Zalkin, H

    1990-01-01

    We have used functional complementation of Escherichia coli pur mutants to clone avian cDNA encoding 5-aminoimidazole ribonucleotide (AIR) carboxylase-5-aminoimidazole-4-N-succinocarboxamide ribonucleotide (SAICAR) synthetase, the bifunctional enzyme catalyzing steps 6 and 7 in the pathway for de novo purine nucleotide synthesis. Mutational analyses have been used to establish the structure-function relationship: NH2-SAICAR synthetase-AIR carboxylase-COOH. The amino acid sequence of the SAICAR synthetase domain is homologous to that of bacterial purC-encoded enzymes, and the sequence of the following AIR carboxylase domain is homologous to that of bacterial purE-encoded enzymes. In E. coli, AIR carboxylase is the product of genes purEK with the purK subunit postulated to have a role in CO2 binding. The avian enzyme lacks sequences corresponding to purK yet functions in E. coli. Functional complementation of E. coli pur mutants can be used to clone additional avian cDNAs for de novo purine nucleotide synthesis. Images PMID:1691501

  13. Simple determination of the CO sub 2 /O sub 2 specificity of Ribulose-1,5-bisphosphate carboxylase/oxygenase by the specific radioactivity of ( sup 14 C) glycerate 3-phosphate

    SciTech Connect

    Genhai Zhu; Jensen, R.G.; Hallick, R.B.; Wildner, G.F. )

    1992-02-01

    A new method is presented for measurement of the CO{sub 2}/O{sub 2} specificity factor of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco). The ({sup 14}C)3-phosphoglycerate (PGA) from the Rubisco carboxylase reaction and its dilution by the Rubisco oxygenase reaction was monitored by directly measuring the specific radioactivity of PGA. {sup 14}CO{sub 2} fixation with Rubisco occurred under two reaction conditions: carboxylase with oxygenase with 40 micromolar CO{sub 2} in O{sub 2}-saturated water and carboxylase only with 160 micromolar CO{sub 2} under N{sub 2}. Detection of the specific radioactivity used the amount of PGA as obtained from the peak area, which was determined by pulsed amperometry following separation by high-performance anion exchange chromatography and the radioactive counts of the ({sup 14}C)PGA in the same peak. The specificity factor of Rubisco from spinach (Spinacia oleracea L.) (93 {plus minus} 4), from the green alga Chlamydomonas reinhardtii (66 {plus minus} 1), and from the photosynthetic bacterium Rhodospirillum rubrum (13) were comparable with the published values measured by different methods.

  14. Ethylene emission and PR protein synthesis in ACC deaminase producing Methylobacterium spp. inoculated tomato plants (Lycopersicon esculentum Mill.) challenged with Ralstonia solanacearum under greenhouse conditions.

    PubMed

    Yim, Woojong; Seshadri, Sundaram; Kim, Kiyoon; Lee, Gillseung; Sa, Tongmin

    2013-06-01

    Bacteria of genus Methylobacterium have been found to promote plant growth and regulate the level of ethylene in crop plants. This work is aimed to test the induction of defense responses in tomato against bacterial wilt by stress ethylene level reduction mediated by the ACC deaminase activity of Methylobacterium strains. Under greenhouse conditions, the disease index value in Methylobacterium sp. inoculated tomato plants was lower than control plants. Plants treated with Methylobacterium sp. challenge inoculated with Ralstonia solanacearum (RS) showed significantly reduced disease symptoms and lowered ethylene emission under greenhouse condition. The ACC and ACO (1-aminocyclopropane-1-carboxylate oxidase) accumulation in tomato leaves were significantly reduced with Methylobacterium strains inoculation. While ACC oxidase gene expression was found higher in plants treated with R. solanacearum than Methylobacterium sp. treatment, PR proteins related to induced systemic resistance like β-1,3-glucanase, PAL, PO and PPO were increased in Methylobacterium sp. inoculated plants. A significant increase in β-1,3-glucanase and PAL gene expression was found in all the Methylobacterium spp. treatments compared to the R. solanacearum treatment. This study confirms the activity of Methylobacterium sp. in increasing the defense enzymes by modulating the ethylene biosynthesis pathway and suggests the use of methylotrophic bacteria as potential biocontrol agents in tomato cultivation.

  15. Bacterial community compositions of tomato (Lycopersicum esculentum Mill.) seeds and plant growth promoting activity of ACC deaminase producing Bacillus subtilis (HYT-12-1) on tomato seedlings.

    PubMed

    Xu, Mingshuang; Sheng, Jiping; Chen, Lin; Men, Yejun; Gan, Lin; Guo, Shuntang; Shen, Lin

    2014-03-01

    Study of endophytic bacteria within plant seeds is very essential and meaningful on account of their heritability and versatility. This study investigated Bacillus bacterial communities within the seeds of four commercial tomato varieties, by 16S rRNA gene PCR-RFLP (restriction fragment length polymorphism). Phylogenetic analysis of 16S rRNA gene sequences indicated that the 22 representative isolates belonged to five species of genus Bacillus and the bacterial compositions showed remarkable differences among tomato varieties. Isolates exhibited multiple plant growth promoting (PGP) traits: 37 % of indole-3-acetic acid production; 37 % of phosphate solubilization; 24 % of siderophores production; 85 % of potential nitrogen fixation and 6 % of 1-aminocyclopropane-1-carboxylate (ACC) deaminase activity. Isolate HYT-12-1 was shown to have highest ACC deaminase activity (112.02 nmol α-ketobutyrate mg⁻¹ protein h⁻¹) among the five ACC deamiase producing strains. 16S rRNA gene sequencing indicated that the isolate HYT-12-1 shared the highest sequence similarity (100 %) with B. subtilis. PGP experiments under gnotobiotic and greenhouse conditions revealed the ability of strain HYT-12-1 to enhance the growth of tomato seedlings. This is the first study to describe endophytic Bacillus communities within tomato seeds, and the results suggest that B. subtilis strain HYT-12-1 would have a great potential for industrial application as biofertilizer in the future.

  16. Functional determinants in transit sequences: import and partial maturation by vascular plant chloroplasts of the ribulose-1,5- bisphosphate carboxylase small subunit of Chlamydomonas

    PubMed Central

    1985-01-01

    The precursor of the ribulose-1,5-bisphosphate carboxylase small subunit and other proteins from Chlamydomonas reinhardtii are efficiently transported into chloroplasts isolated from spinach and pea. Thus, similar determinants specify precursor-chloroplast interactions in the alga and vascular plants. Removal of all or part of its transit sequence was found to block import of the algal small subunit into isolated chloroplasts. Comparison of available sequences revealed a nine amino acid segment conserved in the transit sequences of all small subunit precursors. A protease in the vascular plant chloroplasts recognized this region in the Chlamydomonas precursor and produced an intermediate form of the small subunit. We propose that processing of the small subunit precursor involves at least two proteolytic events; only one of these has been evolutionarily conserved. PMID:3965471

  17. Five palmitoylated polypeptides in the 50 KDa range are not recognized by an antibody against ribulose-biphosphate-carboxylase-oxygenase in Chlamydomonas reinhardtii.

    PubMed

    Picaud, A; Hours, M C; Trémolières, A

    1993-11-30

    After incubation of Chlamydomonas reinhardtii cells with radioactive palmitic acid several labelled bands appeared after gel electrophoresis of delipidated protein extract. Among them, two bands (a major and a minor one) were detected in the 50 KDa range, which is the region where the LSU of the Rubisco (large sub-unit of the ribulose-biphosphate-carboxylase-oxygenase) was also found. Careful analyses by two-dimensional gel electrophoresis have shown that the five palmitate-labelled polypeptides detected in this region do not match with polypeptides immunoreacting with antibody against Rubisco. In addition, polypeptides labelled by palmitate cannot be immunoprecipitated with the same antibody further demonstrating that, in C. reinhardtii, the large sub-unit of Rubisco is not palmitoylated but unindentified proteins.

  18. Acetyl-coenzyme A carboxylase α gene variations may be associated with the direct effects of some antipsychotics on triglyceride levels

    PubMed Central

    Diaz, Francisco J.; Meary, Alexander; Arranz, Maria J.; Ruaño, Gualberto; Windemuth, Andreas; de Leon, Jose

    2009-01-01

    Acetyl-coenzyme A carboxylase α (ACACA) single-nucleotide polymorphism (SNP) (rs2229416) was significantly associated with hypertriglyceridemia, during exploration of antipsychotic direct effects on lipids. Neuropeptide Y (NPY) gene (rs1468271) and ACACB gene (rs2241220) SNPs were significantly associated with severe hypercholesterolemia. In the same sample (173 patients on olanzapine, quetiapine, chlorpromazine or mirtazapine [increasing the risk of hyperlipidemia] and 184 controls taking other antipsychotics), three (rs1266175, rs12453407 and rs9906543) of eight additional ACACA SNPs were significantly associated with hypertriglyceridemia in those taking drugs of interest, but not in controls. Five other ACACA SNPs, three additional NPY SNPs, or seven additional ACACB SNPs were not significant. PMID:19846279

  19. Direct and selective small-molecule inhibition of photosynthetic PEP carboxylase: New approach to combat C4 weeds in arable crops.

    PubMed

    Paulus, Judith Katharina; Förster, Kerstin; Groth, Georg

    2014-06-05

    Phosphoenolpyruvate carboxylase (PEPC) is a key enzyme of C4 photosynthesis. Besides, non-photosynthetic isoforms of PEPC are found in bacteria and all types of plants, although not in animals or fungi. A single residue in the allosteric feedback inhibitor site of PEPC was shown to adjust the affinity of the photosynthetic and non-photosynthetic isoforms for feedback inhibition by metabolites of the C4 pathway. Here, we applied computational screening and biochemical analyses to identify molecules that selectively inhibit C4 PEPC, but have no effect on the activity of non-photosynthetic PEPCs. We found two types of selective inhibitors, catechins and quinoxalines. Binding constants in the lower μM range and a strong preference for C4 PEPC qualify the quinoxaline compounds as potential selective herbicides to combat C4 weeds.

  20. Determination of 3-hydroxyisovalerylcarnitine and other acylcarnitine levels using liquid chromatography-tandem mass spectrometry in serum and urine of a patient with multiple carboxylase deficiency.

    PubMed

    Maeda, Yasuhiro; Ito, Tetsuya; Ohmi, Hironori; Yokoi, Kyoko; Nakajima, Yoko; Ueta, Akihito; Kurono, Yukihisa; Togari, Hajime; Sugiyama, Naruji

    2008-07-15

    Due to its increased concentration in blood, 3-hydroxyisovalerylcarnitine (C5OH-I) is an important indicator for the diagnosis of organic acidemias in newborns. However, C5OH-I has not been used as a standard in tandem mass spectrometric (MS/MS) assays because its isolation is difficult. We developed a new synthesis of C5OH-I and investigated its behavior by MS/MS. A method using the multiple reaction monitoring (MRM) mode of MS/MS with HPLC was developed which provides high accuracy, precision and reproducibility. Acylcarnitine profiles in the serum and urine of a patient with multiple carboxylase deficiency (MCD) showed increased levels compared to a healthy patient.

  1. Cloning, expression, purification and physical and kinetic characterization of the phosphoenolpyruvate carboxylase from orange (Citrus sinensis osbeck var. Valencia) fruit juice sacs.

    PubMed

    Perotti, Valeria E; Figueroa, Carlos M; Andreo, Carlos S; Iglesias, Alberto A; Podestá, Florencio E

    2010-11-01

    Phosphoenolpyruvate (PEP) carboxylase (PEPCase) from orange fruit juice sacs has been cloned and heterogously expressed in high yield. The purified recombinant enzyme displays properties typical of plant PEPCase, including activation by sugar phosphates and inhibition by malate and citrate. Malate inhibition is weak in the physiological pH range, and the enzyme is also poorly affected by Glu and Asp, known inhibitors of C(3) plants PEPCases. However, it is strongly inhibited by citrate. Orange fruit PEPCase phosphorylation by mammalian protein kinase A decreased inhibition by malate. The enzyme presents an unusual high molecular mass in the absence of PEP, while in its presence it displays a more common tetrameric arrangement. The overall properties of the enzyme suggest that it is suited for organic acid synthesis and NADH reoxidation in the mature fruit. The present study provides the first analysis of a recombinant fruit PEPCase.

  2. The bacterial-type phosphoenolpyruvate carboxylase isozyme from developing castor oil seeds is subject to in vivo regulatory phosphorylation at serine-451.

    PubMed

    Dalziel, Katie J; O'Leary, Brendan; Brikis, Carolyne; Rao, Srinath K; She, Yi-Min; Cyr, Terry; Plaxton, William C

    2012-04-05

    Phosphoenolpyruvate carboxylase (PEPC) is a tightly controlled anaplerotic enzyme situated at a pivotal branch point of plant carbohydrate-metabolism. In developing castor oil seeds (COS) a novel allosterically-densensitized 910-kDa Class-2 PEPC hetero-octameric complex arises from a tight interaction between 107-kDa plant-type PEPC and 118-kDa bacterial-type PEPC (BTPC) subunits. Mass spectrometry and immunoblotting with anti-phosphoSer451 specific antibodies established that COS BTPC is in vivo phosphorylated at Ser451, a highly conserved target residue that occurs within an intrinsically disordered region. This phosphorylation was enhanced during COS development or in response to depodding. Kinetic characterization of a phosphomimetic (S451D) mutant indicated that Ser451 phosphorylation inhibits the catalytic activity of BTPC subunits within the Class-2 PEPC complex.

  3. Oxidative Stress Induces Partial Degradation of the Large Subunit of Ribulose-1,5-Bisphosphate Carboxylase/Oxygenase in Isolated Chloroplasts of Barley.

    PubMed Central

    Desimone, M.; Henke, A.; Wagner, E.

    1996-01-01

    The effects of oxidative stress on the degradation of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco; EC 4.1.1.39) were studied in isolated chloroplasts from barley (Hordeum vulgare L. cv Angora). Active oxygen (AO) was generated by varying the light intensity, the oxygen concentration, or the addition of herbicides or ADP-FeCl3-ascorbate to the medium. Oxidative treatments stimulated association of Rubisco with the insoluble fraction of chloroplasts and partial proteolysis of the large subunit (LSU). The most prominent degradation product of the LSU of Rubisco showed an apparent molecular mass of 36 kD. The data suggest that an increase in the amount of AO photogenerated by O2 reduction at photosystem I triggers Rubisco degradation. A possible relationship between AO-mediated denaturation of Rubisco and proteolysis of the LSU is discussed. PMID:12226330

  4. New insights into the post-translational modification of multiple phosphoenolpyruvate carboxylase isoenzymes by phosphorylation and monoubiquitination during sorghum seed development and germination

    PubMed Central

    Ruiz-Ballesta, Isabel; Baena, Guillermo; Gandullo, Jacinto; Wang, Liqun; She, Yi-Min; Plaxton, William Charles; Echevarría, Cristina

    2016-01-01

    Phosphoenolpyruvate carboxylase (PEPC; E.C. 4.1.1.31) was characterized in developing and germinating sorghum seeds, focusing on the transcript and polypeptide abundance of multiple plant-type phosphoenolpyruvate carboxylase (PTPC) genes, and the post-translational modification of each isoenzyme by phosphorylation versus monoubiquitination during germination. We observed high levels of SbPPC4 (Sb07g014960) transcripts during early development (stage I), and extensive transcript abundance of SbPPC2 (Sb02g021090) and SbPPC3 (Sb04g008720) throughout the entire life cycle of the seed. Although tandem mass spectrometry (MS) analysis of immunopurified PTPC indicated that four different PTPC isoenzymes were expressed in the developing and germinating seeds, SbPPC3 was the most abundant isozyme of the developing seed, and of the embryo and the aleurone layer of germinating seeds. In vivo phosphorylation of the different PTPC isoenzymes at their conserved N-terminal seryl phosphorylation site during germination was also established by MS/MS analysis. Furthermore, three of the four isoenzymes were partially monoubiquitinated, with MS/MS pinpointing SbPPC2 and SbPPC3 monoubiquitination at the conserved Lys-630 and Lys-624 residues, respectively. Our results demonstrate that monoubiquitination and phosphorylation simultaneously occur in vivo with different PTPC isozymes during seed germination. In addition, we show that PTPC monoubiquitination in germinating sorghum seeds always increases at stage II (emergence of the radicle), is maintained during the aerobic period of rapid cell division and reserve mobilization, and remains relatively constant until stage IV–V when coleoptiles initiate the formation of the photosynthetic tissues. PMID:27194739

  5. Genes encoding the alpha-carboxyltransferase subunit of acetyl-CoA carboxylase from Brassica napus and parental species: cloning, expression patterns, and evolution.

    PubMed

    Li, Zhi-Guo; Yin, Wei-Bo; Guo, Huan; Song, Li-Ying; Chen, Yu-Hong; Guan, Rong-Zhan; Wang, Jing-Qiao; Wang, Richard R-C; Hu, Zan-Min

    2010-05-01

    Heteromeric acetyl coenzyme A carboxylase (ACCase), a rate-limiting enzyme in fatty acid biosynthesis in dicots, is a multi-enzyme complex consisting of biotin carboxylase, biotin carboxyl carrier protein, and carboxyltransferase (alpha-CT and beta-CT). In the present study, four genes encoding alpha-CT were cloned from Brassica napus, and two were cloned from each of the two parental species, B. rapa and B. oleracea. Comparative and cluster analyses indicated that these genes were divided into two major groups. The major divergence between group-1 and group-2 occurred in the second intron. Group-2 alpha-CT genes represented the ancestral form in the genus Brassica. The divergence of group-1 and group-2 genes occurred in their common ancestor 12.96-17.78 million years ago (MYA), soon after the divergence of Arabidopsis thaliana and Brassica (15-20 MYA). This time of divergence is identical to that reported for the paralogous subgenomes of diploid Brassica species (13-17 MYA). Real-time reverse transcription PCR revealed that the expression patterns of the two groups of genes were similar in different organs, except in leaves. To better understand the regulation and evolution of alpha-CT genes, promoter regions from two sets of orthologous gene copies from B. napus, B. rapa, and B. oleracea were cloned and compared. The function of the promoter of gene Bnalpha-CT-1-1 in group-1 and gene Bnalpha-CT-2-1 in group-2 was examined by assaying beta-glucuronidase activity in transgenic A. thaliana. Our results will be helpful in elucidating the evolution and regulation of ACCase in oilseed rape.

  6. Assessment of 2013 AHA/ACC ASCVD risk scores with behavioral characteristics of an urban cohort in India

    PubMed Central

    Menon, Vidya P.; Edathadathil, Fabia; Sathyapalan, Dipu; Moni, Merlin; Don, Ann; Balachandran, Sabarish; Pushpa, Binny; Prasanna, Preetha; Sivaram, Nithu; Nair, Anupama; Vinod, Nithu; Jayaprasad, Rekha; Menon, Veena

    2016-01-01

    Abstract Cardiovascular diseases (CVDs) are the leading cause of death and disability in India. Early and sustained exposure to behavioral risk factors leads to development of CVDs. The aim of this study was to determine the baseline risk of a “hard CVD event” in subjects attending comprehensive health clinic and assess behavioral characteristics in “at risk” population. Using WHO STEPwise approach to Surveillance modified questionnaire, prevalence of noncommunicable diseases (NCDs) and risk factors was estimated in this cross-sectional study of 4507 subjects. Baseline cardiovascular risk was determined using Framingham risk score (FRS) and American College of Cardiology (ACC)/American Heart Association (AHA) atherosclerotic cardiovascular disease (ASCVD) algorithms. Modifiable behavior associated with high CVD risk was assessed. Among 40 to 59-year olds, ASCVD risk tool derived both a 10-year and lifetime risk score, which were used to stratify the cohort into 3 risk groups, namely, a high 10-year and high lifetime, a low 10-year and high lifetime, and a low 10-year and low lifetime risks. Dyslipidemia (30.6%), hypertension (25.5%), diabetes mellitus (20%), and obstructive airway disorders (17.6%) were most prevalent NCDs in our cohort. The ASCVD score stratified 26.1% subjects into high 10-yr and 59.5% into high lifetime risk while FRS classified 17.2% into high 10-year risk. Compared with FRS, the ASCVD risk estimator identified a larger proportion of subjects “at risk” of developing CVD. A high prevalence of alcohol use (38.4%), decreased intake of fruits and vegetables (96.2%) and low physical activity (58%) were observed in “at risk” population. Logistic regression analysis showed that in 40 to 59-year group, regular and occasional drinkers were 8.5- and 3.1-fold more likely to be in high 10-year and high lifetime ASCVD risk category than in low 10-year and low lifetime risk group. Similarly, regular drinkers and occasional drinkers were 2

  7. High rate GPS positioning , JASON altimetry and marine gravimetry : monitoring the Antarctic Circumpolar Current (ACC) through the DRAKE campaigns.

    NASA Astrophysics Data System (ADS)

    Melachroinos, S. A.; Biancale, R.; Menard, Y.; Sarrailh, M.

    2008-12-01

    The Drake campaign which took place from Jan 14, 2006 - 08 Feb, 2006 has been a very successful mission in collecting a wide range of GPS and marine gravity data all along JASON altimetry ground track n° 104. The same campaign will be repeated in 2009 along 028 and 104 JASON-2 ground track. The Drake Passage (DP) chokepoint is not only well suited geographically, as the Antarctic Circumpolar Current (ACC) is constricted to its narrowest extent of 700 km, but observations and models suggest that dynamical balances are particular effective in this area. Furthermore the space geodesy observations and their products provided from several altimetry missions (currently operating ENVISAT, JASON 1 and 2, GFO, ERS and other plannified for the future such as Altika, SWOT) require the cross comparison with independent geodetic techniques at the DP. The current experiment comprises a kinematic GPS and marine gravimetry Cal/Val geodetic approach and it aims to : validate with respect to altimetry data and surface models such a kinematic high frequency GPS technique for measuring sea state and sea surface height (SSH), compare the GPS SSH profiles with altimetry mean dynamic topography (MDT) and mean sea surface (MSS) models, give recommendations for future "offshore" Cal/Val activities on the ground tracks of altimeter satellites such as JASON-2, GFO, Altika using the GNSS technology etc. The GPS observations are collected from GPS antennas installed on a wave-rider buoy , aboard the R/V "Polarstern" and from continuous geodetic reference stations in the proximity. We also analyse problems related to the ship's attitude variations in roll, pitch and yaw and a way to correct them. We also give emphasis on the impact of the ship's acceleration profiles on the so called "squat effect" and ways to deal with it. The project will in particular benefit the GOCE mission by proposing to integrate GOCE in the ocean circulation study and validate GOCE products with our independent

  8. Complete plastid genome sequence of Primula sinensis (Primulaceae): structure comparison, sequence variation and evidence for accD transfer to nucleus

    PubMed Central

    Liu, Tong-Jian; Zhang, Cai-Yun; Yan, Hai-Fei; Zhang, Lu

    2016-01-01

    Species-rich genus Primula L. is a typical plant group with which to understand genetic variance between species in different levels of relationships. Chloroplast genome sequences are used to be the information resource for quantifying this difference and reconstructing evolutionary history. In this study, we reported the complete chloroplast genome sequence of Primula sinensis and compared it with other related species. This genome of chloroplast showed a typical circular quadripartite structure with 150,859 bp in sequence length consisting of 37.2% GC base. Two inverted repeated regions (25,535 bp) were separated by a large single-copy region (82,064 bp) and a small single-copy region (17,725 bp). The genome consists of 112 genes, including 78 protein-coding genes, 30 tRNA genes and four rRNA genes. Among them, seven coding genes, seven tRNA genes and four rRNA genes have two copies due to their locations in the IR regions. The accD and infA genes lacking intact open reading frames (ORF) were identified as pseudogenes. SSR and sequence variation analyses were also performed on the plastome of Primula sinensis, comparing with another available plastome of P. poissonii. The four most variable regions, rpl36–rps8, rps16–trnQ, trnH–psbA and ndhC–trnV, were identified. Phylogenetic relationship estimates using three sub-datasets extracted from a matrix of 57 protein-coding gene sequences showed the identical result that was consistent with previous studies. A transcript found from P. sinensis transcriptome showed a high similarity to plastid accD functional region and was identified as a putative plastid transit peptide at the N-terminal region. The result strongly suggested that plastid accD has been functionally transferred to the nucleus in P. sinensis. PMID:27375965

  9. ACC synthase genes are polymorphic in watermelon (Citrullus spp.) and differentially expressed in flowers and in response to auxin and gibberellin.

    PubMed

    Salman-Minkov, Ayelet; Levi, Amnon; Wolf, Shmuel; Trebitsh, Tova

    2008-05-01

    The flowering pattern of watermelon species (Citrullus spp.) is either monoecious or andromonoecious. Ethylene is known to play a critical role in floral sex determination of cucurbit species. In contrast to its feminizing effect in cucumber and melon, in watermelon ethylene promotes male flower development. In cucumber, the rate-limiting enzyme of ethylene biosynthesis, 1-aminocyclopropane-1-carboxylate (ACC) synthase (ACS), regulates unisexual flower development. To investigate the role of ethylene in flower development, we isolated four genomic sequences of ACS from watermelon (CitACS1-4). Both CitACS1 and CitACS3 are expressed in floral tissue. CitACS1 is also expressed in vegetative tissue and it may be involved in cell growth processes. Expression of CitACS1 is up-regulated by exogenous treatment with auxin, gibberellin or ACC, the immediate precursor of ethylene. No discernible differential floral sex-dependent expression pattern was observed for this gene. The CitACS3 gene is expressed in open flowers and in young staminate floral buds (male or hermaphrodite), but not in female flowers. CitACS3 is also up-regulated by ACC, and is likely to be involved in ethylene-regulated anther development. The expression of CitACS2 was not detected in vegetative or reproductive organs but was up-regulated by auxin. CitACS4 transcript was not detected under our experimental conditions. Restriction fragment length polymorphism (RFLP) and sequence tagged site (STS) marker analyses of the CitACS genes showed polymorphism among and within the different Citrullus groups, including watermelon cultivars, Citrullus lanatus var. lanatus, the central subspecies Citrullus lanatus var. citroides, and the desert species Citrullus colocynthis (L).

  10. The effects of in-vehicle tasks and time-gap selection while reclaiming control from adaptive cruise control (ACC) with bus simulator.

    PubMed

    Lin, Tsang-Wei; Hwang, Sheue-Ling; Su, Jau-Ming; Chen, Wan-Hui

    2008-05-01

    This research aimed to find out the effects of in-vehicle distractions and time-gap settings with a fix-based bus driving simulator in a following scenario. Professional bus drivers were recruited to perform in-vehicle tasks while driving with adaptive cruise control (ACC) of changeable time-gap settings in freeway traffic. Thirty subjects were divided equally into three groups for different in-vehicle task modes (between subjects), including no task distraction, hands-free, and manual modes. Further, time-gap settings for the experimental ACC were: shorter than 1.0 s, 1.0-1.5 s, 1.5-2.0 s, and longer than 2.0 s (within subjects). Longitudinal (mean headway, forward collision rate, and response time) and lateral control (mean lateral lane position and its standard deviation) performance was assessed. In the results, longitudinal control performance was worsened by both shorter time-gaps and heavier in-vehicle tasks. But the interaction indicated that the harm by heavier in-vehicle distraction could be improved by longer time-gaps. As for the lateral control, it would only be negatively affected by shorter time-gap settings. This research indicates the effects of time-gaps and in-vehicle distraction, as well as the interaction. Proper time-gap selection under different in-vehicle distractions can help avoid accidents and keep safe.

  11. Evolution of C4 phosphoenolpyruvate carboxylase in Flaveria, a conserved serine residue in the carboxyl-terminal part of the enzyme is a major determinant for C4-specific characteristics.

    PubMed

    Bläsing, O E; Westhoff, P; Svensson, P

    2000-09-08

    C4 phosphoenolpyruvate carboxylases have evolved from ancestral C3 isoforms during the evolution of angiosperms and gained distinct kinetic and regulatory properties compared with the C3 isozymes. To identify amino acid residues and/or domains responsible for these C4-specific properties the C4 phosphoenolpyruvate carboxylase of Flaveria trinervia (C4) was compared with its orthologue in the closely related C3 plant Flaveria pringlei. Reciprocal enzyme chimera were constructed and the kinetic constants, K(0.5) and k(cat), as well as the Hill coefficient, h, were determined for the substrate phosphoenolpyruvate both in the presence and absence of the activator glucose 6-phosphate. By this approach two regions were identified which determined most of the kinetic differences of the C4 and C3 ppcA phosphoenolpyruvate carboxylases with respect to the substrate PEP. In addition, the experiments suggest that the two regions do not act additively but interact with each other. The region between amino acids 296 and 437 is essential for activation by glucose 6-phosphate. The carboxyl-terminal segment between amino acids 645 and 966 contains a C4 conserved serine or a C3 invariant alanine at position 774 in the respective enzyme isoform. Site-directed mutagenesis shows that this position is a key determinant for the kinetic properties of the two isozymes.

  12. A signature of the oxygenase intermediate in catalysis by ribulose-bisphosphate carboxylase/oxygenase as provided by a site-directed mutant.

    PubMed

    Chen, Y R; Hartman, F C

    1995-05-19

    An uncharacterized minor transient product, observed in our earlier studies of substrate turnover by the E48Q mutant of Rhodospirillum rubrum ribulose-bisphosphate carboxylase/oxygenase (Lee, E. H., Harpel, M. R., Chen, Y.-R., and Hartman, F. C. (1993) J. Biol. Chem. 268, 26583-26591), becomes a major product when it is trapped and stabilized with borate as an additive to the reaction mixture. Chemical characterization establishes this novel product as D-glycero-2,3-pentodiulose 1,5-bisphosphate, thereby demonstrating oxidation of the C-3 hydroxyl of D-ribulose 1,5-bisphosphate to a carbonyl. As the formation of the novel oxidation product is oxygen-dependent and generates hydrogen peroxide, its precursor must be a peroxy derivative of ribulose bisphosphate. Thus, discovery of the dicarbonyl bisphosphate lends direct support to the long standing, but heretofore unproven, postulate that the normal pathway for oxidative cleavage of ribulose bisphosphate by the wild-type enzyme entails a peroxy intermediate. Our results also suggest that stabilization of the peroxy intermediate by the wild-type enzyme promotes carbon-carbon scission as opposed to elimination of hydrogen peroxide.

  13. Assembly of in vitro synthesized large subunits into ribulose-bisphosphate carboxylase/oxygenase. Formation and discharge of an L8-like species.

    PubMed

    Hubbs, A E; Roy, H

    1993-06-25

    Ribulose-bisphosphate carboxylase/oxygenase (Rubisco) from higher plants consists of eight approximately 53-kDa large subunits and eight approximately 14-kDa small subunits. Cytosolic ribosomes synthesize the small subunits as precursors, which enter the chloroplast, undergo proteolytic processing, and assemble with large subunits. Large subunits, synthesized in the chloroplast, first form a complex with the chloroplast chaperonin 60 (Cpn60(14)). In the presence of ATP, large subunits dissociate from Cpn60(14) and assemble into Rubisco. We now describe partial characterization of a new species, Z, containing radiotracer-labeled, newly synthesized pea Rubisco large subunits. Rubisco assembly occurs in low salt in the presence of small subunits and ATP. As with Rubisco assembly, the formation of Z is ATP-dependent and is inhibited by high chloride. Once formed, Z is stable except in high chloride. Z does not appear to interact directly with small subunits. However, after Z formation, Rubisco assembly occurs in an ATP-independent reaction that requires KCl and small subunits. These results are consistent with the hypothesis that Z is a large subunit containing structure that can contribute large subunits to Rubisco under appropriate conditions. Z shares some physical characteristics with reported cyanobacterial L8 core particles. However, formation of Rubisco from Z in the absence of ATP and the presence of small subunits appears to require conditions that otherwise destabilize Z.

  14. Structural characterization and the determination of negative cooperativity in the tight binding of 2-carboxyarabinitol bisphosphate to higher plant ribulose bisphosphate carboxylase.

    PubMed

    Johal, S; Partridge, B E; Chollet, R

    1985-08-15

    When CO2/Mg2+-activated spinach leaf ribulose-1,5-bisphosphate carboxylase (EC 4.1.1.39) is incubated with the transition-state analog 2-carboxyarabinitol 1,5-bisphosphate, an essentially irreversible complex is formed. The extreme stability of this quaternary complex has allowed the use of native analytical isoelectric focusing, anion-exchange chromatography, and nondenaturing polyacrylamide gel electrophoresis to probe the mechanism of the binding process and the effects of ligand tight-binding on the structure of the protein molecule. Changes in the chromatographic and electrophoretic properties of the enzyme upon tight binding of the inhibitor reveal that the ligand induces a conformational reorganization which extends to the surface of the protein molecule and, at saturation, results in a 16% decrease in apparent molecular weight. Analysis of ligand binding by isoelectric focusing shows that (i) incubating the protein with a stoichiometric molar concentration of ligand (site basis) results in an apparently charge homogeneous enzyme population with an isoelectric point of 4.9, and (ii) substoichiometric levels of ligand produce differential effects on each of the charge microheterogeneous native enzyme forms. These stoichiometry-dependent changes in electrofocusing band patterns were employed as a probe of cooperativity in the ligand tight-binding process. The tight-binding reaction was shown to be negatively cooperative.

  15. Electrophoretic assay for ribulose 1,5-bisphosphate carboxylase/oxygenase in guard cells and other leaf cells of Vicia faba L

    SciTech Connect

    Tarczynski, M.C.; Outlaw, W.H. Jr.; Arold, N.; Neuhoff, V.; Hampp, R. Max-Planck-Institute fuer Experimentelle Medizin, Goettingen Universitaet Tuebingen )

    1989-04-01

    The ribulose 1,5-bisphosphate carboxylase/oxygenase (Rubisco) contents of guard cells and other cells of Vicia faba L. leaflet were determined. To prevent proteolysis, proteins of frozen protoplast preparations or of cells excised from freeze-dried leaf were extracted directly in a sodium-dodecyl-sulfate-containing solution which was heated immediately after sample addition. Protein profiles of the different cell types were obtained by electrophoresis of the extracts and subsequent densitometry of the stained protein bands. About one-third of the protein of palisade parenchyma and of spongy parenchyma was Rubisco large subunit. Using chlorophyll (Chl):protein ratios previously obtained, we calculate mesophyll contained ca. 22 millimoles Rubisco per mole Chl. In contrast, guard-cell protoplast preparations were calculated to contain from 0.7 to 2.2 millimoles Rubisco per mole Chl. The upper end of this range is an overestimate resulting from contamination by mesophyll and to the method of peak integration. Extracts of excised guard cells were calculated to contain 0.05 to 0.17 millimole Rubisco per mole Chl. We conclude that Rubisco is absent, or virtually so, in guard cells of V. faba.

  16. Effect of CO{sub 2} concentration on carbonic anhydrase and ribulose-1,5-bisphosphate carboxylase/oxygenase expression in pea

    SciTech Connect

    Majeau, N.; Coleman, J.R.

    1996-10-01

    The effect of external CO{sub 2} concentration on the expression of carbonic anhydrase (CA) and ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) was examined in pea (Pisum sativum cv Little Marvel) leaves. Enzyme activities and their transcript levels were reduced in plants grown at 1000 {mu}L/L CO{sub 2} compared with plants grown in ambient air. Growth at 160 {mu}L/L CO{sub 2} also appeared to reduce steady-state transcript levels for the rbcS, the gene encoding the small subunit of Rubisco, and for ca, the gene encoding CA; however, rbcS transcripts were reduced to a greater extent at this concentration. Rubisco activity was slightly lower in plants grown at 160 {mu}L/L CO{sub 2}, and CA activity was significantly higher than that observed in air-grown plants. Transfer of plants from 1000 {mu}L/L to air levels of CO{sub 2} resulted in a rapid increase in both ca and rbcS transcript abundance in fully expanded leaves, followed by an increase in enzyme activity. Plants transferred from air to high-CO{sub 2} concentrations appeared to modulate transcript abundance and enzyme activity less quickly. Foliar carbohydrate levels were also examined in plants grown continuously at high and ambient CO{sub 2}, and following changes in growth conditions that rapidly altered ca and rbcS transcript abundance and enzyme activities. 39 refs., 2 figs., 3 tabs.

  17. Diel Rhythms in Ribulose-1,5-Bisphosphate Carboxylase/Oxygenase and Glutamine Synthetase Gene Expression in a Natural Population of Marine Picoplanktonic Cyanobacteria (Synechococcus spp.)

    PubMed Central

    Wyman, Michael

    1999-01-01

    Diel periodicity in the expression of key genes involved in carbon and nitrogen assimilation in marine Synechococcus spp. was investigated in a natural population growing in the surface waters of a cyclonic eddy in the northeast Atlantic Ocean. Synechococcus sp. cell concentrations within the upper mixed layer showed a net increase of three- to fourfold during the course of the experiment (13 to 22 July 1991), the population undergoing approximately one synchronous division per day. Consistent with the observed temporal pattern of phycoerythrin (CpeBA) biosynthesis, comparatively little variation was found in cpeBA mRNA abundance during either of the diel cycles investigated. In marked contrast, the relative abundance of transcripts originating from the genes encoding the large subunit of ribulose bisphosphate carboxylase/oxygenase (rbcL) and glutamine synthetase (glnA) showed considerable systematic temporal variation and oscillated during the course of each diel cycle in a reciprocal rhythm. Whereas activation of rbcL transcription was clearly not light dependent, expression of glnA appeared sensitive to endogenous changes in the physiological demands for nitrogen that arise as a natural consequence of temporal periodicity in photosynthetic carbon assimilation. The data presented support the hypothesis that a degree of temporal separation may exist between the most active periods of carbon and nitrogen assimilation in natural populations of marine Synecoccoccus spp. PMID:10427062

  18. Isoleucine 309 acts as a C4 catalytic switch that increases ribulose-1,5-bisphosphate carboxylase/oxygenase (rubisco) carboxylation rate in Flaveria.

    PubMed

    Whitney, Spencer M; Sharwood, Robert E; Orr, Douglas; White, Sarah J; Alonso, Hernan; Galmés, Jeroni

    2011-08-30

    Improving global yields of important agricultural crops is a complex challenge. Enhancing yield and resource use by engineering improvements to photosynthetic carbon assimilation is one potential solution. During the last 40 million years C(4) photosynthesis has evolved multiple times, enabling plants to evade the catalytic inadequacies of the CO(2)-fixing enzyme, ribulose-1,5-bisphosphate carboxylase/oxygenase (rubisco). Compared with their C(3) ancestors, C(4) plants combine a faster rubisco with a biochemical CO(2)-concentrating mechanism, enabling more efficient use of water and nitrogen and enhanced yield. Here we show the versatility of plastome manipulation in tobacco for identifying sequences in C(4)-rubisco that can be transplanted into C(3)-rubisco to improve carboxylation rate (V(C)). Using transplastomic tobacco lines expressing native and mutated rubisco large subunits (L-subunits) from Flaveria pringlei (C(3)), Flaveria floridana (C(3)-C(4)), and Flaveria bidentis (C(4)), we reveal that Met-309-Ile substitutions in the L-subunit act as a catalytic switch between C(4) ((309)Ile; faster V(C), lower CO(2) affinity) and C(3) ((309)Met; slower V(C), higher CO(2) affinity) catalysis. Application of this transplastomic system permits further identification of other structural solutions selected by nature that can increase rubisco V(C) in C(3) crops. Coengineering a catalytically faster C(3) rubisco and a CO(2)-concentrating mechanism within C(3) crop species could enhance their efficiency in resource use and yield.

  19. Preliminary X-ray crystallographic study of wild-type and mutant ribulose-1,5-bisphosphate carboxylase/oxygenase from Chlamydomonas reinhardtii.

    PubMed

    Yen, A; Haas, E J; Selbo, K M; Ross 2nd, C R; Spreitzer, R J; Stezowski, J J

    1998-07-01

    Ribulose-1,5-bisphosphate carboxylase/oxygenase is the key enzyme for photosynthesis. The wild-type and mutant (amino-acid substitutions in the catalytically important loop 6 region) enzymes from Chlamydomonas reinhardtii, a unicellular green alga, were crystallized. Wild-type, single-mutant (V331A) and two double-mutant (V331A/T342I and V331A/G344S) proteins were activated with cofactors CO2 and Mg2+, complexed with the substrate analog 2'-carboxyarabinitol-1,5-bisphosphate, and crystallized in apparently isomorphous forms. Unit-cell determinations have been completed for three of the enzymes. They display orthorhombic symmetry with similar cell parameters: wild type a = 130.4, b = 203. 3, c = 208.5 A; single mutant (V331A) a = 128.0, b = 203.0, c = 207. 0A; and double mutant (V331A/T342I) a = 130.0, b = 202.1, c = 209.7 A. Crystals of the wild-type and single-mutant (V331A) enzymes diffracted to approximately 2.8 A. A small crystal of the double-mutant (V331A/T342I) enzyme diffracted to approximately 6 A. A partial data set (68% complete) of the wild-type protein has been collected at room temperature to about 3.5 A.

  20. Diversity of ribulose-1,5-bisphosphate carboxylase/oxygenase large-subunit genes in the MgCl2-dominated deep hypersaline anoxic basin discovery.

    PubMed

    van der Wielen, Paul W J J

    2006-06-01

    Partial sequences of the form I (cbbL) and form II (cbbM) of the ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO) large subunit genes were obtained from the brine and interface of the MgCl2-dominated deep hypersaline anoxic basin Discovery. CbbL and cbbM genes were found in both brine and interface of the Discovery Basin but were absent in the overlying seawater. The diversity of both genes in the brine and interface was low, which might caused by the extreme saline conditions in Discovery of approximately 5 M MgCl2. None of the retrieved sequences were closely related to sequences deposited in the GenBank database. A phylogenetic analysis demonstrated that the cbbL sequences were affiliated with a Thiobacillus sp. or with one of the RuBisCO genes from Hydrogenovibrio marinus. The cbbM sequences clustered with thiobacilli or formed a new group with no close relatives. The results implicate that bacteria with the potential for carbon dioxide fixation and chemoautotrophy are present in the Discovery Basin. This is the first report demonstrating that RuBisCO genes are present under hypersaline conditions of 5 M MgCl2.

  1. Quantitative analyses of ribulose-1,5-bisphosphate carboxylase/oxygenase (RubisCO) large-subunit genes (cbbL) in typical paddy soils.

    PubMed

    Xiao, Ke-Qing; Bao, Peng; Bao, Qiong-Li; Jia, Yan; Huang, Fu-Yi; Su, Jian-Qiang; Zhu, Yong-Guan

    2014-01-01

    The Calvin cycle is known to be the major pathway for CO2 fixation, but our current understanding of its occurrence and importance in paddy soils is poor. In this study, the diversity of three ribulose-1,5-bisphosphate carboxylase/oxygenase large-subunit genes (cbbLG, cbbLR, cbbM) was investigated by clone library, T-RFLP, qPCR, and enzyme assay in five paddy soils in China. The cbbLG sequences revealed a relatively low level of diversity and were mostly related to the sequences of species from Thiobacillus. In contrast, highly diverse cbbLR and cbbM sequences were dispersed on the phylogenetic trees, and most of them were distantly related to known sequences, even forming separate clusters. Abundances of three cbbL genes ranged from 10(6) to 10(9) copies g(-1) soil, and cbbLR outnumbered cbbM and cbbLG in all soil samples, indicating that cbbLR may play a more important role than other two cbbL genes. Soil properties significantly influenced cbbL diversity in five paddy soils, of which clay content, C/N ratio, CEC, pH, and SOC correlated well with variations in microbial composition and abundance. In summary, this study provided a comparison of three cbbL genes, advancing our understanding of their role in carbon sequestration and nutrient turnover in the paddy soil.

  2. Regulation of Ribulose-1,5-Bisphosphate Carboxylase Expression in Second Leaves of Maize Seedlings from Low and High Yield Populations 1

    PubMed Central

    Loza-Tavera, Herminia; Martínez-Barajas, Eleazar; Sánchez-de-Jiménez, Estela

    1990-01-01

    Ribulose-1,5-bisphosphate carboxylase oxygenase (EC 4.1.1.39) (Rubisco) activity, Rubisco-protein, and Rubisco large and small subunit gene (rbcL and rbcS) transcripts were measured at seven stages of development in the second leaf of maize (Zea mays L.) seedlings belonging to low and high yield populations. During the three early stages of development, when the leaf has not yet expanded, it was determined that increments in Rubisco-activity were caused by increases in Rubisco-protein and its mRNAs. Afterward, the rbcS level decreased sharply down to nondetectable levels at the seventh stage, when the leaf was at the beginning of senescence. As a contrast, rbcL transcript decreased slowly and Rubisco-protein accumulated up to the fifth stage, when the leaf reached its maximum expansion. A slight decrease in Rubisco-protein was then observed. These results suggest that at early stages of development Rubisco-activity and Rubisco-protein are regulated mainly at the transcriptional level. At the later phase the regulation seems to be at other biochemical levels. Neither Rubisco activity nor Rubisco-protein showed correlation with yield for both maize populations at this stage of development. Slightly higher levels of both transcripts were observed in the high yield population. Images Figure 1 Figure 6 PMID:16667500

  3. The MDM2–p53–pyruvate carboxylase signalling axis couples mitochondrial metabolism to glucose-stimulated insulin secretion in pancreatic β-cells

    PubMed Central

    Li, Xiaomu; Cheng, Kenneth K. Y.; Liu, Zhuohao; Yang, Jin-Kui; Wang, Baile; Jiang, Xue; Zhou, Yawen; Hallenborg, Philip; Hoo, Ruby L. C.; Lam, Karen S. L.; Ikeda, Yasuhiro; Gao, Xin; Xu, Aimin

    2016-01-01

    Mitochondrial metabolism is pivotal for glucose-stimulated insulin secretion (GSIS) in pancreatic β-cells. However, little is known about the molecular machinery that controls the homeostasis of intermediary metabolites in mitochondria. Here we show that the activation of p53 in β-cells, by genetic deletion or pharmacological inhibition of its negative regulator MDM2, impairs GSIS, leading to glucose intolerance in mice. Mechanistically, p53 activation represses the expression of the mitochondrial enzyme pyruvate carboxylase (PC), resulting in diminished production of the TCA cycle intermediates oxaloacetate and NADPH, and impaired oxygen consumption. The defective GSIS and mitochondrial metabolism in MDM2-null islets can be rescued by restoring PC expression. Under diabetogenic conditions, MDM2 and p53 are upregulated, whereas PC is reduced in mouse β-cells. Pharmacological inhibition of p53 alleviates defective GSIS in diabetic islets by restoring PC expression. Thus, the MDM2–p53–PC signalling axis links mitochondrial metabolism to insulin secretion and glucose homeostasis, and could represent a therapeutic target in diabetes. PMID:27265727

  4. Photosynthetic CO2 fixation and ribulose bisphosphate carboxylase/oxygenase activity of Nostoc sp. strain UCD 7801 in symbiotic association with Anthoceros punctatus.

    PubMed Central

    Steinberg, N A; Meeks, J C

    1989-01-01

    The cyanobacterium Nostoc sp. strain UCD 7801, immediately after separation from pure cultures of a reconstituted symbiotic association with the bryophyte Anthoceros punctatus, exhibited a rate of light-dependent CO2 fixation that was eightfold lower than that measured in the free-living growth state. Ribulose bisphosphate carboxylase/oxygenase (RuBPC/O) specific activity was also eightfold lower in cell extracts of symbiotic strain 7801 relative to that in free-living cultures. The in vitro activity from symbiotic strain 7801 could not be increased by incubation under the standard RuBPC/O activation conditions. Polyclonal antibodies against the RuBPC/O large subunit were used in an enzyme-linked immunosorbent assay to determine that RuBPC/O accounted for 4.3 and 5.2% of the total protein in cell extracts of strain 7801 grown in symbiotic and free-living states, respectively. The results imply that the regulation of RuBPC/O activity in the symbiotic growth state is by a posttranslational mechanism rather than by an alteration in RuBPC/O protein synthesis. The amount of carboxyarabinitol bisphosphate required to irreversibly inhibit RuBPC/O activity of sybiotic cell extracts was 80% of that required for extracts of free-living cultures. This result indicates that any covalent modification of RuBPC/O in symbiotically associated Nostoc cells did not interfere with the ribulose bisphosphate binding site, since inactive enzyme also bound carboxyarabinitol bisphosphate. Images PMID:2509431

  5. Mapping of a locus correcting lack of phosphoribosylaminoimidazole carboxylase activity in Chinese hamster ovary cell Ade-D mutants to human chromosome 4.

    PubMed

    Barton, J W; Hart, I M; Patterson, D

    1991-02-01

    The human phosphoribosylaminoimidazole (AIR) carboxylase locus has been until this report one of the genes encoding purine biosynthetic enzymes that had not been assigned to an individual human chromosome. Characterization of Chinese hamster ovary (CHO) cell mutant Ade-D showed that the cell line was unable to produce IMP and accumulated AIR. CHO Ade-D cells were fused with normal human lymphocytes utilizing inactivated Sendai virus and the resulting hybrid cell lines were selected for purine prototrophy. Cytogenetic analysis showed a 100% concordance value for chromosome 4. Two of the isolated subclones contained only the long arm of chromosome 4 translocated onto a CHO chromosome, providing evidence for a regional assignment of the Ade-D gene to the long arm of chromosome 4. Two of the subclones containing chromosome 4 were subjected to the BrdU visible light segregation. All of the isolated purine auxotrophic cell lines showed a loss of the q arm of chromosome 4. The localization of the Ade-D locus to the long arm of chromosome 4 may reveal further clustering of the mammalian purine genes since the Ade-A locus has previously been regionally assigned to 4pter-q21.

  6. Comparison of Patterns of Accumulation of Ribulose Bisphosphate Carboxylase Antigen and Catalytic Activity and Measurement of Antigen Half-Life during the Cell Cycle of Chlorella sorokiniana1

    PubMed Central

    Toman, P. David; Schmidt, Robert R.

    1985-01-01

    By use of specific immunochemical procedures, ribulose-1,5-bisphosphate carboxylase (RuBPCase), antigen and catalytic activity were shown to have coincident step-patterns of accumulation during the cell cycle of Chlorella sorokiniana. Pulse-chase studies, employing radioactive sulfate, were performed during the period of rapid accumulation of enzyme activity and during the period of constant enzyme activity in the cell cycle. No degradation of RuBPCase antigen could be detected during either of these cell cycle periods. Thus, the step-pattern of accumulation of RuBPCase activity resulted from periodic synthesis of an enzyme that was stable under steady-state cell cycle conditions. Although inhibition of protein synthesis by cycloheximide, at different times in the cell cycle in the light, resulted in rapid decay of RuBPCase activity, this loss in activity occurred without detectable loss in enzyme antigen. When synchronous cells were placed into the dark, to slow the rate of protein synthesis in the absence of cycloheximide, the levels of enzyme antigen and activity decreased by 30 and 50%, respectively, during the 10-hour dark period. Thus, in C. sorokiniana changes in RuBPCase activity do not necessarily reflect parallel changes in enzyme antigen, particularly when cell growth is perturbed by changes from steady-state cultural conditions. PMID:16664496

  7. Expression of recombinant cytoplasmic yeast pyruvate carboxylase for the improvement of the production of human erythropoietin by recombinant BHK-21 cells.

    PubMed

    Irani, Noushin; Beccaria, Alejandro José; Wagner, Roland

    2002-02-28

    Recently, a recombinant yeast pyruvate carboxylase expressed in the cytoplasm of BHK-21 cells was shown to reconstitute the missing link between glycolysis and TCA, thus increasing the flux of glucose into the TCA and resulting in a higher intracellular ATP content. Now, these metabolically engineered cells have been additionally transfected with a plasmid bearing the gene for human erythropoietin. EPO yield and substrate-specific productivity of the recombinant BHK-21 cells have been compared to control cells without the PYC2-gene but transfected with the plasmid coding for the expression of the selection genes and EPO. PYC2-expressing clones showed a 2-fold higher glucose-specific productivity and a 2-fold higher product concentration in a continuously perfused bioreactor. Moreover, the PYC2 expression enabled the cells to become more resistant to low glucose concentrations in the culture medium. They could produce at nearly maximum productivity under glucose-limiting conditions of 0.05-1 gl(-1) that guaranteed a reduced accumulation of lactate in fed-batch production systems. Due to the fact that PYC2-expressing cells are characterized by reduced glucose consumption, a prolonged production phase in bioreactors can be maintained. Based on the demand not to fall short of 80% cell viability for the production, EPO could be produced for 2 days (30%) longer compared to the control due to a more economic exploitation of glucose, and the prolonged viability period of the cells using a batch cultivation driven by glutamine limitation.

  8. CO2 reduction and organic compounds production by photosynthetic bacteria with surface displayed carbonic anhydrase and inducible expression of phosphoenolpyruvate carboxylase.

    PubMed

    Park, Ju-Yong; Kim, Yang-Hoon; Min, Jiho

    2017-01-01

    In Rhodobacter sphaeroides, carbonic anhydrase (CA; EC 4.2.1.1) is a zinc-containing metalloenzyme that catalyzes the reversible hydration of CO2 to HCO3(-) while phosphoenolpyruvate carboxylase (PEPC; 4.1.1.31), an enzyme involved in the carbon metabolism that catalyzed the fixation of CO2 to PEP, is a key factor for biological fixation of CO2 and enhances the production of organic compounds. In this study, the recombinant R. sphaeroides with highly-expressed CA was developed based on a surface displayed system of CA (pJY-OmpCA) on the outer membrane of R. sphaeroides using outer membrane protein (Omp) in R. sphaeroides, Finally, two more different recombinant R. sphaeroides were developed, which transformed with a two-vector system harboring cytosolic expressed CA (pJY-OmpCA-CA)or PEPC (pJY-OMPCA-PEPC) in R. sphaeroides with surface displayed CA on the outer membrane. In case of recombinant R. sphaeroides with the pJY-OmpCA-PEPC, it has shown the highest CO2 reduction efficiency and the production of several organic compounds (carotenoids, polyhydroxybutyrate, malic acid, succinic acid). It means that the surface displayed CA on the R. sphaeroides would accelerate the CO2-bicabonate conversion on the bacterial outer membrane. Moreover, inducible over-expression of PEPC with surface-displayed CA was successfully used to facilitate a rapider CO2 reduction and quicker production of organic compounds.

  9. A potential role for RNA turnover in the light regulation of plant gene expression: ribulose-1,5-bisphosphate carboxylase small subunit in soybean.

    PubMed Central

    Shirley, B W; Meagher, R B

    1990-01-01

    Post-transcriptional regulation of the genes encoding the small subunit (rbcS) of ribulose-1,5-bisphosphate carboxylase was examined in soybean seedlings. Substantial discrepancies were detected between relative in vitro transcription rates and steady-state RNA levels in light- and dark-grown seedling leaves, indicating that rbcS RNA may be degraded more rapidly in light than in darkness. Additional data imply that the turnover mechanism is rapidly induced by light, maintained for some time in darkness, and that it may be negatively controlled by far-red light. The proposed RNA turnover system does not affect all RNAs equally since a soybean actin gene showed equivalent in vitro transcription rates and RNA levels in light and darkness. Soybean rbcS genes may be subject to a novel mode of control in which light-induced expression is accompanied by an increased rate of RNA degradation. Models for the specific regulation of rbcS RNA stability in response to light are presented. Images PMID:2356127

  10. Engineering the α-ketoglutarate overproduction from raw glycerol by overexpression of the genes encoding NADP+-dependent isocitrate dehydrogenase and pyruvate carboxylase in Yarrowia lipolytica.

    PubMed

    Yovkova, Venelina; Otto, Christina; Aurich, Andreas; Mauersberger, Stephan; Barth, Gerold

    2014-03-01

    To establish and develop a biotechnological process of α-ketoglutaric acid (KGA) production by Yarrowia lipolytica, it is necessary to increase the KGA productivity and to reduce the amounts of by-products, e.g. pyruvic acid (PA) as major by-product and fumarate, malate and succinate as minor by-products. The aim of this study was the improvement of KGA overproduction with Y. lipolytica by a gene dose-dependent overexpression of genes encoding NADP(+)-dependent isocitrate dehydrogenase (IDP1) and pyruvate carboxylase (PYC1) under KGA production conditions from the renewable carbon source raw glycerol. Recombinant Y. lipolytica strains were constructed, which harbour multiple copies of the respective IDP1, PYC1 or IDP1 and PYC1 genes together. We demonstrated that a selective increase in IDP activity in IDP1 multicopy transformants changes the produced amount of KGA. Overexpression of the gene IDP1 in combination with PYC1 had the strongest effect on increasing the amount of secreted KGA. About 19% more KGA compared to strain H355 was produced in bioreactor experiments with raw glycerol as carbon source. The applied cultivation conditions with this strain significantly reduced the main by-product PA and increased the KGA selectivity to more than 95% producing up to 186 g l(-1) KGA. This proved the high potential of this multicopy transformant for developing a biotechnological KGA production process.

  11. 2-(4-Bromoacetamido)anilino-2-deoxypentitol 1,5-bisphosphate, a new affinity label for ribulose bisphosphate carboxylase/oxygenase from Rhodospirillum rubrum. Determination of reaction parameters and characterization of an active site peptide.

    PubMed

    Herndon, C S; Hartman, F C

    1984-03-10

    A new affinity label for ribulose bisphosphate carboxylase/oxygenase from Rhodospirillum rubrum, 2-(4-bromoacetamido)anilino-2-deoxypentitol 1,5-bisphosphate, has been prepared, Reductive amination of ribulose-P2 with p-phenylenediamine in the presence of sodium cyanoborohydride yielded an epimeric mixture which was resolved by chromatography on quaternary aminoethyl-Sephadex. Subsequent bromoacetylation of the isolated amino bisphosphates gave reagents A and B (ribo and arabino epimers of 2-(4-bromoacetamido) anilino-2-deoxypentitol 1,5-bisphosphate) which were competitive inhibitors of the carboxylase with Ki values of 705 and 104 microM, respectively. Reagent A exhibited no time-dependent effects on the carboxylase in either the deactivated or activated state. Incubation of the enzyme with reagent B in the presence of the essential activators CO2 and Mg2+, however, resulted in an irreversible, time-dependent loss of activity, with a Kinact of 125 microM and a minimal half-time of 7.3 min. Covalent incorporation of [14C]reagent B was directly proportional to the loss of activity, with total inactivation correlating with an incorporation of 1.1 mol of reagent/mol of subunit. Inclusion of the competitive inhibitor 2-carboxyribitol 1,5-bisphosphate protected against inactivation with a concomitant reduction in incorporation. Neither reagent affected the activity of spinach carboxylase. Fractionation of [14C]reagent B-modified enzyme on DEAE-cellulose, subsequent to carboxymethylation and tryptic digestion, revealed two major radioactive peaks of approximately equal area. Digestion of each peak with alkaline phosphatase and rechromatography on DEAE-cellulose resulted in pure peptides I and II. The peptides were identical except in the site of labeling: peptide I contained a modified cysteinyl residue while peptide II contained a modified histidyl residue. Automated Edman degradation established the sequence as (sequence in text) which is located near the NH2 terminus

  12. Comparison of statin eligibility according to the Adult Treatment Panel III, ACC/AHA blood cholesterol guideline, and presence of carotid plaque by ultrasound in Mexican mestizo patients with rheumatoid arthritis.

    PubMed

    Galarza-Delgado, Dionicio A; Azpiri-Lopez, Jose R; Colunga-Pedraza, Iris J; Cardenas-de la Garza, Jesus A; Vera-Pineda, Raymundo; Garcia-Colunga, Judith I; Arvizu-Rivera, Rosa I; Martinez-Moreno, Adrian; Villarreal-Perez, Jesus Z; Elizondo-Riojas, Guillermo; Garza Elizondo, Mario A

    2016-11-01

    Atherosclerotic cardiovascular disease (ASCVD) is the leading cause of death in rheumatoid arthritis (RA) patients. Guidelines of the American College of Cardiology and the American Heart Association (ACC/AHA) 2013 and the Adult Treatment Panel III (ATP-III) differ in their strategies to recommend initiation of statin therapy. The presence of carotid plaque (CP) by carotid ultrasound is an indication to begin statin therapy. We aimed to compare the recommendation to initiate statin therapy according to the ACC/AHA 2013 guidelines, ATP-III guidelines, and CP by carotid ultrasound. We then carried out an observational, cross-sectional study of 62 statin-naive Mexican mestizo RA patients, aged 40 to 75, who fulfilled the 1987 or 2010 ACR/European League Against Rheumatism (EULAR) classification criteria. CP was evaluated with B-mode ultrasound. Cohen's kappa (k) was used to assess agreement between ACC/AHA 2013 guidelines, ATP-III guidelines, and the presence of CP, considering a p < 0.05 as statistically significant. Agreement was classified as slight (0.01-0.20), fair (0.21-0.40), moderate (0.41-0.60), substantial (0.61-0.80), and an almost perfect agreement (0.81-1.00). Slight agreement (k = 0.096) was found when comparing statin recommendation between CP and ATP-III. Fair agreement (k = 0.242) was revealed between ACC/AHA 2013 and ATP-III. Comparison between ACC/AHA 2013 and CP showed moderate agreement (k = 0.438). ACC/AHA 2013 guidelines could be an adequate and cost-effective tool to evaluate the need of statin therapy in Mexican mestizo RA patients, with moderate agreement with the presence of CP by ultrasound.

  13. Determination of l-arginine and NG, NG - and NG, NG' -dimethyl-L-arginine in plasma by liquid chromatography as AccQ-Fluor fluorescent derivatives.

    PubMed

    Heresztyn, Tamila; Worthley, Matthew I; Horowitz, John D

    2004-06-15

    A new HPLC assay for the detection of L-arginine, NG, NG-dimethyl-L-arginine (ADMA) and NG, NG' -dimethyl-L-arginine (SDMA) in plasma using the derivatisation reagent AccQ-Fluor (6-aminoquinolyl-N-hydroxysuccinimidyl carbamate) is described. The fluorescent derivatives produced are extremely stable enabling routine processing of large numbers of samples. Arginine and its metabolites are extracted from plasma on strong cation exchange (SCX) cartridges with NG-monomethyl-L-arginine (NMMA) as internal standard, derivatised and separated on a C18 column with acetonitrile in 0.1M sodium acetate buffer pH 6. Separation of the stereoisomers ADMA and SDMA was excellent and improvements to the solid phase extraction (SPE) procedure enabled good recovery (>80%) of arginine, ADMA and SDMA. The utility of the method is exemplified by comparison of plasma concentrations of ADMA, SDMA and arginine in healthy volunteers and diabetic/ischaemic patients.

  14. Isolation, characterization, and use for plant growth promotion under salt stress, of ACC deaminase-producing halotolerant bacteria derived from coastal soil.

    PubMed

    Siddikee, Md Ashaduzzaman; Chauhan, Puneet S; Anandham, R; Han, Gwang-Hyun; Sa, Tongmin

    2010-11-01

    In total, 140 halotolerant bacterial strains were isolated from both the soil of barren fields and the rhizosphere of six naturally growing halophytic plants in the vicinity of the Yellow Sea, near the city of Incheon in the Republic of Korea. All of these strains were characterized for multiple plant growth promoting traits, such as the production of indole acetic acid (IAA), nitrogen fixation, phosphorus (P) and zinc (Zn) solubilization, thiosulfate (S2O3) oxidation, the production of ammonia (NH3), and the production of extracellular hydrolytic enzymes such as protease, chitinase, pectinase, cellulase, and lipase under in vitro conditions. From the original 140 strains tested, on the basis of the latter tests for plant growth promotional activity, 36 were selected for further examination. These 36 halotolerant bacterial strains were then tested for 1- aminocyclopropane-1-carboxylic acid (ACC) deaminase activity. Twenty-five of these were found to be positive, and to be exhibiting significantly varying levels of activity. 16S rRNA gene sequencing analyses of the 36 halotolerant strains showed that they belong to 10 different bacterial genera: Bacillus, Brevibacterium, Planococcus, Zhihengliuella, Halomonas, Exiguobacterium, Oceanimonas, Corynebacterium, Arthrobacter, and Micrococcus. Inoculation of the 14 halotolerant bacterial strains to ameliorate salt stress (150 mM NaCl) in canola plants produced an increase in root length of between 5.2% and 47.8%, and dry weight of between 16.2% and 43%, in comparison with the uninoculated positive controls. In particular, three of the bacteria, Brevibacterium epidermidis RS15, Micrococcus yunnanensis RS222, and Bacillus aryabhattai RS341, all showed more than 40% increase in root elongation and dry weight when compared with uninoculated saltstressed canola seedlings. These results indicate that certain halotolerant bacteria, isolated from coastal soils, have a real potential to enhance plant growth under saline stress

  15. Ozone-induced ethylene emission accelerates the loss of ribulose-1,5-bisphosphate carboxylase/oxygenase and nuclear-encoded mRNAs in senescing potato leaves

    SciTech Connect

    Glick, R.E.; Schlagnhaufer, C.D.; Arteca, R.N.

    1995-11-01

    The relationships among O{sub 3}-induced accelerated senescence, induction of ethylene, and changes in specific mRNA and protein levels were investigated in potato (Solanum tuberosum L. cv Norland) plants. When plants were exposed to 0.08 {mu}L L{sup -1} O{sub 3} for 5 h d{sup -1}, steady-state levels of rbcS mRNA declined at least 5-fold in expanding leaves after 3 d of O{sub 3} exposure and ethylene levels increased 6- to 10-fold. The expression of OIP-1, a 1-aminocyclo-propane-1-carboxylate synthase cDNA from potato, correlated with increased production of ethylene and decreased levels of rbcS mRNA in foliage of plants treated with O{sub 3}. In plants exposed to 0.30 {mu}L L{sup -1} O{sub 3} for 4 h, rbcS transcript levels were reduced 4-fold, whereas nuclear run-on experiments revealed that rbcS mRNA may be due, in part, to posttranscriptional regulation. The levels of transcripts for other chloroplast proteins, glyceraldehyde-3-phosphate dehydrogenase, and a photosystem II chlorophyll a/b-binding protein decreased in O{sub 3}-treated plants, in parallel with the decrease in rbcS mRNA. The steady-state mRNA level of a cytosolic glyceraldehyde-3-phosphate dehydrogenase increased in O{sub 3}-treated plants. The induction of ethylene and changes in transcript levels preceded visible leaf damage and decreases in ribulose-1,5-biphosphate carboxylase/oxygenase protein levels. 40 refs., 6 figs.

  16. Determination of methylmalonyl coenzyme A by ultra high-performance liquid chromatography tandem mass spectrometry for measuring propionyl coenzyme A carboxylase activity in patients with propionic acidemia.

    PubMed

    Gotoh, Kana; Nakajima, Yoko; Tajima, Go; Watanabe, Yoriko; Hotta, Yuji; Kataoka, Tomoya; Kawade, Yoshihiro; Sugiyama, Naruji; Ito, Tetsuya; Kimura, Kazunori; Maeda, Yasuhiro

    2017-03-01

    Propionic acidemia (PA) is an inherited metabolic disease caused by low activity of propionyl coenzyme A (CoA) carboxylase (PCC), which metabolizes propionyl-CoA into methylmalonyl-CoA. Although many patients with PA have been identified by tandem mass spectrometry since the test was first included in neonatal mass screening in the 1990s, the disease severity varies. Thus, determining the specific level of PCC activity is considered to be helpful to grasp the severity of PA. We developed a new PCC assay method by the determination of methylmalonyl-CoA, which is formed by an enzyme reaction using peripheral lymphocytes, based on ultra high-performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS). With methylmalonyl-CoA concentrations of 0.05, 0.5, and 5μmol/L, the intra-assay coefficients of variation (CVs) were 8.2%, 8.7%, and 5.1%, respectively, and the inter-assay CVs were 13.6%, 10.5%, and 5.9%, respectively. The PCC activities of 20 healthy individuals and 6 PA patients were investigated with this assay. Methylmalonyl-CoA was not detected in one PA patient with a severe form of the disease, but the remaining PA patients with mild disease showed residual activities (3.3-7.8%). These results demonstrate that determination of PCC activity with this assay would be useful to distinguish between mild and severe cases of PA to help choose an appropriate treatment plan.

  17. Cloning and characterization of ribulose-1,5-bisphosphate carboxylase/oxygenase small subunit (RbcS) cDNA from green microalga Ankistrodesmus convolutus.

    PubMed

    Thanh, Tran; Chi, Vu Thi Quynh; Abdullah, Mohd Puad; Omar, Hishamuddin; Noroozi, Mostafa; Napis, Suhaimi

    2011-11-01

    An initial study on gene cloning and characterization of unicellular green microalga Ankistrodesmus convolutus was carried out to isolate and characterize the full-length cDNA of ribulose-1,5-bisphosphate carboxylase/oxygenase small subunit (RbcS) as a first step towards elucidating the structure of A. convolutus RbcS gene. The full-length of A. convolutus RbcS cDNA (AcRbcS) contained 28 bp of 5' untranslated region (UTR), 225 bp of 3' non-coding region, and an open reading frame of 165 amino acids consisting of a chloroplast transit peptide with 24 amino acids and a mature protein of 141 amino acids. The amino acid sequence has high identity to those of other green algae RbcS genes. The AcRbcS contained a few conserved domains including protein kinase C phosphorylation site, tyrosine kinase phosphorylation site and N-myristoylation sites. The AcRbcS was successfully expressed in Escherichia coli and a ~21 kDa of anticipated protein band was observed on SDS-PAGE. From the phylogenetic analysis of RbcS protein sequences, it was found that the RbcS of A. convolutus has closer genetic relationship with green microalgae species compared to those of green seaweed and green macroalgae species. Southern hybridization analysis revealed that the AcRbcS is a member of a small multigene family comprising of two to six members in A. convolutus genome. Under different illumination conditions, RT-PCR analysis showed that AcRbcS transcription was reduced in the dark, and drastically recovered in the light condition. Results presented in this paper established a good foundation for further study on the photosynthetic process of A. convolutus and other green algae species where little information is known on Rubisco small subunit.

  18. Characterization of ribulose-1, 5-bisphosphate carboxylase/oxygenase and transcriptional analysis of its related genes in Saccharina japonica (Laminariales, Phaeophyta)

    NASA Astrophysics Data System (ADS)

    Shao, Zhanru; Liu, Fuli; Li, Qiuying; Yao, Jianting; Duan, Delin

    2014-03-01

    Saccharina japonica is a common macroalga in sublittoral communities of cold seawater environments, and consequently may have highly efficient ribulose-1, 5-bisphosphate carboxylase/oxygenase (Rubisco) activity for carbon assimilation. In our study, we cloned the full-length Rubisco gene from S. japonica ( SJ-rbc). It contained an open reading frame for a large subunit gene ( SJ — rbcL) of 1 467 bp, a small subunit gene ( SJ-rbcS) of 420 bp, and a SJ-rbcL/S intergenic spacer of 269 bp. The deduced peptides of SJ-rbcL and SJ-rbcS were 488 and 139 amino acids with theoretical molecular weights and isoelectric points of 53.97 kDa, 5.81 and 15.84 kDa, 4.71, respectively. After induction with 1 mmol/L isopropyl- β-D-thiogalactopyranoside for 5 h and purification by Ni2+ affinity chromatography, electrophoresis and western blot detection demonstrated successful expression of the 55 kDa SJ-rbcL protein. Real-time quantitative PCR showed that the mRNA levels of SJ-rbcL in gametophytes increased when transferred into normal growth conditions and exhibited diurnal variations: increased expression during the day but suppressed expression at night. This observation implied that Rubisco played a role in normal gametophytic growth and development. In juvenile sporophytes, mRNA levels of SJ-rbcL, carbonic anhydrase, Calvin-Benson-Bassham cycle-related enzyme, and chloroplast light-harvesting protein were remarkably increased under continuous light irradiance. Similarly, expression of these genes was up-regulated under blue light irradiance at 350 μmol/(m2·s). Our results indicate that long-term white light and short-term blue light irradiance enhances juvenile sporophytic growth by synergistic effects of various photosynthetic elements.

  19. ATP and magnesium promote cotton short-form ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) activase hexamer formation at low micromolar concentrations.

    PubMed

    Kuriata, Agnieszka M; Chakraborty, Manas; Henderson, J Nathan; Hazra, Suratna; Serban, Andrew J; Pham, Tuong V T; Levitus, Marcia; Wachter, Rebekka M

    2014-11-25

    We report a fluorescence correlation spectroscopy (FCS) study of the assembly pathway of the AAA+ protein ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) activase (Rca), a ring-forming ATPase responsible for activation of inhibited Rubisco complexes for biological carbon fixation. A thermodynamic characterization of simultaneously populated oligomeric states appears critical in understanding Rca structure and function. Using cotton β-Rca, we demonstrate that apparent diffusion coefficients vary as a function of concentration, nucleotide, and cation. Using manual fitting procedures, we provide estimates for the equilibrium constants for the stepwise assembly and find that in the presence of ATPγS, the Kd for hexamerization is 10-fold lower than with ADP (∼0.1 vs ∼1 μM). Hexamer fractions peak at 30 μM and dominate at 8-70 μM Rca, where they comprise 60-80% of subunits with ATPγS, compared with just 30-40% with ADP. Dimer fractions peak at 1-4 μM Rca, where they comprise 15-18% with ATPγS and 26-28% with ADP. At 30 μM Rca, large aggregates begin to form that comprise ∼10% of total protein with ATPγS and ∼25% with ADP. FCS data collected on the catalytically impaired WalkerB-D173N variant in the presence of ATP provided strong support for these results. Titration with free magnesium ions lead to the disaggregation of larger complexes in favor of hexameric forms, suggesting that a second magnesium binding site with a Kd value of 1-3 mM mediates critical subunit contacts. We propose that closed-ring toroidal hexameric forms are stabilized by binding of Mg·ATP plus Mg2+, whereas Mg·ADP promotes continuous assembly to supramolecular aggregates such as spirals.

  20. Discrimination in the dark. Resolving the interplay between metabolic and physical constraints to phosphoenolpyruvate carboxylase activity during the crassulacean acid metabolism cycle.

    PubMed

    Griffiths, Howard; Cousins, Asaph B; Badger, Murray R; von Caemmerer, Susanne

    2007-02-01

    A model defining carbon isotope discrimination (delta13C) for crassulacean acid metabolism (CAM) plants was experimentally validated using Kalanchoe daigremontiana. Simultaneous measurements of gas exchange and instantaneous CO2 discrimination (for 13C and 18O) were made from late photoperiod (phase IV of CAM), throughout the dark period (phase I), and into the light (phase II). Measurements of CO2 response curves throughout the dark period revealed changing phosphoenolpyruvate carboxylase (PEPC) capacity. These systematic changes in PEPC capacity were tracked by net CO2 uptake, stomatal conductance, and online delta13C signal; all declined at the start of the dark period, then increased to a maximum 2 h before dawn. Measurements of delta13C were higher than predicted from the ratio of intercellular to external CO2 (p(i)/p(a)) and fractionation associated with CO2 hydration and PEPC carboxylations alone, such that the dark period mesophyll conductance, g(i), was 0.044 mol m(-2) s(-1) bar(-1). A higher estimate of g(i) (0.085 mol m(-2) s(-1) bar(-1)) was needed to account for the modeled and measured delta18O discrimination throughout the dark period. The differences in estimates of g(i) from the two isotope measurements, and an offset of -5.5 per thousand between the 18O content of source and transpired water, suggest spatial variations in either CO2 diffusion path length and/or carbonic anhydrase activity, either within individual cells or across a succulent leaf. Our measurements support the model predictions to show that internal CO2 diffusion limitations within CAM leaves increase delta13C discrimination during nighttime CO2 fixation while reducing delta13C during phase IV. When evaluating the phylogenetic distribution of CAM, carbon isotope composition will reflect these diffusive limitations as well as relative contributions from C3 and C4 biochemistry.

  1. Tissue-specific expression and post-translational modifications of plant- and bacterial-type phosphoenolpyruvate carboxylase isozymes of the castor oil plant, Ricinus communis L.

    PubMed

    O'Leary, Brendan; Fedosejevs, Eric T; Hill, Allyson T; Bettridge, James; Park, Joonho; Rao, Srinath K; Leach, Craig A; Plaxton, William C

    2011-11-01

    This study employs transcript profiling together with immunoblotting and co-immunopurification to assess the tissue-specific expression, protein:protein interactions, and post-translational modifications (PTMs) of plant- and bacterial-type phosphoenolpyruvate carboxylase (PEPC) isozymes (PTPC and BTPC, respectively) in the castor plant, Ricinus communis. Previous studies established that the Class-1 PEPC (PTPC homotetramer) of castor oil seeds (COS) is activated by phosphorylation at Ser-11 and inhibited by monoubiquitination at Lys-628 during endosperm development and germination, respectively. Elimination of photosynthate supply to developing COS by depodding caused the PTPC of the endosperm and cotyledon to be dephosphorylated, and then subsequently monoubiquitinated in vivo. PTPC monoubiquitination rather than phosphorylation is widespread throughout the castor plant and appears to be the predominant PTM of Class-1 PEPC that occurs in planta. The distinctive developmental patterns of PTPC phosphorylation versus monoubiquitination indicates that these two PTMs are mutually exclusive. By contrast, the BTPC: (i) is abundant in the inner integument, cotyledon, and endosperm of developing COS, but occurs at low levels in roots and cotyledons of germinated COS, (ii) shows a unique developmental pattern in leaves such that it is present in leaf buds and young expanding leaves, but undetectable in fully expanded leaves, and (iii) tightly interacts with co-expressed PTPC to form the novel and allosterically-desensitized Class-2 PEPC heteromeric complex. BTPC and thus Class-2 PEPC up-regulation appears to be a distinctive feature of rapidly growing and/or biosynthetically active tissues that require a large anaplerotic flux from phosphoenolpyruvate to replenish tricarboxylic acid cycle C-skeletons being withdrawn for anabolism.

  2. The non-photosynthetic phosphoenolpyruvate carboxylases of the C4 dicot Flaveria trinervia -- implications for the evolution of C4 photosynthesis.

    PubMed

    Bläsing, Oliver E; Ernst, Karin; Streubel, Monika; Westhoff, Peter; Svensson, Per

    2002-07-01

    C4 phospho enolpyruvate carboxylases (PEPCase; EC 4.1.1.3) have evolved from ancestral non-photosynthetic (C3) isoforms during the evolution of angiosperms and thereby gained distinct kinetic and regulatory properties. In order to obtain insight into this evolutionary process we have studied the C3 isoforms, ppcB and ppcC, of the C4 dicot Flaveria trinervia (Spreng.) C. Mohr and compared them with the C4 enzyme of this species, ppcA, and its orthologue in the C3 species F. pringlei Gandoger. Phylogenetic analyses indicate that the ppcB PEPCase is the closest relative of the ppcA enzyme. In addition, the presence of ppcB also in the closely related C3 species F. pringlei suggests that this gene was present already in the ancestral C3 species and consequently that ppcA has evolved by gene duplication of ppcB. Investigation of the enzymatic properties of the ppcB and ppcC enzymes showed low and similar K(0.5)-PEP values and limited activation by glucose-6-phosphate, typical of non-photosynthetic PEPCases, at pH 8.0. However, at the more physiological pH of 7.6, the ppcC enzyme displayed a substantially higher K(0.5)-PEP than the ppcB counterpart, indicating their involvement in different metabolic pathways. This indication was strengthened by malate inhibition studies in which the ppcC enzyme showed 10 times higher tolerance to the inhibitor. The ppcA enzyme was, however, by far the most tolerant enzyme towards malate. Interestingly, the increased malate tolerance was correlated with a decrease in enzyme efficiency displayed by the turnover constant k(cat). We therefore suggest that the increased malate tolerance, which is imperative for an efficient C4 cycle, is connected with a decreased enzyme efficiency that in turn is compensated by increased enzyme expression.

  3. Photoaffinity labeling of ribulose-1,5-bisphosphate carboxylase/oxygenase activase with ATP gamma-benzophenone. Identification of the ATP gamma-phosphate binding domain.

    PubMed

    Salvucci, M E; Rajagopalan, K; Sievert, G; Haley, B E; Watt, D S

    1993-07-05

    The phosphate-binding domain of the ATP-binding site of tobacco Rubisco (ribulose-1,5-bisphosphate carboxylase/oxygenase) activase was elucidated by photo-affinity labeling with a monoanhydride of ADP with N-(4-(benzoyl)phenylmethyl)phosphoramide ([gamma-32P]ATP gamma BP). Covalent incorporation of [gamma-32P]ATP gamma BP into the 42-kDa Rubisco activase subunit was dependent upon irradiation with ultraviolet light. Photolabelling of Rubisco activase with ATP gamma BP exhibited saturation kinetics; the apparent Kd for photolabeling was 5 microM. Two lines of evidence showed that ATP gamma BP modified Rubisco activase at the ATP-binding domain. First, physiological concentrations of ATP and ADP afforded complete protection against photolabeling of Rubisco activase by ATP gamma BP. Second, photolysis of Rubisco activase in the presence of ATP gamma BP decreased both the ATPase and the Rubisco activating activities. Inactivation of enzyme activity was dependent on ATP gamma BP concentration and could be prevented by including ADP during photolabeling. The region of Rubisco activase that was modified by ATP gamma BP was identified by isolating photolabeled peptides. Sequence analysis showed that ATP gamma BP modified Rubisco activase in two distinct regions; one region, S117-A136, is adjacent to the P-loop and the other region, V223-T234, exhibits homology to a region of adenylate kinase that ligates the essential metal ion. Photolabeling of these two regions of Rubisco activase was consistent with modification of the ATP gamma-phosphate-binding domain of Rubisco activase with ATP gamma BP.

  4. A mechanism for intergenomic integration: abundance of ribulose bisphosphate carboxylase small-subunit protein influences the translation of the large-subunit mRNA.

    PubMed

    Rodermel, S; Haley, J; Jiang, C Z; Tsai, C H; Bogorad, L

    1996-04-30

    Multimeric protein complexes in chloroplasts and mitochondria are generally composed of products of both nuclear and organelle genes of the cell. A central problem of eukaryotic cell biology is to identify and understand the molecular mechanisms for integrating the production and accumulation of the products of the two separate genomes. Ribulose bisphosphate carboxylase (Rubisco) is localized in the chloroplasts of photosynthetic eukaryotic cells and is composed of small subunits (SS) and large subunits (LS) coded for by nuclear rbcS and chloroplast rbcL genes, respectively. Transgenic tobacco plants containing antisense rbcS DNA have reduced levels of rbcS mRNA, normal levels of rbcL mRNA, and coordinately reduced LS and SS proteins. Our previous experiments indicated that the rate of translation of rbcL mRNA might be reduced in some antisense plants; direct evidence is presented here. After a short-term pulse there is less labeled LS protein in the transgenic plants than in wild-type plants, indicating that LS accumulation is controlled in the mutants at the translational and/or posttranslational levels. Consistent with a primary restriction at translation, fewer rbcL mRNAs are associated with polysomes of normal size and more are free or are associated with only a few ribosomes in the antisense plants. Effects of the rbcS antisense mutation on mRNA and protein accumulation, as well as on the distribution of mRNAs on polysomes, appear to be minimal for other chloroplast and nuclear photosynthetic genes. Our results suggest that SS protein abundance specifically contributes to the regulation of LS protein accumulation at the level of rbcL translation initiation.

  5. Lysine residues involved in the hysteresis and in the regulatory sites of spinach ribulose 1,5-bisphosphate carboxylase/oxygenase.

    PubMed

    Yokota, A; Tokai, H

    1993-11-01

    Lysine residues have been suggested to be involved in the hysteretic decrease of the activity of spinach ribulose 1,5-bisphosphate carboxylase/oxygenase (RuBisCO) and the binding of ribulose 1,5-bisphosphate to its regulatory sites [Yokota, A. & Tsujimoto, N. (1992) Eur. J. Biochem. 204, 901-909]. This work identifies the lysine residues and investigates the effects of their chemical modification on the course of RuBisCO reaction. The carbamylated form of RuBisCO reacted with trinitrobenzene sulfonate in three phases; an initial rapid, second slow, and final non-specific reaction. Lys-334 in loop 6, Lys-21, and Lys-128, all from the large subunits, were trinitrophenylated in the first 60-min reaction. Lys-305 of the large subunits was labeled in the next step. The modification of these residues was strongly suppressed in the enzyme form that had undergone hysteretic conformational change after binding 2-carboxyarabinitol 1,5-bisphosphate at its catalytic sites. Instead, Lys-450 of the large subunits and Lys-71 from the small subunits were newly modified in the quaternary complex. A higher concentration of 2-carboxyarabinitol 1,5-bisphosphate reduced the trinitrophenylation of the two residues to half. The modification of the carbamylated form of the enzyme for 30 min was expected to arylate Lys-21, Lys-128, and Lys-334 at random, and the course of the reaction of the partially modified enzyme was expected to deviate from that of the unmodified enzyme. Experimental results showed that this was the case.(ABSTRACT TRUNCATED AT 250 WORDS)

  6. CO2-responsive expression and gene organization of three ribulose-1,5-bisphosphate carboxylase/oxygenase enzymes and carboxysomes in Hydrogenovibrio marinus strain MH-110.

    PubMed

    Yoshizawa, Yoichi; Toyoda, Koichi; Arai, Hiroyuki; Ishii, Masaharu; Igarashi, Yasuo

    2004-09-01

    Hydrogenovibrio marinus strain MH-110, an obligately lithoautotrophic hydrogen-oxidizing bacterium, fixes CO2 by the Calvin-Benson-Bassham cycle. Strain MH-110 possesses three different sets of genes for ribulose-1,5-bisphosphate carboxylase/oxygenase (RubisCO): CbbLS-1 and CbbLS-2, which belong to form I (L8S8), and CbbM, which belongs to form II (Lx). In this paper, we report that the genes for CbbLS-1 (cbbLS-1) and CbbM (cbbM) are both followed by the cbbQO genes and preceded by the cbbR genes encoding LysR-type regulators. In contrast, the gene for CbbLS-2 (cbbLS-2) is followed by genes encoding carboxysome shell peptides. We also characterized the three RubisCOs in vivo by examining their expression profiles in environments with different CO2 availabilities. Immunoblot analyses revealed that when strain MH-110 was cultivated in 15% CO2, only the form II RubisCO, CbbM, was expressed. When strain MH-110 was cultivated in 2% CO2, CbbLS-1 was expressed in addition to CbbM. In the 0.15% CO2 culture, the expression of CbbM decreased and that of CbbLS-1 disappeared, and CbbLS-2 was expressed. In the atmospheric CO2 concentration of approximately 0.03%, all three RubisCOs were expressed. Transcriptional analyses of mRNA by reverse transcription-PCR showed that the regulation was at the transcriptional level. Electron microscopic observation of MH-110 cells revealed the formation of carboxysomes in the 0.15% CO2 concentration. The results obtained here indicate that strain MH-110 adapts well to various CO2 concentrations by using different types of RubisCO enzymes.

  7. Phylogeny and functional expression of ribulose 1,5-bisphosphate carboxylase/oxygenase from the autotrophic ammonia-oxidizing bacterium Nitrosospira sp. isolate 40KI.

    PubMed

    Utåker, Janne B; Andersen, Kjell; Aakra, Agot; Moen, Birgitte; Nes, Ingolf F

    2002-01-01

    The autotrophic ammonia-oxidizing bacteria (AOB), which play an important role in the global nitrogen cycle, assimilate CO(2) by using ribulose 1,5-bisphosphate carboxylase/oxygenase (RubisCO). Here we describe the first detailed study of RubisCO (cbb) genes and proteins from the AOB. The cbbLS genes from Nitrosospira sp. isolate 40KI were cloned and sequenced. Partial sequences of the RubisCO large subunit (CbbL) from 13 other AOB belonging to the beta and gamma subgroups of the class Proteobacteria are also presented. All except one of the beta-subgroup AOB possessed a red-like type I RubisCO with high sequence similarity to the Ralstonia eutropha enzyme. All of these new red-like RubisCOs had a unique six-amino-acid insert in CbbL. Two of the AOB, Nitrosococcus halophilus Nc4 and Nitrosomonas europaea Nm50, had a green-like RubisCO. With one exception, the phylogeny of the AOB CbbL was very similar to that of the 16S rRNA gene. The presence of a green-like RubisCO in N. europaea was surprising, as all of the other beta-subgroup AOB had red-like RubisCOs. The green-like enzyme of N. europaea Nm50 was probably acquired by horizontal gene transfer. Functional expression of Nitrosospira sp. isolate 40KI RubisCO in the chemoautotrophic host R. eutropha was demonstrated. Use of an expression vector harboring the R. eutropha cbb control region allowed regulated expression of Nitrosospira sp. isolate 40KI RubisCO in an R. eutropha cbb deletion strain. The Nitrosospira RubisCO supported autotrophic growth of R. eutropha with a doubling time of 4.6 h. This expression system may allow further functional analysis of AOB cbb genes.

  8. Analysis of facultative lithotroph distribution and diversity on volcanic deposits by use of the large subunit of ribulose 1,5-bisphosphate carboxylase/oxygenase.

    PubMed

    Nanba, K; King, G M; Dunfield, K

    2004-04-01

    A 492- to 495-bp fragment of the gene coding for the large subunit of the form I ribulose 1,5-bisphosphate carboxylase/oxygenase (RubisCO) (rbcL) was amplified by PCR from facultatively lithotrophic aerobic CO-oxidizing bacteria, colorless and purple sulfide-oxidizing microbial mats, and genomic DNA extracts from tephra and ash deposits from Kilauea volcano, for which atmospheric CO and hydrogen have been previously documented as important substrates. PCR products from the mats and volcanic sites were used to construct rbcL clone libraries. Phylogenetic analyses showed that the rbcL sequences from all isolates clustered with form IC rbcL sequences derived from facultative lithotrophs. In contrast, the microbial mat clone sequences clustered with sequences from obligate lithotrophs representative of form IA rbcL. Clone sequences from volcanic sites fell within the form IC clade, suggesting that these sites were dominated by facultative lithotrophs, an observation consistent with biogeochemical patterns at the sites. Based on phylogenetic and statistical analyses, clone libraries differed significantly among volcanic sites, indicating that they support distinct lithotrophic assemblages. Although some of the clone sequences were similar to known rbcL sequences, most were novel. Based on nucleotide diversity and average pairwise difference, a forested site and an 1894 lava flow were found to support the most diverse and least diverse lithotrophic populations, respectively. These indices of diversity were not correlated with rates of atmospheric CO and hydrogen uptake but were correlated with estimates of respiration and microbial biomass.

  9. Ribulose-1,5-bisphosphate carboxylase/oxygenase genes as a functional marker for chemolithoautotrophic halophilic sulfur-oxidizing bacteria in hypersaline habitats.

    PubMed

    Tourova, Tatjana P; Kovaleva, Olga L; Sorokin, Dimitry Yu; Muyzer, Gerard

    2010-07-01

    The presence and diversity of the cbb genes encoding the large subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO) (a key enzyme of the Calvin-Benson cycle of autotrophic CO(2) assimilation) were investigated in pure cultures of seven genera of halophilic chemolithoautotrophic sulfur-oxidizing bacteria (SOB) and in sediments from a hypersaline lake in which such bacteria have been recently discovered. All of the halophilic SOB strains (with the exception of Thiohalomonas nitratireducens) possessed the cbbL gene encoding RuBisCO form I, while the cbbM gene encoding RuBisCO form II was detected only in some of the pure cultures. The general topologies of the CbbL/CbbM trees and the 16S rRNA gene tree were different, but both markers showed that the halophilic SOB genera formed independent lineages in the Gammaproteobacteria. In some cases, such as with several strains of the genus Thiohalospira and with Thioalkalibacter halophilus, the cbbL clustering was incongruent with the positions of these strains on the ribosomal tree. In the cbbM tree, the clustering of Thiohalospira and Thiohalorhabdus strains was incongruent with their branching in both cbbL and 16S rRNA gene trees. cbbL and cbbM genes related to those found in the analysed halophilic SOB were also detected in a sediment from a hypersaline lake in Kulunda Steppe (Russia). Most of the cbbL and cbbM genes belonged to members of the genus Thiohalorhabdus. In the cbbL clone library, sequences related to those of Halothiobacillus and Thiohalospira were detected as minor components. Some of the environmental cbbM sequences belonged to as yet unknown phylotypes, representing deep lineages of halophilic autotrophs.

  10. Deduced amino acid sequence, functional expression, and unique enzymatic properties of the form I and form II ribulose bisphosphate carboxylase/oxygenase from the chemoautotrophic bacterium Thiobacillus denitrificans.

    PubMed

    Hernandez, J M; Baker, S H; Lorbach, S C; Shively, J M; Tabita, F R

    1996-01-01

    The cbbL cbbS and cbbM genes of Thiobacillus denitrificans, encoding form I and form II ribulose 1,5-bisphosphate carboxylase/oxygenase (RubisCO), respectively, were found to complement a RubisCO-negative mutant of Rhodobacter sphaeroides to autotrophic growth. Endogenous T. denitrificans promoters were shown to function in R. sphaeroides, resulting in high levels of cbbL cbbS and cbbM expression in the R. sphaeroides host. This expression system provided high levels of both T. denitrificans enzymes, each of which was highly purified. The deduced amino acid sequence of the form I enzyme indicated that the large subunit was closely homologous to previously sequenced form I RubisCO enzymes from sulfur-oxidizing bacteria. The form I T. denitrificans enzyme possessed a very low substrate specificity factor and did not exhibit fallover, and yet this enzyme showed a poor ability to recover from incubation with ribulose 1,5-bisphosphate. The deduced amino acid sequence of the form II T. denitrificans enzyme resembled those of other form II RubisCO enzymes. The substrate specificity factor was characteristically low, and the lack of fallover and the inhibition by ribulose 1,5-bisphosphate were similar to those of form II RubisCO obtained from nonsulfur purple bacteria. Both form I and form II RubisCO from T. denitrificans possessed high KCO2 values, suggesting that this organism might suffer in environments containing low levels of dissolved CO2. These studies present the initial description of the kinetic properties of form I and form II RubisCO from a chemoautotrophic bacterium that synthesizes both types of enzyme.

  11. Reciprocal Control of Anaplerotic Phosphoenolpyruvate Carboxylase by in Vivo Monoubiquitination and Phosphorylation in Developing Proteoid Roots of Phosphate-Deficient Harsh Hakea1[W][OA

    PubMed Central

    Shane, Michael W.; Fedosejevs, Eric T.; Plaxton, William C.

    2013-01-01

    Accumulating evidence indicates important functions for phosphoenolpyruvate (PEP) carboxylase (PEPC) in inorganic phosphate (Pi)-starved plants. This includes controlling the production of organic acid anions (malate, citrate) that are excreted in copious amounts by proteoid roots of nonmycorrhizal species such as harsh hakea (Hakea prostrata). This, in turn, enhances the bioavailability of mineral-bound Pi by solubilizing Al3+, Fe3+, and Ca2+ phosphates in the rhizosphere. Harsh hakea thrives in the nutrient-impoverished, ancient soils of southwestern Australia. Proteoid roots from Pi-starved harsh hakea were analyzed over 20 d of development to correlate changes in malate and citrate exudation with PEPC activity, posttranslational modifications (inhibitory monoubiquitination versus activatory phosphorylation), and kinetic/allosteric properties. Immature proteoid roots contained an equivalent ratio of monoubiquitinated 110-kD and phosphorylated 107-kD PEPC polypeptides (p110 and p107, respectively). PEPC purification, immunoblotting, and mass spectrometry indicated that p110 and p107 are subunits of a 430-kD heterotetramer and that they both originate from the same plant-type PEPC gene. Incubation with a deubiquitinating enzyme converted the p110:p107 PEPC heterotetramer of immature proteoid roots into a p107 homotetramer while significantly increasing the enzyme’s activity under suboptimal but physiologically relevant assay conditions. Proteoid root maturation was paralleled by PEPC activation (e.g. reduced Km [PEP] coupled with elevated I50 [malate and Asp] values) via in vivo deubiquitination of p110 to p107, and subsequent phosphorylation of the deubiquitinated subunits. This novel mechanism of posttranslational control is hypothesized to contribute to the massive synthesis and excretion of organic acid anions that dominates the carbon metabolism of the mature proteoid roots. PMID:23407057

  12. Analysis of Facultative Lithotroph Distribution and Diversity on Volcanic Deposits by Use of the Large Subunit of Ribulose 1,5-Bisphosphate Carboxylase/Oxygenase†

    PubMed Central

    Nanba, K.; King, G. M.; Dunfield, K.

    2004-01-01

    A 492- to 495-bp fragment of the gene coding for the large subunit of the form I ribulose 1,5-bisphosphate carboxylase/oxygenase (RubisCO) (rbcL) was amplified by PCR from facultatively lithotrophic aerobic CO-oxidizing bacteria, colorless and purple sulfide-oxidizing microbial mats, and genomic DNA extracts from tephra and ash deposits from Kilauea volcano, for which atmospheric CO and hydrogen have been previously documented as important substrates. PCR products from the mats and volcanic sites were used to construct rbcL clone libraries. Phylogenetic analyses showed that the rbcL sequences from all isolates clustered with form IC rbcL sequences derived from facultative lithotrophs. In contrast, the microbial mat clone sequences clustered with sequences from obligate lithotrophs representative of form IA rbcL. Clone sequences from volcanic sites fell within the form IC clade, suggesting that these sites were dominated by facultative lithotrophs, an observation consistent with biogeochemical patterns at the sites. Based on phylogenetic and statistical analyses, clone libraries differed significantly among volcanic sites, indicating that they support distinct lithotrophic assemblages. Although some of the clone sequences were similar to known rbcL sequences, most were novel. Based on nucleotide diversity and average pairwise difference, a forested site and an 1894 lava flow were found to support the most diverse and least diverse lithotrophic populations, respectively. These indices of diversity were not correlated with rates of atmospheric CO and hydrogen uptake but were correlated with estimates of respiration and microbial biomass. PMID:15066819

  13. Expressed genes for plant-type ribulose 1,5-bisphosphate carboxylase/oxygenase in the photosynthetic bacterium Chromatium vinosum, which possesses two complete sets of the genes.

    PubMed Central

    Viale, A M; Kobayashi, H; Akazawa, T

    1989-01-01

    Two sets of genes for the large and small subunits of ribulose 1,5-bisphosphate carboxylase/oxygenase (RuBisCO) were detected in the photosynthetic purple sulfur bacterium Chromatium vinosum by hybridization analysis with RuBisCO gene probes, cloned by using the lambda Fix vector, and designated rbcL-rbcS and rbcA-rbcB. rbcL and rbcA encode the large subunits, and rbcS and rbcB encode the small subunits. rbcL-rbcS was the same as that reported previously (A. M. Viale, H. Kobayashi, T. Takabe, and T. Akazawa, FEBS Lett. 192:283-288, 1985). A DNA fragment bearing rbcA-rbcB was subcloned in plasmid vectors and sequenced. We found that rbcB was located 177 base pairs downstream of the rbcA coding region, and both genes were preceded by plausible procaryotic ribosome-binding sites. rbcA and rbcD encoded polypeptides of 472 and 118 amino acids, respectively. Edman degradation analysis of the subunits of RuBisCO isolated from C. vinosum showed that rbcA-rbcB encoded the enzyme present in this bacterium. The large- and small-subunit polypeptides were posttranslationally processed to remove 2 and 1 amino acid residues from their N-termini, respectively. Among hetero-oligomeric RuBisCOs, the C. vinosum large subunit exhibited higher homology to that from cyanobacteria, eucaryotic algae, and higher plants (71.6 to 74.2%) than to that from the chemolithotrophic bacterium Alcaligenes eutrophus (56.6%). A similar situation has been observed for the C. vinosum small subunit, although the homology among small subunits from different organisms was lower than that among the large subunits. Images PMID:2708310

  14. Multiple E-Boxes in the Distal Promoter of the Rat Pyruvate Carboxylase Gene Function as a Glucose-Responsive Element

    PubMed Central

    Muangsawat, Sureeporn; Boonsaen, Thirajit; MacDonald, Michael J.; Jitrapakdee, Sarawut

    2014-01-01

    Pyruvate carboxylase (PC) is an anaplerotic enzyme that regulates glucose-induced insulin secretion in pancreatic islets. Dysregulation of its expression is associated with type 2 diabetes. Herein we describe the molecular mechanism underlying the glucose-mediated transcriptional regulation of the PC gene. Incubation of the rat insulin cell line INS-1 832/13 with glucose resulted in a 2-fold increase in PC mRNA expression. Transient transfections of the rat PC promoter-luciferase reporter construct in the above cell line combined with mutational analysis indicated that the rat PC gene promoter contains the glucose-responsive element (GRE), comprising three canonical E-boxes (E1, E3 and E4) and one E-box-like element (E2) clustering between nucleotides –546 and –399, upstream of the transcription start site. Mutation of any of these E-boxes resulted in a marked reduction of glucose-mediated transcriptional induction of the reporter gene. Electrophoretic mobility shift assays revealed that the upstream stimulatory factors 1 and 2 (USF1 and USF2) bind to E1, the Specificity Protein-1 (Sp1) binds to E2, USF2 and the carbohydrate responsive element binding protein (ChREBP) binds to E4, while unknown factors binds to E3. High glucose promotes the recruitment of Sp1 to E2 and, USF2 and ChREBP to E4. Silencing the expression of Sp1, USF2 and ChREBP by their respective siRNAs in INS-1 832/13 cells blunted glucose-induced expression of endogenous PC. We conclude that the glucose-mediated transcriptional activation of the rat PC gene is regulated by at least these three transcription factors. PMID:25054881

  15. Species having C4 single-cell-type photosynthesis in the Chenopodiaceae family evolved a photosynthetic phosphoenolpyruvate carboxylase like that of Kranz-type C4 species.

    PubMed

    Lara, María Valeria; Chuong, Simon D X; Akhani, Hossein; Andreo, Carlos Santiago; Edwards, Gerald E

    2006-10-01

    Spatial and temporal regulation of phosphoenolpyruvate carboxylase (PEPC) is critical to the function of C(4) photosynthesis. The photosynthetic isoform of PEPC in the cytosol of mesophyll cells in Kranz-type C(4) photosynthesis has distinctive kinetic and regulatory properties. Some species in the Chenopodiaceae family perform C(4) photosynthesis without Kranz anatomy by spatial separation of initial fixation of atmospheric CO(2) via PEPC from C(4) acid decarboxylation and CO(2) donation to Rubisco within individual chlorenchyma cells. We studied molecular and functional features of PEPC in two single-cell functioning C(4) species (Bienertia sinuspersici, Suaeda aralocaspica) as compared to Kranz type (Haloxylon persicum, Salsola richteri, Suaeda eltonica) and C(3) (Suaeda linifolia) chenopods. It was found that PEPC from both types of C(4) chenopods displays higher specific activity than that of the C(3) species and shows kinetic and regulatory characteristics similar to those of C(4) species in other families in that they are subject to light/dark regulation by phosphorylation and display differential malate sensitivity. Also, the deduced amino acid sequence from leaf cDNA indicates that the single-cell functioning C(4) species possesses a Kranz-type C(4) isoform with a Ser in the amino terminal. A phylogeny of PEPC shows that isoforms in the two single-cell functioning C(4) species are in a clade with the C(3) and Kranz C(4) Suaeda spp. with high sequence homology. Overall, this study indicates that B. sinuspersici and S. aralocaspica have a C(4)-type PEPC similar to that in Kranz C(4) plants, which likely is required for effective function of C(4) photosynthesis.

  16. Enhanced drought tolerance in transgenic rice over-expressing of maize C4 phosphoenolpyruvate carboxylase gene via NO and Ca(2+).

    PubMed

    Qian, Baoyun; Li, Xia; Liu, Xiaolong; Chen, Pingbo; Ren, Chengang; Dai, Chuanchao

    2015-03-01

    We determined the effects of endogenous nitric oxide and Ca(2+) on photosynthesis and gene expression in transgenic rice plants (PC) over-expressing the maize C4pepc gene, which encodes phosphoenolpyruvate carboxylase (PEPC) under drought. In this study, seedlings were subjected to PEG 6000 treatments using PC and wild type (WT; Kitaake). The results showed that, compared with WT, PC had higher relative water content (RWC) and net photosynthetic rate (Pn) under drought. During a 2-day re-watering treatment, Pn recovered faster in PC than in WT. Further analyses showed that, under the drought treatment, the amount of endogenous hydrogen peroxide (H2O2) increased in WT mainly via NADPH oxidase. While in PC, the endogenous nitric oxide (NO) content increased via nitrate reductase and nitric oxide synthase on day 2 of the drought treatment and day 1 of the re-watering treatment. After 2 days of drought treatment, PC also showed higher PEPC activity, calcium content, phospholipase D (PLD) activity, C4-pepc and NAC6 transcript levels, and protein kinase activity as compared with PC without treatment. These changes did not occur in WT. Correlation analysis also proved NO associated with these indicators in PC. Based on these results, there was a particular molecular mechanism of drought tolerance in PC. The mechanism is related to the signaling processes via NO and Ca(2+) involving the protein kinase and the transcription factor, resulted in up-regulation of PEPC activity and its gene expression, such as C4pepc. Some genes encode antioxidant system, cu/zn-sod as well, which promote antioxidant system to clear MDA and superoxide anion radical, thereby conferring drought tolerance.

  17. Phylogeny and Functional Expression of Ribulose 1,5-Bisphosphate Carboxylase/Oxygenase from the Autotrophic Ammonia-Oxidizing Bacterium Nitrosospira sp.Isolate 40KI

    PubMed Central

    Utåker, Janne B.; Andersen, Kjell; Aakra, Ågot; Moen, Birgitte; Nes, Ingolf F.

    2002-01-01

    The autotrophic ammonia-oxidizing bacteria (AOB), which play an important role in the global nitrogen cycle, assimilate CO2 by using ribulose 1,5-bisphosphate carboxylase/oxygenase (RubisCO). Here we describe the first detailed study of RubisCO (cbb) genes and proteins from the AOB. The cbbLS genes from Nitrosospira sp. isolate 40KI were cloned and sequenced. Partial sequences of the RubisCO large subunit (CbbL) from 13 other AOB belonging to the β and γ subgroups of the class Proteobacteria are also presented. All except one of the β-subgroup AOB possessed a red-like type I RubisCO with high sequence similarity to the Ralstonia eutropha enzyme. All of these new red-like RubisCOs had a unique six-amino-acid insert in CbbL. Two of the AOB, Nitrosococcus halophilus Nc4 and Nitrosomonas europaea Nm50, had a green-like RubisCO. With one exception, the phylogeny of the AOB CbbL was very similar to that of the 16S rRNA gene. The presence of a green-like RubisCO in N. europaea was surprising, as all of the other β-subgroup AOB had red-like RubisCOs. The green-like enzyme of N. europaea Nm50 was probably acquired by horizontal gene transfer. Functional expression of Nitrosospira sp. isolate 40KI RubisCO in the chemoautotrophic host R. eutropha was demonstrated. Use of an expression vector harboring the R. eutropha cbb control region allowed regulated expression of Nitrosospira sp. isolate 40KI RubisCO in an R. eutropha cbb deletion strain. The Nitrosospira RubisCO supported autotrophic growth of R. eutropha with a doubling time of 4.6 h. This expression system may allow further functional analysis of AOB cbb genes. PMID:11751824

  18. 1α,25-dihydroxyvitamin D inhibits de novo fatty acid synthesis and lipid accumulation in metastatic breast cancer cells through down-regulation of pyruvate carboxylase.

    PubMed

    Wilmanski, Tomasz; Buhman, Kimberly; Donkin, Shawn S; Burgess, John R; Teegarden, Dorothy

    2017-02-01

    Both increased de novo fatty acid synthesis and higher neutral lipid accumulation are a common phenotype observed in aggressive breast cancer cells, making lipid metabolism a promising target for breast cancer prevention. In the present studies, we demonstrate a novel effect of the active metabolite of vitamin D, 1α,25-dihydroxyvitamin D (1,25(OH)₂D) on lipid metabolism in malignant breast epithelial cells. Treatment of MCF10CA1a breast epithelial cells with 1,25(OH)₂D (10 nM) for 5 and 7 days decreased the level of triacylglycerol, the most abundant form of neutral lipids, by 20%(±3.9) and 50%(±5.9), respectively. In addition, 1,25(OH)₂D treatment for 5 days decreased palmitate synthesis from glucose, the major fatty acid synthesized de novo (48%±5.5 relative to vehicle). We have further identified the anaplerotic enzyme pyruvate carboxylase (PC) as a target of 1,25(OH)₂D-mediated regulation and hypothesized that 1,25(OH)₂D regulates breast cancer cell lipid metabolism through inhibition of PC. PC mRNA expression was down-regulated with 1,25(OH)₂D treatment at 2 (73%±6 relative to vehicle) and 5 (56%±8 relative to vehicle) days. Decrease in mRNA abundance corresponded with a decrease in PC protein expression at 5 days of treatment (54%±12 relative to vehicle). Constitutive overexpression of PC in MCF10CA1a cells using a pCMV6-PC plasmid inhibited the effect of 1,25(OH)₂D on both TAG accumulation and de novo palmitate synthesis from glucose. Together, these studies demonstrate a novel mechanism through which 1,25(OH)₂D regulates lipid metabolism in malignant breast epithelial cells.

  19. Pyramiding expression of maize genes encoding phosphoenolpyruvate carboxylase (PEPC) and pyruvate orthophosphate dikinase (PPDK) synergistically improve the photosynthetic characteristics of transgenic wheat.

    PubMed

    Zhang, HuiFang; Xu, WeiGang; Wang, HuiWei; Hu, Lin; Li, Yan; Qi, XueLi; Zhang, Lei; Li, ChunXin; Hua, Xia

    2014-09-01

    Using particle bombardment transformation, we introduced maize pepc cDNA encoding phosphoenolpyruvate carboxylase (PEPC) and ppdk cDNA encoding pyruvate orthophosphate dikinase (PPDK) into the C3 crop wheat to generate transgenic wheat lines carrying cDNA of pepc (PC lines), ppdk (PK lines) or both (PKC lines). The integration, transcription, and expression of the foreign genes were confirmed by Southern blot, Real-time quantitative reverse transcription PCR (Q-RT-PCR), and Western blot analysis. Q-RT-PCR results indicated that the average relative expression levels of pepc and ppdk in the PKC lines reached 10 and 4.6, respectively, compared to their expressions in untransformed plants (set to 1). The enzyme activities of PEPC and PPDK in the PKC lines were 4.3- and 2.1-fold higher, respectively, than in the untransformed control. The maximum daily net photosynthetic rates of the PKC, PC, and PK lines were enhanced by 26.4, 13.3, and 4.5%, respectively, whereas the diurnal accumulations of photosynthesis were 21.3, 13.9, and 6.9%, respectively, higher than in the control. The Fv/Fm of the transgenic plants decreased less than in the control under high temperature and high light conditions (2 weeks after anthesis), suggesting that the transgenic wheat transports more absorbed light energy into a photochemical reaction. The exogenous maize C4-specific pepc gene was more effective than ppdk at improving the photosynthetic performance and yield characteristics of transgenic wheat, while the two genes showed a synergistic effect when they were transformed into the same genetic background, because the PKC lines exhibited improved photosynthetic and physiological traits.

  20. Deduced amino acid sequence, functional expression, and unique enzymatic properties of the form I and form II ribulose bisphosphate carboxylase/oxygenase from the chemoautotrophic bacterium Thiobacillus denitrificans.

    PubMed Central

    Hernandez, J M; Baker, S H; Lorbach, S C; Shively, J M; Tabita, F R

    1996-01-01

    The cbbL cbbS and cbbM genes of Thiobacillus denitrificans, encoding form I and form II ribulose 1,5-bisphosphate carboxylase/oxygenase (RubisCO), respectively, were found to complement a RubisCO-negative mutant of Rhodobacter sphaeroides to autotrophic growth. Endogenous T. denitrificans promoters were shown to function in R. sphaeroides, resulting in high levels of cbbL cbbS and cbbM expression in the R. sphaeroides host. This expression system provided high levels of both T. denitrificans enzymes, each of which was highly purified. The deduced amino acid sequence of the form I enzyme indicated that the large subunit was closely homologous to previously sequenced form I RubisCO enzymes from sulfur-oxidizing bacteria. The form I T. denitrificans enzyme possessed a very low substrate specificity factor and did not exhibit fallover, and yet this enzyme showed a poor ability to recover from incubation with ribulose 1,5-bisphosphate. The deduced amino acid sequence of the form II T. denitrificans enzyme resembled those of other form II RubisCO enzymes. The substrate specificity factor was characteristically low, and the lack of fallover and the inhibition by ribulose 1,5-bisphosphate were similar to those of form II RubisCO obtained from nonsulfur purple bacteria. Both form I and form II RubisCO from T. denitrificans possessed high KCO2 values, suggesting that this organism might suffer in environments containing low levels of dissolved CO2. These studies present the initial description of the kinetic properties of form I and form II RubisCO from a chemoautotrophic bacterium that synthesizes both types of enzyme. PMID:8550452

  1. Identification and expression of a soybean nodule-enhanced PEP-carboxylase kinase gene (NE-PpcK) that shows striking up-/down-regulation in vivo.

    PubMed

    Xu, Wenxin; Zhou, You; Chollet, Raymond

    2003-05-01

    Various isoforms of plant phosphoenolpyruvate carboxylase (PEPC (Ppc)) are controlled post-translationally by an intricate interaction between allosteric regulation and reversible protein phosphorylation. In leaves and root nodules of legumes, these changes in PEPC phosphorylation state are governed primarily by PEPC-kinase (PpcK), a novel, 'minimal but functional' Ser/Thr kinase. To date, this plant-specific kinase has been investigated in molecular terms exclusively in non-leguminous plants, such as Crassulacean-acid-metabolism (CAM) species and Arabidopsis. As an important extension of our earlier biochemical studies on this dedicated kinase and PEPC phosphorylation in soybean (Glycine max) nodules, we now report the molecular cloning of the first legume PpcK from a soybean nodule cDNA library, which encodes a functional, 31.0 kDa PpcK polypeptide. Besides displaying organ, developmental, and spatial expression properties that are strikingly up-regulated in mature nodules, the expression pattern of this transcript is distinct from that of a second soybean PpcK isogene (GmPpcK). The steady-state abundance of this former, nodule-enhanced transcript (NE-PpcK) is markedly influenced by photosynthate supply from the shoots. This latter up-/down-regulation of NE-PpcK transcript level occurs in vivo in concert with the corresponding changes in the nodule PpcK activity, the phosphorylation-state of PEPC, and the abundance of a previously identified, nodule-enhanced transcript (GmPEPC7) that encodes the target enzyme (NE-Ppc). Furthermore, genomic Southern analysis and inspection of the public database indicate that there are at least three distinct PpcK and Ppc isogenes in soybean. Collectively, these and recent findings with Arabidopsis implicate the existence of multiple PpcK-Ppc'expression-partners' in plants, exemplified by NE-PpcK and NE-Ppc in the soybean nodule.

  2. Bacterial-type Phosphoenolpyruvate Carboxylase (PEPC) Functions as a Catalytic and Regulatory Subunit of the Novel Class-2 PEPC Complex of Vascular Plants*

    PubMed Central

    O'Leary, Brendan; Rao, Srinath K.; Kim, Julia; Plaxton, William C.

    2009-01-01

    Phosphoenolpyruvate carboxylase (PEPC) is a tightly regulated anaplerotic enzyme situated at a major branch point of the plant C metabolism. Two distinct oligomeric classes of PEPC occur in the triglyceride-rich endosperm of developing castor oil seeds (COS). Class-1 PEPC is a typical homotetramer composed of identical 107-kDa plant-type PEPC (PTPC) subunits (encoded by RcPpc3), whereas the novel Class-2 PEPC 910-kDa hetero-octameric complex arises from a tight interaction between Class-1 PEPC and distantly related 118-kDa bacterial-type PEPC (BTPC) polypeptides (encoded by RcPpc4). Here, COS BTPC was expressed from full-length RcPpc4 cDNA in Escherichia coli as an active PEPC that exhibited unusual properties relative to PTPCs, including a tendency to form large aggregates, enhanced thermal stability, a high Km(PEP), and insensitivity to metabolite effectors. A chimeric 900-kDa Class-2 PEPC hetero-octamer having a 1:1 stoichiometry of BTPC:PTPC subunits was isolated from a mixture of clarified extracts containing recombinant RcPPC4 and an Arabidopsis thaliana Class-1 PEPC (the PTPC, AtPPC3). The purified Class-2 PEPC exhibited biphasic PEP saturation kinetics with high and low affinity sites attributed to its AtPPC3 and RcPPC4 subunits, respectively. The RcPPC4 subunits: (i) catalyzed the majority of the Class-2 PEPC Vmax, particularly in the presence of the inhibitor l-malate, and (ii) also functioned as Class-2 PEPC regulatory subunits by modulating PEP binding and catalytic potential of its AtPPC3 subunits. BTPCs appear to associate with PTPCs to form stable Class-2 PEPC complexes in vivo that are hypothesized to maintain high flux from PEP under physiological conditions that would otherwise inhibit Class-1 PEPCs. PMID:19605358

  3. Bacterial-type phosphoenolpyruvate carboxylase (PEPC) functions as a catalytic and regulatory subunit of the novel class-2 PEPC complex of vascular plants.

    PubMed

    O'Leary, Brendan; Rao, Srinath K; Kim, Julia; Plaxton, William C

    2009-09-11

    Phosphoenolpyruvate carboxylase (PEPC) is a tightly regulated anaplerotic enzyme situated at a major branch point of the plant C metabolism. Two distinct oligomeric classes of PEPC occur in the triglyceride-rich endosperm of developing castor oil seeds (COS). Class-1 PEPC is a typical homotetramer composed of identical 107-kDa plant-type PEPC (PTPC) subunits (encoded by RcPpc3), whereas the novel Class-2 PEPC 910-kDa hetero-octameric complex arises from a tight interaction between Class-1 PEPC and distantly related 118-kDa bacterial-type PEPC (BTPC) polypeptides (encoded by RcPpc4). Here, COS BTPC was expressed from full-length RcPpc4 cDNA in Escherichia coli as an active PEPC that exhibited unusual properties relative to PTPCs, including a tendency to form large aggregates, enhanced thermal stability, a high K(m)((PEP)), and insensitivity to metabolite effectors. A chimeric 900-kDa Class-2 PEPC hetero-octamer having a 1:1 stoichiometry of BTPC:PTPC subunits was isolated from a mixture of clarified extracts containing recombinant RcPPC4 and an Arabidopsis thaliana Class-1 PEPC (the PTPC, AtPPC3). The purified Class-2 PEPC exhibited biphasic PEP saturation kinetics with high and low affinity sites attributed to its AtPPC3 and RcPPC4 subunits, respectively. The RcPPC4 subunits: (i) catalyzed the majority of the Class-2 PEPC V(max), particularly in the presence of the inhibitor l-malate, and (ii) also functioned as Class-2 PEPC regulatory subunits by modulating PEP binding and catalytic potential of its AtPPC3 subunits. BTPCs appear to associate with PTPCs to form stable Class-2 PEPC complexes in vivo that are hypothesized to maintain high flux from PEP under physiological conditions that would otherwise inhibit Class-1 PEPCs.

  4. Decline of Activity and Quantity of Ribulose Bisphosphate Carboxylase/Oxygenase and Net Photosynthesis in Ozone-Treated Potato Foliage 1

    PubMed Central

    Dann, Michael S.; Pell, Eva J.

    1989-01-01

    The effect of ozone (O3) on ribulose bisphosphate carboxylase/oxygenase (Rubisco) activity and quantity and net photosynthesis in greenhouse-grown Solanum tuberosum L. cv `Norland' foliage was studied in relation to oxidant-induced premature senescence. Plants, 26 days old, were exposed to 0.06 to 0.08 microliters per liter O3 from 1000 to 1600 hours for 4 days in a controlled environment chamber. On day 5, plants were exposed to a 6-hour simulated inversion in which O3 peaked at 0.12 microliters per liter. Net photosynthesis declined in response to O3 but recovered to near control levels 3 days after the exposure ended. Rubisco activity and quantity in control potato foliage increased and then decreased during the 12-day interval of the study. In some experiments foliage studied was physiologically mature and Rubisco activity had peaked when O3 exposure commenced. In those cases, O3 accelerated the decline in Rubisco activity. When less mature foliage was treated with O3, the leaves never achieved the maximal level of Rubisco activity observed in control foliage and also exhibited more rapid decline in initial and total activity. Percent activation of Rubisco (initial/total activity) was not affected significantly by treatment. Quantity of Rubisco decreased in concert with activity. The decrease in activities is most likely due to a decrease in available protein rather than a decrease in the percentage of Rubisco activated in vivo. The reduction in the quantity of Rubisco, an important foliar storage protein, could contribute to premature senescence associated with toxicity of this air pollutant. PMID:16667037

  5. Status of the substrate binding sites of ribulose bisphosphate carboxylase as determined with 2-C-carboxyarabinitol 1,5-bisphosphate. [Spinacia oleracea

    SciTech Connect

    Zhu, Genhai; Jensen, R.G. )

    1990-05-01

    The properties of the tight and specific binding of 2-C-carboxy-D-arabinitol 1,5-bisphosphate (CABP), which occurs only to reaction sites of ribulose 1,5-bisphosphate carboxylase (Rubisco) that are activated by CO{sub 2} and Mg{sup 2+}, were studied. With fully active purified spinach (Spinacia oleracea) Rubisco the rate of tight binding of ({sup 14}C)CABP fit a multiple exponential rate equation with half of the sites binding with a rate constant of 40 per minute and the second half of the sites binding at 3.2 per minute. This suggests that after CABP binds to one site of a dimer of Rubisco large subunits, binding to the second site is considerably slower, indicating negative cooperativity as previously reported. The rate of CABP binding to partially activated Rubisco was complete within 2 to 5 minutes, with slower binding to inactive sites as they formed the carbamate and bound Mg{sup 2+}. Addition of ({sup 14}C)CABP and EDTA stopped binding of Mg{sup 2+} and allowed tight binding of the radiolabel only to sites which were CO{sub 2}/Mg{sup 2+}-activated at that moment. The rate of CO{sub 2} fixation was proportional to the CO{sub 2}/Mg{sup 2+}-activated sites. During light-dependent CO{sub 2} fixation with isolated spinach chloroplasts, the amount of carbamylation was proportional to Rubisco activity either initially upon lysis of the plastids or following total activation with Mg{sup 2+} and CO{sub 2}. Lysis of chloroplasts in media with ({sup 14}C)CABP plus EDTA estimated those carbamylated sites having Mg{sup 2+}. The loss of Rubisco activation during illumination was partially due to the lack of Mg{sup 2+} to stabilize the carbamylated sites.

  6. Structure-Function Studies with the Unique Hexameric Form II Ribulose-1,5-bisphosphate Carboxylase/Oxygenase (Rubisco) from Rhodopseudomonas palustris*

    PubMed Central

    Satagopan, Sriram; Chan, Sum; Perry, L. Jeanne; Tabita, F. Robert

    2014-01-01

    The first x-ray crystal structure has been solved for an activated transition-state analog-bound form II ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco). This enzyme, from Rhodopseudomonas palustris, assembles as a unique hexamer with three pairs of catalytic large subunit homodimers around a central 3-fold symmetry axis. This oligomer arrangement is unique among all known Rubisco structures, including the form II homolog from Rhodospirillum rubrum. The presence of a transition-state analog in the active site locked the activated enzyme in a “closed” conformation and revealed the positions of critical active site residues during catalysis. Functional roles of two form II-specific residues (Ile165 and Met331) near the active site were examined via site-directed mutagenesis. Substitutions at these residues affect function but not the ability of the enzyme to assemble. Random mutagenesis and suppressor selection in a Rubisco deletion strain of Rhodobacter capsulatus identified a residue in the amino terminus of one subunit (Ala47) that compensated for a negative change near the active site of a neighboring subunit. In addition, substitution of the native carboxyl-terminal sequence with the last few dissimilar residues from the related R. rubrum homolog increased the enzyme's kcat for carboxylation. However, replacement of a longer carboxyl-terminal sequence with termini from either a form III or a form I enzyme, which varied both in length and sequence, resulted in complete loss of function. From these studies, it is evident that a number of subtle interactions near the active site and the carboxyl terminus account for functional differences between the different forms of Rubiscos found in nature. PMID:24942737

  7. Bacterial- and plant-type phosphoenolpyruvate carboxylase isozymes from developing castor oil seeds interact in vivo and associate with the surface of mitochondria.

    PubMed

    Park, Joonho; Khuu, Nicholas; Howard, Alexander S M; Mullen, Robert T; Plaxton, William C

    2012-07-01

    Phosphoenolpyruvate carboxylase (PEPC) from developing castor oil seeds (COS) exists as two distinct oligomeric isoforms. The typical class-1 PEPC homotetramer consists of 107-kDa plant-type PEPC (PTPC) subunits, whereas the allosterically desensitized 910-kDa class-2 PEPC hetero-octamer arises from the association of class-1 PEPC with 118-kDa bacterial-type PEPC (BTPC) subunits. The in vivo interaction and subcellular location of COS BTPC and PTPC were assessed by imaging fluorescent protein (FP)-tagged PEPCs in tobacco suspension-cultured cells. The BTPC-FP mainly localized to cytoplasmic punctate/globular structures, identified as mitochondria by co-immunostaining of endogenous cytochrome oxidase. Inhibition of respiration with KCN resulted in proportional decreases and increases in mitochondrial versus cytosolic BTPC-FP, respectively. The FP-PTPC and NLS-FP-PTPC (containing an appended nuclear localization signal, NLS) localized to the cytosol and nucleus, respectively, but both co-localized with mitochondrial-associated BTPC when co-expressed with BTPC-FP. Transmission electron microscopy of immunogold-labeled developing COS revealed that BTPC and PTPC are localized at the mitochondrial (outer) envelope, as well as the cytosol. Moreover, thermolysin-sensitive BTPC and PTPC polypeptides were detected on immunoblots of purified COS mitochondria. Overall, our results demonstrate that: (i) COS BTPC and PTPC interact in vivo as a class-2 PEPC complex that associates with the surface of mitochondria, (ii) BTPC's unique and divergent intrinsically disordered region mediates its interaction with PTPC, whereas (iii) the PTPC-containing class-1 PEPC is entirely cytosolic. We hypothesize that mitochondrial-associated class-2 PEPC facilitates rapid refixation of respiratory CO(2) while sustaining a large anaplerotic flux to replenish tricarboxylic acid cycle C-skeletons withdrawn for biosynthesis.

  8. Soybean ribulose bisphosphate carboxylase small subunit; mechanisms and determinants of RNA turnover: Annual progress report for the period June 1, 1987 through May 31, 1988

    SciTech Connect

    Meagher, R.

    1988-01-01

    SRS1 and SRS4 are two closely related soybean ribulose -1,5-bisphosphate carboxylase small subunit (SSU) genes. The promoters from both SRS1 and SRS4 can be fused to a neomycin phosphotransferase gene and these fusions produce similar levels of kanamycin resistance in transgenic petunia plants. The expression of SRS1 and SRS4 has been shown to be controlled at the level of transcription, and this transcriptional control is phytochrome mediated. Together these genes account for 2-3% of the total transcription in light-grown soybean seedlings or expanding soybean leaves. Recent experiments analyzing transcription rates of SRS1 and SRS4, steady state levels of their total and poly A+ RNA and frequency of their cDNAs in a soybean RNA library have led us to hypothesize that the expression of these two genes may also be controlled at the level of RNA turnover. Despite the 30-50 fold difference in transcription of these genes in seedlings grown in light, the steady state levels of RNA are only 4-8 fold higher in the light. When plants are shifted from darkness to light, accumulation of RNA lags far behind the striking transcriptional induction. In plants shifted from light to darkness, SRS1 transcription takes 24 hours to drop to dark-grown levels, and the steady state RNA levels take 72 hours to decay to dark-grown levels. On the other hand light-grown plants treated with far-red light shut down SRS1 transcription immediately, and the steady state levels of SSU RNA also drop rapidly. We have evidence suggesting striking differential turnover of the RNA products of SRS1 and SRS4, the SRS1 RNA being perhaps 5-10 times more stable than the SRS4 RNA.

  9. Phosphorylation of bacterial-type phosphoenolpyruvate carboxylase at Ser425 provides a further tier of enzyme control in developing castor oil seeds.

    PubMed

    O'Leary, Brendan; Rao, Srinath K; Plaxton, William C

    2011-01-01

    PEPC [PEP (phosphoenolpyruvate) carboxylase] is a tightly controlled anaplerotic enzyme situated at a pivotal branch point of plant carbohydrate metabolism. Two distinct oligomeric PEPC classes were discovered in developing COS (castor oil seeds). Class-1 PEPC is a typical homotetramer of 107 kDa PTPC (plant-type PEPC) subunits, whereas the novel 910-kDa Class-2 PEPC hetero-octamer arises from a tight interaction between Class-1 PEPC and 118 kDa BTPC (bacterial-type PEPC) subunits. Mass spectrometric analysis of immunopurified COS BTPC indicated that it is subject to in vivo proline-directed phosphorylation at Ser425. We show that immunoblots probed with phosphorylation site-specific antibodies demonstrated that Ser425 phosphorylation is promoted during COS development, becoming maximal at stage IX (maturation phase) or in response to depodding. Kinetic analyses of a recombinant, chimaeric Class-2 PEPC containing phosphomimetic BTPC mutant subunits (S425D) indicated that Ser425 phosphorylation results in significant BTPC inhibition by: (i) increasing its Km(PEP) 3-fold, (ii) reducing its I50 (L-malate and L-aspartate) values by 4.5- and 2.5-fold respectively, while (iii) decreasing its activity within the physiological pH range. The developmental pattern and kinetic influence of Ser425 BTPC phosphorylation is very distinct from the in vivo phosphorylation/activation of COS Class-1 PEPC's PTPC subunits at Ser11. Collectively, the results establish that BTPC's phospho-Ser425 content depends upon COS developmental and physiological status and that Ser425 phosphorylation attenuates the catalytic activity of BTPC subunits within a Class-2 PEPC complex. To the best of our knowledge, this study provides the first evidence for protein phosphorylation as a mechanism for the in vivo control of vascular plant BTPC activity.

  10. Coimmunopurification of phosphorylated bacterial- and plant-type phosphoenolpyruvate carboxylases with the plastidial pyruvate dehydrogenase complex from developing castor oil seeds.

    PubMed

    Uhrig, R Glen; O'Leary, Brendan; Spang, H Elizabeth; MacDonald, Justin A; She, Yi-Min; Plaxton, William C

    2008-03-01

    The phosphoenolpyruvate carboxylase (PEPC) interactome of developing castor oil seed (COS; Ricinus communis) endosperm was assessed using coimmunopurification (co-IP) followed by proteomic analysis. Earlier studies suggested that immunologically unrelated 107-kD plant-type PEPCs (p107/PTPC) and 118-kD bacterial-type PEPCs (p118/BTPC) are subunits of an unusual 910-kD hetero-octameric class 2 PEPC complex of developing COS. The current results confirm that a tight physical interaction occurs between p118 and p107 because p118 quantitatively coimmunopurified with p107 following elution of COS extracts through an anti-p107-IgG immunoaffinity column. No PEPC activity or immunoreactive PEPC polypeptides were detected in the corresponding flow-through fractions. Although BTPCs lack the N-terminal phosphorylation motif characteristic of PTPCs, Pro-Q Diamond phosphoprotein staining, immunoblotting with phospho-serine (Ser)/threonine Akt substrate IgG, and phosphate-affinity PAGE established that coimmunopurified p118 was multiphosphorylated at unique Ser and/or threonine residues. Tandem mass spectrometric analysis of an endoproteinase Lys-C p118 peptide digest demonstrated that Ser-425 is subject to in vivo proline-directed phosphorylation. The co-IP of p118 with p107 did not appear to be influenced by their phosphorylation status. Because p118 phosphorylation was unchanged 48 h following elimination of photosynthate supply due to COS depodding, the signaling mechanisms responsible for photosynthate-dependent p107 phosphorylation differ from those controlling p118's in vivo phosphorylation. A 110-kD PTPC coimmunopurified with p118 and p107 when depodded COS was used. The plastidial pyruvate dehydrogenase complex (PDC(pl)) was identified as a novel PEPC interactor. Thus, a putative metabolon involving PEPC and PDC(pl) could function to channel carbon from phosphoenolpyruvate to acetyl-coenzyme A and/or to recycle CO(2) from PDC(pl) to PEPC.

  11. Characterization of ribulose-1,5-bisphosphate carboxylase/oxygenase carrying ribulose 1,5-bisphosphate at its regulatory sites and the mechanism of interaction of this form of the enzyme with ribulose-1,5-bisphosphate-carboxylase/oxygenase activase.

    PubMed

    Yokota, A; Tsujimoto, N

    1992-03-01

    Ribulose-1,5-bisphosphate carboxylase/oxygenase [Rbu(1,5)P2CO] from plant sources shows a biphasic reaction course when assayed with more than 2 mM ribulose 1,5-bisphosphate [Rbu(1,5)P2]. In the burst, Rbu(1,5)P2CO has its substrate-binding sites occupied with Rbu(1,5)P2 for the initial few minutes, then both substrate-binding and regulatory sites are occupied by Rbu(1,5)P2 in the subsequent linear phase, at physiological concentrations of Rbu(1,5)P2 [A. Yokota (1991) J. Biochem. (Tokyo) 110, 246-252]. This study attempts the characterization of spinach Rbu(1,5)P2CO carrying Rbu(1,5)P2 at the regulatory sites and the interaction of Rbu(1,5)P2CO activase with Rbu(1,5)P2CO purified with poly(ethylene glycol) 4000 without denaturation. Binding of Rbu(1,5)P2 to the regulatory sites strongly influences the temperature dependence of the carboxylase activity of Rbu(1,5)P2CO. The activation energy of Rbu(1,5)P2CO with Rbu(1,5)P2 at the regulatory sites was 40% larger than that without Rbu(1,5)P2 over 30 degrees C, although the binding did not affect the activation energy below this temperature. This caused the almost linear reaction course of the carboxylase reaction at 50 degrees C. The optimum pH for the activity of Rbu(1,5)P2CO carrying Rbu(1,5)P2 at the sites was 8.0-8.2, and increased by about pH 0.2 from that of Rbu(1,5)P2CO without Rbu(1,5)P2. The ratio of the activity of the former form to that of the latter increased with increasing pH with an inflection point at pH 8.1. The increase in the ratio was accompanied by a decrease in the hysteric conformational change of Rbu(1,5)P2CO. The ATP-hydrolyzing activity inherent to Rbu(1,5)P2CO activase was stimulated about twofold by 3-5 mM Rbu(1,5)P2. Rbu(1,5)P2CO in the inactive complex with Rbu(1,5)P2 experienced hysteresis and bound Rbu(1,5)P2 at the regulatory sites during activation in the presence of Rbu(1,5)P2CO activase. Evidence was obtained that Rbu(1,5)P2CO activase promoted the activation of Rbu(1,5)P2CO through

  12. In vivo transfer of plasmid-encoded ACC-1 AmpC from Klebsiella pneumoniae to Escherichia coli in an infant and selection of impermeability to imipenem in K. pneumoniae.

    PubMed

    Bidet, Philippe; Burghoffer, Béatrice; Gautier, Valérie; Brahimi, Naïma; Mariani-Kurkdjian, Patricia; El-Ghoneimi, Alaa; Bingen, Edouard; Arlet, Guillaume

    2005-08-01

    We describe in vivo selection of a Klebsiella pneumoniae strain with diminished imipenem susceptibility attributable to plasmid-encoded ACC-1 beta-lactamase production and loss of a 36-kDa major outer membrane protein, together with transfer of this plasmid from K. pneumoniae to Escherichia coli in a Tunisian infant.

  13. 2013 AHA/ACC guideline on lifestyle management to reduce cardiovascular risk: A report of the American College of Cardiology/American Heart Association task force on practice guidelines

    Technology Transfer Automated