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Sample records for 2-c-methyl-d-erythritol 4-phosphate mep

  1. The Mycobacterium tuberculosis MEP (2C-methyl-D-erythritol 4-phosphate) pathway as a new drug target

    PubMed Central

    Eoh, Hyungjin; Brennan, Patrick J.; Crick, Dean C.

    2009-01-01

    Tuberculosis (TB) is still a major public health problem, compounded by the human immunodeficiency virus (HIV)-TB co-infection and recent emergence of multidrug-resistant (MDR) and extensive drug resistant (XDR)-TB. Novel anti-TB drugs are urgently required. In this context, the 2C-methyl-D-erythritol 4-phosphate (MEP) pathway of Mycobacterium tuberculosis has drawn attention; it is one of several pathways vital for M. tuberculosis viability and the human host lacks homologous enzymes. Thus, the MEP pathway promises bacterium-specific drug targets and the potential for identification of lead compounds unencumbered by target-based toxicity. Indeed, fosmidomycin is now known to inhibit the second step in the MEP pathway. This review describes the cardinal features of the main enzymes of the MEP pathway in M. tuberculosis and how these can be manipulated in high throughput screening campaigns in the search for new anti-infectives against TB. PMID:18793870

  2. Novel bioassay for the discovery of inhibitors of the 2-C-Methyl-D-Erythritol 4-Phosphate (MEP) and terpenoid pathways leading to carotenoid biosynthesis

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The 2-C-methyl-D-erythritol 4-phosphate (MEP) pathway leads to the synthesis of isopentenyl-phosphate (IPP) in plastids. It is a major branch point providing precursors for the synthesis of carotenoids, tocopherols, plastoquinone and the phytyl chain of chlorophylls, as well as the hormones abscisi...

  3. Cloning and characterization of the 2-C-methyl-D-erythritol 4-phosphate (MEP) pathway genes of a natural-rubber producing plant, Hevea brasiliensis.

    PubMed

    Sando, Tomoki; Takeno, Shinya; Watanabe, Norie; Okumoto, Hiroshi; Kuzuyama, Tomohisa; Yamashita, Atsushi; Hattori, Masahira; Ogasawara, Naotake; Fukusaki, Eiichiro; Kobayashi, Akio

    2008-11-01

    Natural rubber is synthesized as rubber particles in the latex, the fluid cytoplasm of laticifers, of Hevea brasiliensis. Although it has been found that natural rubber is biosynthesized through the mevalonate pathway, the involvement of an alternative 2-C-methyl-D-erythritol 4-phosphate (MEP) pathway is uncertain. We obtained all series of the MEP pathway candidate genes by analyzing expressed sequence tag (EST) information and degenerate PCR in H. brasiliensis. Complementation experiments with Escherichia coli mutants were performed to confirm the functions of the MEP pathway gene products of H. brasiliensis together with those of Arabidopsis thaliana, and it was found that 1-deoxy-D-xylulose-5-phosphate reductoisomerase, 2-C-methyl-D-erythritol 4-phosphate cytidylyltransferase, and 2-C-methyl-D-erythritol 2,4-cyclodiphosphate synthase of H. brasiliensis were functionally active in the E. coli mutants. Gene expression analysis revealed that the expression level of the HbDXS2 gene in latex was relatively high as compared to those of other MEP pathway genes. However, a feeding experiment with [1-(13)C] 1-deoxy-D-xylulose triacetate, an intermediate derivative of the MEP pathway, indicated that the MEP pathway is not involved in rubber biosynthesis, but is involved in carotenoids biosynthesis in H. brasiliensis.

  4. Development of inhibitors of the 2C-methyl-D-erythritol 4-phosphate (MEP) pathway enzymes as potential anti-infective agents.

    PubMed

    Masini, Tiziana; Hirsch, Anna K H

    2014-12-11

    Important pathogens such as Mycobacterium tuberculosis and Plasmodium falciparum, the causative agents of tuberculosis and malaria, respectively, and plants, utilize the 2C-methyl-D-erythritol 4-phosphate (MEP, 5) pathway for the biosynthesis of isopentenyl diphosphate (1) and dimethylallyl diphosphate (2), the universal precursors of isoprenoids, while humans exclusively utilize the alternative mevalonate pathway for the synthesis of 1 and 2. This distinct distribution, together with the fact that the MEP pathway is essential in numerous organisms, makes the enzymes of the MEP pathway attractive drug targets for the development of anti-infective agents and herbicides. Herein, we review the inhibitors reported over the past 2 years, in the context of the most important older developments and with a particular focus on the results obtained against enzymes of pathogenic organisms. We will also discuss new discoveries in terms of structural and mechanistic features, which can help to guide a rational development of inhibitors.

  5. Novel Bioassay for the Discovery of Inhibitors of the 2-C-Methyl-D-erythritol 4-Phosphate (MEP) and Terpenoid Pathways Leading to Carotenoid Biosynthesis

    PubMed Central

    Corniani, Natália; Velini, Edivaldo D.; Silva, Ferdinando M. L.; Nanayakkara, N. P. Dhammika; Witschel, Matthias; Dayan, Franck E.

    2014-01-01

    The 2-C-methyl-d-erythritol 4-phosphate (MEP) pathway leads to the synthesis of isopentenyl diphosphate in plastids. It is a major branch point providing precursors for the synthesis of carotenoids, tocopherols, plastoquinone and the phytyl chain of chlorophylls, as well as the hormones abscisic acid and gibberellins. Consequently, disruption of this pathway is harmful to plants. We developed an in vivo bioassay that can measure the carbon flow through the carotenoid pathway. Leaf cuttings are incubated in the presence of a phytoene desaturase inhibitor to induce phytoene accumulation. Any compound reducing the level of phytoene accumulation is likely to interfere with either one of the steps in the MEP pathway or the synthesis of geranylgeranyl diphosphate. This concept was tested with known inhibitors of steps of the MEP pathway. The specificity of this in vivo bioassay was also verified by testing representative herbicides known to target processes outside of the MEP and carotenoid pathways. This assay enables the rapid screen of new inhibitors of enzymes preceding the synthesis of phytoene, though there are some limitations related to the non-specific effect of some inhibitors on this assay. PMID:25077957

  6. Novel bioassay for the discovery of inhibitors of the 2-C-methyl-D-erythritol 4-phosphate (MEP) and terpenoid pathways leading to carotenoid biosynthesis.

    PubMed

    Corniani, Natália; Velini, Edivaldo D; Silva, Ferdinando M L; Nanayakkara, N P Dhammika; Witschel, Matthias; Dayan, Franck E

    2014-01-01

    The 2-C-methyl-D-erythritol 4-phosphate (MEP) pathway leads to the synthesis of isopentenyl diphosphate in plastids. It is a major branch point providing precursors for the synthesis of carotenoids, tocopherols, plastoquinone and the phytyl chain of chlorophylls, as well as the hormones abscisic acid and gibberellins. Consequently, disruption of this pathway is harmful to plants. We developed an in vivo bioassay that can measure the carbon flow through the carotenoid pathway. Leaf cuttings are incubated in the presence of a phytoene desaturase inhibitor to induce phytoene accumulation. Any compound reducing the level of phytoene accumulation is likely to interfere with either one of the steps in the MEP pathway or the synthesis of geranylgeranyl diphosphate. This concept was tested with known inhibitors of steps of the MEP pathway. The specificity of this in vivo bioassay was also verified by testing representative herbicides known to target processes outside of the MEP and carotenoid pathways. This assay enables the rapid screen of new inhibitors of enzymes preceding the synthesis of phytoene, though there are some limitations related to the non-specific effect of some inhibitors on this assay.

  7. Homology modeling of Mycobacterium tuberculosis 2C-methyl-D-erythritol-4-phosphate cytidylyltransferase, the third enzyme in the MEP pathway for isoprenoid biosynthesis.

    PubMed

    Obiol-Pardo, Cristian; Cordero, Alex; Rubio-Martinez, Jaime; Imperial, Santiago

    2010-06-01

    Tuberculosis is one of the leading infectious diseases in humans. Discovering new treatments for this disease is urgently required, especially in view of the emergence of multiple drug resistant organisms and to reduce the total duration of current treatments. The synthesis of isoprenoids in Mycobacterium tuberculosis has been reported as an interesting pathway to target, and particular attention has been focused on the methylerythritol phosphate (MEP) pathway comprising the early steps of isoprenoid biosynthesis. In this context we have studied the enzyme 2C-methyl-D-erythritol-4-phosphate cytidylyltransferase (CMS), the third enzyme in the MEP pathway, since the lack of a resolved structure of this protein in M. tuberculosis has seriously limited its use as a drug target. We performed homology modeling of M. tuberculosis CMS in order to provide a reliable model for use in structure-based drug design. After evaluating the quality of the model, we performed a thorough study of the catalytic site and the dimerization interface of the model, which suggested the most important sites (conserved and non-conserved) that could be useful for drug discovery and mutagenesis studies. We found that the metal coordination of CDP-methylerythritol in M. tuberculosis CMS differs substantially with respect to the Escherichia coli variant, consistent with the fact that the former is able to utilize several metal ions for catalysis. Moreover, we propose that electrostatic interactions could explain the higher affinity of the MEP substrate compared with the cytosine 5'-triphosphate substrate in the M. tuberculosis enzyme as reported previously.

  8. Cloning and functional characterization of 2-C-methyl-D-erythritol 4-phosphate cytidyltransferase (GbMECT) gene from Ginkgo biloba.

    PubMed

    Kim, Sang-Min; Kuzuyama, Tomohisa; Chang, Yung-Jin; Kwon, Hyung-Jin; Kim, Soo-Un

    2006-07-01

    2-C-methyl-D-erythritol 4-phosphate cytidyltransferase (MECT), the third enzyme of the 2-C-methyl-D-erythritol 4-phosphate (MEP) pathway, catalyzes formation of 4-(cytidine 5'-diphospho)-2-C-methyl-D-erythritol from MEP. GbMECT, presumably involved in ginkgolide biosynthesis, was cloned and characterized from Ginkgo biloba embryonic roots. The protein containing the N-terminal chloroplast transit peptide consisted of 327 amino acid residues. Complementation of GbMECT with Escherichia coli NMW33, ygbP (EcMECT) knock-out mutant, rescued the mutant, confirming the function of the protein. Transcription levels of GbMECT remained generally constant in embryonic roots and leaves for 1 month. Full 88 N-terminal residues were necessary to deliver the protein into the chloroplast as shown by protein-targeting analysis with GFP as a reporter protein in Arabidopsis thaliana protoplasts.

  9. 2C-Methyl-d-erythritol 4-phosphate enhances and sustains cyclodiphosphate synthase IspF activity.

    PubMed

    Bitok, J Kipchirchir; Meyers, Caren Freel

    2012-10-19

    There is significant progress toward understanding catalysis throughout the essential MEP pathway to isoprenoids in human pathogens; however, little is known about pathway regulation. The present study begins by testing the hypothesis that isoprenoid biosynthesis is regulated via feedback inhibition of the fifth enzyme cyclodiphosphate synthase IspF by downstream isoprenoid diphosphates. Here, we demonstrate recombinant E. coli IspF is not inhibited by downstream metabolites isopentenyl diphosphate (IDP), dimethylallyl diphosphate (DMADP), geranyl diphosphate (GDP), and farnesyl diphosphate (FDP) under standard assay conditions. However, 2C-methyl-d-erythritol 4-phosphate (MEP), the product of reductoisomerase IspC and first committed MEP pathway intermediate, activates and sustains this enhanced IspF activity, and the IspF-MEP complex is inhibited by FDP. We further show that the methylerythritol scaffold itself, which is unique to this pathway, drives the activation and stabilization of active IspF. Our results suggest a novel feed-forward regulatory mechanism for 2C-methyl-d-erythritol 2,4-cyclodiphosphate (MEcDP) production and support an isoprenoid biosynthesis regulatory mechanism via feedback inhibition of the IspF-MEP complex by FDP. The results have important implications for development of inhibitors against the IspF-MEP complex, which may be the physiologically relevant form of the enzyme. PMID:22839733

  10. 2C-Methyl-d-erythritol 4-phosphate enhances and sustains cyclodiphosphate synthase IspF activity.

    PubMed

    Bitok, J Kipchirchir; Meyers, Caren Freel

    2012-10-19

    There is significant progress toward understanding catalysis throughout the essential MEP pathway to isoprenoids in human pathogens; however, little is known about pathway regulation. The present study begins by testing the hypothesis that isoprenoid biosynthesis is regulated via feedback inhibition of the fifth enzyme cyclodiphosphate synthase IspF by downstream isoprenoid diphosphates. Here, we demonstrate recombinant E. coli IspF is not inhibited by downstream metabolites isopentenyl diphosphate (IDP), dimethylallyl diphosphate (DMADP), geranyl diphosphate (GDP), and farnesyl diphosphate (FDP) under standard assay conditions. However, 2C-methyl-d-erythritol 4-phosphate (MEP), the product of reductoisomerase IspC and first committed MEP pathway intermediate, activates and sustains this enhanced IspF activity, and the IspF-MEP complex is inhibited by FDP. We further show that the methylerythritol scaffold itself, which is unique to this pathway, drives the activation and stabilization of active IspF. Our results suggest a novel feed-forward regulatory mechanism for 2C-methyl-d-erythritol 2,4-cyclodiphosphate (MEcDP) production and support an isoprenoid biosynthesis regulatory mechanism via feedback inhibition of the IspF-MEP complex by FDP. The results have important implications for development of inhibitors against the IspF-MEP complex, which may be the physiologically relevant form of the enzyme.

  11. The diversion of 2-C-methyl-D-erythritol-2,4-cyclodiphosphate from the 2-C-methyl-D-erythritol 4-phosphate pathway to hemiterpene glycosides mediates stress responses in Arabidopsis thaliana.

    PubMed

    González-Cabanelas, Diego; Wright, Louwrance P; Paetz, Christian; Onkokesung, Nawaporn; Gershenzon, Jonathan; Rodríguez-Concepción, Manuel; Phillips, Michael A

    2015-04-01

    2-C-Methyl-D-erythritol-2,4-cyclodiphosphate (MEcDP) is an intermediate of the plastid-localized 2-C-methyl-D-erythritol-4-phosphate (MEP) pathway which supplies isoprenoid precursors for photosynthetic pigments, redox co-factor side chains, plant volatiles, and phytohormones. The Arabidopsis hds-3 mutant, defective in the 1-hydroxy-2-methyl-2-(E)-butenyl-4-diphosphate synthase step of the MEP pathway, accumulates its substrate MEcDP as well as the free tetraol 2-C-methyl-D-erythritol (ME) and glucosylated ME metabolites, a metabolic diversion also occurring in wild type plants. MEcDP dephosphorylation to the free tetraol precedes glucosylation, a process which likely takes place in the cytosol. Other MEP pathway intermediates were not affected in hds-3. Isotopic labeling, dark treatment, and inhibitor studies indicate that a second pool of MEcDP metabolically isolated from the main pathway is the source of a signal which activates salicylic acid induced defense responses before its conversion to hemiterpene glycosides. The hds-3 mutant also showed enhanced resistance to the phloem-feeding aphid Brevicoryne brassicae due to its constitutively activated defense response. However, this MEcDP-mediated defense response is developmentally dependent and is repressed in emerging seedlings. MEcDP and ME exogenously applied to adult leaves mimics many of the gene induction effects seen in the hds-3 mutant. In conclusion, we have identified a metabolic shunt from the central MEP pathway that diverts MEcDP to hemiterpene glycosides via ME, a process linked to balancing plant responses to biotic stress.

  12. Heterologous expression and characterization of bacterial 2-C-methyl-D-erythritol-4-phosphate pathway in Saccharomyces cerevisiae.

    PubMed

    Carlsen, Simon; Ajikumar, Parayil Kumaran; Formenti, Luca Riccardo; Zhou, Kang; Phon, Too Heng; Nielsen, Michael Lynge; Lantz, Anna Eliasson; Kielland-Brandt, Morten C; Stephanopoulos, Gregory

    2013-07-01

    Transfer of a biosynthetic pathway between evolutionary distant organisms can create a metabolic shunt capable of bypassing the native regulation of the host organism, hereby improving the production of secondary metabolite precursor molecules for important natural products. Here, we report the engineering of Escherichia coli genes encoding the 2-C-methyl-D-erythritol-4-phosphate (MEP) pathway into the genome of Saccharomyces cerevisiae and the characterization of intermediate metabolites synthesized by the MEP pathway in yeast. Our UPLC-MS analysis of the MEP pathway metabolites from engineered yeast showed that the pathway is active until the synthesis of 2-C-methyl-D-erythritol-2,4-cyclodiphosphate, but appears to lack functionality of the last two steps of the MEP pathway, catalyzed by the [4Fe-4S] iron sulfur cluster proteins encoded by ispG and ispH. In order to functionalize the last two steps of the MEP pathway, we co-expressed the genes for the E. coli iron sulfur cluster (ISC) assembly machinery. By deleting ERG13, thereby incapacitating the mevalonate pathway, in conjunction with labeling experiments with U-¹³C₆ glucose and growth experiments, we found that the ISC assembly machinery was unable to functionalize ispG and ispH. However, we have found that leuC and leuD, encoding the heterodimeric iron-sulfur cluster protein, isopropylmalate isomerase, can complement the S. cerevisiae leu1 auxotrophy. To our knowledge, this is the first time a bacterial iron-sulfur cluster protein has been functionally expressed in the cytosol of S. cerevisiae under aerobic conditions and shows that S. cerevisiae has the capability to functionally express at least some bacterial iron-sulfur cluster proteins in its cytosol.

  13. Deuterium-labelled isotopomers of 2-C-methyl-D-erythritol as tools for the elucidation of the 2-C-methyl-D-erythritol 4-phosphate pathway for isoprenoid biosynthesis.

    PubMed Central

    Charon, L; Hoeffler, J F; Pale-Grosdemange, C; Lois, L M; Campos, N; Boronat, A; Rohmer, M

    2000-01-01

    Escherichia coli synthesizes its isoprenoids via the mevalonate-independent 2-C-methyl-D-erythritol 4-phosphate (MEP) pathway. The MC4100dxs::CAT strain, defective in deoxyxylulose-5-phosphate synthase, which is the first enzyme in this metabolic route, exclusively synthesizes its isoprenoids from exogenous 2-C-methyl-D-erythritol (ME) added to the culture medium. The fate of the hydrogen atoms in the MEP pathway was followed by the incorporation of [1,1-(2)H(2)]ME and [3,5,5,5-(2)H(4)]ME. The two C-1 hydrogen atoms of ME were found without any loss in the prenyl chain of menaquinone and/or ubiquinone on the carbon atoms derived from C-4 of isopentenyl diphosphate (IPP) and on the E-methyl group of dimethylallyl diphosphate (DMAPP), the C-5 hydrogen atoms on the methyl groups derived from IPP C-5 methyl group and the Z-methyl group of DMAPP. This showed that no changes in the oxidation state of these carbon atoms occurred in the reaction sequence between MEP and IPP. Furthermore, no deuterium scrambling was observed between the carbon atoms derived from C-4 and C-5 of IPP or DMAPP, suggesting a completely stereoselective IPP isomerase or no significant activity of this enzyme. The C-3 deuterium atom of [3,5,5,5-(2)H(4)]ME was preserved only in the DMAPP starter unit and was completely missing from all those derived from IPP. This finding, aided by the non-essential role of the IPP isomerase gene, suggests the presence in E. coli of two different routes towards IPP and DMAPP, starting from a common intermediate derived from MEP. PMID:10698701

  14. LytB, a novel gene of the 2-C-methyl-D-erythritol 4-phosphate pathway of isoprenoid biosynthesis in Escherichia coli.

    PubMed

    Altincicek, B; Kollas, A; Eberl, M; Wiesner, J; Sanderbrand, S; Hintz, M; Beck, E; Jomaa, H

    2001-06-15

    The mevalonate-independent 2-C-methyl-D-erythritol 4-phosphate (MEP) pathway for isoprenoid biosynthesis is essential in many eubacteria, plants, and the malaria parasite. Using genetically engineered Escherichia coli cells able to utilize exogenously provided mevalonate for isoprenoid biosynthesis by the mevalonate pathway we demonstrate that the lytB gene is involved in the trunk line of the MEP pathway. Cells deleted for the essential lytB gene were viable only if the medium was supplemented with mevalonate or the cells were complemented with an episomal copy of lytB.

  15. Plasmodium IspD (2-C-Methyl-D-erythritol 4-Phosphate Cytidyltransferase), an Essential and Druggable Antimalarial Target

    PubMed Central

    Imlay, Leah S.; Armstrong, Christopher M.; Masters, Mary Clare; Li, Ting; Price, Kathryn E.; Edwards, Rachel L.; Mann, Katherine M.; Li, Lucy X.; Stallings, Christina L.; Berry, Neil G.; O’Neill, Paul M.; Odom, Audrey R.

    2015-01-01

    As resistance to current therapies spreads, novel antimalarials are urgently needed. In this work, we examine the potential for therapeutic intervention via the targeting of Plasmodium IspD (2-C-methyl-D-erythritol 4-phosphate cytidyltransferase), the second dedicated enzyme of the essential methylerythritol phosphate (MEP) pathway for isoprenoid biosynthesis. Enzymes of this pathway represent promising therapeutic targets because the pathway is not present in humans. The Malaria Box compound, MMV008138, inhibits Plasmodium falciparum growth, and PfIspD has been proposed as a candidate intracellular target. We find that PfIspD is the sole intracellular target of MMV008138 and characterize the mode of inhibition and target-based resistance, providing chemical validation of this target. Additionally, we find that the Pf ISPD genetic locus is refractory to disruption in malaria parasites, providing independent genetic validation for efforts targeting this enzyme. This work provides compelling support for IspD as a druggable target for the development of additional, much-needed antimalarial agents. PMID:26783558

  16. The Plastidial 2-C-Methyl-d-Erythritol 4-Phosphate Pathway Provides the Isoprenyl Moiety for Protein Geranylgeranylation in Tobacco BY-2 Cells[C][W

    PubMed Central

    Gerber, Esther; Hemmerlin, Andréa; Hartmann, Michael; Heintz, Dimitri; Hartmann, Marie-Andrée; Mutterer, Jérôme; Rodríguez-Concepción, Manuel; Boronat, Albert; Van Dorsselaer, Alain; Rohmer, Michel; Crowell, Dring N.; Bach, Thomas J.

    2009-01-01

    Protein farnesylation and geranylgeranylation are important posttranslational modifications in eukaryotic cells. We visualized in transformed Nicotiana tabacum Bright Yellow-2 (BY-2) cells the geranylgeranylation and plasma membrane localization of GFP-BD-CVIL, which consists of green fluorescent protein (GFP) fused to the C-terminal polybasic domain (BD) and CVIL isoprenylation motif from the Oryza sativa calmodulin, CaM61. Treatment with fosmidomycin (Fos) or oxoclomazone (OC), inhibitors of the plastidial 2-C-methyl-d-erythritol 4-phosphate (MEP) pathway, caused mislocalization of the protein to the nucleus, whereas treatment with mevinolin, an inhibitor of the cytosolic mevalonate pathway, did not. The nuclear localization of GFP-BD-CVIL in the presence of MEP pathway inhibitors was completely reversed by all-trans-geranylgeraniol (GGol). Furthermore, 1-deoxy-d-xylulose (DX) reversed the effects of OC, but not Fos, consistent with the hypothesis that OC blocks 1-deoxy-d-xylulose 5-phosphate synthesis, whereas Fos inhibits its conversion to 2-C-methyl-d-erythritol 4-phosphate. By contrast, GGol and DX did not rescue the nuclear mislocalization of GFP-BD-CVIL in the presence of a protein geranylgeranyltransferase type 1 inhibitor. Thus, the MEP pathway has an essential role in geranylgeranyl diphosphate (GGPP) biosynthesis and protein geranylgeranylation in BY-2 cells. GFP-BD-CVIL is a versatile tool for identifying pharmaceuticals and herbicides that interfere either with GGPP biosynthesis or with protein geranylgeranylation. PMID:19136647

  17. The plastidial 2-C-methyl-D-erythritol 4-phosphate pathway provides the isoprenyl moiety for protein geranylgeranylation in tobacco BY-2 cells.

    PubMed

    Gerber, Esther; Hemmerlin, Andréa; Hartmann, Michael; Heintz, Dimitri; Hartmann, Marie-Andrée; Mutterer, Jérôme; Rodríguez-Concepción, Manuel; Boronat, Albert; Van Dorsselaer, Alain; Rohmer, Michel; Crowell, Dring N; Bach, Thomas J

    2009-01-01

    Protein farnesylation and geranylgeranylation are important posttranslational modifications in eukaryotic cells. We visualized in transformed Nicotiana tabacum Bright Yellow-2 (BY-2) cells the geranylgeranylation and plasma membrane localization of GFP-BD-CVIL, which consists of green fluorescent protein (GFP) fused to the C-terminal polybasic domain (BD) and CVIL isoprenylation motif from the Oryza sativa calmodulin, CaM61. Treatment with fosmidomycin (Fos) or oxoclomazone (OC), inhibitors of the plastidial 2-C-methyl-d-erythritol 4-phosphate (MEP) pathway, caused mislocalization of the protein to the nucleus, whereas treatment with mevinolin, an inhibitor of the cytosolic mevalonate pathway, did not. The nuclear localization of GFP-BD-CVIL in the presence of MEP pathway inhibitors was completely reversed by all-trans-geranylgeraniol (GGol). Furthermore, 1-deoxy-d-xylulose (DX) reversed the effects of OC, but not Fos, consistent with the hypothesis that OC blocks 1-deoxy-d-xylulose 5-phosphate synthesis, whereas Fos inhibits its conversion to 2-C-methyl-d-erythritol 4-phosphate. By contrast, GGol and DX did not rescue the nuclear mislocalization of GFP-BD-CVIL in the presence of a protein geranylgeranyltransferase type 1 inhibitor. Thus, the MEP pathway has an essential role in geranylgeranyl diphosphate (GGPP) biosynthesis and protein geranylgeranylation in BY-2 cells. GFP-BD-CVIL is a versatile tool for identifying pharmaceuticals and herbicides that interfere either with GGPP biosynthesis or with protein geranylgeranylation.

  18. GcpE is involved in the 2-C-methyl-D-erythritol 4-phosphate pathway of isoprenoid biosynthesis in Escherichia coli.

    PubMed

    Altincicek, B; Kollas, A K; Sanderbrand, S; Wiesner, J; Hintz, M; Beck, E; Jomaa, H

    2001-04-01

    In a variety of organisms, including plants and several eubacteria, isoprenoids are synthesized by the mevalonate-independent 2-C-methyl-D-erythritol 4-phosphate (MEP) pathway. Although different enzymes of this pathway have been described, the terminal biosynthetic steps of the MEP pathway have not been fully elucidated. In this work, we demonstrate that the gcpE gene of Escherichia coli is involved in this pathway. E. coli cells were genetically engineered to utilize exogenously provided mevalonate for isoprenoid biosynthesis by the mevalonate pathway. These cells were then deleted for the essential gcpE gene and were viable only if the medium was supplemented with mevalonate or the cells were complemented with an episomal copy of gcpE.

  19. Francisella tularensis 2-C-Methyl-D-Erythritol 4-Phosphate Cytidylyltransferase: Kinetic Characterization and Phosphoregulation

    PubMed Central

    Tsang, Arthur; Seidle, Heather; Jawaid, Safdar; Zhou, Weidong; Smith, Clint; Couch, Robin D.

    2011-01-01

    Deliberate and natural outbreaks of infectious disease, the prevalence of antibiotic resistant strains, and the ease by which antibiotic resistant bacteria can be intentionally engineered all underscore the necessity of effective vaccines and continued development of novel antimicrobial/antiviral therapeutics. Isoprenes, a group of molecules fundamentally involved in a variety of crucial biological functions, are derived from either the mevalonic acid (MVA) or methylerythritol phosphate (MEP) pathway. While mammals utilize the MVA pathway, many bacteria utilize the MEP pathway, highlighting the latter as an attractive target for antibiotic development. In this report we describe the cloning and characterization of Francisella tularensis MEP cytidylyltransferase, a MEP pathway enzyme and potential target for antibiotic development. Size exclusion chromatography indicates the protein exists as a dimer in solution. Enzyme assays produced an apparent , , , , and a . The enzyme exhibits a strict preference for Mg+2 as a divalent cation and CTP as the nucleotide. Titanium dioxide chromatography-tandem mass spectrometry identified Thr141 as a site of phosphorylation. T141D and T141E site-directed mutants are catalytically inactive, suggesting a mechanism for post-translational control of metabolic flux through the F. tularensis MEP pathway. Overall, our study suggests that MEP cytidylyltransferase is an excellent target for the development of novel antibiotics against F. tularensis. PMID:21694781

  20. [Cloning and expression analysis of 2-C-methyl-D-erythritol 4-phosphate cytidylyltransferase gene in Tripterygium wilfordii].

    PubMed

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

    2015-11-01

    To clone the 2-C-methyl-D-erythritol 4-phosphate cytidylyltransferase (TwMCT) full length cDNA from Tripterygium wilfordii, the specific primers were designed according to the transcriptome data and the LCPCR were carried out. After a series of bioinformatics analysis on the TwMCT, the MeJA induced expression content were investigated by real-time fluorescence quantification polymerase chain reaction (RT-qPCR). The result showed that the full of TwMCTcDNA was 1 318 bp nucleotides encoding 311 amino acids. The molecular weight of the deduced TwMCT protein was about 34.14 kDa and the theoretical isoelectric point was 8.65. Result of the RT-qPCR analysis indicated that the content of TwMCT mRNA expression in T. wilfordii suspension cell was rising after treating with MeJA and reached the maximum in 24 h. Cloning and analyzing TwMCT gene from T. wilfordii provided gene element for studying the function and expression regulation of secondary metabolites.

  1. Analysis of the isoprenoid biosynthesis pathways in Listeria monocytogenes reveals a role for the alternative 2-C-methyl-D-erythritol 4-phosphate pathway in murine infection.

    PubMed

    Begley, Máire; Bron, Peter A; Heuston, Sinead; Casey, Pat G; Englert, Nadine; Wiesner, Jochen; Jomaa, Hassan; Gahan, Cormac G M; Hill, Colin

    2008-11-01

    Most bacteria synthesize isoprenoids through one of two essential pathways which provide the basic building block, isopentyl diphosphate (IPP): either the classical mevalonate pathway or the alternative non-mevalonate 2-C-methyl-D-erythritol 4-phosphate (MEP) pathway. However, postgenomic analyses of the Listeria monocytogenes genome revealed that this pathogen possesses the genetic capacity to produce the complete set of enzymes involved in both pathways. The nonpathogenic species Listeria innocua naturally lacks the last two genes (gcpE and lytB) of the MEP pathway, and bioinformatic analyses strongly suggest that the genes have been lost through evolution. In the present study we show that heterologous expression of gcpE and lytB in L. innocua can functionally restore the MEP pathway in this organism and confer on it the ability to induce Vgamma9 Vdelta2 T cells. We have previously confirmed that both pathways are functional in L. monocytogenes and can provide sufficient IPP for normal growth in laboratory media (M. Begley, C. G. Gahan, A. K. Kollas, M. Hintz, C. Hill, H. Jomaa, and M. Eberl, FEBS Lett. 561:99-104, 2004). Here we describe a targeted mutagenesis strategy to create a double pathway mutant in L. monocytogenes which cannot grow in the absence of exogenously provided mevalonate, confirming the requirement for at least one intact pathway for growth. In addition, murine studies revealed that mutants lacking the MEP pathway were impaired in virulence relative to the parent strain during intraperitoneal infection, while mutants lacking the classical mevalonate pathway were not impaired in virulence potential. In vivo bioluminescence imaging also confirmed in vivo expression of the gcpE gene (MEP pathway) during murine infection.

  2. A 1-deoxy-D-xylulose 5-phosphate reductoisomerase catalyzing the formation of 2-C-methyl-D-erythritol 4-phosphate in an alternative nonmevalonate pathway for terpenoid biosynthesis.

    PubMed

    Takahashi, S; Kuzuyama, T; Watanabe, H; Seto, H

    1998-08-18

    Several eubacteria including Esherichia coli use an alternative nonmevalonate pathway for the biosynthesis of isopentenyl diphosphate instead of the ubiquitous mevalonate pathway. In the alternative pathway, 2-C-methyl-D-erythritol or its 4-phosphate, which is proposed to be formed from 1-deoxy-D-xylulose 5-phosphate via intramolecular rearrangement followed by reduction process, is one of the biosynthetic precursors of isopentenyl diphosphate. To clone the gene(s) responsible for synthesis of 2-C-methyl-D-erythritol 4-phosphate, we prepared and selected E. coli mutants with an obligatory requirement for 2-C-methylerythritol for growth and survival. All the DNA fragments that complemented the defect in synthesizing 2-C-methyl-D-erythritol 4-phosphate of these mutants contained the yaeM gene, which is located at 4.2 min on the chromosomal map of E. coli. The gene product showed significant homologies to hypothetical proteins with unknown functions present in Haemophilus influenzae, Synechocystis sp. PCC6803, Mycobacterium tuberculosis, Helicobacter pyroli, and Bacillus subtilis. The purified recombinant yaeM gene product was overexpressed in E. coli and found to catalyze the formation of 2-C-methyl-D-erythritol 4-phosphate from 1-deoxy-D-xylulose 5-phosphate in the presence of NADPH. Replacement of NADPH with NADH decreased the reaction rate to about 1% of the original rate. The enzyme required Mn2+, Co2+, or Mg2+ as well. These data clearly show that the yaeM gene encodes an enzyme, designated 1-deoxy-D-xylulose 5-phosphate reductoisomerase, that synthesizes 2-C-methyl-D-erythritol 4-phosphate from 1-deoxy-D-xylulose 5-phosphate, in a single step by intramolecular rearrangement and reduction and that this gene is responsible for terpenoid biosynthesis in E. coli. PMID:9707569

  3. A 1-deoxy-d-xylulose 5-phosphate reductoisomerase catalyzing the formation of 2-C-methyl-d-erythritol 4-phosphate in an alternative nonmevalonate pathway for terpenoid biosynthesis

    PubMed Central

    Takahashi, Shunji; Kuzuyama, Tomohisa; Watanabe, Hiroyuki; Seto, Haruo

    1998-01-01

    Several eubacteria including Esherichia coli use an alternative nonmevalonate pathway for the biosynthesis of isopentenyl diphosphate instead of the ubiquitous mevalonate pathway. In the alternative pathway, 2-C-methyl-d-erythritol or its 4-phosphate, which is proposed to be formed from 1-deoxy-d-xylulose 5-phosphate via intramolecular rearrangement followed by reduction process, is one of the biosynthetic precursors of isopentenyl diphosphate. To clone the gene(s) responsible for synthesis of 2-C-methyl-d-erythritol 4-phosphate, we prepared and selected E. coli mutants with an obligatory requirement for 2-C-methylerythritol for growth and survival. All the DNA fragments that complemented the defect in synthesizing 2-C-methyl-d-erythritol 4-phosphate of these mutants contained the yaeM gene, which is located at 4.2 min on the chromosomal map of E. coli. The gene product showed significant homologies to hypothetical proteins with unknown functions present in Haemophilus influenzae, Synechocystis sp. PCC6803, Mycobacterium tuberculosis, Helicobacter pyroli, and Bacillus subtilis. The purified recombinant yaeM gene product was overexpressed in E. coli and found to catalyze the formation of 2-C-methyl-d-erythritol 4-phosphate from 1-deoxy-d-xylulose 5-phosphate in the presence of NADPH. Replacement of NADPH with NADH decreased the reaction rate to about 1% of the original rate. The enzyme required Mn2+, Co2+, or Mg2+ as well. These data clearly show that the yaeM gene encodes an enzyme, designated 1-deoxy-d-xylulose 5-phosphate reductoisomerase, that synthesizes 2-C-methyl-d-erythritol 4-phosphate from 1-deoxy-d-xylulose 5-phosphate, in a single step by intramolecular rearrangement and reduction and that this gene is responsible for terpenoid biosynthesis in E. coli. PMID:9707569

  4. Cloning and characterization of 2-C-methyl-D-erythritol-4-phosphate pathway genes for isoprenoid biosynthesis from Indian ginseng, Withania somnifera.

    PubMed

    Gupta, Parul; Agarwal, Aditya Vikram; Akhtar, Nehal; Sangwan, Rajender Singh; Singh, Surya Pratap; Trivedi, Prabodh Kumar

    2013-02-01

    Withania somnifera (L.) is one of the most valuable medicinal plants used in Ayurvedic and other indigenous medicines. Pharmaceutical activities of this herb are associated with presence of secondary metabolites known as withanolides, a class of phytosteroids synthesized via mevalonate (MVA) and 2-C-methyl-D-erythritol-4-phosphate pathways. Though the plant has been well characterized in terms of phytochemical profiles as well as pharmaceutical activities, not much is known about the genes responsible for biosynthesis of these compounds. In this study, we have characterized two genes encoding 1-deoxy-D-xylulose-5-phosphate synthase (DXS; EC 2.2.1.7) and 1-deoxy-D-xylulose-5-phosphate reductase (DXR; EC 1.1.1.267) enzymes involved in the biosynthesis of isoprenoids. The full-length cDNAs of W. somnifera DXS (WsDXS) and DXR (WsDXR) of 2,154 and 1,428 bps encode polypeptides of 717 and 475 amino acids residues, respectively. The expression analysis suggests that WsDXS and WsDXR are differentially expressed in different tissues (with maximal expression in flower and young leaf), chemotypes of Withania, and in response to salicylic acid, methyl jasmonate, as well as in mechanical injury. Analysis of genomic organization of WsDXS shows close similarity with tomato DXS in terms of exon-intron arrangements. This is the first report on characterization of isoprenoid biosynthesis pathway genes from Withania.

  5. Measurement of carbon flux through the MEP pathway for isoprenoid synthesis by (31)P-NMR spectroscopy after specific inhibition of 2-C-methyl-d-erythritol 2,4-cyclodiphosphate reductase. Effect of light and temperature.

    PubMed

    Mongélard, Gaëlle; Seemann, Myriam; Boisson, Anne-Marie; Rohmer, Michel; Bligny, Richard; Rivasseau, Corinne

    2011-08-01

    The methylerythritol 4-phosphate (MEP) and the mevalonate pathways are the unique synthesis routes for the precursors of all isoprenoids. An original mean to measure the carbon flux through the MEP pathway in plants is proposed by using cadmium as a total short-term inhibitor of 2-C-methyl-d-erythritol 2,4-cyclodiphosphate (MEcDP) reductase (GcpE) and measuring the accumulation rate of its substrate MEcDP by (31) P-NMR spectroscopy. The MEP pathway metabolic flux was determined in spinach (Spinacia oleracea), pea (Pisum sativum), Oregon grape (Mahonia aquifolium) and boxwood (Buxus sempervirens) leaves. In spinach, flux values were compared with the synthesis rate of major isoprenoids. The flux increases with light intensity (fourfold in the 200-1200 µmol m(-2) s(-1) PPFR range) and temperature (sevenfold in the 25-37 °C range). The relationship with the light and the temperature dependency of isoprenoid production downstream of the MEP pathway is discussed.

  6. Accumulation of 2-C-methyl-D-erythritol 2,4-cyclodiphosphate in illuminated plant leaves at supraoptimal temperatures reveals a bottleneck of the prokaryotic methylerythritol 4-phosphate pathway of isoprenoid biosynthesis.

    PubMed

    Rivasseau, Corinne; Seemann, Myriam; Boisson, Anne-Marie; Streb, Peter; Gout, Elisabeth; Douce, Roland; Rohmer, Michel; Bligny, Richard

    2009-01-01

    Metabolic profiling using phosphorus nuclear magnetic resonance ((31)P-NMR) revealed that the leaves of different herbs and trees accumulate 2-C-methyl-D-erythritol 2,4-cyclodiphosphate (MEcDP), an intermediate of the methylerythritol 4-phosphate (MEP) pathway, during bright and hot days. In spinach (Spinacia oleracea L.) leaves, its accumulation closely depended on irradiance and temperature. MEcDP was the only (31)P-NMR-detected MEP pathway intermediate. It remained in chloroplasts and was a sink for phosphate. The accumulation of MEcDP suggested that its conversion rate into 4-hydroxy-3-methylbut-2-enyl diphosphate, catalysed by (E)-4-hydroxy-3-methylbut-2-enyl diphosphate synthase (GcpE), was limiting under oxidative stress. Indeed, O(2) and ROS produced by photosynthesis damage this O(2)-hypersensitive [4Fe-4S]-protein. Nevertheless, as isoprenoid synthesis was not inhibited, damages were supposed to be continuously repaired. On the contrary, in the presence of cadmium that reinforced MEcDP accumulation, the MEP pathway was blocked. In vitro studies showed that Cd(2+) does not react directly with fully assembled GcpE, but interferes with its reconstitution from recombinant GcpE apoprotein and prosthetic group. Our results suggest that MEcDP accumulation in leaves may originate from both GcpE sensitivity to oxidative environment and limitations of its repair. We propose a model wherein GcpE turnover represents a bottleneck of the MEP pathway in plant leaves simultaneously exposed to high irradiance and hot temperature.

  7. Analysis of the expression of CLA1, a gene that encodes the 1-deoxyxylulose 5-phosphate synthase of the 2-C-methyl-D-erythritol-4-phosphate pathway in Arabidopsis.

    PubMed

    Estévez, J M; Cantero, A; Romero, C; Kawaide, H; Jiménez, L F; Kuzuyama, T; Seto, H; Kamiya, Y; León, P

    2000-09-01

    The discovery of the 2-C-methyl-D-erythritol-4-phosphate pathway for the biosynthesis of isoprenoids raises the important question of the nature and regulation of the enzymes involved in this pathway. CLA1, a gene previously isolated from Arabidopsis, encodes the first enzyme of the 2-C-methyl-D-erythritol-4-phosphate pathway, 1-deoxy-D-xylulose-5-phosphate synthase. We demonstrate this enzyme activity by complementation of the cla1-1 mutant phenotype and by direct enzymatic assays. Based on mRNA and protein expression patterns this enzyme is expressed mainly in developing photosynthetic and non-photosynthetic tissues. The beta-glucuronidase expression pattern driven from the CLA1 gene regulatory region supports the northern and protein data while also showing that this gene has some level of expression in most tissues of the plant. A mutation in the CLA1 gene interferes with the normal development of chloroplasts and etioplasts, but does not seem to affect amyloplast structure. Microscopic analysis also shows a pleiotropic effect of the CLA1 gene mutation in mesophyll tissue formation.

  8. 2C-Methyl- D- erythritol 2,4-cyclodiphosphate synthase from Stevia rebaudiana Bertoni is a functional gene.

    PubMed

    Kumar, Hitesh; Singh, Kashmir; Kumar, Sanjay

    2012-12-01

    Stevia [Stevia rebaudiana (Bertoni)] is a perennial herb which accumulates sweet diterpenoid steviol glycosides (SGs) in its leaf tissue. SGs are synthesized by 2C-methyl-D-erythritol 4-phosphate (MEP) pathway. Of the various enzymes of the MEP pathway, 2C-methyl-D-erythritol 2,4-cyclodiphosphate synthase (MDS) (encoded by MDS) catalyzes the cyclization of 4-(cytidine 5' diphospho)-2C-methyl-D-erythritol 2-phosphate into 2C-methyl-D-erythritol 2,4-cyclodiphosphate. Complementation of the MDS knockout mutant strain of Escherichia coli, EB370 with putative MDS of stevia (SrMDS) rescued the lethal mutant, suggesting SrMDS to be a functional gene. Experiments conducted in plant growth chamber and in the field suggested SrMDS to be a light regulated gene. Indole 3-acetic acid (IAA; 50, 100 μM) down-regulated the expression of SrMDS at 4 h of the treatment, whereas, abscisic acid did not modulate its expression. A high expression of SrMDS was observed during the light hours of the day as compared to the dark hours. The present work established functionality of SrMDS and showed the role of light and IAA in regulating expression of SrMDS.

  9. Characterization of the Mycobacterium tuberculosis 4-diphosphocytidyl-2-C-methyl-D-erythritol synthase: potential for drug development.

    PubMed

    Eoh, Hyungjin; Brown, Amanda C; Buetow, Lori; Hunter, William N; Parish, Tanya; Kaur, Devinder; Brennan, Patrick J; Crick, Dean C

    2007-12-01

    Mycobacterium tuberculosis utilizes the methylerythritol phosphate (MEP) pathway for biosynthesis of isopentenyl diphosphate and its isomer, dimethylallyl diphosphate, precursors of all isoprenoid compounds. This pathway is of interest as a source of new drug targets, as it is absent from humans and disruption of the responsible genes has shown a lethal phenotype for Escherichia coli. In the MEP pathway, 4-diphosphocytidyl-2-C-methyl-D-erythritol is formed from 2-C-methyl-D-erythritol 4-phosphate (MEP) and CTP in a reaction catalyzed by a 4-diphosphocytidyl-2-C-methyl-D-erythritol synthase (IspD). In the present work, we demonstrate that Rv3582c is essential for M. tuberculosis: Rv3582c has been cloned and expressed, and the encoded protein has been purified. The purified M. tuberculosis IspD protein was capable of catalyzing the formation of 4-diphosphocytidyl-2-C-methyl-D-erythritol in the presence of MEP and CTP. The enzyme was active over a broad pH range (pH 6.0 to 9.0), with peak activity at pH 8.0. The activity was absolutely dependent upon divalent cations, with 20 mM Mg2+ being optimal, and replacement of CTP with other nucleotide 5'-triphosphates did not support activity. Under the conditions tested, M. tuberculosis IspD had Km values of 58.5 microM for MEP and 53.2 microM for CTP. Calculated kcat and kcat/Km values were 0.72 min(-1) and 12.3 mM(-1) min(-1) for MEP and 1.0 min(-1) and 18.8 mM(-1) min(-1) for CTP, respectively.

  10. Synthesis of 4-diphosphocytidyl-2-C-methyl-D-erythritol 2-phosphate and kinetic studies of Mycobacterium tuberculosis IspF.

    PubMed

    Narayanasamy, Prabagaran; Eoh, Hyungjin; Brennan, Patrick J; Crick, Dean C

    2010-02-26

    Many pathogenic bacteria utilize the 2-C-methyl-D-erythritol 4-phosphate (MEP) pathway for the biosynthesis of isopentenyl diphosphate and dimethylallyl diphosphate, two major building blocks of isoprenoid compounds. The fifth enzyme in the MEP pathway, 2-C-methyl-D-erythritol 2,4-cyclodiphosphate (ME-CPP) synthase (IspF), catalyzes the conversion of 4-diphosphocytidyl-2-C-methyl-D-erythritol 2-phosphate (CDP-ME2P) to ME-CPP with a corresponding release of cytidine 5-monophosphate (CMP). Because there is no ortholog of IspF in human cells, IspF is of interest as a potential drug target. However, study of IspF has been hindered by a lack of enantiopure CDP-ME2P. Herein, we report the first, to our knowledge, synthesis of enantiomerically pure CDP-ME2P from commercially available D-arabinose. Cloned, expressed, and purified M. tuberculosis IspF was able to utilize the synthetic CDP-ME2P as a substrate, a result confirmed by mass spectrometry. A convenient, sensitive, in vitro IspF assay was developed by coupling the CMP released during production of ME-CPP to mononucleotide kinase, which can be used for high throughput screening.

  11. Expression and characterization of soluble 4-diphosphocytidyl-2-C-methyl-D-erythritol kinase from bacterial pathogens.

    PubMed

    Eoh, Hyungjin; Narayanasamy, Prabagaran; Brown, Amanda C; Parish, Tanya; Brennan, Patrick J; Crick, Dean C

    2009-12-24

    Many bacterial pathogens utilize the 2-C-methyl-D-erythritol 4-phosphate pathway for biosynthesizing isoprenoid precursors, a pathway that is vital for bacterial survival and absent from human cells, providing a potential source of drug targets. However, the characterization of 4-diphosphocytidyl-2-C-methyl-D-erythritol (CDP-ME) kinase (IspE) has been hindered due to a lack of enantiopure CDP-ME and difficulty in obtaining pure IspE. Here, enantiopure CDP-ME was chemically synthesized and recombinant IspE from bacterial pathogens were purified and characterized. Although gene disruption was not possible in Mycobacterium tuberculosis, IspE is essential in Mycobacterium smegmatis. The biochemical and kinetic characteristics of IspE provide the basis for development of a high throughput screen and structural characterization.

  12. 2-C-methyl-D-erythritol is a major carbohydrate in petals of Phlox subulata possibly involved in flower development.

    PubMed

    Enomoto, Hiroyuki; Kohata, Katsunori; Nakayama, Masayoshi; Yamaguchi, Yuichi; Ichimura, Kazuo

    2004-08-01

    2-C-methyl-D-erythritol, a soluble carbohydrate that is not ubiquitously found in higher plants, was detected in the ethanol extract from Phlox subulata petals and isolated using HPLC. The isolated compound was identified by 1H-NMR, 13C-NMR and Cl-MS spectra. 2-C-methyl-D-erythritol was a major soluble carbohydrate in petals, leaves and stems. In petals, the concentration of 2-C-methyl-D-erythritol markedly increased during flower development and opening and was similar in concentration to glucose, a ubiquitous metabolic sugar. This suggests that 2-C-methyl-D-erythritol may contribute to flower opening in association with glucose in the P. subulata.

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

    PubMed

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

    2015-11-01

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

  14. Quantifying the Metabolites of the Methylerythritol 4-Phosphate (MEP) Pathway in Plants and Bacteria by Liquid Chromatography-Triple Quadrupole Mass Spectrometry.

    PubMed

    González-Cabanelas, D; Hammerbacher, A; Raguschke, B; Gershenzon, J; Wright, L P

    2016-01-01

    The 2-C-methyl-d-erythritol 4-phosphate (MEP) pathway occurs in the plastids of higher plants and in most economically important prokaryotes where it is responsible for the biosynthesis of the isoprenoid building blocks, isopentenyl diphosphate and dimethylallyl diphosphate. These five-carbon compounds are the substrates for the enormous variety of terpenoid products, including many essential metabolites and substances of commercial value. Increased knowledge of the regulation of the MEP pathway is critical to understanding many aspects of plant and microbial metabolism as well as in developing biotechnological platforms for producing these commercially valuable isoprenoids. To achieve this goal, researchers must have the ability to investigate the in vivo kinetics of the pathway by accurately measuring the concentrations of MEP pathway metabolites. However, the low levels of these metabolites complicate their accurate determination without suitable internal standards. This chapter describes a sensitive method to accurately determine the concentrations of MEP pathway metabolites occurring at trace amounts in biological samples using liquid chromatography coupled to triple quadrupole mass spectrometry. In addition, simple protocols are given for producing stable isotope-labeled internal standards for these analyses. PMID:27480689

  15. Characterization of Aquifex aeolicus 4-diphosphocytidyl-2C-methyl-d-erythritol kinase – ligand recognition in a template for antimicrobial drug discovery

    PubMed Central

    Sgraja, Tanja; Alphey, Magnus S; Ghilagaber, Stephanos; Marquez, Rudi; Robertson, Murray N; Hemmings, Jennifer L; Lauw, Susan; Rohdich, Felix; Bacher, Adelbert; Eisenreich, Wolfgang; Illarionova, Victoria; Hunter, William N

    2008-01-01

    4-Diphosphocytidyl-2C-methyl-d-erythritol kinase (IspE) catalyses the ATP-dependent conversion of 4-diphosphocytidyl-2C-methyl-d-erythritol (CDPME) to 4-diphosphocytidyl-2C-methyl-d-erythritol 2-phosphate with the release of ADP. This reaction occurs in the non-mevalonate pathway of isoprenoid precursor biosynthesis and because it is essential in important microbial pathogens and absent from mammals it represents a potential target for anti-infective drugs. We set out to characterize the biochemical properties, determinants of molecular recognition and reactivity of IspE and report the cloning and purification of recombinant Aquifex aeolicus IspE (AaIspE), kinetic data, metal ion, temperature and pH dependence, crystallization and structure determination of the enzyme in complex with CDP, CDPME and ADP. In addition, 4-fluoro-3,5-dihydroxy-4-methylpent-1-enylphosphonic acid (compound 1) was designed to mimic a fragment of the substrate, a synthetic route to 1 was elucidated and the complex structure determined. Surprisingly, this ligand occupies the binding site for the ATP α-phosphate not the binding site for the methyl-d-erythritol moiety of CDPME. Gel filtration and analytical ultracentrifugation indicate that AaIspE is a monomer in solution. The enzyme displays the characteristic α/β galacto-homoserine-mevalonate-phosphomevalonate kinase fold, with the catalytic centre positioned in a deep cleft between the ATP- and CDPME-binding domains. Comparisons indicate a high degree of sequence conservation on the IspE active site across bacterial species, similarities in structure, specificity of substrate recognition and mechanism. The biochemical characterization, attainment of well-ordered and reproducible crystals and the models resulting from the analyses provide reagents and templates to support the structure-based design of broad-spectrum antimicrobial agents. PMID:18422643

  16. Effect of oxidative stress on the biosynthesis of 2-C-methyl-D-erythritol-2,4-cyclopyrophosphate and isoprenoids by several bacterial strains.

    PubMed

    Ostrovsky, D; Diomina, G; Lysak, E; Matveeva, E; Ogrel, O; Trutko, S

    1998-12-01

    In this study, the gram-negative bacteria Xanthomonas campestris, Xanthomonas maltophilia, and Pseudomonas putida, facultative parasites of plants and animals, were shown to accumulate 2-C-methyl-d-erythritol-2,4-cyclopyrophosphate (MEC) in response to benzyl-viologen-induced oxidative stress. Corynebacterium ammoniagenes mutants capable of accumulating MEC in the absence of an exogenous oxidative stress inducer were obtained. Isoprenoid synthesis and MEC synthesis in these and other bacteria were shown to be alternative processes, while biosynthesis of brominated polyene xanthomonadin (an antioxidant pigment of X. campestris) increased concomitantly with the accumulation of MEC.

  17. [Screening of potential antibiotics, inhibitors of the nonmevalonate pathway of isoprenoid biosynthesis--2-C-methyl-D-erythritol-2,4-cyclodiphosphate derivatives].

    PubMed

    Ershov, Iu V; Mazikin, K V; Ostrovskiĭ, D N

    2010-01-01

    The recently discovered nonmevalonate pathway of isoprenoid biosynthesis is a prospective target in screening of new antibiotics. Because of the absence of the pathway in the animal cells, the specific inhibitors of the pathway will be a new class of antibiotics against many pathogens (which cause, e.g., malaria, tuberculosis, etc), combining high efficiency and low toxicity. Several derivatives of 2-C-methyl-D-erythritol-2,4-cyclodiphosphate (MEC) were synthesized. 4-Phospho-methyl-D-erythritol-1,2-cyclophosphate, benzyl ether and benzyliden derivative of MEC inhibited the 14C-MEC incorporation into isoprenoids of chromoplasts from red pepper with IC50 of 1.7-5 MM. Some inhibition (about 10%) was also observed with the use of dimethyl ether and isopropyliden derivative of MEC.

  18. Structure of 2C-methyl-d-erythritol 2,4- cyclodiphosphate synthase: An essential enzyme for isoprenoid biosynthesis and target for antimicrobial drug development

    PubMed Central

    Kemp, Lauris E.; Bond, Charles S.; Hunter, William N.

    2002-01-01

    The crystal structure of the zinc enzyme Escherichia coli 2C-methyl-d-erythritol 2,4-cyclodiphosphate synthase in complex with cytidine 5′-diphosphate and Mn2+ has been determined to 1.8-Å resolution. This enzyme is essential in E. coli and participates in the nonmevalonate pathway of isoprenoid biosynthesis, a critical pathway present in some bacterial and apicomplexans but distinct from that used by mammals. Our analysis reveals a homotrimer, built around a β prism, carrying three active sites, each of which is formed in a cleft between pairs of subunits. Residues from two subunits recognize and bind the nucleotide in an active site that contains a Zn2+ with tetrahedral coordination. A Mn2+, with octahedral geometry, is positioned between the α and β phosphates acting in concert with the Zn2+ to align and polarize the substrate for catalysis. A high degree of sequence conservation for the enzymes from E. coli, Plasmodium falciparum, and Mycobacterium tuberculosis suggests similarities in secondary structure, subunit fold, quaternary structure, and active sites. Our model will therefore serve as a template to facilitate the structure-based design of potential antimicrobial agents targeting two of the most serious human diseases, tuberculosis and malaria. PMID:11997478

  19. Mathematical modelling of the diurnal regulation of the MEP pathway in Arabidopsis.

    PubMed

    Pokhilko, Alexandra; Bou-Torrent, Jordi; Pulido, Pablo; Rodríguez-Concepción, Manuel; Ebenhöh, Oliver

    2015-05-01

    Isoprenoid molecules are essential elements of plant metabolism. Many important plant isoprenoids, such as chlorophylls, carotenoids, tocopherols, prenylated quinones and hormones are synthesised in chloroplasts via the 2-C-methyl-d-erythritol 4-phosphate (MEP) pathway. Here we develop a mathematical model of diurnal regulation of the MEP pathway in Arabidopsis thaliana. We used both experimental and theoretical approaches to integrate mechanisms potentially involved in the diurnal control of the pathway. Our data show that flux through the MEP pathway is accelerated in light due to the photosynthesis-dependent supply of metabolic substrates of the pathway and the transcriptional regulation of key biosynthetic genes by the circadian clock. We also demonstrate that feedback regulation of both the activity and the abundance of the first enzyme of the MEP pathway (1-deoxy-D-xylulose 5-phosphate synthase, DXS) by pathway products stabilizes the flux against changes in substrate supply and adjusts the flux according to product demand under normal growth conditions. These data illustrate the central relevance of photosynthesis, the circadian clock and feedback control of DXS for the diurnal regulation of the MEP pathway.

  20. Structure of 2C-Methyl-D-erythritol-2,4-cyclodiphosphate Synthase from Shewanella oneidensis at 1.6 angstrom: Identification of Farnesyl pyrophosphate Trapped in a Hydrophobic Cavity

    SciTech Connect

    Ni, Shuisong; Robinson, Howard; Marsing, Gregory C.; Bussiere, Dirksen E.; Kennedy, Michael A.

    2004-11-01

    1. Introduction Enzymes in the non-mevalonate pathway for isoprenoid synthesis have gained recent attention because of their potential value as targets for antibiotic drug development. 2C-methyl-D-erythritol-2,4 cyclophosphate (MECDP) synthase is the fifth enzyme in the seven enzyme non-mevalonate pathway for synthesis of isopentenyl diphosphate. Four groups have published structures of MECDP synthase at resolutions varying from 1.6Å to 2.8Å, either in the presence or absence of substrate from Escherichia coli (Richard et al., 2002; Kemp et al., 2002; Steinbacher et al., 2002) or from Thermus thermophilus (Kishida et al., 2003). Among these structures, the protein always exists as a homotrimer either with a crystallographic or a non-crystallographic three-fold symmetry axis and an active site formed in a cleft between adjacent monomers. While the overall shape of the proteins is highly similar among these structures, each of the four reported structures contain different combinations of metal ions in the active site including a Zn2+ ion only (Steinbacher et al., 2002), a Mn2+ ion only (Richard et al., 2002), Zn2+ and Mn2+ ions (Kemp et al., 2002) or two Mg2+ ions (Kishida et al., 2003). Furthermore, two of the structures are reported to contain a hydrophobic channel along the three-fold symmetry axis that is capped by a cluster of three arginine side chains (one from each monomer) at one end of the cavity and a cluster of three glutamic acid side chains (one from each monomer) at the other side of the cavity. In a 1.8Å resolution structure, Kemp et al. (2002) reported a sulfate ion coordinated to the arginine cap and solvent trapped in a hydrophobic cavity. In a lower 2.8Å resolution structure, Richard et al. (2002) concluded that geranyl diphosphate, GPP, was most likely trapped by the arginine cap and hydrophobic cavity (Richard et al., 2002), however, the low resolution of the data together with the presence of the crystallographic symmetry axis prohibited a

  1. Cloning and Expression Analysis of MEP Pathway Enzyme-encoding Genes in Osmanthus fragrans

    PubMed Central

    Xu, Chen; Li, Huogeng; Yang, Xiulian; Gu, Chunsun; Mu, Hongna; Yue, Yuanzheng; Wang, Lianggui

    2016-01-01

    The 2-C-methyl-d-erythritol 4-phosphate (MEP) pathway is responsible for the biosynthesis of many crucial secondary metabolites, such as carotenoids, monoterpenes, plastoquinone, and tocopherols. In this study, we isolated and identified 10 MEP pathway genes in the important aromatic plant sweet osmanthus (Osmanthus fragrans). Multiple sequence alignments revealed that 10 MEP pathway genes shared high identities with other reported proteins. The genes showed distinctive expression profiles in various tissues, or at different flower stages and diel time points. The qRT-PCR results demonstrated that these genes were highly expressed in inflorescences, which suggested a tissue-specific transcript pattern. Our results also showed that OfDXS1, OfDXS2, and OfHDR1 had a clear diurnal oscillation pattern. The isolation and expression analysis provides a strong foundation for further research on the MEP pathway involved in gene function and molecular evolution, and improves our understanding of the molecular mechanism underlying this pathway in plants. PMID:27690108

  2. Combination of Entner-Doudoroff Pathway with MEP Increases Isoprene Production in Engineered Escherichia coli

    PubMed Central

    Liu, Huaiwei; Sun, Yuanzhang; Ramos, Kristine Rose M.; Nisola, Grace M.; Valdehuesa, Kris Niño G.; Lee, Won–Keun; Park, Si Jae; Chung, Wook-Jin

    2013-01-01

    Embden-Meyerhof pathway (EMP) in tandem with 2-C-methyl-D-erythritol 4-phosphate pathway (MEP) is commonly used for isoprenoid biosynthesis in E. coli. However, this combination has limitations as EMP generates an imbalanced distribution of pyruvate and glyceraldehyde-3-phosphate (G3P). Herein, four glycolytic pathways—EMP, Entner-Doudoroff Pathway (EDP), Pentose Phosphate Pathway (PPP) and Dahms pathway were tested as MEP feeding modules for isoprene production. Results revealed the highest isoprene production from EDP containing modules, wherein pyruvate and G3P were generated simultaneously; isoprene titer and yield were more than three and six times higher than those of the EMP module, respectively. Additionally, the PPP module that generates G3P prior to pyruvate was significantly more effective than the Dahms pathway, in which pyruvate production precedes G3P. In terms of precursor generation and energy/reducing-equivalent supply, EDP+PPP was found to be the ideal feeding module for MEP. These findings may launch a new direction for the optimization of MEP-dependent isoprenoid biosynthesis pathways. PMID:24376679

  3. Reconstruction and Evaluation of the Synthetic Bacterial MEP Pathway in Saccharomyces cerevisiae

    PubMed Central

    Partow, Siavash; Siewers, Verena; Daviet, Laurent; Schalk, Michel; Nielsen, Jens

    2012-01-01

    Isoprenoids, which are a large group of natural and chemical compounds with a variety of applications as e.g. fragrances, pharmaceuticals and potential biofuels, are produced via two different metabolic pathways, the mevalonate (MVA) pathway and the 2-C-methyl-D-erythritol 4-phosphate (MEP) pathway. Here, we attempted to replace the endogenous MVA pathway in Saccharomyces cerevisiae by a synthetic bacterial MEP pathway integrated into the genome to benefit from its superior properties in terms of energy consumption and productivity at defined growth conditions. It was shown that the growth of a MVA pathway deficient S. cerevisiae strain could not be restored by the heterologous MEP pathway even when accompanied by the co-expression of genes erpA, hISCA1 and CpIscA involved in the Fe-S trafficking routes leading to maturation of IspG and IspH and E. coli genes fldA and fpr encoding flavodoxin and flavodoxin reductase believed to be responsible for electron transfer to IspG and IspH. PMID:23285068

  4. Reconstruction and evaluation of the synthetic bacterial MEP pathway in Saccharomyces cerevisiae.

    PubMed

    Partow, Siavash; Siewers, Verena; Daviet, Laurent; Schalk, Michel; Nielsen, Jens

    2012-01-01

    Isoprenoids, which are a large group of natural and chemical compounds with a variety of applications as e.g. fragrances, pharmaceuticals and potential biofuels, are produced via two different metabolic pathways, the mevalonate (MVA) pathway and the 2-C-methyl-D-erythritol 4-phosphate (MEP) pathway. Here, we attempted to replace the endogenous MVA pathway in Saccharomyces cerevisiae by a synthetic bacterial MEP pathway integrated into the genome to benefit from its superior properties in terms of energy consumption and productivity at defined growth conditions. It was shown that the growth of a MVA pathway deficient S. cerevisiae strain could not be restored by the heterologous MEP pathway even when accompanied by the co-expression of genes erpA, hISCA1 and CpIscA involved in the Fe-S trafficking routes leading to maturation of IspG and IspH and E. coli genes fldA and fpr encoding flavodoxin and flavodoxin reductase believed to be responsible for electron transfer to IspG and IspH. PMID:23285068

  5. Feedback inhibition of deoxy-D-xylulose-5-phosphate synthase regulates the methylerythritol 4-phosphate pathway.

    PubMed

    Banerjee, Aparajita; Wu, Yan; Banerjee, Rahul; Li, Yue; Yan, Honggao; Sharkey, Thomas D

    2013-06-01

    The 2-C-methyl-D-erythritol 4-phosphate (MEP) pathway leads to the biosynthesis of isopentenyl diphosphate (IDP) and dimethylallyl diphosphate (DMADP), the precursors for isoprene and higher isoprenoids. Isoprene has significant effects on atmospheric chemistry, whereas other isoprenoids have diverse roles ranging from various biological processes to applications in commercial uses. Understanding the metabolic regulation of the MEP pathway is important considering the numerous applications of this pathway. The 1-deoxy-D-xylulose-5-phosphate synthase (DXS) enzyme was cloned from Populus trichocarpa, and the recombinant protein (PtDXS) was purified from Escherichia coli. The steady-state kinetic parameters were measured by a coupled enzyme assay. An LC-MS/MS-based assay involving the direct quantification of the end product of the enzymatic reaction, 1-deoxy-D-xylulose 5-phosphate (DXP), was developed. The effect of different metabolites of the MEP pathway on PtDXS activity was tested. PtDXS was inhibited by IDP and DMADP. Both of these metabolites compete with thiamine pyrophosphate for binding with the enzyme. An atomic structural model of PtDXS in complex with thiamine pyrophosphate and Mg(2+) was built by homology modeling and refined by molecular dynamics simulations. The refined structure was used to model the binding of IDP and DMADP and indicated that IDP and DMADP might bind with the enzyme in a manner very similar to the binding of thiamine pyrophosphate. The feedback inhibition of PtDXS by IDP and DMADP constitutes an important mechanism of metabolic regulation of the MEP pathway and indicates that thiamine pyrophosphate-dependent enzymes may often be affected by IDP and DMADP.

  6. The 2-C-methylerythritol 4-phosphate pathway in melon is regulated by specialized isoforms for the first and last steps.

    PubMed

    Saladié, Montserrat; Wright, Louwrance P; Garcia-Mas, Jordi; Rodriguez-Concepcion, Manuel; Phillips, Michael A

    2014-09-01

    The 2-C-methyl-d-erythritol-4-phosphate (MEP) pathway provides the precursors for the biosynthesis of plastidial isoprenoids, which include the carotenoid pigments of many fruits. We have analysed the genes encoding the seven enzymes of the MEP pathway in melon (Cucumis melo L.) and determined that the first one, 1-deoxyxylulose 5-phosphate synthase (DXS), and the last one, 1-hydroxy-2-methyl-2-(E)-butenyl 4-diphosphate reductase (HDR), are represented in the genome as a small gene family and paralogous pair, respectively. In the case of DXS, three genes encode functional DXS activities which fall into previously established type I (CmDXS1) and II (CmDXS2a and CmDXS2b) categories, while a fourth DXS-like gene belonging to the type III group did not encode a protein with DXS activity. Their expression patterns and phylogenies suggest that CmDXS1 is functionally specialized for developmental and photosynthetic processes, while CmDXS2a and CmDXS2b are induced in flowers and ripening fruit of orange- (but not white-) fleshed varieties, coinciding with β-carotene accumulation. This is the first instance connecting type II DXS genes to specialized isoprenoid biosynthesis in the fruit of an agronomically important species. Two HDR paralogues were shown to encode functional enzymes, although only CmHDR1 was highly expressed in the tissues and developmental stages tested. Phylogenetic analysis showed that in cucurbits such as melon, these HDR paralogues probably arose through individual gene duplications in a common angiosperm ancestor, mimicking a prior division in gymnosperms, while other flowering plants, including apple, soy, canola, and poplar, acquired HDR duplicates recently as homoeologues through large-scale genome duplications. We report the influence of gene duplication history on the regulation of the MEP pathway in melon and the role of specialized MEP-pathway isoforms in providing precursors for β-carotene production in orange-fleshed melon varieties.

  7. PEG and ABA trigger methyl jasmonate accumulation to induce the MEP pathway and increase tanshinone production in Salvia miltiorrhiza hairy roots.

    PubMed

    Yang, Dongfeng; Ma, Pengda; Liang, Xiao; Wei, Zheng; Liang, Zongsuo; Liu, Yan; Liu, Fenghua

    2012-10-01

    Tanshinones, a group of active ingredients in Salvia miltiorrhiza, are derived from at least two biosynthetic pathways, which are the mevalonate (MVA) pathway in the cytosol and the 2-C-methyl-d-erythritol-4-phosphate (MEP) pathway in the plastids. Abscisic acid (ABA) and methyl jasmonate (MJ) are two well-known plant hormones induced by water stress. In this study, effects of polyethylene glycol (PEG), ABA and MJ on tanshinone production in S. miltiorrhiza hairy roots were investigated, and the role of MJ in PEG- and ABA-induced tanshinone production was further elucidated. The results showed that tanshinone production was significantly enhanced by treatments with PEG, ABA and MJ. The mRNA levels of 3-hydroxy-3-methylglutaryl co-enzyme A reductase (HMGR), 1-deoxy-d-xylulose 5-phosphate reductoisomerase (DXR) and 1-deoxy-d-xylulose 5-phosphate synthase (DXS), as well as the enzyme activities of HMGR and DXS were stimulated by all three treatments. PEG and ABA triggered MJ accumulation. Effects of PEG and ABA on tanshinone production were completely abolished by the ABA biosynthesis inhibitor [tungstate (TUN)] and the MJ biosynthesis inhibitor [ibuprofen (IBU)], while effects of MJ were almost unaffected by TUN. In addition, MJ-induced tanshinone production was completely abolished by the MEP pathway inhibitor [fosmidomycin (FOS)], but was just partially arrested by the MVA pathway inhibitor [mevinolin (MEV)]. In conclusion, a signal transduction model was proposed that exogenous applications of PEG and ABA triggered endogenous MJ accumulation by activating ABA signaling pathway to stimulate tanshinone production, while exogenous MJ could directly induce tanshinone production mainly via the MEP pathway in S. miltiorrhiza hairy roots.

  8. The 2-C-methylerythritol 4-phosphate pathway in melon is regulated by specialized isoforms for the first and last steps.

    PubMed

    Saladié, Montserrat; Wright, Louwrance P; Garcia-Mas, Jordi; Rodriguez-Concepcion, Manuel; Phillips, Michael A

    2014-09-01

    The 2-C-methyl-d-erythritol-4-phosphate (MEP) pathway provides the precursors for the biosynthesis of plastidial isoprenoids, which include the carotenoid pigments of many fruits. We have analysed the genes encoding the seven enzymes of the MEP pathway in melon (Cucumis melo L.) and determined that the first one, 1-deoxyxylulose 5-phosphate synthase (DXS), and the last one, 1-hydroxy-2-methyl-2-(E)-butenyl 4-diphosphate reductase (HDR), are represented in the genome as a small gene family and paralogous pair, respectively. In the case of DXS, three genes encode functional DXS activities which fall into previously established type I (CmDXS1) and II (CmDXS2a and CmDXS2b) categories, while a fourth DXS-like gene belonging to the type III group did not encode a protein with DXS activity. Their expression patterns and phylogenies suggest that CmDXS1 is functionally specialized for developmental and photosynthetic processes, while CmDXS2a and CmDXS2b are induced in flowers and ripening fruit of orange- (but not white-) fleshed varieties, coinciding with β-carotene accumulation. This is the first instance connecting type II DXS genes to specialized isoprenoid biosynthesis in the fruit of an agronomically important species. Two HDR paralogues were shown to encode functional enzymes, although only CmHDR1 was highly expressed in the tissues and developmental stages tested. Phylogenetic analysis showed that in cucurbits such as melon, these HDR paralogues probably arose through individual gene duplications in a common angiosperm ancestor, mimicking a prior division in gymnosperms, while other flowering plants, including apple, soy, canola, and poplar, acquired HDR duplicates recently as homoeologues through large-scale genome duplications. We report the influence of gene duplication history on the regulation of the MEP pathway in melon and the role of specialized MEP-pathway isoforms in providing precursors for β-carotene production in orange-fleshed melon varieties. PMID

  9. The 2-C-methylerythritol 4-phosphate pathway in melon is regulated by specialized isoforms for the first and last steps

    PubMed Central

    Saladié, Montserrat; Wright, Louwrance P.; Garcia-Mas, Jordi; Rodriguez-Concepcion, Manuel; Phillips, Michael A.

    2014-01-01

    The 2-C-methyl-d-erythritol-4-phosphate (MEP) pathway provides the precursors for the biosynthesis of plastidial isoprenoids, which include the carotenoid pigments of many fruits. We have analysed the genes encoding the seven enzymes of the MEP pathway in melon (Cucumis melo L.) and determined that the first one, 1-deoxyxylulose 5-phosphate synthase (DXS), and the last one, 1-hydroxy-2-methyl-2-(E)-butenyl 4-diphosphate reductase (HDR), are represented in the genome as a small gene family and paralogous pair, respectively. In the case of DXS, three genes encode functional DXS activities which fall into previously established type I (CmDXS1) and II (CmDXS2a and CmDXS2b) categories, while a fourth DXS-like gene belonging to the type III group did not encode a protein with DXS activity. Their expression patterns and phylogenies suggest that CmDXS1 is functionally specialized for developmental and photosynthetic processes, while CmDXS2a and CmDXS2b are induced in flowers and ripening fruit of orange- (but not white-) fleshed varieties, coinciding with β-carotene accumulation. This is the first instance connecting type II DXS genes to specialized isoprenoid biosynthesis in the fruit of an agronomically important species. Two HDR paralogues were shown to encode functional enzymes, although only CmHDR1 was highly expressed in the tissues and developmental stages tested. Phylogenetic analysis showed that in cucurbits such as melon, these HDR paralogues probably arose through individual gene duplications in a common angiosperm ancestor, mimicking a prior division in gymnosperms, while other flowering plants, including apple, soy, canola, and poplar, acquired HDR duplicates recently as homoeologues through large-scale genome duplications. We report the influence of gene duplication history on the regulation of the MEP pathway in melon and the role of specialized MEP-pathway isoforms in providing precursors for β-carotene production in orange-fleshed melon varieties. PMID

  10. Enzyme Inhibitor Studies Reveal Complex Control of Methyl-D-Erythritol 4-Phosphate (MEP) Pathway Enzyme Expression in Catharanthus roseus

    PubMed Central

    Han, Mei; Heppel, Simon C.; Su, Tao; Bogs, Jochen; Zu, Yuangang; An, Zhigang; Rausch, Thomas

    2013-01-01

    In Catharanthus roseus, the monoterpene moiety exerts a strong flux control for monoterpene indole alkaloid (MIA) formation. Monoterpene synthesis depends on the methyl-D-erythritol 4-phosphate (MEP) pathway. Here, we have explored the regulation of this pathway in response to developmental and environmental cues and in response to specific enzyme inhibitors. For the MEP pathway entry enzyme 1-deoxy-D-xylulose 5-phosphate synthase (DXS), a new (type I) DXS isoform, CrDXS1, has been cloned, which, in contrast to previous reports on type II CrDXS, was not transcriptionally activated by the transcription factor ORCA3. Regulation of the MEP pathway in response to metabolic perturbations has been explored using the enzyme inhibitors clomazone (precursor of 5-ketochlomazone, inhibitor of DXS) and fosmidomycin (inhibitor of deoxyxylulose 5-phosphate reductoisomerase (DXR)), respectively. Young leaves of non-flowering plants were exposed to both inhibitors, adopting a non-invasive in vivo technique. Transcripts and proteins of DXS (3 isoforms), DXR, and hydroxymethylbutenyl diphosphate synthase (HDS) were monitored, and protein stability was followed in isolated chloroplasts. Transcripts for DXS1 were repressed by both inhibitors, whereas transcripts for DXS2A&B, DXR and HDS increased after clomazone treatment but were barely affected by fosmidomycin treatment. DXS protein accumulated in response to both inhibitors, whereas DXR and HDS proteins were less affected. Fosmidomycin-induced accumulation of DXS protein indicated substantial posttranscriptional regulation. Furthermore, fosmidomycin effectively protected DXR against degradation in planta and in isolated chloroplasts. Thus our results suggest that DXR protein stability may be affected by substrate binding. In summary, the present results provide novel insight into the regulation of DXS expression in C. roseus in response to MEP-pathway perturbation. PMID:23650515

  11. Enzyme inhibitor studies reveal complex control of methyl-D-erythritol 4-phosphate (MEP) pathway enzyme expression in Catharanthus roseus.

    PubMed

    Han, Mei; Heppel, Simon C; Su, Tao; Bogs, Jochen; Zu, Yuangang; An, Zhigang; Rausch, Thomas

    2013-01-01

    In Catharanthus roseus, the monoterpene moiety exerts a strong flux control for monoterpene indole alkaloid (MIA) formation. Monoterpene synthesis depends on the methyl-D-erythritol 4-phosphate (MEP) pathway. Here, we have explored the regulation of this pathway in response to developmental and environmental cues and in response to specific enzyme inhibitors. For the MEP pathway entry enzyme 1-deoxy-D-xylulose 5-phosphate synthase (DXS), a new (type I) DXS isoform, CrDXS1, has been cloned, which, in contrast to previous reports on type II CrDXS, was not transcriptionally activated by the transcription factor ORCA3. Regulation of the MEP pathway in response to metabolic perturbations has been explored using the enzyme inhibitors clomazone (precursor of 5-ketochlomazone, inhibitor of DXS) and fosmidomycin (inhibitor of deoxyxylulose 5-phosphate reductoisomerase (DXR)), respectively. Young leaves of non-flowering plants were exposed to both inhibitors, adopting a non-invasive in vivo technique. Transcripts and proteins of DXS (3 isoforms), DXR, and hydroxymethylbutenyl diphosphate synthase (HDS) were monitored, and protein stability was followed in isolated chloroplasts. Transcripts for DXS1 were repressed by both inhibitors, whereas transcripts for DXS2A&B, DXR and HDS increased after clomazone treatment but were barely affected by fosmidomycin treatment. DXS protein accumulated in response to both inhibitors, whereas DXR and HDS proteins were less affected. Fosmidomycin-induced accumulation of DXS protein indicated substantial posttranscriptional regulation. Furthermore, fosmidomycin effectively protected DXR against degradation in planta and in isolated chloroplasts. Thus our results suggest that DXR protein stability may be affected by substrate binding. In summary, the present results provide novel insight into the regulation of DXS expression in C. roseus in response to MEP-pathway perturbation.

  12. Expression of the cytoplasmic mevalonate pathway in chloroplasts to reduce substrate limitations for cytoplasmically-produced terpenoid secondary products

    Technology Transfer Automated Retrieval System (TEKTRAN)

    All products of isoprenoid metabolism originate with the C5 non-allylic substrate, isopentenyl pyrophosphate (IPP). IPP is produced in plants by two distinct pathways, the mevalonate pathway (MEV) in the cytosol and the 2 C methyl-D-erythritol 4 phosphate (MEP) pathway in plastids. A multi-gene a...

  13. Isoprenoid biosynthesis in plant chloroplasts via the MEP pathway: direct thylakoid/ferredoxin-dependent photoreduction of GcpE/IspG.

    PubMed

    Seemann, Myriam; Tse Sum Bui, Bernadette; Wolff, Murielle; Miginiac-Maslow, Myroslawa; Rohmer, Michel

    2006-03-01

    In the methylerythritol phosphate pathway for isoprenoid biosynthesis, the GcpE/IspG enzyme catalyzes the conversion of 2-C-methyl-d-erythritol 2,4-cyclodiphosphate into (E)-4-hydroxy-3-methylbut-2-enyl diphosphate. This reaction requires a double one-electron transfer involving a [4Fe-4S] cluster. A thylakoid preparation from spinach chloroplasts was capable in the presence of light to act as sole electron donor for the plant GcpE Arabidopsis thaliana in the absence of any pyridine nucleotide. This is in sharp contrast with the bacterial Escherichia coli GcpE, which requires flavodoxin/flavodoxin reductase and NADPH as reducing system and represents the first proof that the electron flow from photosynthesis can directly act in phototrophic organisms as reducer in the 2-C-methyl-d-erythritol 4-phosphate pathway, most probably via ferredoxin, in the absence of any reducing cofactor. In the dark, the plant GcpE catalysis requires in addition of ferredoxin NADP(+)/ferredoxin oxido-reductase and NADPH as electron shuttle.

  14. Absence of substrate channeling between active sites in the Agrobacterium tumefaciens IspDF and IspE enzymes of the methyl erythritol phosphate pathway.

    PubMed

    Lherbet, Christian; Pojer, Florence; Richard, Stéphane B; Noel, Joseph P; Poulter, C D

    2006-03-21

    The conversion of 2-C-methyl-d-erythritol 4-phosphate (MEP) to 2-C-methyl-d-erythritol 2,4-cyclodiphosphate (cMEDP) in the MEP entry into the isoprenoid biosynthetic pathway occurs in three consecutive steps catalyzed by the IspD, IspE, and IspF enzymes, respectively. In Agrobacterium tumefaciens the ispD and ispF genes are fused to encode a bifunctional enzyme that catalyzes the first (synthesis of 4-diphosphocytidyl-2-C-methyl d-erythritol) and third (synthesis of 2-C-methyl-d-erythritol 2,4-cyclodiphosphate) steps. Sedimentation velocity experiments indicate that the bifunctional IspDF enzyme and the IspE protein associate in solution, raising the possibility of substrate channeling among the active sites in these two proteins. Kinetic evidence for substrate channeling was sought by measuring the time courses for product formation during incubations of MEP, CTP, and ATP with the IspDF and IspE proteins with and without an excess of the inactive IspE(D152A) mutant in the presence or absence of 30% (v/v) glycerol. The time dependencies indicate that the enzyme-generated intermediates are not transferred from the IspD active site in IspDF to the active site of IspE or from the active site in IspE to the active site of the IspF module of IspDF.

  15. The methylerythritol phosphate pathway is functionally active in all intraerythrocytic stages of Plasmodium falciparum.

    PubMed

    Cassera, María B; Gozzo, Fabio C; D'Alexandri, Fabio L; Merino, Emilio F; del Portillo, Hernando A; Peres, Valnice J; Almeida, Igor C; Eberlin, Marcos N; Wunderlich, Gerhard; Wiesner, Jochen; Jomaa, Hassan; Kimura, Emilia A; Katzin, Alejandro M

    2004-12-10

    Two genes encoding the enzymes 1-deoxy-D-xylulose-5-phosphate synthase and 1-deoxy-D-xylulose-5-phosphate reductoisomerase have been recently identified, suggesting that isoprenoid biosynthesis in Plasmodium falciparum depends on the methylerythritol phosphate (MEP) pathway, and that fosmidomycin could inhibit the activity of 1-deoxy-D-xylulose-5-phosphate reductoisomerase. The metabolite 1-deoxy-D-xylulose-5-phosphate is not only an intermediate of the MEP pathway for the biosynthesis of isopentenyl diphosphate but is also involved in the biosynthesis of thiamin (vitamin B1) and pyridoxal (vitamin B6) in plants and many microorganisms. Herein we report the first isolation and characterization of most downstream intermediates of the MEP pathway in the three intraerythrocytic stages of P. falciparum. These include, 1-deoxy-D-xylulose-5-phosphate, 2-C-methyl-D-erythritol-4-phosphate, 4-(cytidine-5-diphospho)-2-C-methyl-D-erythritol, 4-(cytidine-5-diphospho)-2-C-methyl-D-erythritol-2-phosphate, and 2-C-methyl-D-erythritol-2,4-cyclodiphosphate. These intermediates were purified by HPLC and structurally characterized via biochemical and electrospray mass spectrometric analyses. We have also investigated the effect of fosmidomycin on the biosynthesis of each intermediate of this pathway and isoprenoid biosynthesis (dolichols and ubiquinones). For the first time, therefore, it is demonstrated that the MEP pathway is functionally active in all intraerythrocytic forms of P. falciparum, and de novo biosynthesis of pyridoxal in a protozoan is reported. Its absence in the human host makes both pathways very attractive as potential new targets for antimalarial drug development.

  16. Remodeling the isoprenoid pathway in tobacco by expressing the cytoplasmic mevalonate pathway in chloroplasts.

    PubMed

    Kumar, Shashi; Hahn, Frederick M; Baidoo, Edward; Kahlon, Talwinder S; Wood, Delilah F; McMahan, Colleen M; Cornish, Katrina; Keasling, Jay D; Daniell, Henry; Whalen, Maureen C

    2012-01-01

    Metabolic engineering to enhance production of isoprenoid metabolites for industrial and medical purposes is an important goal. The substrate for isoprenoid synthesis in plants is produced by the mevalonate pathway (MEV) in the cytosol and by the 2-C-methyl-d-erythritol 4-phosphate (MEP) pathway in plastids. A multi-gene approach was employed to insert the entire cytosolic MEV pathway into the tobacco chloroplast genome. Molecular analysis confirmed the site-specific insertion of seven transgenes and homoplasmy. Functionality was demonstrated by unimpeded growth on fosmidomycin, which specifically inhibits the MEP pathway. Transplastomic plants containing the MEV pathway genes accumulated higher levels of mevalonate, carotenoids, squalene, sterols, and triacyglycerols than control plants. This is the first time an entire eukaryotic pathway with six enzymes has been transplastomically expressed in plants. Thus, we have developed an important tool to redirect metabolic fluxes in the isoprenoid biosynthesis pathway and a viable multigene strategy for engineering metabolism in plants.

  17. Synthesis and biological evaluation with plant cells of new fosmidomycin analogues containing a benzoxazolone or oxazolopyridinone ring.

    PubMed

    Courtois, Martine; Mincheva, Zoia; Andreu, Françoise; Rideau, Marc; Viaud-Massuard, Marie-Claude

    2004-12-01

    Fosmidomycin, 3-(N-formyl-N-hydroxyamido) propylphosphonic acid sodium salt, is an efficient inhibitor of 1-deoxy-D-xylulose-5-phosphate (DOXP) reductoisomerase, the second enzyme of the 2C-methyl-D-erythritol-4-phosphate (MEP) pathway notably present in Plasmodium species. We have synthesized a new series of analogues of fosmidomycin, containing a benzoxazolone, benzoxazolethione or oxazolopyridinone ring. As the MEP pathway is involved in the biosynthesis of all isoprenoids, accumulation of ajmalicine in Catharanthus roseus cells was chosen as a marker of monoterpenoid indole alkaloid (MIA) production. None of the twelve studied phosphonic esters 3 and phosphonic acids 4 affected periwinkle cell growth, but some of them (3c, 3e, 3g and 3h) showed a significant inhibition of ajmalicine accumulation: 45-85% at 125 microM. Surprisingly, this effect disappeared by conversion of 3c and 3g into the corresponding acids 4c and 4g, respectively.

  18. Validation of a homology model of Mycobacterium tuberculosis DXS: rationalization of observed activities of thiamine derivatives as potent inhibitors of two orthologues of DXS.

    PubMed

    Masini, T; Lacy, B; Monjas, L; Hawksley, D; de Voogd, A R; Illarionov, B; Iqbal, A; Leeper, F J; Fischer, M; Kontoyianni, M; Hirsch, A K H

    2015-12-14

    The enzyme DXS catalyzes the first, rate-limiting step of the 2-C-methyl-d-erythritol-4-phosphate (MEP, 1) pathway using thiamine diphosphate (ThDP) as cofactor; the DXS-catalyzed reaction constitutes also the first step in vitamin B1 and B6 metabolism in bacteria. DXS is the least studied among the enzymes of this pathway in terms of crystallographic information, with only one complete crystal structure deposited in the Protein Data Bank (Deinococcus radiodurans DXS, PDB: ). We synthesized a series of thiamine and ThDP derivatives and tested them for their biochemical activity against two DXS orthologues, namely D. radiodurans DXS and Mycobacterium tuberculosis DXS. These experimental results, combined with advanced docking studies, led to the development and validation of a homology model of M. tuberculosis DXS, which, in turn, will guide medicinal chemists in rationally designing potential inhibitors for M. tuberculosis DXS.

  19. 1-Deoxy-D-xylulose-5-phosphate synthase, a limiting enzyme for plastidic isoprenoid biosynthesis in plants.

    PubMed

    Estévez, J M; Cantero, A; Reindl, A; Reichler, S; León, P

    2001-06-22

    The initial step of the plastidic 2C-methyl-D-erythritol 4-phosphate (MEP) pathway that produces isopentenyl diphosphate is catalyzed by 1-deoxy-d-xylulose-5-phosphate synthase. To investigate whether or not 1-deoxy-d-xylulose-5-phosphate synthase catalyzes a limiting step in the MEP pathway in plants, we produced transgenic Arabidopsis plants that over- or underexpress this enzyme. Compared with non-transgenic wild-type plants, the transgenic plants accumulate different levels of various isoprenoids such as chlorophylls, tocopherols, carotenoids, abscisic acid, and gibberellins. Phenotypically, the transgenic plants had slight alterations in growth and germination rates. Because the levels of several plastidic isoprenoids correlate with changes in 1-deoxy-D-xylulose-5-phosphate synthase levels, we conclude that this enzyme catalyzes one of the rate-limiting steps of the MEP biosynthetic pathway. Furthermore, since the product of the MEP pathway is isopentenyl diphosphate, our results suggest that in plastids the pool of isopentenyl diphosphate is limiting to isprenoid production.

  20. Cross-talk between the cytosolic mevalonate and the plastidial methylerythritol phosphate pathways in tobacco bright yellow-2 cells.

    PubMed

    Hemmerlin, Andréa; Hoeffler, Jean-François; Meyer, Odile; Tritsch, Denis; Kagan, Isabelle A; Grosdemange-Billiard, Catherine; Rohmer, Michel; Bach, Thomas J

    2003-07-18

    In plants, two pathways are utilized for the synthesis of isopentenyl diphosphate, the universal precursor for isoprenoid biosynthesis. The key enzyme of the cytoplasmic mevalonic acid (MVA) pathway is 3-hydroxy-3-methylglutaryl-coenzyme A reductase (HMGR). Treatment of Tobacco Bright Yellow-2 (TBY-2) cells by the HMGR-specific inhibitor mevinolin led to growth reduction and induction of apparent HMGR activity, in parallel to an increase in protein representing two HMGR isozymes. Maximum induction was observed at 24 h. 1-Deoxy-d-xylulose (DX), the dephosphorylated first precursor of the plastidial 2-C-methyl-d-erythritol 4-phosphate (MEP) pathway, complemented growth inhibition by mevinolin in the low millimolar concentration range. Furthermore, DX partially re-established feedback repression of mevinolin-induced HMGR activity. Incorporation studies with [1,1,1,4-2H4]DX showed that sterols, normally derived from MVA, in the presence of mevinolin are synthesized via the MEP pathway. Fosmidomycin, an inhibitor of 1-deoxy-d-xylulose-5-phosphate reductoisomerase, the second enzyme of the MEP pathway, was utilized to study the reverse complementation. Growth inhibition by fosmidomycin of TBY-2 cells could be partially overcome by MVA. Chemical complementation was further substantiated by incorporation of [2-13C]MVA into plastoquinone, representative of plastidial isoprenoids. Best rates of incorporation of exogenous stably labeled precursors were observed in the presence of both inhibitors, thereby avoiding internal isotope dilution.

  1. Improving peppermint essential oil yield and composition by metabolic engineering.

    PubMed

    Lange, Bernd Markus; Mahmoud, Soheil Seyed; Wildung, Mark R; Turner, Glenn W; Davis, Edward M; Lange, Iris; Baker, Raymond C; Boydston, Rick A; Croteau, Rodney B

    2011-10-11

    Peppermint (Mentha × piperita L.) was transformed with various gene constructs to evaluate the utility of metabolic engineering for improving essential oil yield and composition. Oil yield increases were achieved by overexpressing genes involved in the supply of precursors through the 2C-methyl-D-erythritol 4-phosphate (MEP) pathway. Two-gene combinations to enhance both oil yield and composition in a single transgenic line were assessed as well. The most promising results were obtained by transforming plants expressing an antisense version of (+)-menthofuran synthase, which is critical for adjusting the levels of specific undesirable oil constituents, with a construct for the overexpression of the MEP pathway gene 1-deoxy-D-xylulose 5-phosphate reductoisomerase (up to 61% oil yield increase over wild-type controls with low levels of the undesirable side-product (+)-menthofuran and its intermediate (+)-pulegone). Elite transgenic lines were advanced to multiyear field trials, which demonstrated consistent oil yield increases of up to 78% over wild-type controls and desirable effects on oil composition under commercial growth conditions. The transgenic expression of a gene encoding (+)-limonene synthase was used to accumulate elevated levels of (+)-limonene, which allows oil derived from transgenic plants to be recognized during the processing of commercial formulations containing peppermint oil. Our study illustrates the utility of metabolic engineering for the sustainable agricultural production of high quality essential oils at a competitive cost. PMID:21963983

  2. Improving peppermint essential oil yield and composition by metabolic engineering.

    PubMed

    Lange, Bernd Markus; Mahmoud, Soheil Seyed; Wildung, Mark R; Turner, Glenn W; Davis, Edward M; Lange, Iris; Baker, Raymond C; Boydston, Rick A; Croteau, Rodney B

    2011-10-11

    Peppermint (Mentha × piperita L.) was transformed with various gene constructs to evaluate the utility of metabolic engineering for improving essential oil yield and composition. Oil yield increases were achieved by overexpressing genes involved in the supply of precursors through the 2C-methyl-D-erythritol 4-phosphate (MEP) pathway. Two-gene combinations to enhance both oil yield and composition in a single transgenic line were assessed as well. The most promising results were obtained by transforming plants expressing an antisense version of (+)-menthofuran synthase, which is critical for adjusting the levels of specific undesirable oil constituents, with a construct for the overexpression of the MEP pathway gene 1-deoxy-D-xylulose 5-phosphate reductoisomerase (up to 61% oil yield increase over wild-type controls with low levels of the undesirable side-product (+)-menthofuran and its intermediate (+)-pulegone). Elite transgenic lines were advanced to multiyear field trials, which demonstrated consistent oil yield increases of up to 78% over wild-type controls and desirable effects on oil composition under commercial growth conditions. The transgenic expression of a gene encoding (+)-limonene synthase was used to accumulate elevated levels of (+)-limonene, which allows oil derived from transgenic plants to be recognized during the processing of commercial formulations containing peppermint oil. Our study illustrates the utility of metabolic engineering for the sustainable agricultural production of high quality essential oils at a competitive cost.

  3. Accumulation of brachycerine, an antioxidant glucosidic indole alkaloid, is induced by abscisic acid, heavy metal, and osmotic stress in leaves of Psychotria brachyceras.

    PubMed

    do Nascimento, Naíla Cannes; Menguer, Paloma Koprovski; Henriques, Amélia Teresinha; Fett-Neto, Arthur Germano

    2013-12-01

    Psychotria brachyceras Muell. Arg. produces the antioxidant monoterpene indole alkaloid (MIA) brachycerine, which, besides retaining a glucose residue, has its terpenoid moiety derived not from secologanin, but probably from epiloganin, representing a new subclass of MIAs. In this work we showed that osmotic stress agents, such as sodium chloride, sorbitol and polyethylene glycol (PEG), induced brachycerine accumulation in leaf disks of P. brachyceras. Other oxidative stress inducers, such as exposure to aluminum and silver, also increased brachycerine content. Abscisic acid (ABA) treatment was shown to increase brachycerine yield, suggesting its involvement in brachycerine induction during osmotic stress. Ascorbate peroxidase activity was induced in PEG-treated leaf disks, whereas superoxide dismutase (SOD) activity remained unaltered. Assays with specific inhibitors of the cytosolic mevalonate (MVA) and plastidic 2-C-methyl-D-erythritol 4-phosphate (MEP) pathways showed that the terpenoid moiety of brachycerine derived predominantly from the MEP pathway. These results suggest a potential involvement of brachycerine in plant defense against osmotic/oxidative stress damage, possibly contributing to detoxification of hydroxyl radical and superoxide anion as a SOD-like molecule.

  4. Functional characterization of the three genes encoding 1-deoxy-D-xylulose 5-phosphate synthase in maize.

    PubMed

    Cordoba, Elizabeth; Porta, Helena; Arroyo, Analilia; San Román, Carolina; Medina, Luis; Rodríguez-Concepción, Manuel; León, Patricia

    2011-03-01

    The 1-deoxy-D-xylulose 5-phosphate synthase (DXS) enzyme catalyses the first biosynthetic step of the 2-C-methyl-D-erythritol 4-phosphate (MEP) pathway. In plants the MEP pathway is involved in the synthesis of the common precursors to the plastidic isoprenoids, isopentenyl diphosphate and dimethylallyl diphosphate, in plastids. DXS is recognized as limiting this pathway and is a potential target for manipulation to increase various isoprenoids such as carotenoids. In Zea mays three dxs genes exist that encode plastid-targeted functional enzymes. Evidence is provided that these genes represent phylogenetically distinctive clades conserved among plants preceding monocot-dicot divergence. There is differential accumulation for each dxs gene transcript, during development and in response to external signals such as light. At the protein level, the analysis demonstrates that in Z. mays, DXS protein is feedback regulated in response to the inhibition of the pathway flow. The results support that the multilevel regulation of DXS activity is conserved in evolution.

  5. Crystal structure of 1-deoxy-d-xylulose 5-phosphate reductoisomerase from the hyperthermophile Thermotoga maritima for insights into the coordination of conformational changes and an inhibitor binding.

    PubMed

    Takenoya, Mihoko; Ohtaki, Akashi; Noguchi, Keiichi; Endo, Kiwamu; Sasaki, Yasuyuki; Ohsawa, Kanju; Yajima, Shunsuke; Yohda, Masafumi

    2010-06-01

    Isopentenyl diphosphate is a precursor of various isoprenoids and is produced by the 2-C-methyl-d-erythritol 4-phosphate (MEP) pathway in plastids of plants, protozoa and many eubacteria. A key enzyme in the MEP pathway, 1-deoxy-d-xylulose 5-phosphate reductoisomerase (DXR), has been shown to be the target of fosmidomycin, which works as an antimalarial, antibacterial and herbicidal compound. In this paper, we report studies of kinetics and the crystal structures of the thermostable DXR from the hyperthermophile Thermotoga maritima. Unlike the mesophilic DXRs, Thermotoga DXR (tDXR) showed activity only with Mg(2+) at its growth temperature. We solved the crystal structures of tDXR with and without fosmidomycin. The structure without fosmidomycin but unexpectedly bound with 2-methyl-2,4-pentanediol (MPD), revealing a new extra space available for potential drug design. This structure adopted the closed form by rigid domain rotation but without the flexible loop over the active site, which was considered as a novel conformation. Further, the conserved Asp residue responsible for cation binding seemed to play an important role in adjusting the position of fosmidomycin. Taken together, our kinetic and the crystal structures illustrate the binding mode of fosmidomycin that leads to its slow, tight binding according to the conformational changes of DXR.

  6. Molecular cloning, functional characterization and expression of potato (Solanum tuberosum) 1-deoxy-d-xylulose 5-phosphate synthase 1 (StDXS1) in response to Phytophthora infestans.

    PubMed

    Henriquez, Maria Antonia; Soliman, Atta; Li, Genyi; Hannoufa, Abdelali; Ayele, Belay T; Daayf, Fouad

    2016-02-01

    1-Deoxy-D-xylulose 5-phosphate synthase (DXS) catalyzes the initial step of the plastidial 2C-methyl-D-erythritol-4-phosphate (DOXP-MEP) pathway involved in isoprenoid biosynthesis. In this study, we cloned the complete cDNA of potato DXS gene that was designated StDXS1. StDXS1 cDNA encodes for 719 amino acid residues, with MW of 77.8 kDa, and is present in one copy in the potato genome. Phylogenetic analysis and protein sequence alignments assigned StDXS1 to a group with DXS homologues from closely related species and exhibited homodomain identity with known DXS proteins from other plant species. Late blight symptoms occurred in parallel with a reduction in StDXS1 transcript levels, which may be associated with the levels of isoprenoids that contribute to plant protection against pathogens. Subcellular localization indicated that StDXS1 targets the chloroplasts where isoprenoids are synthesized. Arabidopsis expressing StDXS1 showed a higher accumulation of carotenoids and chlorophyll as compared to wild type controls. Lower levels of ABA and GA were detected in the transgenic DXS lines as compared to control plants, which reflected on higher germination rates of the transgenic DXS lines. No changes were detected in JA or SA contents. Selected downstream genes in the DOXP-MEP pathway, especially GGPPS genes, were up-regulated in the transgenic lines.

  7. Temperature controls on the basal emission rate of isoprene in a tropical tree Ficus septica: exploring molecular regulatory mechanisms.

    PubMed

    Mutanda, Ishmael; Inafuku, Masashi; Saitoh, Seikoh; Iwasaki, Hironori; Fukuta, Masakazu; Watanabe, Keiichi; Oku, Hirosuke

    2016-10-01

    Isoprene emission from plants is very sensitive to environmental temperature both at short-term and long-term scales. Our previous study demonstrated suppression of isoprene emission by cold temperatures in a high emitting tropical tree Ficus septica and revealed a strong correlation of emission to isoprene synthase (IspS) protein levels. When challenged with decreasing daily temperatures from 30 to 12 °C, F. septica completely stopped isoprene emission at 12 °C, only to recover on the second day after re-exposure to 30 °C. Here, we explored this regulation of isoprene emission in response to environmental temperature by a comprehensive analysis of transcriptome data, gene expressions and metabolite pools of the 2-C-methyl-D-erythritol 4-phosphate (MEP) pathway. MEP pathway genes and metabolites dynamics did not support substrate-level limitations as major control over observed basal emission, but transcriptome data, network inferences and putative regulatory elements on IspS promoter suggested transcriptional regulation of IspS gene through circadian rhythm and phytohormone signalling processes. Expression levels of 29 genes involved in these pathways were examined by quantitative real-time PCR. We propose that temperature controls over basal isoprene emission at a time-scale of hours to few days are regulated by phytohormone-mediated transcriptional modulation of IspS gene under synchronization by the circadian clock. PMID:27425779

  8. Engineering the lactococcal mevalonate pathway for increased sesquiterpene production.

    PubMed

    Song, Adelene A; Abdullah, Janna Ong; Abdullah, Mohd P; Shafee, Norazizah; Othman, Roohaida; Noor, Normah Mohd; Rahim, Raha A

    2014-06-01

    Isoprenoids are a large, diverse group of secondary metabolites which has recently raised a renewed research interest due to genetic engineering advances, allowing specific isoprenoids to be produced and characterized in heterologous hosts. Many researches on metabolic engineering of heterologous hosts for increased isoprenoid production are focussed on Escherichia coli and yeasts. E. coli, as most prokaryotes, use the 2-C-methyl-d-erythritol-4-phosphate (MEP) pathway for isoprenoid production. Yeasts on the other hand, use the mevalonate pathway which is commonly found in eukaryotes. However, Lactococcus lactis is an attractive alternative host for heterologous isoprenoid production. Apart from being food-grade, this Gram-positive prokaryote uses the mevalonate pathway for isoprenoid production instead of the MEP pathway. Previous studies have shown that L. lactis is able to produce sesquiterpenes through heterologous expression of plant sesquiterpene synthases. In this work, we analysed the gene expression of the lactococcal mevalonate pathway through RT-qPCR to successfully engineer L. lactis as an efficient host for isoprenoid production. We then overexpressed the mvk gene singly or co-expressed with the mvaA gene as an attempt to increase β-sesquiphellandrene production in L. lactis. It was observed that co-expression of mvk with mvaA doubled the amount of β-sesquiphellandrene produced. PMID:24828482

  9. Design of novel ligands of CDP-methylerythritol kinase by mimicking direct protein-protein and solvent-mediated interactions.

    PubMed

    Giménez-Oya, Victor; Villacañas, Oscar; Obiol-Pardo, Cristian; Antolin-Llovera, Meritxell; Rubio-Martinez, Jaime; Imperial, Santiago

    2011-01-01

    The methylerythritol 4-phosphate (MEP) pathway for the biosynthesis of the isoprenoid universal building blocks (isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP)) is present in most of human pathogens and is absent in animals, turning it into a promising therapeutic druggable pathway. Two different strategies, a pharmacophore-directed virtual screening and a protein-protein interaction (PPI)-mimicking cyclic peptide were used to search for compounds that bind to the PPI surface of the 4-(cytidine 5-diphospho)-2C-methyl-D-erythritol kinase (CMK), which catalyzes the fourth step of the MEP pathway. A significant part of the pharmacophore hypothesis used in this study was designed by mimicking water-mediated PPI relevant in the CMK homodimer complex stabilization. After database search and with the aid of docking and molecular dynamics (MD) simulations, a 7H-furo[3,2-g]chromen-7-one derivative and a cyclic peptide were chosen as candidates to be ligands of CMK. Their binding affinities were measured using surface plasmon resonance (SPR) technology.

  10. Structural and functional studies of mycobacterial IspD enzymes.

    PubMed

    Björkelid, Christofer; Bergfors, Terese; Henriksson, Lena M; Stern, Ana Laura; Unge, Torsten; Mowbray, Sherry L; Jones, T Alwyn

    2011-05-01

    A number of pathogens, including the causative agents of tuberculosis and malaria, synthesize isopentenyl diphosphate via the 2-C-methyl-D-erythritol 4-phosphate (MEP) pathway rather than the classical mevalonate pathway found in humans. As part of a structure-based drug-discovery program against tuberculosis, IspD, the enzyme that carries out the third step in the MEP pathway, was targeted. Constructs of both the Mycobacterium smegmatis and the Mycobacterium tuberculosis enzymes that were suitable for structural and inhibitor-screening studies were engineered. Two crystal structures of the M. smegmatis enzyme were produced, one in complex with CTP and the other in complex with CMP. In addition, the M. tuberculosis enzyme was crystallized in complex with CTP. Here, the structure determination and crystallographic refinement of these crystal forms and the enzymatic characterization of the M. tuberculosis enzyme construct are reported. A comparison with known IspD structures allowed the definition of the structurally conserved core of the enzyme. It indicates potential flexibility in the enzyme and in particular in areas close to the active site. These well behaved constructs provide tools for future target-based screening of potential inhibitors. The conserved nature of the extended active site suggests that any new inhibitor will potentially exhibit broad-spectrum activity. PMID:21543842

  11. Role of reactive oxygen species and proline cycle in anthraquinone accumulation in Rubia tinctorum cell suspension cultures subjected to methyl jasmonate elicitation.

    PubMed

    Perassolo, María; Quevedo, Carla Verónica; Busto, Víctor Daniel; Giulietti, Ana María; Talou, Julián Rodríguez

    2011-07-01

    Elicitors are compounds or factors capable of triggering a defense response in plants. This kind of response involves signal transduction pathways, second messengers and events such as Reactive Oxygen Species (ROS) generation, proline accumulation and secondary metabolite production. Anthraquinone (AQs) biosynthesis in Rubia tinctorum L. involves different metabolic routes, including shikimate and 2-C-methyl-d-erythritol-4-phosphate (MEP) pathways. It has been proposed that the proline cycle could be coupled with the pentose phosphate pathway (PPP), since the NADP+ generated by this cycle could act as a cofactor of the first enzymes of the PPP. The end-product of this pathway is erithrose-4-phosphate, which becomes the substrate of the shikimate pathway. The aim of this work was to study the effect of methyl jasmonate (MeJ), a well-known endogenous elicitor, on the PPP, the proline cycle and AQs production in R. tinctorum cell suspension cultures, and to elucidate the role of ROS in MeJ elicitation. Treatment with MeJ resulted in AQs as well as proline accumulation, which was mimicked by the treatment with a H₂O₂-generating system. Both MeJ-induced effects were abolished in the presence of diphenyliodonium (DPI), a NADPH oxidase inhibitor (main source of ROS). Treatment with the elicitor failed to induce PPP; therefore, this route did not turn out to be limiting the carbon flux to the shikimate pathway.

  12. Isoprenoid biosynthesis authenticates the classification of the green alga Mesostigma viride as an ancient streptophyte.

    PubMed

    Grauvogel, Carina; Petersen, Jörn

    2007-07-01

    Land plants harbor two essential and completely different metabolic pathways for isoprenoid synthesis. The cytosolic mevalonate pathway (MVA) is shared with heterotrophic eukaryotes, whereas the plastidial 2-C-methyl-d-erythritol 4-phosphate (MEP) pathway has a cyanobacterial origin and was recruited after primary endosymbiosis. Terrestrial plants and green algae have a common evolutionary ancestry, but biochemical as well as genome analyses indicate that the cytosolic MVA pathway is generally absent from Chlorophyta. We investigated the distribution of genes for both pathways in the green alga Mesostigma viride, a key species at the basis of streptophycean (charophycean green algae, land plant) evolution. Ten of altogether twelve generally weakly expressed genes for isoprenoid biosynthesis, including three for the cytosolic MVA pathway, were amplified using a reverse transcription PCR approach with individually designed degenerate primers. Two full length cDNA clones for the first enzyme of the MVA pathway (HMGS) were additionally established from the charophycean green alga Chara vulgaris by library screening. The presence of the MVA pathway in these advanced green algae indicates a universal distribution among Streptophyta, and our phylogenetic HMGS analyses substantiate the recent classification of Mesostigma basal to charophytes and land plants. We identified each of the five cytosolic MVA genes/cDNAs in the genome of the rhodophyte Galdieria sulphuraria and, furthermore, amplified four of them from the glaucophyte Cyanophora paradoxa. Our data indicate that the MVA pathway is a characteristic trait of Plantae in general and propose that it was specifically lost in a common ancestor of Chlorophyta.

  13. Enhanced Diterpene Tanshinone Accumulation and Bioactivity of Transgenic Salvia miltiorrhiza Hairy Roots by Pathway Engineering.

    PubMed

    Shi, Min; Luo, Xiuqin; Ju, Guanhua; Li, Leilei; Huang, Shengxiong; Zhang, Tong; Wang, Huizhong; Kai, Guoyin

    2016-03-30

    Tanshinones are health-promoting diterpenoids found in Salvia miltiorrhiza and have wide applications. Here, SmGGPPS (geranylgeranyl diphosphate synthase) and SmDXSII (1-deoxy-D-xylulose-5-phosphate synthase) were introduced into hairy roots of S. miltiorrhiza. Overexpression of SmGGPPS and SmDXSII in hairy roots produces higher levels of tanshinone than control and single-gene transformed lines; tanshinone production in the double-gene transformed line GDII10 reached 12.93 mg/g dry weight, which is the highest tanshinone content that has been achieved through genetic engineering. Furthermore, transgenic hairy root lines showed higher antioxidant and antitumor activities than control lines. In addition, contents of chlorophylls, carotenoids, indoleacetic acid, and gibberellins were significantly elevated in transgenic Arabidopsis thaliana plants. These results demonstrate a promising method to improve the production of diterpenoids including tanshinone as well as other natural plastid-derived isoprenoids in plants by genetic manipulation of the 2-C-methyl-D-erythritol-4-phosphate (MEP) pathway.

  14. Functional Expression of an Orchid Fragrance Gene in Lactococcus lactis

    PubMed Central

    Song, Adelene Ai Lian; Abdullah, Janna O.; Abdullah, Mohd Puad; Shafee, Norazizah; Rahim, Raha A.

    2012-01-01

    Vanda Mimi Palmer (VMP), an orchid hybrid of Vanda tesselata and Vanda Tan Chay Yan is a highly scented tropical orchid which blooms all year round. Previous studies revealed that VMP produces a variety of isoprenoid volatiles during daylight. Isoprenoids are well known to contribute significantly to the scent of most fragrant plants. They are a large group of secondary metabolites which may possess valuable characteristics such as flavor, fragrance and toxicity and are produced via two pathways, the mevalonate (MVA) pathway or/and the 2-C-methyl-D-erythritol-4-phosphate (MEP) pathway. In this study, a sesquiterpene synthase gene denoted VMPSTS, previously isolated from a floral cDNA library of VMP was cloned and expressed in Lactococcus lactis to characterize the functionality of the protein. L. lactis, a food grade bacterium which utilizes the mevalonate pathway for isoprenoid production was found to be a suitable host for the characterization of plant terpene synthases. Through recombinant expression of VMPSTS, it was revealed that VMPSTS produced multiple sesquiterpenes and germacrene D dominates its profile. PMID:22408409

  15. Disruption of the 1-deoxy-D-xylulose-5-phosphate reductoisomerase (DXR) gene results in albino, dwarf and defects in trichome initiation and stomata closure in Arabidopsis.

    PubMed

    Xing, Shufan; Miao, Jin; Li, Shuang; Qin, Genji; Tang, Si; Li, Haoni; Gu, Hongya; Qu, Li-Jia

    2010-06-01

    1-Deoxy-D-xylulose-5-phosphate reductoisomerase (DXR) is an important enzyme involved in the 2-C-methyl-D-erythritol-4-phosphate (MEP) pathway which provides the basic five-carbon units for isoprenoid biosynthesis. To investigate the role of the MEP pathway in plant development and metabolism, we carried out detailed analyses on a dxr mutant (GK_215C01) and two DXR transgenic co-suppression lines, OX-DXR-L2 and OX-DXR-L7. We found that the dxr mutant was albino and dwarf. It never bolted, had significantly reduced number of trichomes and most of the stomata could not close normally in the leaves. The two co-suppression lines produced more yellow inflorescences and albino sepals with no trichomes. The transcription levels of genes involved in trichome initiation were found to be strongly affected, including GLABRA1, TRANSPARENT TESTA GLABROUS 1, TRIPTYCHON and SPINDLY, expression of which is regulated by gibberellic acids (GAs). Exogenous application of GA(3) could partially rescue the dwarf phenotype and the trichome initiation of dxr, whereas exogenous application of abscisic acid (ABA) could rescue the stomata closure defect, suggesting that lower levels of both GA and ABA contribute to the phenotype in the dxr mutants. We further found that genes involved in the biosynthetic pathways of GA and ABA were coordinately regulated. These results indicate that disruption of the plastidial MEP pathway leads to biosynthetic deficiency of photosynthetic pigments, GAs and ABA, and thus the developmental abnormalities, and that the flux from the cytoplasmic mevalonate pathway is not sufficient to rescue the deficiency caused by the blockage of the plastidial MEP pathway. These results reveal a critical role for the MEP biosynthetic pathway in controlling the biosynthesis of isoprenoids.

  16. Bisphosphonate inhibitors reveal a large elasticity of plastidic isoprenoid synthesis pathway in isoprene-emitting hybrid aspen.

    PubMed

    Rasulov, Bahtijor; Talts, Eero; Kännaste, Astrid; Niinemets, Ülo

    2015-06-01

    Recently, a feedback inhibition of the chloroplastic 1-deoxy-D-xylulose 5-phosphate (DXP)/2-C-methyl-D-erythritol 4-phosphate (MEP) pathway of isoprenoid synthesis by end products dimethylallyl diphosphate (DMADP) and isopentenyl diphosphate (IDP) was postulated, but the extent to which DMADP and IDP can build up is not known. We used bisphosphonate inhibitors, alendronate and zoledronate, that inhibit the consumption of DMADP and IDP by prenyltransferases to gain insight into the extent of end product accumulation and possible feedback inhibition in isoprene-emitting hybrid aspen (Populus tremula × Populus tremuloides). A kinetic method based on dark release of isoprene emission at the expense of substrate pools accumulated in light was used to estimate the in vivo pool sizes of DMADP and upstream metabolites. Feeding with fosmidomycin, an inhibitor of DXP reductoisomerase, alone or in combination with bisphosphonates was used to inhibit carbon input into DXP/MEP pathway or both input and output. We observed a major increase in pathway intermediates, 3- to 4-fold, upstream of DMADP in bisphosphonate-inhibited leaves, but the DMADP pool was enhanced much less, 1.3- to 1.5-fold. In combined fosmidomycin/bisphosphonate treatment, pathway intermediates accumulated, reflecting cytosolic flux of intermediates that can be important under strong metabolic pull in physiological conditions. The data suggested that metabolites accumulated upstream of DMADP consist of phosphorylated intermediates and IDP. Slow conversion of the huge pools of intermediates to DMADP was limited by reductive energy supply. These data indicate that the DXP/MEP pathway is extremely elastic, and the presence of a significant pool of phosphorylated intermediates provides an important valve for fine tuning the pathway flux.

  17. S-Carvone Suppresses Cellulase-Induced Capsidiol Production in Nicotiana tabacum by Interfering with Protein Isoprenylation1[C][W

    PubMed Central

    Huchelmann, Alexandre; Gastaldo, Clément; Veinante, Mickaël; Zeng, Ying; Heintz, Dimitri; Tritsch, Denis; Schaller, Hubert; Rohmer, Michel; Bach, Thomas J.; Hemmerlin, Andréa

    2014-01-01

    S-Carvone has been described as a negative regulator of mevalonic acid (MVA) production by interfering with 3-hydroxy-3-methyl glutaryl coenzyme A reductase (HMGR) activity, a key player in isoprenoid biosynthesis. The impact of this monoterpene on the production of capsidiol in Nicotiana tabacum, an assumed MVA-derived sesquiterpenoid phytoalexin produced in response to elicitation by cellulase, was investigated. As expected, capsidiol production, as well as early stages of elicitation such as hydrogen peroxide production or stimulation of 5-epi-aristolochene synthase activity, were repressed. Despite the lack of capsidiol synthesis, apparent HMGR activity was boosted. Feeding experiments using (1-13C)Glc followed by analysis of labeling patterns by 13C-NMR, confirmed an MVA-dependent biosynthesis; however, treatments with fosmidomycin, an inhibitor of the MVA-independent 2-C-methyl-d-erythritol 4-phosphate (MEP) isoprenoid pathway, unexpectedly down-regulated the biosynthesis of this sesquiterpene as well. We postulated that S-carvone does not directly inhibit the production of MVA by inactivating HMGR, but possibly targets an MEP-derived isoprenoid involved in the early steps of the elicitation process. A new model is proposed in which the monoterpene blocks an MEP pathway–dependent protein geranylgeranylation necessary for the signaling cascade. The production of capsidiol was inhibited when plants were treated with some inhibitors of protein prenylation or by further monoterpenes. Moreover, S-carvone hindered isoprenylation of a prenylable GFP indicator protein expressed in N. tabacum cell lines, which can be chemically complemented with geranylgeraniol. The model was further validated using N. tabacum cell extracts or recombinant N. tabacum protein prenyltransferases expressed in Escherichia coli. Our study endorsed a reevaluation of the effect of S-carvone on plant isoprenoid metabolism. PMID:24367019

  18. Bisphosphonate Inhibitors Reveal a Large Elasticity of Plastidic Isoprenoid Synthesis Pathway in Isoprene-Emitting Hybrid Aspen1

    PubMed Central

    2015-01-01

    Recently, a feedback inhibition of the chloroplastic 1-deoxy-d-xylulose 5-phosphate (DXP)/2-C-methyl-d-erythritol 4-phosphate (MEP) pathway of isoprenoid synthesis by end products dimethylallyl diphosphate (DMADP) and isopentenyl diphosphate (IDP) was postulated, but the extent to which DMADP and IDP can build up is not known. We used bisphosphonate inhibitors, alendronate and zoledronate, that inhibit the consumption of DMADP and IDP by prenyltransferases to gain insight into the extent of end product accumulation and possible feedback inhibition in isoprene-emitting hybrid aspen (Populus tremula × Populus tremuloides). A kinetic method based on dark release of isoprene emission at the expense of substrate pools accumulated in light was used to estimate the in vivo pool sizes of DMADP and upstream metabolites. Feeding with fosmidomycin, an inhibitor of DXP reductoisomerase, alone or in combination with bisphosphonates was used to inhibit carbon input into DXP/MEP pathway or both input and output. We observed a major increase in pathway intermediates, 3- to 4-fold, upstream of DMADP in bisphosphonate-inhibited leaves, but the DMADP pool was enhanced much less, 1.3- to 1.5-fold. In combined fosmidomycin/bisphosphonate treatment, pathway intermediates accumulated, reflecting cytosolic flux of intermediates that can be important under strong metabolic pull in physiological conditions. The data suggested that metabolites accumulated upstream of DMADP consist of phosphorylated intermediates and IDP. Slow conversion of the huge pools of intermediates to DMADP was limited by reductive energy supply. These data indicate that the DXP/MEP pathway is extremely elastic, and the presence of a significant pool of phosphorylated intermediates provides an important valve for fine tuning the pathway flux. PMID:25926480

  19. Enhanced flux through the methylerythritol 4-phosphate pathway in Arabidopsis plants overexpressing deoxyxylulose 5-phosphate reductoisomerase.

    PubMed

    Carretero-Paulet, Lorenzo; Cairó, Albert; Botella-Pavía, Patricia; Besumbes, Oscar; Campos, Narciso; Boronat, Albert; Rodríguez-Concepción, Manuel

    2006-11-01

    The methylerythritol 4-phosphate (MEP) pathway synthesizes the precursors for an astonishing diversity of plastid isoprenoids, including the major photosynthetic pigments chlorophylls and carotenoids. Since the identification of the first two enzymes of the pathway, deoxyxylulose 5-phoshate (DXP) synthase (DXS) and DXP reductoisomerase (DXR), they both were proposed as potential control points. Increased DXS activity has been shown to up-regulate the production of plastid isoprenoids in all systems tested, but the relative contribution of DXR to the supply of isoprenoid precursors is less clear. In this work, we have generated transgenic Arabidopsis thaliana plants with altered DXS and DXR enzyme levels, as estimated from their resistance to clomazone and fosmidomycin, respectively. The down-regulation of DXR resulted in variegation, reduced pigmentation and defects in chloroplast development, whereas DXR-overexpressing lines showed an increased accumulation of MEP- derived plastid isoprenoids such as chlorophylls, carotenoids, and taxadiene in transgenic plants engineered to produce this non-native isoprenoid. Changes in DXR levels in transgenic plants did not result in changes in DXS gene expression or enzyme accumulation, confirming that the observed effects on plastid isoprenoid levels in DXR-overexpressing lines were not an indirect consequence of altering DXS levels. The results indicate that the biosynthesis of MEP (the first committed intermediate of the pathway) limits the production of downstream isoprenoids in Arabidopsis chloroplasts, supporting a role for DXR in the control of the metabolic flux through the MEP pathway.

  20. An account of cloned genes of Methyl-erythritol-4-phosphate pathway of isoprenoid biosynthesis in plants.

    PubMed

    Ganjewala, Deepak; Kumar, Shiv; Luthra, Rajesh

    2009-01-01

    Isoprenoids, also known as terpenoids, are biosynthesized by the condensation of the two C5 unit isopentenyl diphosphate (IPP) and isomer dimethylallyl diphosphate (DMAPP). Generally, plants use two separate pathways plastidial Methyl-erythritol-4-phosphate (MEP) and cytosolic acetate-mevalonate (MVA) pathways for formation of IPP. The genes, enzymes and intermediates of the MEP pathway have been unravelled in plants over the past few years. Interestingly, MEP pathway enzymes are encoded by nuclear genes but they function in plastids to produce precursors for isoprenes, monoterpenes, carotenoids, abscisic acid, gibberellins, and the side chain of chlorophylls, tocopherols, phylloquinones, and plastoquinone. In Arabidopsis thaliana, a complete set of genes of MEP pathway homologous to the E. coli MEP pathway genes have been identified. Although, these genes have been cloned and characterized from several other plants but overall information about them at one place is not available so far. Though, a range of reviews are available about their roles in isoprenoid biosynthesis and regulation. Therefore, we decided to compile the data on cloned and characterized genes of MEP pathway in plants. Also, we summarize the results of the previously published reports, particularly those which were based on incorporation of 13C-glucose or by application of specific inhibitors such as mevinolin and fosmidomycin to look into the MEP pathway in plants. In addition, we searched for the two key enzymes DXS and HMGR that could be assigned for the acetate-MVA and MEP pathway with the help of bioinformatics tools. Presence or absence of these enzymes can be correlated with respective isoprenoid biosynthetic pathways in plants.

  1. The mitochondrial PPR protein LOVASTATIN INSENSITIVE 1 plays regulatory roles in cytosolic and plastidial isoprenoid biosynthesis through RNA editing.

    PubMed

    Tang, Jianwei; Kobayashi, Keiko; Suzuki, Masashi; Matsumoto, Shogo; Muranaka, Toshiya

    2010-02-01

    Unlike animals, plants synthesize isoprenoids via two pathways, the cytosolic mevalonate (MVA) pathway and the plastidial 2-C-methyl-d-erythritol-4-phosphate (MEP) pathway. Little information is known about the mechanisms that regulate these complex biosynthetic networks over multiple organelles. To understand such regulatory mechanisms of the biosynthesis of isoprenoids in plants, we previously characterized the Arabidopsis mutant, lovastatin insensitive 1 (loi1), which is resistant to lovastatin and clomazone, specific inhibitors of the MVA and MEP pathways, respectively. LOI1 encodes a pentatricopeptide repeat (PPR) protein localized in mitochondria that is thought to have RNA binding ability and function in post-transcriptional regulation of mitochondrial gene expression. LOI1 belongs to the DYW subclass of PPR proteins, which is hypothesized to be correlated with RNA editing. As a result of analysis of RNA editing of mitochondrial genes in loi1, a defect in RNA editing of three genes, nad4, ccb203 and cox3, was identified in loi1. These genes are related to the respiratory chain. Wild type (WT) treated with some respiration inhibitors mimicked the loi1 phenotype. Interestingly, HMG-CoA reductase activity of WT treated with lovastatin combined with antimycin A, an inhibitor of complex III in the respiratory chain, was higher than that of WT treated with only lovastatin, despite the lack of alteration of transcript or protein levels of HMGR. These results suggest that HMGR enzyme activity is regulated through the respiratory cytochrome pathway. Although various mechanisms exist for isoprenoid biosynthesis, our studies demonstrate the novel possibility that mitochondrial respiration plays potentially regulatory roles in isoprenoid biosynthesis.

  2. Complete blockage of the mevalonate pathway results in male gametophyte lethality.

    PubMed

    Suzuki, Masashi; Nakagawa, Shoko; Kamide, Yukiko; Kobayashi, Keiko; Ohyama, Kiyoshi; Hashinokuchi, Hiromi; Kiuchi, Reiko; Saito, Kazuki; Muranaka, Toshiya; Nagata, Noriko

    2009-01-01

    Plants have two isoprenoid biosynthetic pathways: the cytosolic mevalonate (MVA) pathway and the plastidic 2-C-methyl-D-erythritol 4-phosphate (MEP) pathway. Since the discovery of the MEP pathway, possible metabolic cross-talk between these pathways has prompted intense research. Although many studies have shown the existence of such cross-talk using feeding experiments, it remains to be determined if native cross-talk, rather than exogenously applied metabolites, can compensate for complete blockage of the MVA pathway. Previously, Arabidopsis mutants for HMG1 and HMG2 encoding HMG-CoA reductase (HMGR) were isolated. Although it was shown that HMGR1 is a functional HMGR, the enzyme activity of HMGR2 has not been confirmed. It is demonstrated here that HMG2 encodes a functional reductase with similar activity to HMGR1, using enzyme assays and complementation experiments. To estimate the contribution of native cross-talk, an attempt was made to block the MVA pathway by making double mutants lacking both HMG1 and HMG2, but no double homozygotes were detected in the progeny of self-pollinated HMG1/hmg1 hmg2/hmg2 plants. hmg1 hmg2 male gametophytes appeared to be lethal based on crossing experiments, and microscopy indicated that approximately 50% of the microspores from the HMG1/hmg1 hmg2/hmg2 plant appeared shrunken and exhibited poorly defined endoplasmic reticulum membranes. In situ hybridization showed that HMG1 transcripts were expressed in both the tapetum and microspores, while HMG2 mRNA appeared only in microspores. It is concluded that native cross-talk from the plastid cannot compensate for complete blockage of the MVA pathway, at least during male gametophyte development, because either HMG1 or HMG2 is required for male gametophyte development.

  3. Biosynthesis of sesquiterpenes in grape berry exocarp of Vitis vinifera L.: evidence for a transport of farnesyl diphosphate precursors from plastids to the cytosol.

    PubMed

    May, Bianca; Lange, B Markus; Wüst, Matthias

    2013-11-01

    The participation of the mevalonic acid (MVA) and 1-deoxy-d-xylulose 5-phosphate/2-C-methyl-d-erythritol-4-phosphate (DOXP/MEP) pathways in sesquiterpene biosynthesis of grape berries was investigated. There is an increasing interest in this class of terpenoids, since the oxygenated sesquiterpene rotundone was identified as the peppery aroma impact compound in Australian Shiraz wines. To investigate precursor supply pathway utilization, in vivo feeding experiments were performed with the deuterium labeled, pathway specific, precursors [5,5-(2)H2]-1-deoxy-d-xylulose and [5,5-(2)H2]-mevalonic acid lactone. Head Space-Solid Phase Micro Extraction-Gas Chromatography-Mass Spectrometry (HS-SPME-GC-MS) analysis of the generated volatile metabolites demonstrated that de novo sesquiterpene biosynthesis is mainly located in the grape berry exocarp (skin), with no detectable activity in the mesocarp (flesh) of the Lemberger variety. Interestingly, precursors from both the (primarily) cytosolic MVA and plastidial DOXP/MEP pathways were incorporated into grape sesquiterpenes in the varieties Lemberger, Gewürztraminer and Syrah. Our labeling data provide evidence for a homogenous, cytosolic pool of precursors for sesquiterpene biosynthesis, indicating that a transport of precursors occurs mostly from plastids to the cytosol. The labeling patterns of the sesquiterpene germacrene D were in agreement with a cyclization mechanism analogous to that of a previously cloned enantioselective (R)-germacrene D synthase from Solidago canadensis. This observation was subsequently confirmed by enantioselective GC-MS analysis demonstrating the exclusive presence of (R)-germacrene D, and not the (S)-enantiomer, in grape berries. PMID:23954075

  4. Biosynthesis of sesquiterpenes in grape berry exocarp of Vitis vinifera L.: evidence for a transport of farnesyl diphosphate precursors from plastids to the cytosol.

    PubMed

    May, Bianca; Lange, B Markus; Wüst, Matthias

    2013-11-01

    The participation of the mevalonic acid (MVA) and 1-deoxy-d-xylulose 5-phosphate/2-C-methyl-d-erythritol-4-phosphate (DOXP/MEP) pathways in sesquiterpene biosynthesis of grape berries was investigated. There is an increasing interest in this class of terpenoids, since the oxygenated sesquiterpene rotundone was identified as the peppery aroma impact compound in Australian Shiraz wines. To investigate precursor supply pathway utilization, in vivo feeding experiments were performed with the deuterium labeled, pathway specific, precursors [5,5-(2)H2]-1-deoxy-d-xylulose and [5,5-(2)H2]-mevalonic acid lactone. Head Space-Solid Phase Micro Extraction-Gas Chromatography-Mass Spectrometry (HS-SPME-GC-MS) analysis of the generated volatile metabolites demonstrated that de novo sesquiterpene biosynthesis is mainly located in the grape berry exocarp (skin), with no detectable activity in the mesocarp (flesh) of the Lemberger variety. Interestingly, precursors from both the (primarily) cytosolic MVA and plastidial DOXP/MEP pathways were incorporated into grape sesquiterpenes in the varieties Lemberger, Gewürztraminer and Syrah. Our labeling data provide evidence for a homogenous, cytosolic pool of precursors for sesquiterpene biosynthesis, indicating that a transport of precursors occurs mostly from plastids to the cytosol. The labeling patterns of the sesquiterpene germacrene D were in agreement with a cyclization mechanism analogous to that of a previously cloned enantioselective (R)-germacrene D synthase from Solidago canadensis. This observation was subsequently confirmed by enantioselective GC-MS analysis demonstrating the exclusive presence of (R)-germacrene D, and not the (S)-enantiomer, in grape berries.

  5. Intraerythrocytic stages of Plasmodium falciparum biosynthesize vitamin E.

    PubMed

    Sussmann, Rodrigo A C; Angeli, Cláudia B; Peres, Valnice J; Kimura, Emilia A; Katzin, Alejandro M

    2011-12-15

    The 2-C-methyl-D-erythritol-4-phosphate and shikimate pathways were found to be active in Plasmodium falciparum and both can result in vitamin E biosynthesis in plants and algae. This study biochemically confirmed vitamin E biosynthesis in the malaria parasite, which can be inhibited by usnic acid. Furthermore, we found evidence pointing to a role of this vitamin in infected erythrocytes. These findings not only contribute to current understanding of P. falciparum biology but also reveal a pathway that could serve as a chemotherapeutic target.

  6. Microextraction by packed sorbent (MEPS): a tutorial.

    PubMed

    Abdel-Rehim, Mohamed

    2011-09-01

    This tutorial provides an overview on a new technique for sample preparation, microextraction by packed sorbent (MEPS). Not only the automation process by MEPS is the advantage but also the much smaller volumes of the samples, solvents and dead volumes in the system. Other significant advantages such as the speed and the simplicity of the sample preparation process are provided. In this tutorial the main concepts of MEPS will be elucidated. Different practical aspects in MEPS are addressed. The factors affecting MEPS performance will be discussed. The application of MEPS in clinical and pre-clinical studies for quantification of drugs and metabolites in blood, plasma and urine will be provided. A comparison between MEPS and other extraction techniques such as SPE, LLE, SPME and SBSE will be discussed.

  7. Prerequisite for highly efficient isoprenoid production by cyanobacteria discovered through the over-expression of 1-deoxy-d-xylulose 5-phosphate synthase and carbon allocation analysis.

    PubMed

    Kudoh, Kai; Kawano, Yusuke; Hotta, Shingo; Sekine, Midori; Watanabe, Takafumi; Ihara, Masaki

    2014-07-01

    Cyanobacteria have recently been receiving considerable attention owing to their potential as photosynthetic producers of biofuels and biomaterials. Here, we focused on the production of isoprenoids by cyanobacteria, and aimed to provide insight into metabolic engineering design. To this end, we examined the over-expression of a key enzyme in 2-C-methyl-d-erythritol 4-phosphate (MEP) pathway, 1-deoxy-d-xylulose 5-phosphate synthase (DXS) in the cyanobacterium Synechocystis sp. PCC6803. In the DXS-over-expression strain (Dxs_ox), the mRNA and protein levels of DXS were 4-times and 1.5-times the levels in the wild-type (WT) strain, respectively. The carotenoid content of the Dxs_ox strain (8.4 mg/g dry cell weight [DCW]) was also up to 1.5-times higher than that in the WT strain (5.6 mg/g DCW), whereas the glycogen content dramatically decreased to an undetectable level. These observations suggested that the carotenoid content in the Dxs_ox strain was increased by consuming glycogen, which is a C-storage compound in cyanobacteria. We also quantified the total sugar (145 and 104 mg/g DCW), total fatty acids (31 and 24 mg/g DCW) and total protein (200 and 240 mg/g DCW) content in the WT and Dxs_ox strains, respectively, which were much higher than the carotenoid content. In particular, approximately 54% of the proteins were phycobiliproteins. This study demonstrated the major destinations of carbon flux in cyanobacteria, and provided important insights into metabolic engineering. Target yield can be improved through optimization of gene expression, the DXS protein stabilization, cell propagation depression and restriction of storage compound synthesis.

  8. De novo assembly and transcriptome analysis of the rubber tree (Hevea brasiliensis) and SNP markers development for rubber biosynthesis pathways.

    PubMed

    Mantello, Camila Campos; Cardoso-Silva, Claudio Benicio; da Silva, Carla Cristina; de Souza, Livia Moura; Scaloppi Junior, Erivaldo José; de Souza Gonçalves, Paulo; Vicentini, Renato; de Souza, Anete Pereira

    2014-01-01

    Hevea brasiliensis (Willd. Ex Adr. Juss.) Muell.-Arg. is the primary source of natural rubber that is native to the Amazon rainforest. The singular properties of natural rubber make it superior to and competitive with synthetic rubber for use in several applications. Here, we performed RNA sequencing (RNA-seq) of H. brasiliensis bark on the Illumina GAIIx platform, which generated 179,326,804 raw reads on the Illumina GAIIx platform. A total of 50,384 contigs that were over 400 bp in size were obtained and subjected to further analyses. A similarity search against the non-redundant (nr) protein database returned 32,018 (63%) positive BLASTx hits. The transcriptome analysis was annotated using the clusters of orthologous groups (COG), gene ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG), and Pfam databases. A search for putative molecular marker was performed to identify simple sequence repeats (SSRs) and single nucleotide polymorphisms (SNPs). In total, 17,927 SSRs and 404,114 SNPs were detected. Finally, we selected sequences that were identified as belonging to the mevalonate (MVA) and 2-C-methyl-D-erythritol 4-phosphate (MEP) pathways, which are involved in rubber biosynthesis, to validate the SNP markers. A total of 78 SNPs were validated in 36 genotypes of H. brasiliensis. This new dataset represents a powerful information source for rubber tree bark genes and will be an important tool for the development of microsatellites and SNP markers for use in future genetic analyses such as genetic linkage mapping, quantitative trait loci identification, investigations of linkage disequilibrium and marker-assisted selection.

  9. Helper component-proteinase enhances the activity of 1-deoxy-D-xylulose-5-phosphate synthase and promotes the biosynthesis of plastidic isoprenoids in Potato virus Y-infected tobacco.

    PubMed

    Li, Heng; Ma, Dongyuan; Jin, Yongsheng; Tu, Yayi; Liu, Liping; Leng, Chunxu; Dong, Jiangli; Wang, Tao

    2015-10-01

    Virus-infected plants show strong morphological and physiological alterations. Many physiological processes in chloroplast are affected, including the plastidic isoprenoid biosynthetic pathway [the 2C-methyl-D-erythritol-4-phosphate (MEP) pathway]; indeed, isoprenoid contents have been demonstrated to be altered in virus-infected plants. In this study, we found that the levels of photosynthetic pigments and abscisic acid (ABA) were altered in Potato virus Y (PVY)-infected tobacco. Using yeast two-hybrid assays, we demonstrated an interaction between virus protein PVY helper component-proteinase (HC-Pro) and tobacco chloroplast protein 1-deoxy-D-xylulose-5-phosphate synthase (NtDXS). This interaction was confirmed using bimolecular fluorescence complementation (BiFC) assays and pull-down assays. The Transket_pyr domain (residues 394-561) of NtDXS was required for interaction with HC-Pro, while the N-terminal region of HC-Pro (residues 1-97) was necessary for interaction with NtDXS. Using in vitro enzyme activity assays, PVY HC-Pro was found to promote the synthase activity of NtDXS. We observed increases in photosynthetic pigment contents and ABA levels in transgenic plants with HC-Pro accumulating in the chloroplasts. During virus infection, the enhancement of plastidic isoprenoid biosynthesis was attributed to the enhancement of DXS activity by HC-Pro. Our study reveals a new role of HC-Pro in the host plant metabolic system and will contribute to the study of host-virus relationships.

  10. Functional and evolutionary analysis of DXL1, a non-essential gene encoding a 1-deoxy-D-xylulose 5-phosphate synthase like protein in Arabidopsis thaliana.

    PubMed

    Carretero-Paulet, Lorenzo; Cairó, Albert; Talavera, David; Saura, Andreu; Imperial, Santiago; Rodríguez-Concepción, Manuel; Campos, Narciso; Boronat, Albert

    2013-07-15

    The synthesis of 1-deoxy-D-xylulose 5-phosphate (DXP), catalyzed by the enzyme DXP synthase (DXS), represents a key regulatory step of the 2-C-methyl-D-erythritol 4-phosphate (MEP) pathway for isoprenoid biosynthesis. In plants DXS is encoded by small multigene families that can be classified into, at least, three specialized subfamilies. Arabidopsis thaliana contains three genes encoding proteins with similarity to DXS, including the well-known DXS1/CLA1 gene, which clusters within subfamily I. The remaining proteins, initially named DXS2 and DXS3, have not yet been characterized. Here we report the expression and functional analysis of A. thaliana DXS2. Unexpectedly, the expression of DXS2 failed to rescue Escherichia coli and A. thaliana mutants defective in DXS activity. Coherently, we found that DXS activity was negligible in vitro, being renamed as DXL1 following recent nomenclature recommendation. DXL1 is targeted to plastids as DXS1, but shows a distinct expression pattern. The phenotypic analysis of a DXL1 defective mutant revealed that the function of the encoded protein is not essential for growth and development. Evolutionary analyses indicated that DXL1 emerged from DXS1 through a recent duplication apparently specific of the Brassicaceae lineage. Divergent selective constraints would have affected a significant fraction of sites after diversification of the paralogues. Furthermore, amino acids subjected to divergent selection and likely critical for functional divergence through the acquisition of a novel, although not yet known, biochemical function, were identified. Our results provide with the first evidences of functional specialization at both the regulatory and biochemical level within the plant DXS family.

  11. Enhanced production of steviol glycosides in mycorrhizal plants: a concerted effect of arbuscular mycorrhizal symbiosis on transcription of biosynthetic genes.

    PubMed

    Mandal, Shantanu; Upadhyay, Shivangi; Singh, Ved Pal; Kapoor, Rupam

    2015-04-01

    Stevia rebaudiana (Bertoni) produces steviol glycosides (SGs)--stevioside (stev) and rebaudioside-A (reb-A) that are valued as low calorie sweeteners. Inoculation with arbuscular mycorrhizal fungi (AMF) augments SGs production, though the effect of this interaction on SGs biosynthesis has not been studied at molecular level. In this study transcription profiles of eleven key genes grouped under three stages of the SGs biosynthesis pathway were compared. The transcript analysis showed upregulation of genes encoding 2-C-methyl-D-erythritol-4-phosphate (MEP) pathway enzymes viz.,1-deoxy-D-xylulose 5-phospate synthase (DXS), 1-deoxy-D-xylulose 5-phospate reductoisomerase (DXR) and 2-C-methyl-D-erytrithol 2,4-cyclodiphosphate synthase (MDS) in mycorrhizal (M) plants. Zn and Mn are imperative for the expression of MDS and their enhanced uptake in M plants could be responsible for the increased transcription of MDS. Furthermore, in the second stage of SGs biosynthesis pathway, mycorrhization enhanced the transcription of copalyl diphosphate synthase (CPPS) and kaurenoic acid hydroxylase (KAH). Their expression is decisive for SGs biosynthesis as CPPS regulates flow of metabolites towards synthesis of kaurenoid precursors and KAH directs these towards steviol synthesis instead of gibberellins. In the third stage glucosylation of steviol to reb-A by four specific uridine diphosphate (UDP)-dependent glycosyltransferases (UGTs) occurs. While higher transcription of all the three characterized UGTs in M plants explains augmented production of SGs; higher transcript levels of UGT76G1, specifically improved reb-A to stev ratio implying increased sweetness. The work signifies that AM symbiosis upregulates the transcription of all eleven SGs biosynthesis genes as a result of improved nutrition and enhanced sugar concentration due to increased photosynthesis in M plants. PMID:25734328

  12. Possible cellular regulation schemes of isoprene synthesis and emission under different ambient carbon dioxide levels. (Invited)

    NASA Astrophysics Data System (ADS)

    Noe, S. M.; Schnitzler, J.; Arneth, A.; Monson, R. K.; Niinemets, U.

    2010-12-01

    Research on the effects of higher atmospheric carbon dioxide (CO2) levels on isoprene synthesis and emission leaded to several newly proposed regulation schemes. They can be classified as substrate level control on one side and as energetic cofactor control of the plastidic 2-C-methyl-d-erythritol 4-phosphate (MEP) pathway on the other one. Viewed on a whole cell scale, the precursors of isoprene, such as dimethylallyl pyrophosphate (DMAPP) and isopentenyl pyrophosphate (IPP), can be found in several cellular compartments such as chloroplasts, cytosol and mitochondria. Furthermore, necessary entry points into the isoprene synthesis pathway like phosphoenolpyruvate (PEP) and pyruvate are provided by two processes, photosynthesis and glycolysis, which are as well located in different cellular compartments. These findings imply, that the effect of modulating the isoprene emission under high levels of atmospheric CO2 have to take transport over membranes, possible concurrent pathways, i.e. Shikimi acid pathway or anaplerotic metabolism reactions and the availability of adenosine triphosphate (ATP) and nicotinamide adenine dinucleotide phosphate (NADPH) on a cellular scale into account. In this modeling study we applied box models that include several facets of the proposed regulation and transport schemes. The models have been set up such that at least two cellular compartments, chloroplast and cytosol are taken into account. The boxes itself represent metabolites and several possible regulation schemes have been realized by the formulation of rate equations between those metabolite pools. As many intermediates are not readily available as measured values, the models aim to build a set of tools to simulate possible regulatory schemes and provide parameter estimations for key processes. Inverse modeling techniques allow to assess certain parameter ranges within the proposed regulation schemes by fitting the models to data on isoprene emission and photosynthesis under

  13. Deoxyxylulose 5-Phosphate Synthase Controls Flux through the Methylerythritol 4-Phosphate Pathway in Arabidopsis1[C][W][OPEN

    PubMed Central

    Wright, Louwrance P.; Rohwer, Johann M.; Ghirardo, Andrea; Hammerbacher, Almuth; Ortiz-Alcaide, Miriam; Raguschke, Bettina; Schnitzler, Jörg-Peter; Gershenzon, Jonathan; Phillips, Michael A.

    2014-01-01

    The 2-C-methylerythritol 4-phosphate (MEP) pathway supplies precursors for plastidial isoprenoid biosynthesis including carotenoids, redox cofactor side chains, and biogenic volatile organic compounds. We examined the first enzyme of this pathway, 1-deoxyxylulose 5-phosphate synthase (DXS), using metabolic control analysis. Multiple Arabidopsis (Arabidopsis thaliana) lines presenting a range of DXS activities were dynamically labeled with 13CO2 in an illuminated, climate-controlled, gas exchange cuvette. Carbon was rapidly assimilated into MEP pathway intermediates, but not into the mevalonate pathway. A flux control coefficient of 0.82 was calculated for DXS by correlating absolute flux to enzyme activity under photosynthetic steady-state conditions, indicating that DXS is the major controlling enzyme of the MEP pathway. DXS manipulation also revealed a second pool of a downstream metabolite, 2-C-methylerythritol-2,4-cyclodiphosphate (MEcDP), metabolically isolated from the MEP pathway. DXS overexpression led to a 3- to 4-fold increase in MEcDP pool size but to a 2-fold drop in maximal labeling. The existence of this pool was supported by residual MEcDP levels detected in dark-adapted transgenic plants. Both pools of MEcDP are closely modulated by DXS activity, as shown by the fact that the concentration control coefficient of DXS was twice as high for MEcDP (0.74) as for 1-deoxyxylulose 5-phosphate (0.35) or dimethylallyl diphosphate (0.34). Despite the high flux control coefficient for DXS, its overexpression led to only modest increases in isoprenoid end products and in the photosynthetic rate. Diversion of flux via MEcDP may partly explain these findings and suggests new opportunities to engineer the MEP pathway. PMID:24987018

  14. 32 CFR 1602.16 - MEPS.

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ... 32 National Defense 6 2010-07-01 2010-07-01 false MEPS. 1602.16 Section 1602.16 National Defense Other Regulations Relating to National Defense SELECTIVE SERVICE SYSTEM DEFINITIONS § 1602.16 MEPS. A Military Entrance Processing Station is a military installation to which registrants are ordered to...

  15. 32 CFR 1602.16 - MEPS.

    Code of Federal Regulations, 2012 CFR

    2012-07-01

    ... 32 National Defense 6 2012-07-01 2012-07-01 false MEPS. 1602.16 Section 1602.16 National Defense Other Regulations Relating to National Defense SELECTIVE SERVICE SYSTEM DEFINITIONS § 1602.16 MEPS. A Military Entrance Processing Station is a military installation to which registrants are ordered to...

  16. 32 CFR 1602.16 - MEPS.

    Code of Federal Regulations, 2013 CFR

    2013-07-01

    ... 32 National Defense 6 2013-07-01 2013-07-01 false MEPS. 1602.16 Section 1602.16 National Defense Other Regulations Relating to National Defense SELECTIVE SERVICE SYSTEM DEFINITIONS § 1602.16 MEPS. A Military Entrance Processing Station is a military installation to which registrants are ordered to...

  17. 32 CFR 1602.16 - MEPS.

    Code of Federal Regulations, 2011 CFR

    2011-07-01

    ... 32 National Defense 6 2011-07-01 2011-07-01 false MEPS. 1602.16 Section 1602.16 National Defense Other Regulations Relating to National Defense SELECTIVE SERVICE SYSTEM DEFINITIONS § 1602.16 MEPS. A Military Entrance Processing Station is a military installation to which registrants are ordered to...

  18. 32 CFR 1602.16 - MEPS.

    Code of Federal Regulations, 2014 CFR

    2014-07-01

    ... 32 National Defense 6 2014-07-01 2014-07-01 false MEPS. 1602.16 Section 1602.16 National Defense Other Regulations Relating to National Defense SELECTIVE SERVICE SYSTEM DEFINITIONS § 1602.16 MEPS. A Military Entrance Processing Station is a military installation to which registrants are ordered to...

  19. Characterization of the Arabidopsis clb6 mutant illustrates the importance of posttranscriptional regulation of the methyl-D-erythritol 4-phosphate pathway.

    PubMed

    Guevara-García, Arturo; San Román, Carolina; Arroyo, Analilia; Cortés, María Elena; de la Luz Gutiérrez-Nava, María; León, Patricia

    2005-02-01

    The biosynthesis of isopentenyl diphosphate and dimethylallyl diphosphate, the two building blocks for isoprenoid biosynthesis, occurs by two independent pathways in plants. The mevalonic pathway operates in the cytoplasm, and the methyl-d-erythritol 4-phosphate (MEP) pathway operates in plastids. Plastidic isoprenoids play essential roles in plant growth and development. Plants must regulate the biosynthesis of isoprenoids to fulfill metabolic requirements in specific tissues and developmental conditions. The regulatory events that modulate the plant MEP pathway are not well understood. In this article, we demonstrate that the CHLOROPLAST BIOGENESIS6 (CLB6) gene, previously shown to be required for chloroplast development, encodes 1-hydroxy-2-methyl-butenyl 4-diphosphate reductase, the last-acting enzyme of the MEP pathway. Comparative analysis of the expression levels of all MEP pathway gene transcripts and proteins in the clb6-1 mutant background revealed that posttranscriptional control modulates the levels of different proteins in this central pathway. Posttranscriptional regulation was also found during seedling development and during fosmidomycin inhibition of the pathway. Our results show that the first enzyme of the pathway, 1-deoxy-d-xylulose 5-phosphate synthase, is feedback regulated in response to the interruption of the flow of metabolites through the MEP pathway.

  20. Chlorophyta exclusively use the 1-deoxyxylulose 5-phosphate/2-C-methylerythritol 4-phosphate pathway for the biosynthesis of isoprenoids.

    PubMed

    Schwender, J; Gemünden, C; Lichtenthaler, H K

    2001-02-01

    The biosynthesis of the C5 building block of isoprenoids, isopentenyl diphosphate (IPP), proceeds in higher plants via two basically different pathways; in the cytosolic compartment sterols are formed via mevalonate (MVA), whereas in the plastids the isoprenoids are formed via the 1-deoxyxylulose 5-phosphate/2-C-methylerythritol 4-phosphate pathway (DOXP/MEP pathway). In the present investigation, we found for the Charophyceae, being close relatives to land plants, and in the original green flagellate Mesostignma virilde the same IPP biosynthesis pattern as in higher plants: sterols are formed via MVA, and the phytol-moiety of chlorophylls via the DOXP/MEP pathway. In contrast, representatives of four classes of the Chlorophyta (Chlorophyceae, Ulvophyceae, Trebouxiophyceae, Prasinophyceae) did not incorporate MVA into sterols or phytol. Instead, they incorporated [1-2H1]-1-deoxy-D-xylulose into phytol and sterols. The results indicate that the entire Chlorophyta lineage, which is well separated from the land plant/Charophyceae lineage, is devoid of the acetate/ MVA pathway and uses the DOXP/MEP pathway not only for plastidic, but also for cytosolic isoprenoid formation.

  1. 1-Hydroxy-2-methyl-2-(E)-butenyl 4-diphosphate reductase (IDS) is encoded by multicopy genes in gymnosperms Ginkgo biloba and Pinus taeda.

    PubMed

    Kim, Sang-Min; Kuzuyama, Tomohisa; Kobayashi, Akio; Sando, Tomoki; Chang, Yung-Jin; Kim, Soo-Un

    2008-01-01

    Isoprenoids are synthesized through the condensation of five-carbon intermediates, isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP), derived from two distinct biosynthetic routes: cytosolic mevalonate (MVA) and plastidial 2-C-methyl-D: -erythritol 4-phosphate (MEP) pathways. 1-Hydroxy-2-methyl-2-(E)-butenyl 4-diphosphate reductase (IDS; EC 1.17.1.2), which catalyzes the last step of MEP pathway, was cloned as a multicopy gene from gymnosperms Ginkgo biloba (GbIDS1, GbIDS2, and GbIDS2-1) and Pinus taeda (PtIDS1 and PtIDS2), and characterized. Phylogenetic tree constructed with other plant IDSs demonstrated gymnosperm IDSs were distinctively different from angiosperm IDSs. The gymnosperm IDS clade contained two subclades, one composed of GbIDS1 and PtIDS1, and the other composed of GbIDS2, GbIDS2-1, and PtIDS2. G. biloba IDSs, except GbIDS2-1, successfully complemented Escherichia coli DLYT1, a lytB disruptant, confirming the in vivo competency of isozymes. During the 4 weeks study period, although transcript levels of GbIDS1s were similar both in roots and leaves of cultured G. biloba embryo, the transcripts of GbIDS2 predominantly occurred in the embryo roots, where diterpene ginkgolides are biosynthesized. Levels of PtIDS2 transcripts in the diterpenoid resin-producing wood were 4-5 times higher than those in other tissues. Higher levels of GbIDS1 transcripts were induced by light, whereas those of GbIDS2 were increased by methyl jasmonate treatment. These results strongly imply GbIDS2 and PtIDS2 have high correlation with secondary metabolism. In Arabidopsis transient expression system, N-terminal 100 amino acid residues of GbIDS1 delivered fused GFP protein into chloroplast as well as cytosol and nucleus, whereas those of GbIDS2, GbIDS2-1, and two PtIDSs delivered GFP only into chloroplast.

  2. Plastidic phosphoglucose isomerase is an important determinant of starch accumulation in mesophyll cells, growth, photosynthetic capacity, and biosynthesis of plastidic cytokinins in Arabidopsis.

    PubMed

    Bahaji, Abdellatif; Sánchez-López, Ángela M; De Diego, Nuria; Muñoz, Francisco J; Baroja-Fernández, Edurne; Li, Jun; Ricarte-Bermejo, Adriana; Baslam, Marouane; Aranjuelo, Iker; Almagro, Goizeder; Humplík, Jan F; Novák, Ondřej; Spíchal, Lukáš; Doležal, Karel; Pozueta-Romero, Javier

    2015-01-01

    Phosphoglucose isomerase (PGI) catalyzes the reversible isomerization of glucose-6-phosphate and fructose-6-phosphate. It is involved in glycolysis and in the regeneration of glucose-6-P molecules in the oxidative pentose phosphate pathway (OPPP). In chloroplasts of illuminated mesophyll cells PGI also connects the Calvin-Benson cycle with the starch biosynthetic pathway. In this work we isolated pgi1-3, a mutant totally lacking pPGI activity as a consequence of aberrant intron splicing of the pPGI encoding gene, PGI1. Starch content in pgi1-3 source leaves was ca. 10-15% of that of wild type (WT) leaves, which was similar to that of leaves of pgi1-2, a T-DNA insertion pPGI null mutant. Starch deficiency of pgi1 leaves could be reverted by the introduction of a sex1 null mutation impeding β-amylolytic starch breakdown. Although previous studies showed that starch granules of pgi1-2 leaves are restricted to both bundle sheath cells adjacent to the mesophyll and stomata guard cells, microscopy analyses carried out in this work revealed the presence of starch granules in the chloroplasts of pgi1-2 and pgi1-3 mesophyll cells. RT-PCR analyses showed high expression levels of plastidic and extra-plastidic β-amylase encoding genes in pgi1 leaves, which was accompanied by increased β-amylase activity. Both pgi1-2 and pgi1-3 mutants displayed slow growth and reduced photosynthetic capacity phenotypes even under continuous light conditions. Metabolic analyses revealed that the adenylate energy charge and the NAD(P)H/NAD(P) ratios in pgi1 leaves were lower than those of WT leaves. These analyses also revealed that the content of plastidic 2-C-methyl-D-erythritol 4-phosphate (MEP)-pathway derived cytokinins (CKs) in pgi1 leaves were exceedingly lower than in WT leaves. Noteworthy, exogenous application of CKs largely reverted the low starch content phenotype of pgi1 leaves. The overall data show that pPGI is an important determinant of photosynthesis, energy status, growth

  3. Plastidic Phosphoglucose Isomerase Is an Important Determinant of Starch Accumulation in Mesophyll Cells, Growth, Photosynthetic Capacity, and Biosynthesis of Plastidic Cytokinins in Arabidopsis

    PubMed Central

    De Diego, Nuria; Muñoz, Francisco J.; Baroja-Fernández, Edurne; Li, Jun; Ricarte-Bermejo, Adriana; Baslam, Marouane; Aranjuelo, Iker; Almagro, Goizeder; Humplík, Jan F.; Novák, Ondřej; Spíchal, Lukáš; Doležal, Karel; Pozueta-Romero, Javier

    2015-01-01

    Phosphoglucose isomerase (PGI) catalyzes the reversible isomerization of glucose-6-phosphate and fructose-6-phosphate. It is involved in glycolysis and in the regeneration of glucose-6-P molecules in the oxidative pentose phosphate pathway (OPPP). In chloroplasts of illuminated mesophyll cells PGI also connects the Calvin-Benson cycle with the starch biosynthetic pathway. In this work we isolated pgi1-3, a mutant totally lacking pPGI activity as a consequence of aberrant intron splicing of the pPGI encoding gene, PGI1. Starch content in pgi1-3 source leaves was ca. 10-15% of that of wild type (WT) leaves, which was similar to that of leaves of pgi1-2, a T-DNA insertion pPGI null mutant. Starch deficiency of pgi1 leaves could be reverted by the introduction of a sex1 null mutation impeding β-amylolytic starch breakdown. Although previous studies showed that starch granules of pgi1-2 leaves are restricted to both bundle sheath cells adjacent to the mesophyll and stomata guard cells, microscopy analyses carried out in this work revealed the presence of starch granules in the chloroplasts of pgi1-2 and pgi1-3 mesophyll cells. RT-PCR analyses showed high expression levels of plastidic and extra-plastidic β-amylase encoding genes in pgi1 leaves, which was accompanied by increased β-amylase activity. Both pgi1-2 and pgi1-3 mutants displayed slow growth and reduced photosynthetic capacity phenotypes even under continuous light conditions. Metabolic analyses revealed that the adenylate energy charge and the NAD(P)H/NAD(P) ratios in pgi1 leaves were lower than those of WT leaves. These analyses also revealed that the content of plastidic 2-C-methyl-D-erythritol 4-phosphate (MEP)-pathway derived cytokinins (CKs) in pgi1 leaves were exceedingly lower than in WT leaves. Noteworthy, exogenous application of CKs largely reverted the low starch content phenotype of pgi1 leaves. The overall data show that pPGI is an important determinant of photosynthesis, energy status, growth

  4. Differential Contribution of the First Two Enzymes of the MEP Pathway to the Supply of Metabolic Precursors for Carotenoid and Chlorophyll Biosynthesis in Carrot (Daucus carota).

    PubMed

    Simpson, Kevin; Quiroz, Luis F; Rodriguez-Concepción, Manuel; Stange, Claudia R

    2016-01-01

    Carotenoids and chlorophylls are photosynthetic pigments synthesized in plastids from metabolic precursors provided by the methylerythritol 4-phosphate (MEP) pathway. The first two steps in the MEP pathway are catalyzed by the deoxyxylulose 5-phosphate synthase (DXS) and reductoisomerase (DXR) enzymes. While DXS has been recently shown to be the main flux-controlling step of the MEP pathway, both DXS and DXR enzymes have been proven to be able to promote an increase in MEP-derived products when overproduced in diverse plant systems. Carrot (Daucus carota) produces photosynthetic pigments (carotenoids and chlorophylls) in leaves and in light-exposed roots, whereas only carotenoids (mainly α- and β-carotene) accumulate in the storage root in darkness. To evaluate whether DXS and DXR activities influence the production of carotenoids and chlorophylls in carrot leaves and roots, the corresponding Arabidopsis thaliana genes were constitutively expressed in transgenic carrot plants. Our results suggest that DXS is limiting for the production of both carotenoids and chlorophylls in roots and leaves, whereas the regulatory role of DXR appeared to be minor. Interestingly, increased levels of DXS (but not of DXR) resulted in higher transcript abundance of endogenous carrot genes encoding phytoene synthase, the main rate-determining enzyme of the carotenoid pathway. These results support a central role for DXS on modulating the production of MEP-derived precursors to synthesize carotenoids and chlorophylls in carrot, confirming the pivotal relevance of this enzyme to engineer healthier, carotenoid-enriched products. PMID:27630663

  5. Differential Contribution of the First Two Enzymes of the MEP Pathway to the Supply of Metabolic Precursors for Carotenoid and Chlorophyll Biosynthesis in Carrot (Daucus carota)

    PubMed Central

    Simpson, Kevin; Quiroz, Luis F.; Rodriguez-Concepción, Manuel; Stange, Claudia R.

    2016-01-01

    Carotenoids and chlorophylls are photosynthetic pigments synthesized in plastids from metabolic precursors provided by the methylerythritol 4-phosphate (MEP) pathway. The first two steps in the MEP pathway are catalyzed by the deoxyxylulose 5-phosphate synthase (DXS) and reductoisomerase (DXR) enzymes. While DXS has been recently shown to be the main flux-controlling step of the MEP pathway, both DXS and DXR enzymes have been proven to be able to promote an increase in MEP-derived products when overproduced in diverse plant systems. Carrot (Daucus carota) produces photosynthetic pigments (carotenoids and chlorophylls) in leaves and in light-exposed roots, whereas only carotenoids (mainly α- and β-carotene) accumulate in the storage root in darkness. To evaluate whether DXS and DXR activities influence the production of carotenoids and chlorophylls in carrot leaves and roots, the corresponding Arabidopsis thaliana genes were constitutively expressed in transgenic carrot plants. Our results suggest that DXS is limiting for the production of both carotenoids and chlorophylls in roots and leaves, whereas the regulatory role of DXR appeared to be minor. Interestingly, increased levels of DXS (but not of DXR) resulted in higher transcript abundance of endogenous carrot genes encoding phytoene synthase, the main rate-determining enzyme of the carotenoid pathway. These results support a central role for DXS on modulating the production of MEP-derived precursors to synthesize carotenoids and chlorophylls in carrot, confirming the pivotal relevance of this enzyme to engineer healthier, carotenoid-enriched products. PMID:27630663

  6. Differential Contribution of the First Two Enzymes of the MEP Pathway to the Supply of Metabolic Precursors for Carotenoid and Chlorophyll Biosynthesis in Carrot (Daucus carota)

    PubMed Central

    Simpson, Kevin; Quiroz, Luis F.; Rodriguez-Concepción, Manuel; Stange, Claudia R.

    2016-01-01

    Carotenoids and chlorophylls are photosynthetic pigments synthesized in plastids from metabolic precursors provided by the methylerythritol 4-phosphate (MEP) pathway. The first two steps in the MEP pathway are catalyzed by the deoxyxylulose 5-phosphate synthase (DXS) and reductoisomerase (DXR) enzymes. While DXS has been recently shown to be the main flux-controlling step of the MEP pathway, both DXS and DXR enzymes have been proven to be able to promote an increase in MEP-derived products when overproduced in diverse plant systems. Carrot (Daucus carota) produces photosynthetic pigments (carotenoids and chlorophylls) in leaves and in light-exposed roots, whereas only carotenoids (mainly α- and β-carotene) accumulate in the storage root in darkness. To evaluate whether DXS and DXR activities influence the production of carotenoids and chlorophylls in carrot leaves and roots, the corresponding Arabidopsis thaliana genes were constitutively expressed in transgenic carrot plants. Our results suggest that DXS is limiting for the production of both carotenoids and chlorophylls in roots and leaves, whereas the regulatory role of DXR appeared to be minor. Interestingly, increased levels of DXS (but not of DXR) resulted in higher transcript abundance of endogenous carrot genes encoding phytoene synthase, the main rate-determining enzyme of the carotenoid pathway. These results support a central role for DXS on modulating the production of MEP-derived precursors to synthesize carotenoids and chlorophylls in carrot, confirming the pivotal relevance of this enzyme to engineer healthier, carotenoid-enriched products.

  7. Differential Contribution of the First Two Enzymes of the MEP Pathway to the Supply of Metabolic Precursors for Carotenoid and Chlorophyll Biosynthesis in Carrot (Daucus carota).

    PubMed

    Simpson, Kevin; Quiroz, Luis F; Rodriguez-Concepción, Manuel; Stange, Claudia R

    2016-01-01

    Carotenoids and chlorophylls are photosynthetic pigments synthesized in plastids from metabolic precursors provided by the methylerythritol 4-phosphate (MEP) pathway. The first two steps in the MEP pathway are catalyzed by the deoxyxylulose 5-phosphate synthase (DXS) and reductoisomerase (DXR) enzymes. While DXS has been recently shown to be the main flux-controlling step of the MEP pathway, both DXS and DXR enzymes have been proven to be able to promote an increase in MEP-derived products when overproduced in diverse plant systems. Carrot (Daucus carota) produces photosynthetic pigments (carotenoids and chlorophylls) in leaves and in light-exposed roots, whereas only carotenoids (mainly α- and β-carotene) accumulate in the storage root in darkness. To evaluate whether DXS and DXR activities influence the production of carotenoids and chlorophylls in carrot leaves and roots, the corresponding Arabidopsis thaliana genes were constitutively expressed in transgenic carrot plants. Our results suggest that DXS is limiting for the production of both carotenoids and chlorophylls in roots and leaves, whereas the regulatory role of DXR appeared to be minor. Interestingly, increased levels of DXS (but not of DXR) resulted in higher transcript abundance of endogenous carrot genes encoding phytoene synthase, the main rate-determining enzyme of the carotenoid pathway. These results support a central role for DXS on modulating the production of MEP-derived precursors to synthesize carotenoids and chlorophylls in carrot, confirming the pivotal relevance of this enzyme to engineer healthier, carotenoid-enriched products.

  8. Reconciling medical expenditure estimates from the MEPS and NHEA, 2002.

    PubMed

    Sing, Merrile; Banthin, Jessica S; Selden, Thomas M; Cowan, Cathy A; Keehan, Sean P

    2006-01-01

    The Medical Expenditure Panel Survey (MEPS) and National Health Expenditure Accounts (NHEA) are often used for health care policy analysis and simulations because they contain comprehensive estimates of national health care expenditures. The NHEA are primarily based on aggregate provider revenue data, while MEPS is based on person-level data on health care expenditures. This article compares MEPS and NHEA expenditure estimates for 2002 and discusses the differences. When MEPS and the NHEA are adjusted to be on a consistent basis, their expenditure estimates differ by 13.8 percent.

  9. Imaging phosphatidylinositol 4-phosphate dynamics in living plant cells.

    PubMed

    Vermeer, Joop E M; Thole, Julie M; Goedhart, Joachim; Nielsen, Erik; Munnik, Teun; Gadella, Theodorus W J

    2009-01-01

    Polyphosphoinositides represent a minor group of phospholipids, accounting for less than 1% of the total. Despite their low abundance, these molecules have been implicated in various signalling and membrane trafficking events. Phosphatidylinositol 4-phosphate (PtdIns4P) is the most abundant polyphosphoinositide. (32)Pi-labelling studies have shown that the turnover of PtdIns4P is rapid, but little is known about where in the cell or plant this occurs. Here, we describe the use of a lipid biosensor that monitors PtdIns4P dynamics in living plant cells. The biosensor consists of a fusion between a fluorescent protein and a lipid-binding domain that specifically binds PtdIns4P, i.e. the pleckstrin homology domain of the human protein phosphatidylinositol-4-phosphate adaptor protein-1 (FAPP1). YFP-PH(FAPP1) was expressed in four plant systems: transiently in cowpea protoplasts, and stably in tobacco BY-2 cells, Medicago truncatula roots and Arabidopsis thaliana seedlings. All systems allowed YFP-PH(FAPP1) expression without detrimental effects. Two distinct fluorescence patterns were observed: labelling of motile punctate structures and the plasma membrane. Co-expression studies with organelle markers revealed strong co-labelling with the Golgi marker STtmd-CFP, but not with the endocytic/pre-vacuolar marker GFP-AtRABF2b. Co-expression with the Ptdins3P biosensor YFP-2 x FYVE revealed totally different localization patterns. During cell division, YFP-PH(FAPP1) showed strong labelling of the cell plate, but PtdIns3P was completely absent from the newly formed cell membrane. In root hairs of M. truncatula and A. thaliana, a clear PtdIns4P gradient was apparent in the plasma membrane, with the highest concentration in the tip. This only occurred in growing root hairs, indicating a role for PtdIns4P in tip growth.

  10. Structural basis for Mep2 ammonium transceptor activation by phosphorylation.

    PubMed

    van den Berg, Bert; Chembath, Anupama; Jefferies, Damien; Basle, Arnaud; Khalid, Syma; Rutherford, Julian C

    2016-04-18

    Mep2 proteins are fungal transceptors that play an important role as ammonium sensors in fungal development. Mep2 activity is tightly regulated by phosphorylation, but how this is achieved at the molecular level is not clear. Here we report X-ray crystal structures of the Mep2 orthologues from Saccharomyces cerevisiae and Candida albicans and show that under nitrogen-sufficient conditions the transporters are not phosphorylated and present in closed, inactive conformations. Relative to the open bacterial ammonium transporters, non-phosphorylated Mep2 exhibits shifts in cytoplasmic loops and the C-terminal region (CTR) to occlude the cytoplasmic exit of the channel and to interact with His2 of the twin-His motif. The phosphorylation site in the CTR is solvent accessible and located in a negatively charged pocket ∼30 Å away from the channel exit. The crystal structure of phosphorylation-mimicking Mep2 variants from C. albicans show large conformational changes in a conserved and functionally important region of the CTR. The results allow us to propose a model for regulation of eukaryotic ammonium transport by phosphorylation.

  11. Structural basis for Mep2 ammonium transceptor activation by phosphorylation

    PubMed Central

    van den Berg, Bert; Chembath, Anupama; Jefferies, Damien; Basle, Arnaud; Khalid, Syma; Rutherford, Julian C.

    2016-01-01

    Mep2 proteins are fungal transceptors that play an important role as ammonium sensors in fungal development. Mep2 activity is tightly regulated by phosphorylation, but how this is achieved at the molecular level is not clear. Here we report X-ray crystal structures of the Mep2 orthologues from Saccharomyces cerevisiae and Candida albicans and show that under nitrogen-sufficient conditions the transporters are not phosphorylated and present in closed, inactive conformations. Relative to the open bacterial ammonium transporters, non-phosphorylated Mep2 exhibits shifts in cytoplasmic loops and the C-terminal region (CTR) to occlude the cytoplasmic exit of the channel and to interact with His2 of the twin-His motif. The phosphorylation site in the CTR is solvent accessible and located in a negatively charged pocket ∼30 Å away from the channel exit. The crystal structure of phosphorylation-mimicking Mep2 variants from C. albicans show large conformational changes in a conserved and functionally important region of the CTR. The results allow us to propose a model for regulation of eukaryotic ammonium transport by phosphorylation. PMID:27088325

  12. Structural basis for Mep2 ammonium transceptor activation by phosphorylation.

    PubMed

    van den Berg, Bert; Chembath, Anupama; Jefferies, Damien; Basle, Arnaud; Khalid, Syma; Rutherford, Julian C

    2016-01-01

    Mep2 proteins are fungal transceptors that play an important role as ammonium sensors in fungal development. Mep2 activity is tightly regulated by phosphorylation, but how this is achieved at the molecular level is not clear. Here we report X-ray crystal structures of the Mep2 orthologues from Saccharomyces cerevisiae and Candida albicans and show that under nitrogen-sufficient conditions the transporters are not phosphorylated and present in closed, inactive conformations. Relative to the open bacterial ammonium transporters, non-phosphorylated Mep2 exhibits shifts in cytoplasmic loops and the C-terminal region (CTR) to occlude the cytoplasmic exit of the channel and to interact with His2 of the twin-His motif. The phosphorylation site in the CTR is solvent accessible and located in a negatively charged pocket ∼30 Å away from the channel exit. The crystal structure of phosphorylation-mimicking Mep2 variants from C. albicans show large conformational changes in a conserved and functionally important region of the CTR. The results allow us to propose a model for regulation of eukaryotic ammonium transport by phosphorylation. PMID:27088325

  13. Structural characterization of MepB from Staphylococcus aureus reveals homology to endonucleases

    PubMed Central

    Agah, Sayeh; Poulos, Sandra; Banchs, Christian; Faham, Salem

    2014-01-01

    The MepRAB operon in Staphylococcus aureus has been identified to play a role in drug resistance. Although the functions of MepA and MepR are known, little information is available on the function of MepB. Here we report the X-ray structure of MepB to 2.1 Å revealing its structural similarity to the PD-(D/E)XK family of endonucleases. We further show that MepB binds DNA and RNA, with a higher affinity towards RNA and single stranded DNA than towards double stranded DNA. Notably, the PD-(D/E)XK catalytic active site residues are not conserved in MepB. MepB's association with a drug resistance operon suggests that it plays a role in responding to antimicrobials. This role is likely carried out through MepB's interactions with nucleic acids. PMID:24501097

  14. Reconciling Medical Expenditure Estimates from the MEPS and NHEA, 2007

    PubMed Central

    Bernard, Didem; Cowan, Cathy; Selden, Thomas; Cai, Liming; Catlin, Aaron; Heffler, Stephen

    2012-01-01

    Objective Provide a comparison of health care expenditure estimates for 2007 from the Medical Expenditure Panel Survey (MEPS) and the National Health Expenditure Accounts (NHEA). Reconciling these estimates serves two important purposes. First, it is an important quality assurance exercise for improving and ensuring the integrity of each source's estimates. Second, the reconciliation provides a consistent baseline of health expenditure data for policy simulations. Our results assist researchers to adjust MEPS to be consistent with the NHEA so that the projected costs as well as budgetary and tax implications of any policy change are consistent with national health spending estimates. Data Sources The Medical Expenditure Panel Survey produced by the Agency for Healthcare Research and Quality, and the National Health Center for Health Statistics and the National Health Expenditures produced by the Centers for Medicare & Medicaid Service's Office of the Actuary. Results In this study, we focus on the personal health care (PHC) sector, which includes the goods and services rendered to treat or prevent a specific disease or condition in an individual. The official 2007 NHEA estimate for PHC spending is $1,915 billion and the MEPS estimate is $1,126 billion. Adjusting the NHEA estimates for differences in underlying populations, covered services, and other measurement concepts reduces the NHEA estimate for 2007 to $1,366 billion. As a result, MEPS is $240 billion, or 17.6 percent, less than the adjusted NHEA total. PMID:24800157

  15. Map-based cloning of zb7 encoding an IPP and DMAPP synthase in the MEP pathway of maize.

    PubMed

    Lu, Xiao-Min; Hu, Xiao-Jiao; Zhao, Yuan-Zeng; Song, Wei-Bin; Zhang, Mei; Chen, Zong-Liang; Chen, Wei; Dong, Yong-Bin; Wang, Zhen-Hua; Lai, Jin-Sheng

    2012-09-01

    IspH is a key enzyme in the last step of the methyl-D-erythritol-4-phosphate (MEP) pathway. Loss of function of IspH can often result in complete yellow or albino phenotype in many plants. Here, we report the characterization of a recessive mutant of maize, zebra7 (zb7), showing transverse green/yellow striped leaves in young plants. The yellow bands of the mutant have decreased levels of chlorophylls and carotenoids with delayed chloroplast development. Low temperature suppressed mutant phenotype, while alternate light/dark cycle or high temperature enlarged the yellow section. Map-based cloning demonstrated that zb7 encodes the IspH protein with a mis-sense mutation in a conserved region. Transgenic silencing of Zb7 in maize resulted in complete albino plantlets that are aborted in a few weeks, confirming that Zb7 is important in the early stages of maize chloroplast development. Zb7 is constitutively expressed and its expression subject to a 16-h light/8-h dark cycle regulation. Our results suggest that the less effective or unstable IspH in zb7 mutant, together with its diurnal expression, are mechanistically accounted for the zebra phenotype. The increased IspH mRNA in the leaves of zb7 at the late development stage may explain the restoration of mutant phenotype in mature stages.

  16. Overexpression of SrUGT85C2 from Stevia reduced growth and yield of transgenic Arabidopsis by influencing plastidial MEP pathway.

    PubMed

    Guleria, Praveen; Masand, Shikha; Yadav, Sudesh Kumar

    2014-04-15

    The transcript expression of a gene SrUGT85C2 has been documented for direct relation with steviol glycoside content in Stevia plant. Steviol glycoside and gibberellin biosynthetic routes are divergent branches of methyl erythritol-4 phosphate (MEP) pathway. So, SrUGT85C2 might be an influencing gibberellin content. Hence in the present study, transgenic Arabidopsis thaliana overexpressing SrUGT85C2 cDNA from Stevia rebaudiana was developed to check its effect on gibberellin accumulation and related plant growth parameters. The developed transgenics showed a noteworthy decrease of 78-83% in GA3 content. Moreover, the transgenics showed a gibberellin deficient phenotype comprising stunted hypocotyl length, reduced shoot growth and a significant fall in relative water content. Transgenics also showed 17-37 and 64-76% reduction in chlorophyll a and chlorophyll b contents, respectively. Reduction in photosynthetic pigments could be responsible for the noticed significant decrease in plant biomass. Like steviol glycoside and gibberellin biosynthesis, chlorophyll biosynthesis also occurs from the precursors isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP) of MEP pathway in the plastids. The observed downregulated expression of genes encoding MEP pathway enzymes geranyl geranyl diphosphate synthase (GGDPS), copalyl diphosphate synthase (CDPS), kaurenoic acid oxidase (KAO), chlorophyll synthetase and chlorophyll a oxygenase in transgenics overexpressing SrUGT85C2 might be responsible for the reduction in gibberellins as well as chlorophyll. This study has documented for the first time the regulatory role of SrUGT85C2 in the biosynthesis of steviol glycoside, gibberellins and chlorophyll.

  17. Natural Variation in Monoterpene Synthesis in Kiwifruit: Transcriptional Regulation of Terpene Synthases by NAC and ETHYLENE-INSENSITIVE3-Like Transcription Factors1

    PubMed Central

    Nieuwenhuizen, Niels J.; Chen, Xiuyin; Wang, Mindy Y.; Matich, Adam J.; Perez, Ramon Lopez; Allan, Andrew C.; Green, Sol A.; Atkinson, Ross G.

    2015-01-01

    Two kiwifruit (Actinidia) species with contrasting terpene profiles were compared to understand the regulation of fruit monoterpene production. High rates of terpinolene production in ripe Actinidia arguta fruit were correlated with increasing gene and protein expression of A. arguta terpene synthase1 (AaTPS1) and correlated with an increase in transcript levels of the 2-C-methyl-d-erythritol 4-phosphate pathway enzyme 1-deoxy-d-xylulose-5-phosphate synthase (DXS). Actinidia chinensis terpene synthase1 (AcTPS1) was identified as part of an array of eight tandemly duplicated genes, and AcTPS1 expression and terpene production were observed only at low levels in developing fruit. Transient overexpression of DXS in Nicotiana benthamiana leaves elevated monoterpene synthesis by AaTPS1 more than 100-fold, indicating that DXS is likely to be the key step in regulating 2-C-methyl-d-erythritol 4-phosphate substrate flux in kiwifruit. Comparative promoter analysis identified potential NAC (for no apical meristem [NAM], Arabidopsis transcription activation factor [ATAF], and cup-shaped cotyledon [CUC])-domain transcription factor) and ETHYLENE-INSENSITIVE3-like transcription factor (TF) binding sites in the AaTPS1 promoter, and cloned members of both TF classes were able to activate the AaTPS1 promoter in transient assays. Electrophoretic mobility shift assays showed that AaNAC2, AaNAC3, and AaNAC4 bind a 28-bp fragment of the proximal NAC binding site in the AaTPS1 promoter but not the A. chinensis AcTPS1 promoter, where the NAC binding site was mutated. Activation could be restored by reintroducing multiple repeats of the 12-bp NAC core-binding motif. The absence of NAC transcriptional activation in ripe A. chinensis fruit can account for the low accumulation of AcTPS1 transcript, protein, and monoterpene volatiles in this species. These results indicate the importance of NAC TFs in controlling monoterpene production and other traits in ripening fruits. PMID:25649633

  18. Natural variation in monoterpene synthesis in kiwifruit: transcriptional regulation of terpene synthases by NAC and ETHYLENE-INSENSITIVE3-like transcription factors.

    PubMed

    Nieuwenhuizen, Niels J; Chen, Xiuyin; Wang, Mindy Y; Matich, Adam J; Perez, Ramon Lopez; Allan, Andrew C; Green, Sol A; Atkinson, Ross G

    2015-04-01

    Two kiwifruit (Actinidia) species with contrasting terpene profiles were compared to understand the regulation of fruit monoterpene production. High rates of terpinolene production in ripe Actinidia arguta fruit were correlated with increasing gene and protein expression of A. arguta terpene synthase1 (AaTPS1) and correlated with an increase in transcript levels of the 2-C-methyl-D-erythritol 4-phosphate pathway enzyme 1-deoxy-D-xylulose-5-phosphate synthase (DXS). Actinidia chinensis terpene synthase1 (AcTPS1) was identified as part of an array of eight tandemly duplicated genes, and AcTPS1 expression and terpene production were observed only at low levels in developing fruit. Transient overexpression of DXS in Nicotiana benthamiana leaves elevated monoterpene synthesis by AaTPS1 more than 100-fold, indicating that DXS is likely to be the key step in regulating 2-C-methyl-D-erythritol 4-phosphate substrate flux in kiwifruit. Comparative promoter analysis identified potential NAC (for no apical meristem [NAM], Arabidopsis transcription activation factor [ATAF], and cup-shaped cotyledon [CUC])-domain transcription factor) and ETHYLENE-INSENSITIVE3-like transcription factor (TF) binding sites in the AaTPS1 promoter, and cloned members of both TF classes were able to activate the AaTPS1 promoter in transient assays. Electrophoretic mobility shift assays showed that AaNAC2, AaNAC3, and AaNAC4 bind a 28-bp fragment of the proximal NAC binding site in the AaTPS1 promoter but not the A. chinensis AcTPS1 promoter, where the NAC binding site was mutated. Activation could be restored by reintroducing multiple repeats of the 12-bp NAC core-binding motif. The absence of NAC transcriptional activation in ripe A. chinensis fruit can account for the low accumulation of AcTPS1 transcript, protein, and monoterpene volatiles in this species. These results indicate the importance of NAC TFs in controlling monoterpene production and other traits in ripening fruits.

  19. Phosphatidylinositol(4,5)bisphosphate and Phosphatidylinositol(4)phosphate in Plant Tissues 1

    PubMed Central

    Irvine, R. F.; Letcher, A. J.; Lander, D. J.; Drøbak, B. K.; Dawson, A. P.; Musgrave, A.

    1989-01-01

    Pea (Pisum sativum) leaf discs or swimming suspensions of Chlamydomonas eugametos were radiolabeled with [3H]myo-inositol or [32P]Pi and the lipids were extracted, deacylated, and their glycerol moieties removed. The resulting inositol trisphosphate and bisphosphate fractions were examined by periodate degradation, reduction and dephosphorylation, or by incubation with human red cell membranes. Their likely structures were identified as d-myo-inositol(1,4,5)trisphosphate and d-myo-inositol(1,4,)-bisphosphate. It is concluded that plants contain phosphatidylinositol(4)phosphate and phosphatidylinositol(4,5)bisphosphate; no other polyphosphoinositides were detected. PMID:16666637

  20. Phosphatidylinositol(4,5)bisphosphate and phosphatidylinositol(4)phosphate in plant tissues. [Pisum sativum

    SciTech Connect

    Irvine, R.F.; Letcher, A.J.; Lander, D.J. ); Dawson, A.P. ); Musgrave, A. ); Drobak, B.K. )

    1989-03-01

    Pea (Pisum sativum) leaf discs or swimming suspensions of Chlamydomonas eugametos were radiolabeled with ({sup 3}H)myo-inositol or ({sup 32}P)Pi and the lipids were extracted, deacylated, and their glycerol moieties removed. The resulting inositol trisphosphate and bisphosphate fractions were examined by periodate degradation, reduction and dephosphorylation, or by incubation with human red cell membranes. Their likely structures were identified as D-myo-inositol(1,4,5)trisphosphate and D-myo-inositol(1,4,)-bisphosphate. It is concluded that plants contain phosphatidylinositol(4)phosphate and phosphatidylinositol(4,5)bisphosphate; no other polyphosphoinositides were detected.

  1. Methylerythritol phosphate pathway to isoprenoids: kinetic modeling and in silico enzyme inhibitions in Plasmodium falciparum.

    PubMed

    Singh, Vivek Kumar; Ghosh, Indira

    2013-09-01

    The methylerythritol phosphate (MEP) pathway of Plasmodium falciparum (P. falciparum) has become an attractive target for anti-malarial drug discovery. This study describes a kinetic model of this pathway, its use in validating 1-deoxy-d-xylulose 5-phosphate reductoisomerase (DXR) as drug target from the systemic perspective, and additional target identification, using metabolic control analysis and in silico inhibition studies. In addition to DXR, 1-deoxy-d-xylulose 5-phosphate synthase (DXS) can be targeted because it is the first enzyme of the pathway and has the highest flux control coefficient followed by that of DXR. In silico inhibition of both enzymes caused large decrement in the pathway flux. An added advantage of targeting DXS is its influence on vitamin B1 and B6 biosynthesis. Two more potential targets, 2-C-methyl-d-erythritol 2,4-cyclodiphosphate synthase and 1-hydroxy-2-methyl-2-(E)-butenyl 4-diphosphate synthase, were also identified. Their inhibition caused large accumulation of their substrates causing instability of the system. This study demonstrates that both types of enzyme targets, one acting via flux reduction and the other by metabolite accumulation, exist in P. falciparum MEP pathway. These groups of targets can be exploited for independent anti-malarial drugs.

  2. Cytidine derivatives as IspF inhibitors of Burkolderia pseudomallei

    PubMed Central

    Zhang, Zheng; Jakkaraju, Sriram; Blain, Joy; Gogol, Kenneth; Zhao, Lei; Hartley, Robert C.; Karlsson, Courtney A.; Staker, Bart L.; Stewart, Lance J.; Myler, Peter J.; Clare, Michael; Begley, Darren W.; Horn, James R.; Hagen, Timothy J

    2013-01-01

    Published biological data suggest that the methyl erythritol phosphate (MEP) pathway, a non-mevalonate isoprenoid biosynthetic pathway, is essential for certain bacteria and other infectious disease organisms. One highly conserved enzyme in the MEP pathway is 2C-methyl-D-erythritol 2,4-cyclodiphosphate synthase (IspF). Fragment-bound complexes of IspF from Burkholderia pseudomallei were used to design and synthesize a series of molecules linking the cytidine moiety to different zinc pocket fragment binders. Testing by surface plasmon resonance (SPR) found one molecule in the series to possess binding affinity equal to that of cytidine diphosphate, despite lacking any metal-coordinating phosphate groups. Close inspection of the SPR data suggest different binding stoichiometries between IspF and test compounds. Crystallographic analysis shows important variations between the binding mode of one synthesized compound and the pose of the bound fragment from which it was designed. The binding modes of these molecules add to our structural knowledge base for IspF and suggest future refinements in this compound series. PMID:24157367

  3. A specific process to purify 2-methyl-D-erythritol-4-phosphate enzymatically converted from D-glyceraldehyde-3-phosphate and pyruvate.

    PubMed

    Yang, Shao-Qing; Deng, Jian; Wu, Qian-Qian; Li, Heng; Gao, Wen-Yun

    2015-02-01

    A one-pot enzymatic cascade was established to synthesize MEP, one of the key intermediates in the MEP terpenoid biosynthetic pathway. D-GAP and sodium pyruvate were converted to MEP in a reaction catalyzed by DXP synthase and DXP reductoisomerase (DXR) in the presence of the coenzymes ThPP, NADPH, and Mg2+. The product was then isolated by using a specific two-step purification process and MEP was obtained in a yield of nearly 60% and high purity. Importantly, MEP prepared by this way was totally free from contamination by minor amounts of DXP that was not completely convertible by DXR.

  4. MESA/MEP at American River College: Year One Evaluation Report.

    ERIC Educational Resources Information Center

    Lee, Beth S.; And Others

    In 1989, the Mathematics, Engineering, and Science Achievement (MESA)/Minority Engineering Program (MEP) was initiated at American River College. The MESA/MEP program recruits Black, Hispanic, and Native American students and provides assistance, encouragement, and enrichment programs to help them succeed in the fields of mathematics, engineering,…

  5. 75 FR 33769 - Manufacturing Extension Partnership (MEP) Availability of Funds for Projects To Develop and...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2010-06-15

    ... organizations for projects that address new and emerging competitive needs of manufacturers in areas consistent.... Please see the NIST MEP Web site, http://www.nist.gov/mep for details on these strategies. Funded... region, and contribute to the long-term economic stability of the region. Competitive projects will...

  6. 77 FR 12041 - Applications for New Awards; Migrant Education Program (MEP) Consortium Incentive Grants Program

    Federal Register 2010, 2011, 2012, 2013, 2014

    2012-02-28

    ... Applications for New Awards; Migrant Education Program (MEP) Consortium Incentive Grants Program AGENCY: Office...: Migrant Education Program (MEP) Consortium Incentive Grants Program; Notice inviting applications for new... appropriate entities to improve the delivery of services to migrant children whose education is...

  7. Erythritol feeds the pentose phosphate pathway via three new isomerases leading to D-erythrose-4-phosphate in Brucella

    PubMed Central

    Barbier, Thibault; Collard, François; Zúñiga-Ripa, Amaia; Moriyón, Ignacio; Godard, Thibault; Becker, Judith; Wittmann, Christoph; Van Schaftingen, Emile; Letesson, Jean-Jacques

    2014-01-01

    Erythritol is an important nutrient for several α-2 Proteobacteria, including N2-fixing plant endosymbionts and Brucella, a worldwide pathogen that finds this four-carbon polyol in genital tissues. Erythritol metabolism involves phosphorylation to l-erythritol-4-phosphate by the kinase EryA and oxidation of the latter to l-3-tetrulose 4-phosphate by the dehydrogenase EryB. It is accepted that further steps involve oxidation by the putative dehydrogenase EryC and subsequent decarboxylation to yield triose-phosphates. Accordingly, growth on erythritol as the sole C source should require aldolase and fructose-1,6-bisphosphatase to produce essential hexose-6-monophosphate. However, we observed that a mutant devoid of fructose-1,6-bisphosphatases grew normally on erythritol and that EryC, which was assumed to be a dehydrogenase, actually belongs to the xylose isomerase superfamily. Moreover, we found that TpiA2 and RpiB, distant homologs of triose phosphate isomerase and ribose 5-phosphate isomerase B, were necessary, as previously shown for Rhizobium. By using purified recombinant enzymes, we demonstrated that l-3-tetrulose-4-phosphate was converted to d-erythrose 4-phosphate through three previously unknown isomerization reactions catalyzed by EryC (tetrulose-4-phosphate racemase), TpiA2 (d-3-tetrulose-4-phosphate isomerase; renamed EryH), and RpiB (d-erythrose-4-phosphate isomerase; renamed EryI), a pathway fully consistent with the isotopomer distribution of the erythrose-4-phosphate-derived amino acids phenylalanine and tyrosine obtained from bacteria grown on 13C-labeled erythritol. d-Erythrose-4-phosphate is then converted by enzymes of the pentose phosphate pathway to glyceraldehyde 3-phosphate and fructose 6-phosphate, thus bypassing fructose-1,6-bisphosphatase. This is the first description to our knowledge of a route feeding carbohydrate metabolism exclusively via d-erythrose 4-phosphate, a pathway that may provide clues to the preferential metabolism of

  8. Erythritol feeds the pentose phosphate pathway via three new isomerases leading to D-erythrose-4-phosphate in Brucella.

    PubMed

    Barbier, Thibault; Collard, François; Zúñiga-Ripa, Amaia; Moriyón, Ignacio; Godard, Thibault; Becker, Judith; Wittmann, Christoph; Van Schaftingen, Emile; Letesson, Jean-Jacques

    2014-12-16

    Erythritol is an important nutrient for several α-2 Proteobacteria, including N2-fixing plant endosymbionts and Brucella, a worldwide pathogen that finds this four-carbon polyol in genital tissues. Erythritol metabolism involves phosphorylation to L-erythritol-4-phosphate by the kinase EryA and oxidation of the latter to L-3-tetrulose 4-phosphate by the dehydrogenase EryB. It is accepted that further steps involve oxidation by the putative dehydrogenase EryC and subsequent decarboxylation to yield triose-phosphates. Accordingly, growth on erythritol as the sole C source should require aldolase and fructose-1,6-bisphosphatase to produce essential hexose-6-monophosphate. However, we observed that a mutant devoid of fructose-1,6-bisphosphatases grew normally on erythritol and that EryC, which was assumed to be a dehydrogenase, actually belongs to the xylose isomerase superfamily. Moreover, we found that TpiA2 and RpiB, distant homologs of triose phosphate isomerase and ribose 5-phosphate isomerase B, were necessary, as previously shown for Rhizobium. By using purified recombinant enzymes, we demonstrated that L-3-tetrulose-4-phosphate was converted to D-erythrose 4-phosphate through three previously unknown isomerization reactions catalyzed by EryC (tetrulose-4-phosphate racemase), TpiA2 (D-3-tetrulose-4-phosphate isomerase; renamed EryH), and RpiB (D-erythrose-4-phosphate isomerase; renamed EryI), a pathway fully consistent with the isotopomer distribution of the erythrose-4-phosphate-derived amino acids phenylalanine and tyrosine obtained from bacteria grown on (13)C-labeled erythritol. D-erythrose-4-phosphate is then converted by enzymes of the pentose phosphate pathway to glyceraldehyde 3-phosphate and fructose 6-phosphate, thus bypassing fructose-1,6-bisphosphatase. This is the first description to our knowledge of a route feeding carbohydrate metabolism exclusively via D-erythrose 4-phosphate, a pathway that may provide clues to the preferential metabolism of

  9. Effects of polyamines and calcium and sodium ions on smooth muscle cytoskeleton-associated phosphatidylinositol (4)-phosphate 5-kinase.

    PubMed

    Chen, H; Baron, C B; Griffiths, T; Greeley, P; Coburn, R F

    1998-10-01

    In many different cell types, including smooth muscle cells (Baron et al., 1989, Am. J. Physiol., 256: C375-383; Baron et al., J. Pharmacol. Exp. Ther. 266: 8-15), phosphatidylinositol (4)-phosphate 5-kinase plays a critical role in the regulation of membrane concentrations of phosphatidylinositol (4,5)-bisphosphate and formation of inositol (1,4,5)-trisphosphate. In unstimulated porcine trachealis smooth muscle, 70% of total cellular phosphatidylinositol (4)-phosphate 5-kinase activity was associated with cytoskeletal proteins and only trace activity was detectable in isolated sarcolemma. Using two different preparations, we studied cytoskeleton-associated phosphatidyl inositol (4)-phosphate 5-kinase under conditions that attempted to mimic the ionic and thermal cytoplasmic environment of living cells. The cytoskeleton-associated enzyme, studied using phosphatidylinositol (4)-phosphate substrate concentrations that produced phosphatidylinositol 4,5-bisphosphate at about 10% of the maximal rate, was sensitive to free [Mg2+], had an absolute requirement for phosphatidylserine, phosphatidic acid, or phosphatidylinositol, and included type I isoforms. At 0.5 mM free [Mg2+], physiological spermine concentrations, 0.2-0.4 mM, increased phosphatidylinositol (4)-phosphate 5-kinase activity two to four times compared to controls run without spermine. The EC50 for spermine-evoked increases in activity was 0.17 +/- 0.02 mM. Spermine-evoked enzyme activity was a function of both free [Mg2+] and substrate concentration. Cytoskeleton-associated phosphatidylinositol (4)-phosphate 5-kinase was inhibited by free [Ca2+] over a physiological range for cytoplasm--10(-8) to 10(-5) M, an effect independent of the presence of calmodulin. Na+ over the range 20 to 50 mM also inhibited this enzyme activated by 5 mM Mg2+ but had no effect on spermine-activated enzyme. Na+, Ca2+, and spermine appear to be physiological modulators of smooth muscle cytoskeleton-bound phosphatidylinositol (4

  10. A sugar phosphatase regulates the methylerythritol phosphate (MEP) pathway in malaria parasites

    PubMed Central

    Edwards, Rachel L.; Kelly, Megan L.; Hodge, Dana M.; Tolia, Niraj H.; Odom, Audrey R.

    2014-01-01

    Isoprenoid biosynthesis through the methylerythritol phosphate (MEP) pathway generates commercially important products and is a target for antimicrobial drug development. MEP pathway regulation is poorly understood in microorganisms. We employ a forward genetics approach to understand MEP pathway regulation in the malaria parasite, Plasmodium falciparum. The antimalarial fosmidomycin inhibits the MEP pathway enzyme deoxyxylulose 5-phosphate reductoisomerase (DXR). Fosmidomycin-resistant P. falciparum are enriched for changes in the PF3D7_1033400 locus (hereafter referred to as PfHAD1), encoding a homologue of haloacid dehalogenase (HAD)-like sugar phosphatases. We describe the structural basis for loss-of-function PfHAD1 alleles and find that PfHAD1 dephosphorylates a variety of sugar phosphates, including glycolytic intermediates. Loss of PfHAD1 is required for fosmidomycin resistance. Parasites lacking PfHAD1 have increased MEP pathway metabolites, particularly the DXR substrate, deoxyxylulose 5-phosphate. PfHAD1 therefore controls substrate availability to the MEP pathway. Because PfHAD1 has homologs in plants and bacteria, other HAD proteins may be MEP pathway regulators. PMID:25058848

  11. Histones Cause Aggregation and Fusion of Lipid Vesicles Containing Phosphatidylinositol-4-Phosphate

    PubMed Central

    Lete, Marta G.; Sot, Jesus; Gil, David; Valle, Mikel; Medina, Milagros; Goñi, Felix M.; Alonso, Alicia

    2015-01-01

    In a previous article, we demonstrated that histones (H1 or histone octamers) interact with negatively charged bilayers and induce extensive aggregation of vesicles containing phosphatidylinositol-4-phosphate (PIP) and, to a lesser extent, vesicles containing phosphatidylinositol (PI). Here, we found that vesicles containing PIP, but not those containing PI, can undergo fusion induced by histones. Fusion was demonstrated through the observation of intervesicular mixing of total lipids and inner monolayer lipids, and by ultrastructural and confocal microscopy studies. Moreover, in both PI- and PIP-containing vesicles, histones caused permeabilization and release of vesicular aqueous contents, but the leakage mechanism was different (all-or-none for PI and graded release for PIP vesicles). These results indicate that histones could play a role in the remodeling of the nuclear envelope that takes place during the mitotic cycle. PMID:25692591

  12. Clinical Value of the Assessment of Changes in MEP Duration with Voluntary Contraction

    PubMed Central

    Brum, Marisa; Cabib, Christopher; Valls-Solé, Josep

    2016-01-01

    Transcranial magnetic stimulation (TMS) gives rise to muscle responses, known as motor evoked potentials (MEP), through activation of the motor pathways. Voluntary contraction causes facilitation of MEPs, which consists of shortening MEP latency, increasing MEP amplitude and widening MEP duration. While an increase in excitability of alpha motorneurons and the corticospinal tract can easily explain latency shortening and amplitude increase, other mechanisms have to be accounted for to explain the increase in duration. We measured the increase in duration of the MEP during contraction with respect to rest in a group of healthy volunteers and retrospectively assessed this parameter in patients who were examined in a standardized fashion during the past 5 years. We included 25 healthy subjects, 21 patients with multiple sclerosis, 33 patients with acute stroke, 5 patients with hereditary spastic paraparesis, and 5 patients with signs suggesting psychogenic paresis. We found already significant differences among groups in the MEP duration at rest, patients with MS had a significantly longer duration, and patients with stroke had significantly shorter duration, than the other two groups. The increase in MEP duration during voluntary contraction was different in patients and in healthy subjects. It was significantly shorter in MS and significantly longer in stroke patients. It was absent in the five patients with suspected psychogenic weakness. In patients with HSP, an abnormally increase in duration occurred only in leg muscles. Our results suggest that the increase in duration of the MEP during contraction may reveal the contribution of propriospinal interneurons to the activation of alpha motorneurons. This mechanism may be altered in some diseases and, therefore, the assessment proposed in this work may have clinical applicability for the differential diagnosis of weakness. PMID:26793051

  13. Unmanned Multiple Exploratory Probe System (MEPS) for Mars observation. Volume 2: Calculations and derivations

    NASA Technical Reports Server (NTRS)

    Adams, Daniel E.; Crumbly, Christopher M.; Delp, Steve E.; Guidry, Michelle A.; Lisano, Michael E.; Packard, James D.; Striepe, Scott A.

    1988-01-01

    This volume of the final report on the unmanned Multiple Exploratory Probe System (MEPS) details all calculations, derivations, and computer programs that support the information presented in the first volume.

  14. 78 FR 21109 - Manufacturing Extension Partnership (MEP) Center for Nebraska; Availability of Funds

    Federal Register 2010, 2011, 2012, 2013, 2014

    2013-04-09

    ... National Institute of Standards and Technology Manufacturing Extension Partnership (MEP) Center for... applicants for funding projects that provide manufacturing extension services to primarily small- and medium... obtained by contacting Diane Henderson, National Institute of Standards and Technology,...

  15. 77 FR 12563 - Manufacturing Extension Partnership (MEP) Centers for South Dakota and Kentucky; Availability of...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2012-03-01

    ... National Institute of Standards and Technology Manufacturing Extension Partnership (MEP) Centers for South... eligible proposers for funding projects to provide manufacturing extension services to primarily small- and... Diane Henderson, National Institute of Standards and Technology, Manufacturing Extension...

  16. 77 FR 23462 - Proposed Information Collection; Comment Request; Manufacturing Extension Partnership (MEP...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2012-04-19

    ...; Manufacturing Extension Partnership (MEP) Management Information Reporting AGENCY: National Institute of... McMahon, National Institute of Standards and Technology--Manufacturing Extension Partnership, 100... Deirdre.mcmahon@nist.gov . SUPPLEMENTARY INFORMATION: I. Abstract Sponsored by NIST, the...

  17. Revisit of the Global Surface Energy Balance Using the MEP Model of Surface Heat Fluxes

    NASA Astrophysics Data System (ADS)

    Deng, Y.; Wang, J.; Park, T. W.; Ming, Y.

    2015-12-01

    The recently proposed model of surface heat fluxes, based on the theory of maximum entropy production (MEP), was used to estimate the global evapotranspiration (ET) and heat fluxes. Compared to bulk transfer models, the MEP model has several remote-sensing-friendly features including fewer input variables, automatic closure of surface energy budget, being independent of bulk gradients of temperature and water vapor, not using wind speed and surface roughness as model parameters, and being less sensitive to uncertainties of input variables and model parameters. The MEP model is formulated for the entire range of soil moisture from dryness to saturation over the land surfaces and has even more advantages over water-snow-ice surfaces compared to traditional methods due to its independence of surface humidity data. The MEP model provides the first global maps of water heat fluxes at ocean surfaces as well as conductive heat fluxes at snow/ice covered polar regions. Ten years of Clouds and the Earth's Radiant Energy System (CERES) earth surface radiation fluxes, surface temperature data products supplemented (when needed) by the Modern-Era Retrospective analysis for Research and Applications (MERRA) surface specific humidity data are used to test the MEP model by comparing the MEP based global annual ET and heat fluxes with existing products. The MEP based fluxes over land surfaces agree closely with previous studies. Over the oceans, the MEP modeled ET tends to be lower than previous estimates while those of sensible heat fluxes are in close agreement with previous studies. A counterpart, "off-line" analysis is also carried out using the NOAA GFDL climate model output from a control experiment and a "warming" experiment. Substantial differences in the warming-related changes of ET and Bowen ratio are found over regions such as North Africa and the southwestern U.S. The implications of these differences for understanding trends and variability in regional energy and

  18. MEP and planetary climates: insights from a two-box climate model containing atmospheric dynamics

    PubMed Central

    Jupp, Tim E.; Cox, Peter M.

    2010-01-01

    A two-box model for equator-to-pole planetary heat transport is extended to include simple atmospheric dynamics. The surface drag coefficient CD is treated as a free parameter and solutions are calculated analytically in terms of the dimensionless planetary parameters η (atmospheric thickness), ω (rotation rate) and ξ (advective capability). Solutions corresponding to maximum entropy production (MEP) are compared with solutions previously obtained from dynamically unconstrained two-box models. As long as the advective capability ξ is sufficiently large, dynamically constrained MEP solutions are identical to dynamically unconstrained MEP solutions. Consequently, the addition of a dynamical constraint does not alter the previously obtained MEP results for Earth, Mars and Titan, and an analogous result is presented here for Venus. The rate of entropy production in an MEP state is shown to be independent of rotation rate if the advective capability ξ is sufficiently large (as for the four examples in the solar system), or if the rotation rate ω is sufficiently small. The model indicates, however, that the dynamical constraint does influence the MEP state when ξ is small, which might be the case for some extrasolar planets. Finally, results from the model developed here are compared with previous numerical simulations in which the effect of varying surface drag coefficient on entropy production was calculated. PMID:20368254

  19. MEP and planetary climates: insights from a two-box climate model containing atmospheric dynamics.

    PubMed

    Jupp, Tim E; Cox, Peter M

    2010-05-12

    A two-box model for equator-to-pole planetary heat transport is extended to include simple atmospheric dynamics. The surface drag coefficient CD is treated as a free parameter and solutions are calculated analytically in terms of the dimensionless planetary parameters eta (atmospheric thickness), omega (rotation rate) and xi (advective capability). Solutions corresponding to maximum entropy production (MEP) are compared with solutions previously obtained from dynamically unconstrained two-box models. As long as the advective capability xi is sufficiently large, dynamically constrained MEP solutions are identical to dynamically unconstrained MEP solutions. Consequently, the addition of a dynamical constraint does not alter the previously obtained MEP results for Earth, Mars and Titan, and an analogous result is presented here for Venus. The rate of entropy production in an MEP state is shown to be independent of rotation rate if the advective capability xi is sufficiently large (as for the four examples in the solar system), or if the rotation rate omega is sufficiently small. The model indicates, however, that the dynamical constraint does influence the MEP state when xi is small, which might be the case for some extrasolar planets. Finally, results from the model developed here are compared with previous numerical simulations in which the effect of varying surface drag coefficient on entropy production was calculated. PMID:20368254

  20. Hamsters vaccinated with Ace-mep-7 DNA vaccine produced protective immunity against Ancylostoma ceylanicum infection.

    PubMed

    Wiśniewski, Marcin; Jaros, Sławomir; Bąska, Piotr; Cappello, Michael; Długosz, Ewa; Wędrychowicz, Halina

    2016-04-01

    Hookworms are intestinal nematodes that infect up to 740 million people, mostly in tropical and subtropical regions. Adult worms suck blood from damaged vessels in the gut mucosa, digesting hemoglobin using aspartic-, cysteine- and metalloproteases. Targeting aspartic hemoglobinases using drugs or vaccines is therefore a promising approach to ancylostomiasis control. Based on homology to metalloproteases from other hookworm species, we cloned the Ancylostoma ceylanicum metalloprotease 7 cDNA (Ace-mep-7). The corresponding Ace-MEP-7 protein has a predicted molecular mass of 98.8 kDa. The homology to metallopeptidases from other hookworm species and its predicted transmembrane region support the hypothesis that Ace-MEP-7 may be involved in hemoglobin digestion in the hookworm gastrointestinal tract, especially that our analyses show expression of Ace-mep-7 in the adult stage of the parasite. Immunization of Syrian golden hamsters with Ace-mep-7 cDNA resulted in 50% (p < 0.01) intestinal worm burden reduction. Additionally 78% (p < 0.05) egg count reduction in both sexes was observed. These results suggest that immunization with Ace-mep-7 may contribute to reduction in egg count released into the environment during the A. ceylanicum infection. PMID:26795262

  1. MEP and planetary climates: insights from a two-box climate model containing atmospheric dynamics.

    PubMed

    Jupp, Tim E; Cox, Peter M

    2010-05-12

    A two-box model for equator-to-pole planetary heat transport is extended to include simple atmospheric dynamics. The surface drag coefficient CD is treated as a free parameter and solutions are calculated analytically in terms of the dimensionless planetary parameters eta (atmospheric thickness), omega (rotation rate) and xi (advective capability). Solutions corresponding to maximum entropy production (MEP) are compared with solutions previously obtained from dynamically unconstrained two-box models. As long as the advective capability xi is sufficiently large, dynamically constrained MEP solutions are identical to dynamically unconstrained MEP solutions. Consequently, the addition of a dynamical constraint does not alter the previously obtained MEP results for Earth, Mars and Titan, and an analogous result is presented here for Venus. The rate of entropy production in an MEP state is shown to be independent of rotation rate if the advective capability xi is sufficiently large (as for the four examples in the solar system), or if the rotation rate omega is sufficiently small. The model indicates, however, that the dynamical constraint does influence the MEP state when xi is small, which might be the case for some extrasolar planets. Finally, results from the model developed here are compared with previous numerical simulations in which the effect of varying surface drag coefficient on entropy production was calculated.

  2. In situ localization of gene transcriptions for monoterpene synthesis in irregular parenchymic cells surrounding the secretory cavities in rough lemon (Citrus jambhiri).

    PubMed

    Yamasaki, Yumiko; Akimitsu, Kazuya

    2007-11-01

    A cDNA (RlemispF) encoding 2-C-methyl-d-erythritol 2,4-cyclodiphosphate synthase, an enzyme of the methyl erythritol phosphate (MEP) pathway, and two homologs (RlemTPS1 and RlemTPS2) of citrus monoterpene synthase cDNA were isolated from the rough lemon (Citrus jambhiri). Transient localization of all or a part of RlemispF fused to a green fluorescence protein using particle gun-mediated DNA delivery localized RlemispF in the chloroplast. Transcripts of RlemispF and other monoterpene synthase genes are constitutively expressed in leaves of rough lemon. Transcript accumulations of RlemispF and RlemTPS1 were not induced by microbe attacks, but microbe attack weakly induced RlemTPS2 expression. Wounding decreased RlemispF expression. RlemispF and two different monoterpene synthase genes were specifically expressed in the epithelial tissue cells with dense cytoplasm that surround secretory cavities, which form a broadly round package containing a large volume of essential oils composed of monoterpenes. Interestingly, although expressions of RlemTPS1 and RlemTPS2 were detected at both mature and developing secretory cavities, the RlemispF-expressing cells were found more at around developing secretory cavities.

  3. Two distinct pathways for essential metabolic precursors for isoprenoid biosynthesis

    PubMed Central

    KUZUYAMA, Tomohisa; SETO, Haruo

    2012-01-01

    Isoprenoids are a diverse group of molecules found in all organisms, where they perform such important biological functions as hormone signaling (e.g., steroids) in mammals, antioxidation (e.g., carotenoids) in plants, electron transport (e.g., ubiquinone), and cell wall biosynthesis intermediates in bacteria. All isoprenoids are synthesized by the consecutive condensation of the five-carbon monomer isopentenyl diphosphate (IPP) to its isomer, dimethylallyl diphosphate (DMAPP). The biosynthetic pathway for the formation of IPP from acetyl-CoA (i.e., the mevalonate pathway) had been established mainly in mice and the budding yeast Saccharomyces cerevisiae. Curiously, most prokaryotic microorganisms lack homologs of the genes in the mevalonate pathway, even though IPP and DMAPP are essential for isoprenoid biosynthesis in bacteria. This observation provided an impetus to search for an alternative pathway to synthesize IPP and DMAPP, ultimately leading to the discovery of the mevalonate-independent 2-C-methyl-d-erythritol 4-phosphate pathway. This review article focuses on our significant contributions to a comprehensive understanding of the biosynthesis of IPP and DMAPP. PMID:22450534

  4. Genetic structure and regulation of isoprene synthase in Poplar (Populus spp.).

    PubMed

    Vickers, Claudia E; Possell, Malcolm; Nicholas Hewitt, C; Mullineaux, Philip M

    2010-07-01

    Isoprene is a volatile 5-carbon hydrocarbon derived from the chloroplastic methylerythritol 2-C-methyl-D: -erythritol 4-phosphate isoprenoid pathway. In plants, isoprene emission is controlled by the enzyme isoprene synthase; however, there is still relatively little known about the genetics and regulation of this enzyme. Isoprene synthase gene structure was analysed in three poplar species. It was found that genes encoding stromal isoprene synthase exist as a small gene family, the members of which encode virtually identical proteins and are differentially regulated. Accumulation of isoprene synthase protein is developmentally regulated, but does not differ between sun and shade leaves and does not increase when heat stress is applied. Our data suggest that, in mature leaves, isoprene emission rates are primarily determined by substrate (dimethylallyl diphosphate, DMADP) availability. In immature leaves, where isoprene synthase levels are variable, emission levels are also influenced by the amount of isoprene synthase protein. No thylakoid isoforms could be identified in Populus alba or in Salix babylonica. Together, these data show that control of isoprene emission at the genetic level is far more complicated than previously assumed.

  5. Cloning, Characterization, and Immunolocalization of a Mycorrhiza-Inducible 1-Deoxy-D-Xylulose 5-Phosphate Reductoisomerase in Arbuscule-Containing Cells of Maize1

    PubMed Central

    Hans, Joachim; Hause, Bettina; Strack, Dieter; Walter, Michael H.

    2004-01-01

    Colonization of plant roots by symbiotic arbuscular mycorrhizal fungi frequently leads to the accumulation of several apocarotenoids. The corresponding carotenoid precursors originate from the plastidial 2-C-methyl-d-erythritol 4-phosphate pathway. We have cloned and characterized 1-deoxy-d-xylulose 5-phosphate reductoisomerase (DXR), catalyzing the first committed step of the pathway, from maize (Zea mays). Functional identification was accomplished by heterologous expression of sequences coding for the mature protein in Escherichia coli. DXR is up-regulated in maize roots during mycorrhization as shown at transcript and protein levels, but is also abundant in leaves and young seedlings. Inspection of sequenced genomes and expressed sequence tag (EST) databases argue for a single-copy DXR gene. Immunolocalization studies in mycorrhizal roots using affinity-purified antibodies revealed a DXR localization in plastids around the main symbiotic structures, the arbuscules. DXR protein accumulation is tightly correlated with arbuscule development. The highest level of DXR protein is reached around maturity and initial senescence of these structures. We further demonstrate the formation of a DXR-containing plastidial network around arbuscules, which is highly interconnected in the mature, functional state of the arbuscules. Our findings imply a functional role of a still unknown nature for the apocarotenoids or their respective carotenoid precursors in the arbuscular life cycle. PMID:14764905

  6. A Geranylfarnesyl Diphosphate Synthase Provides the Precursor for Sesterterpenoid (C25) Formation in the Glandular Trichomes of the Mint Species Leucosceptrum canum.

    PubMed

    Liu, Yan; Luo, Shi-Hong; Schmidt, Axel; Wang, Guo-Dong; Sun, Gui-Ling; Grant, Marcus; Kuang, Ce; Yang, Min-Jie; Jing, Shu-Xi; Li, Chun-Huan; Schneider, Bernd; Gershenzon, Jonathan; Li, Sheng-Hong

    2016-03-01

    Plant sesterterpenoids, an important class of terpenoids, are widely distributed in various plants, including food crops. However, little is known about their biosynthesis. Here, we cloned and functionally characterized a plant geranylfarnesyl diphosphate synthase (Lc-GFDPS), the enzyme producing the C25 prenyl diphosphate precursor to all sesterterpenoids, from the glandular trichomes of the woody plant Leucosceptrum canum. GFDPS catalyzed the formation of GFDP after expression in Escherichia coli. Overexpressing GFDPS in Arabidopsis thaliana also gave an extract catalyzing GFDP formation. GFDPS was strongly expressed in glandular trichomes, and its transcript profile was completely in accordance with the sesterterpenoid accumulation pattern. GFDPS is localized to the plastids, and inhibitor studies indicated its use of isoprenyl diphosphate substrates supplied by the 2-C-methyl-D-erythritol 4-phosphate pathway. Application of a jasmonate defense hormone induced GFDPS transcript and sesterterpenoid accumulation, while reducing feeding and growth of the generalist insect Spodoptera exigua, suggesting that these C25 terpenoids play a defensive role. Phylogenetic analysis suggested that GFDPS probably evolved from plant geranylgeranyl diphosphate synthase under the influence of positive selection. The isolation of GFDPS provides a model for investigating sesterterpenoid formation in other species and a tool for manipulating the formation of this group in plants and other organisms. PMID:26941091

  7. Characterization of cytidylyltransferase enzyme activity through high performance liquid chromatography.

    PubMed

    Brault, James P; Friesen, Jon A

    2016-10-01

    The cytidylyltransferases are a family of enzymes that utilize cytidine 5'-triphosphate (CTP) to synthesize molecules that are typically precursors to membrane phospholipids. The most extensively studied cytidylyltransferase is CTP:phosphocholine cytidylyltransferase (CCT), which catalyzes conversion of phosphocholine and CTP to cytidine diphosphocholine (CDP-choline), a step critical for synthesis of the membrane phospholipid phosphatidylcholine (PC). The current method used to determine catalytic activity of CCT measures production of radiolabeled CDP-choline from (14)C-labeled phosphocholine. The goal of this research was to develop a CCT enzyme assay that employed separation of non-radioactive CDP-choline from CTP. A C18 reverse phase column with a mobile phase of 0.1 M ammonium bicarbonate (98%) and acetonitrile (2%) (pH 7.4) resulted in separation of solutions of the substrate CTP from the product CDP-choline. A previously characterized truncated version of rat CCTα (denoted CCTα236) was used to test the HPLC enzyme assay by measuring CDP-choline product formation. The Vmax for CCTα236 was 3850 nmol/min/mg and K0.5 values for CTP and phosphocholine were 4.07 mM and 2.49 mM, respectively. The HPLC method was applied to glycerol 3-phosphate cytidylyltransferase (GCT) and CTP:2-C-methyl-D-erythritol-4-phosphate cytidylyltransferase synthetase (CMS), members of the cytidylyltransferase family that produce CDP-glycerol and CDP-methylerythritol, respectively. PMID:27443959

  8. Enhancement of anthraquinone production in Morinda citrifolia cell suspension cultures after stimulation of the proline cycle with two proline analogs.

    PubMed

    Quevedo, Carla V; Perassolo, María; Giulietti, Ana M; Rodríguez Talou, Julián

    2012-03-01

    Synthesis of anthraquinones (AQs) involves the shikimate and 2-C-methyl-D-erythritol 4-phosphate pathways. The proline cycle is linked to the pentose phosphate pathway (PPP) to generate NADPH needed in the first steps of this pathway. The effect of two proline analogs, azetidine-2-carboxylic acid (A2C) and thiazolidine-4-carboxylic acid (T4C), were evaluated in Morinda citrifolia suspension cultures. Both analogs gave higher proline accumulation after 6 and 10 days (68 and 179% after 6 days with A2C at 25 and 50 μM, respectively, and 111% with T4C added at 100 μM). Induction of the proline cycle increased the AQ content after 6 days (~40% for 50 μM A2C and 100 μM T4C). Whereas A2C (50 μM) increased only AQ production, T4C also enhanced total phenolics. However, no induction of the PPP was observed with any of the treatments. This pathway therefore does not limit the supply of carbon skeletons to secondary metabolic pathways.

  9. Cloning, characterization, and immunolocalization of a mycorrhiza-inducible 1-deoxy-d-xylulose 5-phosphate reductoisomerase in arbuscule-containing cells of maize.

    PubMed

    Hans, Joachim; Hause, Bettina; Strack, Dieter; Walter, Michael H

    2004-02-01

    Colonization of plant roots by symbiotic arbuscular mycorrhizal fungi frequently leads to the accumulation of several apocarotenoids. The corresponding carotenoid precursors originate from the plastidial 2-C-methyl-d-erythritol 4-phosphate pathway. We have cloned and characterized 1-deoxy-d-xylulose 5-phosphate reductoisomerase (DXR), catalyzing the first committed step of the pathway, from maize (Zea mays). Functional identification was accomplished by heterologous expression of sequences coding for the mature protein in Escherichia coli. DXR is up-regulated in maize roots during mycorrhization as shown at transcript and protein levels, but is also abundant in leaves and young seedlings. Inspection of sequenced genomes and expressed sequence tag (EST) databases argue for a single-copy DXR gene. Immunolocalization studies in mycorrhizal roots using affinity-purified antibodies revealed a DXR localization in plastids around the main symbiotic structures, the arbuscules. DXR protein accumulation is tightly correlated with arbuscule development. The highest level of DXR protein is reached around maturity and initial senescence of these structures. We further demonstrate the formation of a DXR-containing plastidial network around arbuscules, which is highly interconnected in the mature, functional state of the arbuscules. Our findings imply a functional role of a still unknown nature for the apocarotenoids or their respective carotenoid precursors in the arbuscular life cycle.

  10. A Geranylfarnesyl Diphosphate Synthase Provides the Precursor for Sesterterpenoid (C25) Formation in the Glandular Trichomes of the Mint Species Leucosceptrum canum.

    PubMed

    Liu, Yan; Luo, Shi-Hong; Schmidt, Axel; Wang, Guo-Dong; Sun, Gui-Ling; Grant, Marcus; Kuang, Ce; Yang, Min-Jie; Jing, Shu-Xi; Li, Chun-Huan; Schneider, Bernd; Gershenzon, Jonathan; Li, Sheng-Hong

    2016-03-01

    Plant sesterterpenoids, an important class of terpenoids, are widely distributed in various plants, including food crops. However, little is known about their biosynthesis. Here, we cloned and functionally characterized a plant geranylfarnesyl diphosphate synthase (Lc-GFDPS), the enzyme producing the C25 prenyl diphosphate precursor to all sesterterpenoids, from the glandular trichomes of the woody plant Leucosceptrum canum. GFDPS catalyzed the formation of GFDP after expression in Escherichia coli. Overexpressing GFDPS in Arabidopsis thaliana also gave an extract catalyzing GFDP formation. GFDPS was strongly expressed in glandular trichomes, and its transcript profile was completely in accordance with the sesterterpenoid accumulation pattern. GFDPS is localized to the plastids, and inhibitor studies indicated its use of isoprenyl diphosphate substrates supplied by the 2-C-methyl-D-erythritol 4-phosphate pathway. Application of a jasmonate defense hormone induced GFDPS transcript and sesterterpenoid accumulation, while reducing feeding and growth of the generalist insect Spodoptera exigua, suggesting that these C25 terpenoids play a defensive role. Phylogenetic analysis suggested that GFDPS probably evolved from plant geranylgeranyl diphosphate synthase under the influence of positive selection. The isolation of GFDPS provides a model for investigating sesterterpenoid formation in other species and a tool for manipulating the formation of this group in plants and other organisms.

  11. Structure of 1-deoxy-d-xylulose 5-phosphate reductoisomerase in a quaternary complex with a magnesium ion, NADPH and the antimalarial drug fosmidomycin

    SciTech Connect

    Yajima, Shunsuke Hara, Kodai; Iino, Daisuke; Sasaki, Yasuyuki; Kuzuyama, Tomohisa; Ohsawa, Kanju; Seto, Haruo

    2007-06-01

    The crystal structure of 1-deoxy-d-xylulose 5-phosphate reductoisomerase (DXR) from Escherichia coli complexed with Mg{sup 2+}, NADPH and fosmidomycin was determined at 2.2 Å resolution. The structure showed a well defined loop conformation at the active site of DXR. The crystal structure of 1-deoxy-d-xylulose 5-phosphate reductoisomerase (DXR) from Escherichia coli complexed with Mg{sup 2+}, NADPH and fosmidomycin was solved at 2.2 Å resolution. DXR is the key enzyme in the 2-C-methyl-d-erythritol 4-phosphate pathway and is an effective target of antimalarial drugs such as fosmidomycin. In the crystal structure, electron density for the flexible loop covering the active site was clearly observed, indicating the well ordered conformation of DXR upon substrate binding. On the other hand, no electron density was observed for the nicotinamide-ribose portion of NADPH and the position of Asp149 anchoring Mg{sup 2+} was shifted by NADPH in the active site.

  12. A functional (E)-4-hydroxy-3-methylbut-2-enyl diphosphate reductase exhibits diurnal regulation of expression in Stevia rebaudiana (Bertoni).

    PubMed

    Kumar, Hitesh; Kumar, Sanjay

    2013-09-15

    The leaves of stevia [Stevia rebaudiana (Bertoni)] are a rich source of steviol glycosides that are used as non-calorific sweetener in many countries around the world. Steviol moiety of steviol glycosides is synthesized via plastidial 2C-methyl-D-erythritol 4-phosphate pathway, where (E)-4-hydroxy-3-methylbut-2-enyl diphosphate reductase (HDR) is the key enzyme. HDR catalyzes the simultaneous conversion of (E)-4-hydroxy-3-methylbut-2-enyl diphosphate into five carbon isoprenoid units, isopentenyl diphosphate and dimethylallyl diphosphate. Stevia HDR (SrHDR) successfully rescued HDR lethal mutant strain MG1655 ara<>ispH upon genetic complementation, suggesting SrHDR to encode a functional protein. The gene exhibited diurnal variation in expression. To identify the possible regulatory elements, upstream region of the gene was cloned and putative cis-acting elements were detected by in silico analysis. Electrophoretic mobility shift assay, using a putative light responsive element GATA showed the binding of nuclear proteins (NP) isolated from leaves during light period of the day, but not with the NP from leaves during the dark period. Data suggested the involvement of GATA box in light mediated gene regulation of SrHDR in stevia.

  13. Multi-Substrate Terpene Synthases: Their Occurrence and Physiological Significance.

    PubMed

    Pazouki, Leila; Niinemets, Ülo

    2016-01-01

    Terpene synthases are responsible for synthesis of a large number of terpenes in plants using substrates provided by two distinct metabolic pathways, the mevalonate-dependent pathway that is located in cytosol and has been suggested to be responsible for synthesis of sesquiterpenes (C15), and 2-C-methyl-D-erythritol-4-phosphate pathway located in plastids and suggested to be responsible for the synthesis of hemi- (C5), mono- (C10), and diterpenes (C20). Recent advances in characterization of genes and enzymes responsible for substrate and end product biosynthesis as well as efforts in metabolic engineering have demonstrated existence of a number of multi-substrate terpene synthases. This review summarizes the progress in the characterization of such multi-substrate terpene synthases and suggests that the presence of multi-substrate use might have been significantly underestimated. Multi-substrate use could lead to important changes in terpene product profiles upon substrate profile changes under perturbation of metabolism in stressed plants as well as under certain developmental stages. We therefore argue that multi-substrate use can be significant under physiological conditions and can result in complicate modifications in terpene profiles. PMID:27462341

  14. Multi-Substrate Terpene Synthases: Their Occurrence and Physiological Significance

    PubMed Central

    Pazouki, Leila; Niinemets, Ülo

    2016-01-01

    Terpene synthases are responsible for synthesis of a large number of terpenes in plants using substrates provided by two distinct metabolic pathways, the mevalonate-dependent pathway that is located in cytosol and has been suggested to be responsible for synthesis of sesquiterpenes (C15), and 2-C-methyl-D-erythritol-4-phosphate pathway located in plastids and suggested to be responsible for the synthesis of hemi- (C5), mono- (C10), and diterpenes (C20). Recent advances in characterization of genes and enzymes responsible for substrate and end product biosynthesis as well as efforts in metabolic engineering have demonstrated existence of a number of multi-substrate terpene synthases. This review summarizes the progress in the characterization of such multi-substrate terpene synthases and suggests that the presence of multi-substrate use might have been significantly underestimated. Multi-substrate use could lead to important changes in terpene product profiles upon substrate profile changes under perturbation of metabolism in stressed plants as well as under certain developmental stages. We therefore argue that multi-substrate use can be significant under physiological conditions and can result in complicate modifications in terpene profiles. PMID:27462341

  15. A Genome-Wide Scenario of Terpene Pathways in Self-pollinated Artemisia annua.

    PubMed

    Ma, Dong-Ming; Wang, Zhilong; Wang, Liangjiang; Alejos-Gonzales, Fatima; Sun, Ming-An; Xie, De-Yu

    2015-11-01

    Scenarios of genes to metabolites in Artemisia annua remain uninvestigated. Here, we report the use of an integrated approach combining metabolomics, transcriptomics, and gene function analyses to characterize gene-to-terpene and terpene pathway scenarios in a self-pollinating variety of this species. Eighty-eight metabolites including 22 sesquiterpenes (e.g., artemisinin), 26 monoterpenes, two triterpenes, one diterpene and 38 other non-polar metabolites were identified from 14 tissues. These metabolites were differentially produced by leaves and flowers at lower to higher positions. Sequences from cDNA libraries of six tissues were assembled into 18 871 contigs and genome-wide gene expression profiles in tissues were strongly associated with developmental stages and spatial specificities. Sequence mining identified 47 genes that mapped to the artemisinin, non-amorphadiene sesquiterpene, monoterpene, triterpene, 2-C-methyl-D-erythritol 4-phosphate and mevalonate pathways. Pearson correlation analysis resulted in network integration that characterized significant correlations of gene-to-gene expression patterns and gene expression-to-metabolite levels in six tissues simultaneously. More importantly, manipulations of amorpha-4,11-diene synthase gene expression not only affected the activity of this pathway toward artemisinin, artemisinic acid, and arteannuin b but also altered non-amorphadiene sesquiterpene and genome-wide volatile profiles. Such gene-to-terpene landscapes associated with different tissues are fundamental to the metabolic engineering of artemisinin. PMID:26192869

  16. Phosphatidylinositol-4-phosphate-dependent membrane traffic is critical for fungal filamentous growth.

    PubMed

    Ghugtyal, Vikram; Garcia-Rodas, Rocio; Seminara, Agnese; Schaub, Sébastien; Bassilana, Martine; Arkowitz, Robert Alan

    2015-07-14

    The phospholipid phosphatidylinositol-4-phosphate [PI(4)P], generated at the Golgi and plasma membrane, has been implicated in many processes, including membrane traffic, yet its role in cell morphology changes, such as the budding to filamentous growth transition, is unknown. We show that Golgi PI(4)P is required for such a transition in the human pathogenic fungus Candida albicans. Quantitative analyses of membrane traffic revealed that PI(4)P is required for late Golgi and secretory vesicle dynamics and targeting and, as a result, is important for the distribution of a multidrug transporter and hence sensitivity to antifungal drugs. We also observed that plasma membrane PI(4)P, which we show is functionally distinct from Golgi PI(4)P, forms a steep gradient concomitant with filamentous growth, despite uniform plasma membrane PI-4-kinase distribution. Mathematical modeling indicates that local PI(4)P generation and hydrolysis by phosphatases are crucial for this gradient. We conclude that PI(4)P-regulated membrane dynamics are critical for morphology changes.

  17. Phosphatidylinositol-4-phosphate-dependent membrane traffic is critical for fungal filamentous growth

    PubMed Central

    Ghugtyal, Vikram; Garcia-Rodas, Rocio; Seminara, Agnese; Schaub, Sébastien; Bassilana, Martine; Arkowitz, Robert Alan

    2015-01-01

    The phospholipid phosphatidylinositol-4-phosphate [PI(4)P], generated at the Golgi and plasma membrane, has been implicated in many processes, including membrane traffic, yet its role in cell morphology changes, such as the budding to filamentous growth transition, is unknown. We show that Golgi PI(4)P is required for such a transition in the human pathogenic fungus Candida albicans. Quantitative analyses of membrane traffic revealed that PI(4)P is required for late Golgi and secretory vesicle dynamics and targeting and, as a result, is important for the distribution of a multidrug transporter and hence sensitivity to antifungal drugs. We also observed that plasma membrane PI(4)P, which we show is functionally distinct from Golgi PI(4)P, forms a steep gradient concomitant with filamentous growth, despite uniform plasma membrane PI-4-kinase distribution. Mathematical modeling indicates that local PI(4)P generation and hydrolysis by phosphatases are crucial for this gradient. We conclude that PI(4)P-regulated membrane dynamics are critical for morphology changes. PMID:26124136

  18. The Salmonella effector SteA binds phosphatidylinositol 4-phosphate for subcellular targeting within host cells.

    PubMed

    Domingues, Lia; Ismail, Ahmad; Charro, Nuno; Rodríguez-Escudero, Isabel; Holden, David W; Molina, María; Cid, Víctor J; Mota, Luís Jaime

    2016-07-01

    Many bacterial pathogens use specialized secretion systems to deliver virulence effector proteins into eukaryotic host cells. The function of these effectors depends on their localization within infected cells, but the mechanisms determining subcellular targeting of each effector are mostly elusive. Here, we show that the Salmonella type III secretion effector SteA binds specifically to phosphatidylinositol 4-phosphate [PI(4)P]. Ectopically expressed SteA localized at the plasma membrane (PM) of eukaryotic cells. However, SteA was displaced from the PM of Saccharomyces cerevisiae in mutants unable to synthesize the local pool of PI(4)P and from the PM of HeLa cells after localized depletion of PI(4)P. Moreover, in infected cells, bacterially translocated or ectopically expressed SteA localized at the membrane of the Salmonella-containing vacuole (SCV) and to Salmonella-induced tubules; using the PI(4)P-binding domain of the Legionella type IV secretion effector SidC as probe, we found PI(4)P at the SCV membrane and associated tubules throughout Salmonella infection of HeLa cells. Both binding of SteA to PI(4)P and the subcellular localization of ectopically expressed or bacterially translocated SteA were dependent on a lysine residue near the N-terminus of the protein. Overall, this indicates that binding of SteA to PI(4)P is necessary for its localization within host cells.

  19. INTRACELLULAR TRANSPORT. Phosphatidylserine transport by ORP/Osh proteins is driven by phosphatidylinositol 4-phosphate.

    PubMed

    Moser von Filseck, Joachim; Čopič, Alenka; Delfosse, Vanessa; Vanni, Stefano; Jackson, Catherine L; Bourguet, William; Drin, Guillaume

    2015-07-24

    In eukaryotic cells, phosphatidylserine (PS) is synthesized in the endoplasmic reticulum (ER) but is highly enriched in the plasma membrane (PM), where it contributes negative charge and to specific recruitment of signaling proteins. This distribution relies on transport mechanisms whose nature remains elusive. Here, we found that the PS transporter Osh6p extracted phosphatidylinositol 4-phosphate (PI4P) and exchanged PS for PI4P between two membranes. We solved the crystal structure of Osh6p:PI4P complex and demonstrated that the transport of PS by Osh6p depends on PI4P recognition in vivo. Finally, we showed that the PI4P-phosphatase Sac1p, by maintaining a PI4P gradient at the ER/PM interface, drove PS transport. Thus, PS transport by oxysterol-binding protein-related protein (ORP)/oxysterol-binding homology (Osh) proteins is fueled by PI4P metabolism through PS/PI4P exchange cycles.

  20. Osh4p exchanges sterols for phosphatidylinositol 4-phosphate between lipid bilayers

    PubMed Central

    de Saint-Jean, Maud; Delfosse, Vanessa; Douguet, Dominique; Chicanne, Gaëtan; Payrastre, Bernard; Bourguet, William

    2011-01-01

    Osh/Orp proteins transport sterols between organelles and are involved in phosphoinositide metabolism. The link between these two aspects remains elusive. Using novel assays, we address the influence of membrane composition on the ability of Osh4p/Kes1p to extract, deliver, or transport dehydroergosterol (DHE). Surprisingly, phosphatidylinositol 4-phosphate (PI(4)P) specifically inhibited DHE extraction because PI(4)P was itself efficiently extracted by Osh4p. We solve the structure of the Osh4p–PI(4)P complex and reveal how Osh4p selectively substitutes PI(4)P for sterol. Last, we show that Osh4p quickly exchanges DHE for PI(4)P and, thereby, can transport these two lipids between membranes along opposite routes. These results suggest a model in which Osh4p transports sterol from the ER to late compartments pinpointed by PI(4)P and, in turn, transports PI(4)P backward. Coupled to PI(4)P metabolism, this transport cycle would create sterol gradients. Because the residues that recognize PI(4)P are conserved in Osh4p homologues, other Osh/Orp are potential sterol/phosphoinositol phosphate exchangers. PMID:22162133

  1. West Nile virus replication requires fatty acid synthesis but is independent on phosphatidylinositol-4-phosphate lipids.

    PubMed

    Martín-Acebes, Miguel A; Blázquez, Ana-Belén; Jiménez de Oya, Nereida; Escribano-Romero, Estela; Saiz, Juan-Carlos

    2011-01-01

    West Nile virus (WNV) is a neurovirulent mosquito-borne flavivirus, which main natural hosts are birds but it also infects equines and humans, among other mammals. As in the case of other plus-stranded RNA viruses, WNV replication is associated to intracellular membrane rearrangements. Based on results obtained with a variety of viruses, different cellular processes have been shown to play important roles on these membrane rearrangements for efficient viral replication. As these processes are related to lipid metabolism, fatty acid synthesis, as well as generation of a specific lipid microenvironment enriched in phosphatidylinositol-4-phosphate (PI4P), has been associated to it in other viral models. In this study, intracellular membrane rearrangements following infection with a highly neurovirulent strain of WNV were addressed by means of electron and confocal microscopy. Infection of WNV, and specifically viral RNA replication, were dependent on fatty acid synthesis, as revealed by the inhibitory effect of cerulenin and C75, two pharmacological inhibitors of fatty acid synthase, a key enzyme of this process. However, WNV infection did not induce redistribution of PI4P lipids, and PI4P did not localize at viral replication complex. Even more, WNV multiplication was not inhibited by the use of the phosphatidylinositol-4-kinase inhibitor PIK93, while infection by the enterovirus Coxsackievirus B5 was reduced. Similar features were found when infection by other flavivirus, the Usutu virus (USUV), was analyzed. These features of WNV replication could help to design specific antiviral approaches against WNV and other related flaviviruses.

  2. Phosphatidylinositol 4-Phosphate 5-Kinase β Controls Recruitment of Lipid Rafts into the Immunological Synapse.

    PubMed

    Kallikourdis, Marinos; Trovato, Anna Elisa; Roselli, Giuliana; Muscolini, Michela; Porciello, Nicla; Tuosto, Loretta; Viola, Antonella

    2016-02-15

    Phosphatidylinositol 4,5-biphosphate (PIP2) is critical for T lymphocyte activation serving as a substrate for the generation of second messengers and the remodeling of actin cytoskeleton necessary for the clustering of lipid rafts, TCR, and costimulatory receptors toward the T:APC interface. Spatiotemporal analysis of PIP2 synthesis in T lymphocytes suggested that distinct isoforms of the main PIP2-generating enzyme, phosphatidylinositol 4-phosphate 5-kinase (PIP5K), play a differential role on the basis of their distinct localization. In this study, we analyze the contribution of PIP5Kβ to T cell activation and show that CD28 induces the recruitment of PIP5Kβ to the immunological synapse, where it regulates filamin A and lipid raft accumulation, as well as T cell activation, in a nonredundant manner. Finally, we found that Vav1 and the C-terminal 83 aa of PIP5Kβ are pivotal for the PIP5Kβ regulatory functions in response to CD28 stimulation.

  3. Phosphatidylinositol-4-phosphate-dependent membrane traffic is critical for fungal filamentous growth.

    PubMed

    Ghugtyal, Vikram; Garcia-Rodas, Rocio; Seminara, Agnese; Schaub, Sébastien; Bassilana, Martine; Arkowitz, Robert Alan

    2015-07-14

    The phospholipid phosphatidylinositol-4-phosphate [PI(4)P], generated at the Golgi and plasma membrane, has been implicated in many processes, including membrane traffic, yet its role in cell morphology changes, such as the budding to filamentous growth transition, is unknown. We show that Golgi PI(4)P is required for such a transition in the human pathogenic fungus Candida albicans. Quantitative analyses of membrane traffic revealed that PI(4)P is required for late Golgi and secretory vesicle dynamics and targeting and, as a result, is important for the distribution of a multidrug transporter and hence sensitivity to antifungal drugs. We also observed that plasma membrane PI(4)P, which we show is functionally distinct from Golgi PI(4)P, forms a steep gradient concomitant with filamentous growth, despite uniform plasma membrane PI-4-kinase distribution. Mathematical modeling indicates that local PI(4)P generation and hydrolysis by phosphatases are crucial for this gradient. We conclude that PI(4)P-regulated membrane dynamics are critical for morphology changes. PMID:26124136

  4. The Clathrin Adaptor Gga2p Is a Phosphatidylinositol 4-phosphate Effector at the Golgi Exit

    PubMed Central

    Demmel, Lars; Gravert, Maike; Ercan, Ebru; Habermann, Bianca; Müller-Reichert, Thomas; Kukhtina, Viktoria; Haucke, Volker; Baust, Thorsten; Sohrmann, Marc; Kalaidzidis, Yannis; Klose, Christian; Beck, Mike; Peter, Matthias

    2008-01-01

    Phosphatidylinositol 4-phosphate (PI(4)P) is a key regulator of membrane transport required for the formation of transport carriers from the trans-Golgi network (TGN). The molecular mechanisms of PI(4)P signaling in this process are still poorly understood. In a search for PI(4)P effector molecules, we performed a screen for synthetic lethals in a background of reduced PI(4)P and found the gene GGA2. Our analysis uncovered a PI(4)P-dependent recruitment of the clathrin adaptor Gga2p to the TGN during Golgi-to-endosome trafficking. Gga2p recruitment to liposomes is stimulated both by PI(4)P and the small GTPase Arf1p in its active conformation, implicating these two molecules in the recruitment of Gga2p to the TGN, which ultimately controls the formation of clathrin-coated vesicles. PI(4)P binding occurs through a phosphoinositide-binding signature within the N-terminal VHS domain of Gga2p resembling a motif found in other clathrin interacting proteins. These data provide an explanation for the TGN-specific membrane recruitment of Gga2p. PMID:18287542

  5. Lovastatin-Induced Phosphatidylinositol-4-Phosphate 5-Kinase Diffusion from Microvilli Stimulates ROMK Channels

    PubMed Central

    Liu, Bing-Chen; Yang, Li-Li; Lu, Xiao-Yu; Song, Xiang; Li, Xue-Chen; Chen, Guangping; Li, Yichao; Yao, Xincheng; Humphrey, Donald R.; Eaton, Douglas C.

    2015-01-01

    We recently showed that lovastatin attenuates cyclosporin A (CsA)-induced damage of cortical collecting duct (CCD) principal cells by reducing intracellular cholesterol. Previous studies showed that, in cell expression models or artificial membranes, exogenous cholesterol directly inhibits inward rectifier potassium channels, including Kir1.1 (Kcnj1; the gene locus for renal outer medullary K+ [ROMK1] channels). Therefore, we hypothesized that lovastatin might stimulate ROMK1 by reducing cholesterol in CCD cells. Western blots showed that mpkCCDc14 cells express ROMK1 channels with molecular masses that approximate the molecular masses of ROMK1 in renal tubules detected before and after treatment with DTT. Confocal microscopy showed that ROMK1 channels were not in the microvilli, where cholesterol-rich lipid rafts are located, but rather, the planar regions of the apical membrane of mpkCCDc14 cells. Furthermore, phosphatidylinositol-4,5-bisphosphate [PI(4,5)P2], an activator of ROMK channels, was detected mainly in the microvilli under resting conditions along with the kinase responsible for PI(4,5)P2 synthesis, phosphatidylinositol-4-phosphate 5-kinase, type I γ [PI(4)P5K I γ], which may explain the low basal open probability and increased sensitivity to tetraethylammonium observed here for this channel. Notably, lovastatin induced PI(4)P5K I γ diffusion into planar regions and elevated PI(4,5)P2 and ROMK1 open probability in these regions through a cholesterol-associated mechanism. However, exogenous cholesterol alone did not induce these effects. These results suggest that lovastatin stimulates ROMK1 channels, at least in part, by inducing PI(4,5)P2 synthesis in planar regions of the renal CCD cell apical membrane, suggesting that lovastatin could reduce cyclosporin-induced nephropathy and associated hyperkalemia. PMID:25349201

  6. Lovastatin-Induced Phosphatidylinositol-4-Phosphate 5-Kinase Diffusion from Microvilli Stimulates ROMK Channels.

    PubMed

    Liu, Bing-Chen; Yang, Li-Li; Lu, Xiao-Yu; Song, Xiang; Li, Xue-Chen; Chen, Guangping; Li, Yichao; Yao, Xincheng; Humphrey, Donald R; Eaton, Douglas C; Shen, Bao-Zhong; Ma, He-Ping

    2015-07-01

    We recently showed that lovastatin attenuates cyclosporin A (CsA)-induced damage of cortical collecting duct (CCD) principal cells by reducing intracellular cholesterol. Previous studies showed that, in cell expression models or artificial membranes, exogenous cholesterol directly inhibits inward rectifier potassium channels, including Kir1.1 (Kcnj1; the gene locus for renal outer medullary K(+) [ROMK1] channels). Therefore, we hypothesized that lovastatin might stimulate ROMK1 by reducing cholesterol in CCD cells. Western blots showed that mpkCCDc14 cells express ROMK1 channels with molecular masses that approximate the molecular masses of ROMK1 in renal tubules detected before and after treatment with DTT. Confocal microscopy showed that ROMK1 channels were not in the microvilli, where cholesterol-rich lipid rafts are located, but rather, the planar regions of the apical membrane of mpkCCDc14 cells. Furthermore, phosphatidylinositol-4,5-bisphosphate [PI(4,5)P2], an activator of ROMK channels, was detected mainly in the microvilli under resting conditions along with the kinase responsible for PI(4,5)P2 synthesis, phosphatidylinositol-4-phosphate 5-kinase, type I γ [PI(4)P5K I γ], which may explain the low basal open probability and increased sensitivity to tetraethylammonium observed here for this channel. Notably, lovastatin induced PI(4)P5K I γ diffusion into planar regions and elevated PI(4,5)P2 and ROMK1 open probability in these regions through a cholesterol-associated mechanism. However, exogenous cholesterol alone did not induce these effects. These results suggest that lovastatin stimulates ROMK1 channels, at least in part, by inducing PI(4,5)P2 synthesis in planar regions of the renal CCD cell apical membrane, suggesting that lovastatin could reduce cyclosporin-induced nephropathy and associated hyperkalemia. PMID:25349201

  7. West Nile Virus Replication Requires Fatty Acid Synthesis but Is Independent on Phosphatidylinositol-4-Phosphate Lipids

    PubMed Central

    Martín-Acebes, Miguel A.; Blázquez, Ana-Belén; Jiménez de Oya, Nereida; Escribano-Romero, Estela; Saiz, Juan-Carlos

    2011-01-01

    West Nile virus (WNV) is a neurovirulent mosquito-borne flavivirus, which main natural hosts are birds but it also infects equines and humans, among other mammals. As in the case of other plus-stranded RNA viruses, WNV replication is associated to intracellular membrane rearrangements. Based on results obtained with a variety of viruses, different cellular processes have been shown to play important roles on these membrane rearrangements for efficient viral replication. As these processes are related to lipid metabolism, fatty acid synthesis, as well as generation of a specific lipid microenvironment enriched in phosphatidylinositol-4-phosphate (PI4P), has been associated to it in other viral models. In this study, intracellular membrane rearrangements following infection with a highly neurovirulent strain of WNV were addressed by means of electron and confocal microscopy. Infection of WNV, and specifically viral RNA replication, were dependent on fatty acid synthesis, as revealed by the inhibitory effect of cerulenin and C75, two pharmacological inhibitors of fatty acid synthase, a key enzyme of this process. However, WNV infection did not induce redistribution of PI4P lipids, and PI4P did not localize at viral replication complex. Even more, WNV multiplication was not inhibited by the use of the phosphatidylinositol-4-kinase inhibitor PIK93, while infection by the enterovirus Coxsackievirus B5 was reduced. Similar features were found when infection by other flavivirus, the Usutu virus (USUV), was analyzed. These features of WNV replication could help to design specific antiviral approaches against WNV and other related flaviviruses. PMID:21949814

  8. Phosphatidylinositol 4-phosphate 5-kinase α activation critically contributes to CD28-dependent signaling responses.

    PubMed

    Muscolini, Michela; Camperio, Cristina; Capuano, Cristina; Caristi, Silvana; Piccolella, Enza; Galandrini, Ricciarda; Tuosto, Loretta

    2013-05-15

    CD28 is one of the most relevant costimulatory receptors that deliver both TCR-dependent and TCR-independent signals regulating a wide range of signaling pathways crucial for cytokine and chemokine gene expressions, T cell survival, and proliferation. Most of the CD28-dependent signaling functions are initiated by the recruitment and activation of class IA PI3Ks, which catalyze the conversion of phosphatidylinositol 4,5-biphosphate (PIP2) into phosphatidylinositol 3,4,5-triphosphate, thus generating the docking sites for key signaling proteins. Hence, PIP2 is a crucial substrate in driving the PI3K downstream signaling pathways, and PIP2 turnover may be an essential regulatory step to ensure the activation of PI3K following CD28 engagement. Despite some data evidence that CD28 augments TCR-induced turnover of PIP2, its direct role in regulating PIP2 metabolism has never been assessed. In this study, we show that CD28 regulates PIP2 turnover by recruiting and activating phosphatidylinositol 4-phosphate 5-kinases α (PIP5Kα) in human primary CD4(+) T lymphocytes. This event leads to the neosynthesis of PIP2 and to its consumption by CD28-activated PI3K. We also evidenced that PIP5Kα activation is required for both CD28 unique signals regulating IL-8 gene expression as well as for CD28/TCR-induced Ca(2+) mobilization, NF-AT nuclear translocation, and IL-2 gene transcription. Our findings elucidate a novel mechanism that involves PIP5Kα as a key modulator of CD28 costimulatory signals.

  9. Elicitor induced activation of the methylerythritol phosphate pathway toward phytoalexins biosynthesis in rice.

    PubMed

    Okada, Atsushi; Shimizu, Takafumi; Okada, Kazunori; Kuzuyama, Tomohisa; Koga, Jinichiro; Shibuya, Naoto; Nojiri, Hideaki; Yamane, Hisakazu

    2007-09-01

    Diterpenoid phytoalexins such as momilactones and phytocassanes are produced via geranylgeranyl diphosphate in suspension-cultured rice cells after treatment with a chitin elicitor. We have previously shown that the production of diterpene hydrocarbons leading to phytoalexins and the expression of related biosynthetic genes are activated in suspension-cultured rice cells upon elicitor treatment. To better understand the elicitor-induced activation of phytoalexin biosynthesis, we conducted microarray analysis using suspension-cultured rice cells collected at various times after treatment with chitin elicitor. Hierarchical cluster analysis revealed two types of early-induced expression (EIE-1, EIE-2) nodes and a late-induced expression (LIE) node that includes genes involved in phytoalexins biosynthesis. The LIE node contains genes that may be responsible for the methylerythritol phosphate (MEP) pathway, a plastidic biosynthetic pathway for isopentenyl diphosphate, an early precursor of phytoalexins. The elicitor-induced expression of these putative MEP pathway genes was confirmed by quantitative reverse-transcription PCR. 1-Deoxy-D: -xylulose 5-phosphate synthase (DXS), 1-deoxy-D: -xylulose 5-phosphate reductoisomerase (DXR), and 4-(cytidine 5'-diphospho)-2-C-methyl-D: -erythritol synthase (CMS), which catalyze the first three committed steps in the MEP pathway, were further shown to have enzymatic activities that complement the growth of E. coli mutants disrupted in the corresponding genes. Application of ketoclomazone and fosmidomycin, inhibitors of DXS and DXR, respectively, repressed the accumulation of diterpene-type phytoalexins in suspension cells treated with chitin elicitor. These results suggest that activation of the MEP pathway is required to supply sufficient terpenoid precursors for the production of phytoalexins in infected rice plants.

  10. 34 CFR 200.89 - MEP allocations; Re-interviewing; Eligibility documentation; and Quality control.

    Code of Federal Regulations, 2012 CFR

    2012-07-01

    ... 34 Education 1 2012-07-01 2012-07-01 false MEP allocations; Re-interviewing; Eligibility documentation; and Quality control. 200.89 Section 200.89 Education Regulations of the Offices of the Department of Education OFFICE OF ELEMENTARY AND SECONDARY EDUCATION, DEPARTMENT OF EDUCATION TITLE I-IMPROVING THE ACADEMIC ACHIEVEMENT OF THE...

  11. 34 CFR 200.89 - MEP allocations; Re-interviewing; Eligibility documentation; and Quality control.

    Code of Federal Regulations, 2011 CFR

    2011-07-01

    ..., the re-interviewing process must include— (A) Selection of a sample of identified migratory children... independent re-interviewers (i.e., interviewers who are neither SEA or local operating agency staff members... operating agency staff members working to administer or operate the State MEP nor any other persons...

  12. 34 CFR 200.89 - MEP allocations; Re-interviewing; Eligibility documentation; and Quality control.

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ..., the re-interviewing process must include— (A) Selection of a sample of identified migratory children... independent re-interviewers (i.e., interviewers who are neither SEA or local operating agency staff members... operating agency staff members working to administer or operate the State MEP nor any other persons...

  13. Unmanned Multiple Exploratory Probe System (MEPS) for Mars observation. Volume 1: Trade analysis and design

    NASA Technical Reports Server (NTRS)

    Adams, Daniel E.; Crumbly, Christopher M.; Delp, Steve E.; Guidry, Michelle A.; Lisano, Michael E.; Packard, James D.; Striepe, Scott A.

    1988-01-01

    This report presents the unmanned Multiple Exploratory Probe Systems (MEPS), a space vehicle designed to observe the planet Mars in preparation for manned missions. The options considered for each major element are presented as a trade analysis, and the final vehicle design is defined.

  14. 34 CFR 200.84 - Responsibilities of SEAs for evaluating the effectiveness of the MEP.

    Code of Federal Regulations, 2012 CFR

    2012-07-01

    ... 34 Education 1 2012-07-01 2012-07-01 false Responsibilities of SEAs for evaluating the effectiveness of the MEP. 200.84 Section 200.84 Education Regulations of the Offices of the Department of Education OFFICE OF ELEMENTARY AND SECONDARY EDUCATION, DEPARTMENT OF EDUCATION TITLE I-IMPROVING...

  15. 34 CFR 200.84 - Responsibilities of SEAs for evaluating the effectiveness of the MEP.

    Code of Federal Regulations, 2011 CFR

    2011-07-01

    ... 34 Education 1 2011-07-01 2011-07-01 false Responsibilities of SEAs for evaluating the effectiveness of the MEP. 200.84 Section 200.84 Education Regulations of the Offices of the Department of Education OFFICE OF ELEMENTARY AND SECONDARY EDUCATION, DEPARTMENT OF EDUCATION TITLE I-IMPROVING...

  16. 34 CFR 200.84 - Responsibilities of SEAs for evaluating the effectiveness of the MEP.

    Code of Federal Regulations, 2014 CFR

    2014-07-01

    ... 34 Education 1 2014-07-01 2014-07-01 false Responsibilities of SEAs for evaluating the effectiveness of the MEP. 200.84 Section 200.84 Education Regulations of the Offices of the Department of Education OFFICE OF ELEMENTARY AND SECONDARY EDUCATION, DEPARTMENT OF EDUCATION TITLE I-IMPROVING...

  17. Prior history of FDI muscle contraction: different effect on MEP amplitude and muscle activity.

    PubMed

    Talis, V L; Kazennikov, O V; Castellote, J M; Grishin, A A; Ioffe, M E

    2014-03-01

    Motor evoked potentials (MEPs) in the right first dorsal interosseous (FDI) muscle elicited by transcranial magnetic stimulation of left motor cortex were assessed in ten healthy subjects during maintenance of a fixed FDI contraction level. Subjects maintained an integrated EMG (IEMG) level with visual feedback and reproduced this level by memory afterwards in the following tasks: stationary FDI muscle contraction at the level of 40 ± 5 % of its maximum voluntary contraction (MVC; 40 % task), at the level of 20 ± 5 % MVC (20 % task), and also when 20 % MVC was preceded by either no contraction (0-20 task), by stronger muscle contraction (40-20 task) or by no contraction with a previous strong contraction (40-0-20 task). The results show that the IEMG level was within the prescribed limits when 20 and 40 % stationary tasks were executed with and without visual feedback. In 0-20, 40-20, and 40-0-20 tasks, 20 % IEMG level was precisely controlled in the presence of visual feedback, but without visual feedback the IEMG and force during 20 % IEMG maintenance were significantly higher in the 40-0-20 task than those in 0-20 and 40-20 tasks. That is, without visual feedback, there were significant variations in muscle activity due to different prehistory of contraction. In stationary tasks, MEP amplitudes in 40 % task were higher than in 20 % task. MEPs did not differ significantly during maintenance of the 20 % level in tasks with different prehistory of muscle contraction with and without visual feedback. Thus, in spite of variations in muscle background activity due to different prehistory of contraction MEPs did not vary significantly. This dissociation suggests that the voluntary maintenance of IEMG level is determined not only by cortical mechanisms, as reflected by corticospinal excitability, but also by lower levels of CNS, where afferent signals and influences from other brain structures and spinal cord are convergent.

  18. Kinetic Characterization and Phosphoregulation of the Francisella tularensis 1-Deoxy-D-Xylulose 5-Phosphate Reductoisomerase (MEP Synthase)

    PubMed Central

    Jawaid, Safdar; Seidle, Heather; Zhou, Weidong; Abdirahman, Hafsa; Abadeer, Maher; Hix, Joseph H.; van Hoek, Monique L.; Couch, Robin D.

    2009-01-01

    Deliberate and natural outbreaks of infectious disease underscore the necessity of effective vaccines and antimicrobial/antiviral therapeutics. The prevalence of antibiotic resistant strains and the ease by which antibiotic resistant bacteria can be intentionally engineered further highlights the need for continued development of novel antibiotics against new bacterial targets. Isoprenes are a class of molecules fundamentally involved in a variety of crucial biological functions. Mammalian cells utilize the mevalonic acid pathway for isoprene biosynthesis, whereas many bacteria utilize the methylerythritol phosphate (MEP) pathway, making the latter an attractive target for antibiotic development. In this report we describe the cloning and characterization of Francisella tularensis MEP synthase, a MEP pathway enzyme and potential target for antibiotic development. In vitro growth-inhibition assays using fosmidomycin, an inhibitor of MEP synthase, illustrates the effectiveness of MEP pathway inhibition with F. tularensis. To facilitate drug development, F. tularensis MEP synthase was cloned, expressed, purified, and characterized. Enzyme assays produced apparent kinetic constants (KMDXP = 104 µM, KMNADPH = 13 µM, kcatDXP = 2 s−1, kcatNADPH = 1.3 s−1), an IC50 for fosmidomycin of 247 nM, and a Ki for fosmidomycin of 99 nM. The enzyme exhibits a preference for Mg+2 as a divalent cation. Titanium dioxide chromatography-tandem mass spectrometry identified Ser177 as a site of phosphorylation. S177D and S177E site-directed mutants are inactive, suggesting a mechanism for post-translational control of metabolic flux through the F. tularensis MEP pathway. Overall, our study suggests that MEP synthase is an excellent target for the development of novel antibiotics against F. tularensis. PMID:20011597

  19. Lipids in salicylic acid-mediated defense in plants: focusing on the roles of phosphatidic acid and phosphatidylinositol 4-phosphate

    PubMed Central

    Zhang, Qiong; Xiao, Shunyuan

    2015-01-01

    Plants have evolved effective defense strategies to protect themselves from various pathogens. Salicylic acid (SA) is an essential signaling molecule that mediates pathogen-triggered signals perceived by different immune receptors to induce downstream defense responses. While many proteins play essential roles in regulating SA signaling, increasing evidence also supports important roles for signaling phospholipids in this process. In this review, we collate the experimental evidence in support of the regulatory roles of two phospholipids, phosphatidic acid (PA), and phosphatidylinositol 4-phosphate (PI4P), and their metabolizing enzymes in plant defense, and examine the possible mechanistic interaction between phospholipid signaling and SA-dependent immunity with a particular focus on the immunity-stimulated biphasic PA production that is reminiscent of and perhaps mechanistically connected to the biphasic reactive oxygen species (ROS) generation and SA accumulation during defense activation. PMID:26074946

  20. Biosynthesis of riboflavin. Studies on the mechanism of L-3,4-dihydroxy-2-butanone 4-phosphate synthase.

    PubMed

    Volk, R; Bacher, A

    1991-11-01

    The riboflavin precursor, L-3,4-dihydroxy-2-butanone 4-phosphate, is formed from D-ribulose 5-phosphate by a single 24-kDa enzyme. Studies with various specifically 13C-labeled D-ribulose 5-phosphates as substrate showed that the carbon atoms 1-3 of the enzyme product correspond to carbon atoms 1-3 of the substrate, whereas C-4 of the product stems from C-5 of the substrate. Carbon atom 4 of the substrate is released as formate together with the hydrogen atom attached to it. The skeletal rearrangement which leads to the loss of C-4 and the direct linkage between C-3 and C-5 of the substrate is an intramolecular reaction. The hydrogen atom at C-3 of the enzyme product is introduced from solvent water. A reaction mechanism which is in agreement with all experimental data is proposed. PMID:1939111

  1. Comparison of Australian and US Cost-Benefit Approaches to MEPS

    SciTech Connect

    McMahon, James E.

    2004-03-12

    The Australian Greenhouse Office contracted with the Collaborative Labeling and Appliance Standards Program (CLASP) for LBNL to compare US and Australian approaches to analyzing costs and benefits of minimum energy performance standards (MEPS). This report compares the approaches for three types of products: household refrigerators and freezers, small electric storage water heaters, and commercial/industrial air conditioners. This report presents the findings of similarities and differences between the approaches of the two countries and suggests changes to consider in the approach taken in Australia. The purpose of the Australian program is to reduce greenhouse gas emissions, while the US program is intended to increase energy efficiency; each program is thus subject to specific constraints. The market and policy contexts are different, with the USA producing most of its own products and conducting pioneering engineering-economic studies to identify maximum energy efficiency levels that are technologically feasible and economically justified. In contrast, Australia imports a large share of its products and adopts MEPS already in place elsewhere. With these differences in circumstances, Australia's analysis approach could be expected to have less analytical detail and still result in MEPS levels that are appropriate for their policy and market context. In practice, the analysis required to meet these different objectives is quite similar. To date, Australia's cost-benefit analysis has served the goals and philosophies of the program well and been highly effective in successfully identifying MEPS that are significantly reducing greenhouse gas emissions while providing economic benefits to consumers. In some cases, however, the experience of the USA--using more extensive data sets and more detailed analysis--suggests possible improvements to Australia's cost-benefit analysis. The principal findings of the comparison are: (1) The Technology and Market Assessments are

  2. Structural basis for competitive inhibition of 3,4-dihydroxy-2-butanone-4-phosphate synthase from Vibrio cholerae.

    PubMed

    Islam, Zeyaul; Kumar, Adarsh; Singh, Suruchi; Salmon, Laurent; Karthikeyan, Subramanian

    2015-05-01

    The riboflavin biosynthesis pathway has been shown to be essential in many pathogens and is absent in humans. Therefore, enzymes involved in riboflavin synthesis are considered as potential antibacterial drug targets. The enzyme 3,4-dihydroxy-2-butanone-4-phosphate synthase (DHBPS) catalyzes one of the two committed steps in the riboflavin pathway and converts d-ribulose 5-phosphate (Ru5P) to l-3,4-dihydroxy-2-butanone 4-phosphate and formate. Moreover, DHBPS is shown to be indispensable for Mycobacterium, Salmonella, and Helicobacter species. Despite the essentiality of this enzyme in bacteria, no inhibitor has been identified hitherto. Here, we describe kinetic and crystal structure characterization of DHBPS from Vibrio cholerae (vDHBPS) with a competitive inhibitor 4-phospho-d-erythronohydroxamic acid (4PEH) at 1.86-Å resolution. In addition, we also report the structural characterization of vDHBPS in its apo form and in complex with its substrate and substrate plus metal ions at 1.96-, 1.59-, and 2.04-Å resolution, respectively. Comparison of these crystal structures suggests that 4PEH inhibits the catalytic activity of DHBPS as it is unable to form a proposed intermediate that is crucial for DHBPS activity. Furthermore, vDHBPS structures complexed with substrate and metal ions reveal that, unlike Candida albicans, binding of substrate to vDHBPS induces a conformational change from an open to closed conformation. Interestingly, the position of second metal ion, which is different from that of Methanococcus jannaschii, strongly supports an active role in the catalytic mechanism. Thus, the kinetic and structural characterization of vDHBPS reveals the molecular mechanism of inhibition shown by 4PEH and that it can be explored further for designing novel antibiotics.

  3. Peptidoglycan-associated outer membrane protein Mep45 of rumen anaerobe Selenomonas ruminantium forms a non-specific diffusion pore via its C-terminal transmembrane domain.

    PubMed

    Kojima, Seiji; Hayashi, Kanako; Tochigi, Saeko; Kusano, Tomonobu; Kaneko, Jun; Kamio, Yoshiyuki

    2016-10-01

    The major outer membrane protein Mep45 of Selenomonas ruminantium, an anaerobic Gram-negative bacterium, comprises two distinct domains: the N-terminal S-layer homologous (SLH) domain that protrudes into the periplasm and binds to peptidoglycan, and the remaining C-terminal transmembrane domain, whose function has been unknown. Here, we solubilized and purified Mep45 and characterized its function using proteoliposomes reconstituted with Mep45. We found that Mep45 forms a nonspecific diffusion channel via its C-terminal region. The channel was permeable to solutes smaller than a molecular weight of roughly 600, and the estimated pore radius was 0.58 nm. Truncation of the SLH domain did not affect the channel property. On the basis of the fact that Mep45 is the most abundant outer membrane protein in S. ruminantium, we conclude that Mep45 serves as a main pathway through which small solutes diffuse across the outer membrane of this bacterium.

  4. MEP Latencies Predict the Neuromodulatory Effect of cTBS Delivered to the Ipsilateral and Contralateral Sensorimotor Cortex

    PubMed Central

    Huang, Gan; Mouraux, André

    2015-01-01

    Background Recently, it was shown that the highly variable after-effect of continuous theta-burst stimulation (cTBS) of the primary motor cortex (M1) can be predicted by the latency of motor-evoked potentials (MEPs) recorded before cTBS. This suggests that at least part of this inter-individual variability is driven by differences in the neuronal populations preferentially activated by transcranial magnetic stimulation (TMS). Methods Here, we recorded MEPs, TMS-evoked brain potentials (TEPs) and somatosensory-evoked potentials (SEPs) to investigate the effects of cTBS delivered over the primary sensorimotor cortex on both the ipsilateral and contralateral M1, and the ipsilateral and contralateral primary somatosensory cortex (S1). Results We confirm that the after-effects of cTBS can be predicted by the latency of MEPs recorded before cTBS. Over the hemisphere onto which cTBS was delivered, short-latency MEPs at baseline were associated with an increase of MEP magnitude (i.e. an excitatory effect of cTBS) whereas late-latency MEPs were associated with reduced MEPs (i.e. an inhibitory effect of cTBS). This relationship was reversed over the contralateral hemisphere, indicating opposite effects of cTBS on the responsiveness of the ipsilateral and contralateral M1. Baseline MEP latencies also predicted changes in the magnitude of the N100 wave of TEPs elicited by stimulation of the ipsilateral and contralateral hemisphere, indicating that this TEP component is specifically dependent on the state of M1. Finally, there was a reverse relationship between MEP latency and the effects of cTBS on the SEP waveforms (50–130 ms), indicating that after-effects of cTBS on S1 are opposite to those on M1. Conclusion Taken together, our results confirm that the variable after-effects of cTBS can be explained by differences in the neuronal populations activated by TMS. Furthermore, our results show that this variability also determines remote effects of cTBS in S1 and the

  5. Nuclear pool of phosphatidylinositol 4 phosphate 5 kinase 1α is modified by polySUMO-2 during apoptosis

    SciTech Connect

    Chakrabarti, Rajarshi; Bhowmick, Debajit; Bhargava, Varsha; Bhar, Kaushik; Siddhanta, Anirban

    2013-09-20

    Highlights: •Nuclear pool of PIP5K is SUMOylated. •Enhancement of SUMOylated nuclear PIP5K during apoptosis. •Nuclear PIP5K is modified by polySUMO-1 during apoptosis. •Nuclear PIP5K is modified by polySUMO-2 chain during apoptosis. -- Abstract: Phosphatidylinositol 4 phosphate 5 kinase 1α (PIP5K) is mainly localized in the cytosol and plasma membrane. Studies have also indicated its prominent association with nuclear speckles. The exact nature of this nuclear pool of PIP5K is not clear. Using biochemical and microscopic techniques, we have demonstrated that the nuclear pool of PIP5K is modified by SUMO-1 in HEK-293 cells stably expressing PIP5K. Moreover, this SUMOylated pool of PIP5K increased during apoptosis. PolySUMO-2 chain conjugated PIP5K was detected by pull-down experiment using affinity-tagged RNF4, a polySUMO-2 binding protein, during late apoptosis.

  6. Co-Encapsulation of Combretastatin-A4 Phosphate and Doxorubicin in Polymersomes for Synergistic Therapy of Nasopharyngeal Epidermal Carcinoma.

    PubMed

    Zhu, Jinfang; Xu, Xiaoping; Hu, Mengying; Qiu, Liyan

    2015-06-01

    In this study, we designed biodegradable polymersomes for co-delivery of an antiangiogenic drug combretastatin-A4 phosphate (CA4P) and doxorubicin (DOX) to collapse tumor neovasculature and inhibit cancer cell proliferation with the aim to achieve synergistic antitumor effects. The polymersomes co-encapsulating DOX and CA4P (Ps-DOX-CA4P) were prepared by solvent evaporation method using methoxy poly(ethylene glycol)-b-polylactide (mPEG-PLA) block copolymers as drug carriers. The resulting Ps-DOX-CA4P has vesicles shape with uniform sizes of about 50 nm and controlled co-encapsulation ratios of DOX to CA4P. More importantly, Ps-DOX-CA4P (1:10) showed strong synergistic cytotoxicity (combination index CI = 0.31) against human nasopharyngeal epidermal carcinoma (KB) cells. Furthermore, Ps-DOX-CA4P accumulated remarkably in KB tissues xenografts in nude mice. Consistent with these observations, Ps-DOX-CA4P (1:10) achieved significant antitumor potency because of fast tumor vasculature disruption and sustained tumor cells proliferation inhibition in vivo. The overall findings indicate that co-delivery of an antiangiogenic drug and a chemotherapeutic agent in polymersomes is a potentially promising strategy for cancer therapy.

  7. Foot-and-mouth disease virus replicates independently of phosphatidylinositol 4-phosphate and type III phosphatidylinositol 4-kinases.

    PubMed

    Berryman, Stephen; Moffat, Katy; Harak, Christian; Lohmann, Volker; Jackson, Terry

    2016-08-01

    Picornaviruses form replication complexes in association with membranes in structures called replication organelles. Common themes to emerge from studies of picornavirus replication are the need for cholesterol and phosphatidylinositol 4-phosphate (PI4P). In infected cells, type III phosphatidylinositol 4-kinases (PI4KIIIs) generate elevated levels of PI4P, which is then exchanged for cholesterol at replication organelles. For the enteroviruses, replication organelles form at Golgi membranes in a process that utilizes PI4KIIIβ. Other picornaviruses, for example the cardioviruses, are believed to initiate replication at the endoplasmic reticulum and subvert PI4KIIIα to generate PI4P. Here we investigated the role of PI4KIII in foot-and-mouth disease virus (FMDV) replication. Our results showed that, in contrast to the enteroviruses and the cardioviruses, FMDV replication does not require PI4KIII (PI4KIIIα and PI4KIIIβ), and PI4P levels do not increase in FMDV-infected cells and PI4P is not seen at replication organelles. These results point to a unique requirement towards lipids at the FMDV replication membranes. PMID:27093462

  8. Phosphatidylinositol 4-phosphate 5-kinase α and Vav1 mutual cooperation in CD28-mediated actin remodeling and signaling functions.

    PubMed

    Muscolini, Michela; Camperio, Cristina; Porciello, Nicla; Caristi, Silvana; Capuano, Cristina; Viola, Antonella; Galandrini, Ricciarda; Tuosto, Loretta

    2015-02-01

    Phosphatidylinositol 4,5-biphosphate (PIP2) is a cell membrane phosphoinositide crucial for cell signaling and activation. Indeed, PIP2 is a pivotal source for second messenger generation and controlling the activity of several proteins regulating cytoskeleton reorganization. Despite its critical role in T cell activation, the molecular mechanisms regulating PIP2 turnover remain largely unknown. In human primary CD4(+) T lymphocytes, we have recently demonstrated that CD28 costimulatory receptor is crucial for regulating PIP2 turnover by allowing the recruitment and activation of the lipid kinase phosphatidylinositol 4-phosphate 5-kinase (PIP5Kα). We also identified PIP5Kα as a key modulator of CD28 costimulatory signals leading to the efficient T cell activation. In this study, we extend these data by demonstrating that PIP5Kα recruitment and activation is essential for CD28-mediated cytoskeleton rearrangement necessary for organizing a complete signaling compartment leading to downstream signaling functions. We also identified Vav1 as the linker molecule that couples the C-terminal proline-rich motif of CD28 to the recruitment and activation of PIP5Kα, which in turn cooperates with Vav1 in regulating actin polymerization and CD28 signaling functions.

  9. Biosynthesis of riboflavin: cloning, sequencing, and expression of the gene coding for 3,4-dihydroxy-2-butanone 4-phosphate synthase of Escherichia coli.

    PubMed Central

    Richter, G; Volk, R; Krieger, C; Lahm, H W; Röthlisberger, U; Bacher, A

    1992-01-01

    3,4-Dihydroxy-2-butanone 4-phosphate is biosynthesized from ribulose 5-phosphate and serves as the biosynthetic precursor for the xylene ring of riboflavin. The gene coding for 3,4-dihydroxy-2-butanone 4-phosphate synthase of Escherichia coli has been cloned and sequenced. The gene codes for a protein of 217 amino acid residues with a calculated molecular mass of 23,349.6 Da. The enzyme was purified to near homogeneity from a recombinant E. coli strain and had a specific activity of 1,700 nmol mg-1 h-1. The N-terminal amino acid sequence and the amino acid composition of the protein were in agreement with the deduced sequence. The molecular mass as determined by ion spray mass spectrometry was 23,351 +/- 2 Da, which is in agreement with the predicted mass. The previously reported loci htrP, "luxH-like," and ribB at 66 min of the E. coli chromosome are all identical to the gene coding for 3,4-dihydroxy-2-butanone 4-phosphate synthase, but their role had not been hitherto determined. Sequence homology indicates that gene luxH of Vibrio harveyi and the central open reading frame of the Bacillus subtilis riboflavin operon code for 3,4-dihydroxy-2-butanone 4-phosphate synthase. Images PMID:1597419

  10. Association of MEP1A gene variants with insulin metabolism in central European women with polycystic ovary syndrome.

    PubMed

    Lam, Uyen D P; Lerchbaum, Elisabeth; Schweighofer, Natascha; Trummer, Olivia; Eberhard, Katharina; Genser, Bernd; Pieber, Thomas R; Obermayer-Pietsch, Barbara

    2014-03-10

    Polycystic ovary syndrome (PCOS) shows not only hyperandrogenemia, hirsutism and fertility problems, but also metabolic disturbances including obesity, cardiovascular events and type-2 diabetes. Accumulating evidence suggests some degree of inflammation associated with prominent aspects of PCOS. We aimed to investigate the association of genetic variants 3'UTR rs17468190 (G/T) of the inflammation-associated gene MEP1A (GenBank ID: NM_005588.2) with metabolic disturbances in PCOS and healthy control women. Genetic variants rs17468190 (G/T) of MEP1A gene were analyzed in 576 PCOS women and 206 controls by using the Taqman fluorogenic 5'-exonuclease assay. This polymorphism was tested for association with anthropometric, metabolic, hormonal, and functional parameters of PCOS. There was a borderline significant difference in genotype distribution between PCOS and control women (p=0.046). In overweight/obese PCOS patients, the variants rs17468190 (G/T) in the MEP1A gene are associated with glucose and insulin metabolism. In a dominant model, the GG genotype of the MEP1A gene was more strongly associated with insulin metabolism in overweight/obese PCOS women (body mass index, BMI>25 kg/m(2)), than in GT+TT genotypes. The MEP1A GG-carriers showed a significantly increased homeostatic model assessment - insulin resistance (HOMA-IR) (p=0.003), elevation of fasting insulin (p=0.004) and stimulated insulin (30 min, p<0.001; 60 min, p=0.009; 120 min, p=0.009) as well as triglyceride (p=0.032) levels. MEP1A is a possible target gene for disease modification in PCOS. It might contribute to the abnormalities of glucose metabolism and insulin sensitivity and serve as a diagnostic or therapeutic target gene for PCOS.

  11. The carotenogenesis pathway via the isoprenoid-beta-carotene interference approach in a new strain of Dunaliella salina isolated from Baja California Mexico.

    PubMed

    Paniagua-Michel, J; Capa-Robles, Willian; Olmos-Soto, Jorge; Gutierrez-Millan, Luis Enrique

    2009-01-01

    D. salina is one of the recognized natural sources to produce beta-carotene, and an useful model for studying the role of inhibitors and enhancers of carotenogenesis. However there is little information in D. salina regarding whether the isoprenoid substrate can be influenced by stress factors (carotenogenic) or selective inhibitors which in turn may further contribute to elucidate the early steps of carotenogenesis and biosynthesis of beta-carotene. In this study, Dunaliella salina (BC02) isolated from La Salina BC Mexico, was subjected to the method of isoprenoids-beta-carotene interference in order to promote the interruption or accumulation of the programmed biosynthesis of carotenoids. When Carotenogenic and non-carotenogenic cells of D. salina BC02 were grown under photoautotrophic growth conditions in the presence of 200 microM fosmidomycin, carotenogenesis and the synthesis of beta-carotene were interrupted after two days in cultured D. salina cells. This result is an indirect consequence of the inhibition of the synthesis of isoprenoids and activity of the recombinant DXR enzyme thereby preventing the conversion of 1-deoxy-D-xylulose 5-phosphate (DXP) to 2-C-methyl-D-erythritol (MEP) and consequently interrupts the early steps of carotenogenesis in D. salina. The effect at the level of proteins and RNA was not evident. Mevinolin treated D. salina cells exhibited carotenogenesis and beta-carotene levels very similar to those of control cell cultures indicating that mevinolin not pursued any indirect action in the biosynthesis of isoprenoids and had no effect at the level of the HMG-CoA reductase, the key enzyme of the Ac/MVA pathway.

  12. A cytosolic Arabidopsis D-xylulose kinase catalyzes the phosphorylation of 1-deoxy-D-xylulose into a precursor of the plastidial isoprenoid pathway.

    PubMed

    Hemmerlin, Andréa; Tritsch, Denis; Hartmann, Michael; Pacaud, Karine; Hoeffler, Jean-François; van Dorsselaer, Alain; Rohmer, Michel; Bach, Thomas J

    2006-10-01

    Plants are able to integrate exogenous 1-deoxy-D-xylulose (DX) into the 2C-methyl-D-erythritol 4-phosphate pathway, implicated in the biosynthesis of plastidial isoprenoids. Thus, the carbohydrate needs to be phosphorylated into 1-deoxy-D-xylulose 5-phosphate and translocated into plastids, or vice versa. An enzyme capable of phosphorylating DX was partially purified from a cell-free Arabidopsis (Arabidopsis thaliana) protein extract. It was identified by mass spectrometry as a cytosolic protein bearing D-xylulose kinase (XK) signatures, already suggesting that DX is phosphorylated within the cytosol prior to translocation into the plastids. The corresponding cDNA was isolated and enzymatic properties of a recombinant protein were determined. In Arabidopsis, xylulose kinases are encoded by a small gene family, in which only two genes are putatively annotated. The additional gene is coding for a protein targeted to plastids, as was proved by colocalization experiments using green fluorescent protein fusion constructs. Functional complementation assays in an Escherichia coli strain deleted in xk revealed that the cytosolic enzyme could exclusively phosphorylate xylulose in vivo, not the enzyme that is targeted to plastids. xk activities could not be detected in chloroplast protein extracts or in proteins isolated from its ancestral relative Synechocystis sp. PCC 6803. The gene encoding the plastidic protein annotated as "xylulose kinase" might in fact yield an enzyme having different phosphorylation specificities. The biochemical characterization and complementation experiments with DX of specific Arabidopsis knockout mutants seedlings treated with oxo-clomazone, an inhibitor of 1-deoxy-D-xylulose 5-phosphate synthase, further confirmed that the cytosolic protein is responsible for the phosphorylation of DX in planta.

  13. A Cytosolic Arabidopsis d-Xylulose Kinase Catalyzes the Phosphorylation of 1-Deoxy-d-Xylulose into a Precursor of the Plastidial Isoprenoid Pathway1

    PubMed Central

    Hemmerlin, Andréa; Tritsch, Denis; Hartmann, Michael; Pacaud, Karine; Hoeffler, Jean-François; van Dorsselaer, Alain; Rohmer, Michel; Bach, Thomas J.

    2006-01-01

    Plants are able to integrate exogenous 1-deoxy-d-xylulose (DX) into the 2C-methyl-d-erythritol 4-phosphate pathway, implicated in the biosynthesis of plastidial isoprenoids. Thus, the carbohydrate needs to be phosphorylated into 1-deoxy-d-xylulose 5-phosphate and translocated into plastids, or vice versa. An enzyme capable of phosphorylating DX was partially purified from a cell-free Arabidopsis (Arabidopsis thaliana) protein extract. It was identified by mass spectrometry as a cytosolic protein bearing d-xylulose kinase (XK) signatures, already suggesting that DX is phosphorylated within the cytosol prior to translocation into the plastids. The corresponding cDNA was isolated and enzymatic properties of a recombinant protein were determined. In Arabidopsis, xylulose kinases are encoded by a small gene family, in which only two genes are putatively annotated. The additional gene is coding for a protein targeted to plastids, as was proved by colocalization experiments using green fluorescent protein fusion constructs. Functional complementation assays in an Escherichia coli strain deleted in xk revealed that the cytosolic enzyme could exclusively phosphorylate xylulose in vivo, not the enzyme that is targeted to plastids. xk activities could not be detected in chloroplast protein extracts or in proteins isolated from its ancestral relative Synechocystis sp. PCC 6803. The gene encoding the plastidic protein annotated as “xylulose kinase” might in fact yield an enzyme having different phosphorylation specificities. The biochemical characterization and complementation experiments with DX of specific Arabidopsis knockout mutants seedlings treated with oxo-clomazone, an inhibitor of 1-deoxy-d-xylulose 5-phosphate synthase, further confirmed that the cytosolic protein is responsible for the phosphorylation of DX in planta. PMID:16920870

  14. [Cloning and analysis of cDNA encoding key enzyme gene (dxr) of the non-MVA pathway in Taxus chinensis cells].

    PubMed

    Zheng, Qing-Ping; Yu, Long-Jiang; Liu, Zhi; Li, Mo-Yi; Xiang, Fu; Yang, Qin

    2004-07-01

    Two distinct routes (classical mevalonate pathway and a novel mevalonate-independent pathway) are utilized by plants for the biosynthesis of isopentenyl diphosphate, the universal precursor of isoprenoids (Fig. 1). Present researches indicated that taxol was synthesized mainly via non-mevalonate pathway, but not genetic evidence was showed. The second step in non-mevalonate pathway involves an intramolecular rearrangement and subsequent reduction of deoxyxylulose phosphate to yield 2-C-methyl-D-erythritol-4-phosphate, and 1-Deoxy-D-xylulose-5-phosphate reductoisomerase (DXR) with responsibility for this reaction was considered as a key enzyme. As a tool for the isolation of genes in terpenoid biosynthesis in plants, total RNA was prepared from Taxus chinensis suspension cells, a cell type highly specialized for diterpene (taxol). A reverse transcription-PCR strategy based on the design of degenerated oligonucleotides was developed for isolating the gene encoding a gymnosperm homolog of this enzyme from Taxus chinensis. Through sequence analysis by Blast P online, the resulting cDNA showed highly homologous to 1-deoxy-D-xylulose 5-phosphate reductoisomerases, with 95% identification compared with Arabidopsis thaliana (Q9XFS9), 94% with Mentha x piperita (Q9XESO), 80% with Synechococcus elongatus (Q8DK30), 78% with Synechocystis sp. PCC 6803 (Q55663) and Nostoc sp. PCC 7120 (Q8YP49), and 73% with Synechococcus leopoliensis (Q9RKT1). Deduced amino acid sequences were also analyzed by PROSITE, ClustalX (1.81) and Phylio (3.6 alpha), and data present evidence for the existence of this deoxyxyluose phosphate reductoisomerase in Taxus chinensis. This is the first report of the dxr gene cloned from gymnosperm. PMID:15968987

  15. Alteration of the flexible loop in 1-deoxy-D-xylulose-5-phosphate reductoisomerase boosts enthalpy-driven inhibition by fosmidomycin.

    PubMed

    Kholodar, Svetlana A; Tombline, Gregory; Liu, Juan; Tan, Zhesen; Allen, C Leigh; Gulick, Andrew M; Murkin, Andrew S

    2014-06-01

    1-Deoxy-d-xylulose-5-phosphate reductoisomerase (DXR), which catalyzes the first committed step in the 2-C-methyl-d-erythritol 4-phosphate pathway of isoprenoid biosynthesis used by Mycobacterium tuberculosis and other infectious microorganisms, is absent in humans and therefore an attractive drug target. Fosmidomycin is a nanomolar inhibitor of DXR, but despite great efforts, few analogues with comparable potency have been developed. DXR contains a strictly conserved residue, Trp203, within a flexible loop that closes over and interacts with the bound inhibitor. We report that while mutation to Ala or Gly abolishes activity, mutation to Phe and Tyr only modestly impacts kcat and Km. Moreover, pre-steady-state kinetics and primary deuterium kinetic isotope effects indicate that while turnover is largely limited by product release for the wild-type enzyme, chemistry is significantly more rate-limiting for W203F and W203Y. Surprisingly, these mutants are more sensitive to inhibition by fosmidomycin, resulting in Km/Ki ratios up to 19-fold higher than that of wild-type DXR. In agreement, isothermal titration calorimetry revealed that fosmidomycin binds up to 11-fold more tightly to these mutants. Most strikingly, mutation strongly tips the entropy-enthalpy balance of total binding energy from 50% to 75% and 91% enthalpy in W203F and W203Y, respectively. X-ray crystal structures suggest that these enthalpy differences may be linked to differences in hydrogen bond interactions involving a water network connecting fosmidomycin's phosphonate group to the protein. These results confirm the importance of the flexible loop, in particular Trp203, in ligand binding and suggest that improved inhibitor affinity may be obtained against the wild-type protein by introducing interactions with this loop and/or the surrounding structured water network.

  16. Metabolic engineering of Salmonella vaccine bacteria to boost human Vγ2Vδ2 T cell immunity.

    PubMed

    Workalemahu, Grefachew; Wang, Hong; Puan, Kia-Joo; Nada, Mohanad H; Kuzuyama, Tomohisa; Jones, Bradley D; Jin, Chenggang; Morita, Craig T

    2014-07-15

    Human Vγ2Vδ2 T cells monitor isoprenoid metabolism by recognizing foreign (E)-4-hydroxy-3-methyl-but-2-enyl pyrophosphate (HMBPP), a metabolite in the 2-C-methyl-D-erythritol-4-phosphate pathway used by most eubacteria and apicomplexan parasites, and self isopentenyl pyrophosphate, a metabolite in the mevalonate pathway used by humans. Whereas microbial infections elicit prolonged expansion of memory Vγ2Vδ2 T cells, immunization with prenyl pyrophosphates or aminobisphosphonates elicit short-term Vγ2Vδ2 expansion with rapid anergy and deletion upon subsequent immunizations. We hypothesized that a live, attenuated bacterial vaccine that overproduces HMBPP would elicit long-lasting Vγ2Vδ2 T cell immunity by mimicking a natural infection. Therefore, we metabolically engineered the avirulent aroA(-) Salmonella enterica serovar Typhimurium SL7207 strain by deleting the gene for LytB (the downstream enzyme from HMBPP) and functionally complementing for this loss with genes encoding mevalonate pathway enzymes. LytB(-) Salmonella SL7207 had high HMBPP levels, infected human cells as efficiently as did the wild-type bacteria, and stimulated large ex vivo expansions of Vγ2Vδ2 T cells from human donors. Importantly, vaccination of a rhesus monkey with live lytB(-) Salmonella SL7207 stimulated a prolonged expansion of Vγ2Vδ2 T cells without significant side effects or anergy induction. These studies provide proof-of-principle that metabolic engineering can be used to derive live bacterial vaccines that boost Vγ2Vδ2 T cell immunity. Similar engineering of metabolic pathways to produce lipid Ags or B vitamin metabolite Ags could be used to derive live bacterial vaccine for other unconventional T cells that recognize nonpeptide Ags.

  17. Stimulation of artemisinin synthesis by combined cerebroside and nitric oxide elicitation in Artemisia annua hairy roots.

    PubMed

    Wang, Jian Wen; Zheng, Li Ping; Zhang, Ben; Zou, Ting

    2009-11-01

    This work examined the accumulation of artemisinin and related secondary metabolism pathways in hairy root cultures of Artemisia annua L. induced by a fungal-derived cerebroside (2S,2'R,3R,3'E,4E,8E)-1-O-beta-D-glucopyranosyl-2-N-(2'-hydroxy-3'-octadecenoyl)-3-hydroxy-9-methyl-4,8-sphingadienine. The presence of the cerebroside induced nitric oxide (NO) burst and artemisinin biosynthesis in the hairy roots. The endogenous NO generation was examined to be involved in the cerebroside-induced biosynthesis of artemisinin by using NO inhibitors, N (omega)-nitro-L-arginine methyl ester and 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide. The gene expression and activity of 3-hydroxy-3-methylglutaryl CoA reductase and 1-deoxy-D-xylulose 5-phosphate synthase were stimulated by the cerebroside, but more strongly by the potentiation of NO. While the mevalonate pathway inhibitor, mevinolin, only partially inhibited the induced artemisinin accumulation, the plastidic 2-C-methyl-D-erythritol 4-phosphate pathway inhibitor, fosmidomycin, nearly arrested artemisinin accumulation induced by cerebroside and the combination elicitation with an NO donor, sodium nitroprusside (SNP). With the potentiation by SNP at 10 microM, the cerebroside elicitor stimulated artemisinin production in 20-day-old hairy root cultures up to 22.4 mg/l, a 2.3-fold increase over the control. These results suggest that cerebroside plays as a novel elicitor and the involvement of NO in the signaling pathway of the elicitor activity for artemisinin biosynthesis.

  18. Comparative Transcriptomics Unravel Biochemical Specialization of Leaf Tissues of Stevia for Diterpenoid Production.

    PubMed

    Kim, Mi Jung; Jin, Jingjing; Zheng, Junshi; Wong, Limsoon; Chua, Nam-Hai; Jang, In-Cheol

    2015-12-01

    Stevia (Stevia rebaudiana) produces not only a group of diterpenoid glycosides known as steviol glycosides (SGs), but also other labdane-type diterpenoids that may be spatially separated from SGs. However, their biosynthetic routes and spatial distribution in leaf tissues have not yet been elucidated. Here, we integrate metabolome and transcriptome analyses of Stevia to explore the biosynthetic capacity of leaf tissues for diterpenoid metabolism. Tissue-specific chemical analyses confirmed that SGs were accumulated in leaf cells but not in trichomes. On the other hand, Stevia leaf trichomes stored other labdane-type diterpenoids such as oxomanoyl oxide and agatholic acid. RNA sequencing analyses from two different tissues of Stevia provided a comprehensive overview of dynamic metabolic activities in trichomes and leaf without trichomes. These metabolite-guided transcriptomics and phylogenetic and gene expression analyses clearly identified specific gene members encoding enzymes involved in the 2-C-methyl-d-erythritol 4-phosphate pathway and the biosynthesis of steviol or other labdane-type diterpenoids. Additionally, our RNA sequencing analysis uncovered copalyl diphosphate synthase (SrCPS) and kaurene synthase1 (SrKS1) homologs, SrCPS2 and KS-like (SrKSL), which were specifically expressed in trichomes. In vitro and in planta assays showed that unlike SrCPS and SrKS1, SrCPS2 synthesized labda-13-en-8-ol diphosphate and successively catalyzed the formation of manoyl oxide and epi-manoyl oxide in combination with SrKSL. Our findings suggest that Stevia may have evolved to use distinct metabolic pathways to avoid metabolic interferences in leaf tissues for efficient production of diverse secondary metabolites. PMID:26438788

  19. Metabolic Engineering of Salmonella Vaccine Bacteria to Boost Human Vγ2Vδ2 T Cell Immunity

    PubMed Central

    Workalemahu, Grefachew; Wang, Hong; Puan, Kia-Joo; Nada, Mohanad H.; Kuzuyama, Tomohisa; Jones, Bradley D.; Jin, Chenggang; Morita, Craig T.

    2014-01-01

    Human Vγ2Vδ2 T cells monitor isoprenoid metabolism by recognizing foreign (E)-4-hydroxy-3-methyl-but-2-enyl pyrophosphate (HMBPP), a metabolite in the 2-C-methyl-D-erythritol-4-phosphate pathway used by most eubacteria and apicomplexan parasites, and self isopentenyl pyrophosphate, a metabolite in the mevalonate pathway used by humans. Whereas microbial infections elicit prolonged expansion of memory Vγ2Vδ2 T cells, immunization with prenyl pyrophosphates or aminobisphosphonates elicit short-term Vγ2Vδ2 expansion with rapid anergy and deletion upon subsequent immunizations. We hypothesized that a live, attenuated bacterial vaccine that overproduces HMBPP would elicit long lasting Vγ2Vδ2 T cell immunity by mimicking a natural infection. Therefore, we metabolically engineered the avirulent aroA− Salmonella enterica serovar Typhimurium SL7207 strain by deleting the gene for LytB (the downstream enzyme from HMBPP) and functionally complementing for this loss with genes encoding mevalonate pathway enzymes. LytB− Salmonella SL7207 had high HMBPP levels, infected human cells as efficiently as the wild-type bacteria, and stimulated large ex vivo expansions of Vγ2Vδ2 T cells from human donors. Importantly, vaccination of a rhesus monkey with live lytB− Salmonella SL7207 stimulated a prolonged expansion of Vγ2Vδ2 T cells without significant side effects or anergy induction. These studies provide proof-of-principle that metabolic engineering can be used to derive live bacterial vaccines that boost Vγ2Vδ2 T cell immunity. Similar engineering of metabolic pathways to produce lipid Ags or B vitamin metabolite Ags could be used to derive live bacterial vaccine for other unconventional T cells that recognize nonpeptide Ags. PMID:24943221

  20. Comparative Transcriptomics Unravel Biochemical Specialization of Leaf Tissues of Stevia for Diterpenoid Production.

    PubMed

    Kim, Mi Jung; Jin, Jingjing; Zheng, Junshi; Wong, Limsoon; Chua, Nam-Hai; Jang, In-Cheol

    2015-12-01

    Stevia (Stevia rebaudiana) produces not only a group of diterpenoid glycosides known as steviol glycosides (SGs), but also other labdane-type diterpenoids that may be spatially separated from SGs. However, their biosynthetic routes and spatial distribution in leaf tissues have not yet been elucidated. Here, we integrate metabolome and transcriptome analyses of Stevia to explore the biosynthetic capacity of leaf tissues for diterpenoid metabolism. Tissue-specific chemical analyses confirmed that SGs were accumulated in leaf cells but not in trichomes. On the other hand, Stevia leaf trichomes stored other labdane-type diterpenoids such as oxomanoyl oxide and agatholic acid. RNA sequencing analyses from two different tissues of Stevia provided a comprehensive overview of dynamic metabolic activities in trichomes and leaf without trichomes. These metabolite-guided transcriptomics and phylogenetic and gene expression analyses clearly identified specific gene members encoding enzymes involved in the 2-C-methyl-d-erythritol 4-phosphate pathway and the biosynthesis of steviol or other labdane-type diterpenoids. Additionally, our RNA sequencing analysis uncovered copalyl diphosphate synthase (SrCPS) and kaurene synthase1 (SrKS1) homologs, SrCPS2 and KS-like (SrKSL), which were specifically expressed in trichomes. In vitro and in planta assays showed that unlike SrCPS and SrKS1, SrCPS2 synthesized labda-13-en-8-ol diphosphate and successively catalyzed the formation of manoyl oxide and epi-manoyl oxide in combination with SrKSL. Our findings suggest that Stevia may have evolved to use distinct metabolic pathways to avoid metabolic interferences in leaf tissues for efficient production of diverse secondary metabolites.

  1. Characterization of the Pseudomonas aeruginosa metalloendopeptidase, Mep72, a member of the Vfr regulon

    PubMed Central

    2013-01-01

    metalloendopeptidase, which we named Mep72. Mep72, which has metalloendopeptidase and carbohydrate-binding domains, produced proteolytic and endopeptidase activities in E. coli. Vfr directly regulates the expression of the PA2782-mep72 operon by binding to its upstream region. However, unlike other Vfr-targeted genes, Vfr binding does not require an intact Vfr consensus binding sequence. PMID:24279383

  2. Local control of phosphatidylinositol 4-phosphate signaling in the Golgi apparatus by Vps74 and Sac1 phosphoinositide phosphatase

    PubMed Central

    Wood, Christopher S.; Hung, Chia-Sui; Huoh, Yu-San; Mousley, Carl J.; Stefan, Christopher J.; Bankaitis, Vytas; Ferguson, Kathryn M.; Burd, Christopher G.

    2012-01-01

    In the Golgi apparatus, lipid homeostasis pathways are coordinated with the biogenesis of cargo transport vesicles by phosphatidylinositol 4-kinases (PI4Ks) that produce phosphatidylinositol 4-phosphate (PtdIns4P), a signaling molecule that is recognized by downstream effector proteins. Quantitative analysis of the intra-Golgi distribution of a PtdIns4P reporter protein confirms that PtdIns4P is enriched on the trans-Golgi cisterna, but surprisingly, Vps74 (the orthologue of human GOLPH3), a PI4K effector required to maintain residence of a subset of Golgi proteins, is distributed with the opposite polarity, being most abundant on cis and medial cisternae. Vps74 binds directly to the catalytic domain of Sac1 (KD = 3.8 μM), the major PtdIns4P phosphatase in the cell, and PtdIns4P is elevated on medial Golgi cisternae in cells lacking Vps74 or Sac1, suggesting that Vps74 is a sensor of PtdIns4P level on medial Golgi cisternae that directs Sac1-mediated dephosphosphorylation of this pool of PtdIns4P. Consistent with the established role of Sac1 in the regulation of sphingolipid biosynthesis, complex sphingolipid homeostasis is perturbed in vps74Δ cells. Mutant cells lacking complex sphingolipid biosynthetic enzymes fail to properly maintain residence of a medial Golgi enzyme, and cells lacking Vps74 depend critically on complex sphingolipid biosynthesis for growth. The results establish additive roles of Vps74-mediated and sphingolipid-dependent sorting of Golgi residents. PMID:22553352

  3. Structural definition of the active site and catalytic mechanism of 3,4-dihydroxy-2-butanone 4-phosphate synthase

    SciTech Connect

    Liao, D.-I.; Zheng, Y.-J.; Viitanen, P.V.; Jordan, D.B.

    2010-03-08

    X-ray crystal structures of L-3,4-dihydroxy-2-butanone-4-phosphate synthase from Magnaporthe grisea are reported for the E-SO{sub 4}{sup 2-}, E-{sub 4}{sup 2-}-Mg{sup 2+}, E-SO{sub 4}{sup 2-}-Mn{sup 2+}, E-SO{sub 4}{sup 2-}-Mn{sup 2+}-glycerol, and E-SO{sub 4}{sup 2-}-Zn{sup 2+} complexes with resolutions that extend to 1.55, 0.98, 1.60, 1.16, and 1.00 {angstrom}, respectively. Active-site residues of the homodimer are fully defined. The structures were used to model the substrate ribulose 5-phosphate in the active site with the phosphate group anchored at the sulfate site and the placement of the ribulose group guided by the glycerol site. The model includes two Mg{sup 2+} cations that bind to the oxygen substituents of the C2, C3, C4, and phosphate groups of the substrate, the side chains of Glu37 and His153, and water molecules. The position of the metal cofactors and the substrate's phosphate group are further stabilized by an extensive hydrogen-bond and salt-bridge network. On the basis of their proximity to the substrate's reaction participants, the imidazole of an Asp99-His136 dyad from one subunit, the side chains of the Asp41, Cys66, and Glu174 residues from the other subunit, and Mg{sup 2+}-activated water molecules are proposed to serve specific roles in the catalytic cycle as general acid-base functionalities. The model suggests that during the 1,2-shift step of the reaction, the substrate's C3 and C4 hydroxyl groups are cis to each other. A cis transition state is calculated to have an activation barrier that is 2 kcal/mol greater than that of the trans transition state in the absence of the enzyme.

  4. Molecular Basis of Phosphatidylinositol 4-Phosphate and ARF1 GTPase Recognition by the FAPP1 Pleckstrin Homology (PH) Domain

    SciTech Connect

    He, J.; Heroux, A.; Scott, J. L.; Roy, S.; Lenoir, M.; Overduin, M.; Stahelin, R. V.; Kutateladze, T. G.

    2011-05-27

    Four-phosphate-adaptor protein 1 (FAPP1) regulates secretory transport from the trans-Golgi network (TGN) to the plasma membrane. FAPP1 is recruited to the Golgi through binding of its pleckstrin homology (PH) domain to phosphatidylinositol 4-phosphate (PtdIns(4)P) and a small GTPase ADP-ribosylation factor 1 (ARF1). Despite the critical role of FAPP1 in membrane trafficking, the molecular basis of its dual function remains unclear. Here, we report a 1.9 {angstrom} resolution crystal structure of the FAPP1 PH domain and detail the molecular mechanisms of the PtdIns(4)P and ARF1 recognition. The FAPP1 PH domain folds into a seven-stranded {beta}-barrel capped by an {alpha}-helix at one edge, whereas the opposite edge is flanked by three loops and the {beta}4 and {beta}7 strands that form a lipid-binding pocket within the {beta}-barrel. The ARF1-binding site is located on the outer side of the {beta}-barrel as determined by NMR resonance perturbation analysis, mutagenesis, and measurements of binding affinities. The two binding sites have little overlap, allowing FAPP1 PH to associate with both ligands simultaneously and independently. Binding to PtdIns(4)P is enhanced in an acidic environment and is required for membrane penetration and tubulation activity of FAPP1, whereas the GTP-bound conformation of the GTPase is necessary for the interaction with ARF1. Together, these findings provide structural and biochemical insight into the multivalent membrane anchoring by the PH domain that may augment affinity and selectivity of FAPP1 toward the TGN membranes enriched in both PtdIns(4)P and GTP-bound ARF1.

  5. Complementation of growth factor receptor-dependent mitogenic signaling by a truncated type I phosphatidylinositol 4-phosphate 5-kinase.

    PubMed

    Davis, J N; Rock, C O; Cheng, M; Watson, J B; Ashmun, R A; Kirk, H; Kay, R J; Roussel, M F

    1997-12-01

    Substitution of phenylalanine for tyrosine at codon 809 (Y809F) of the human colony-stimulating factor 1 (CSF-1) receptor (CSF-1R) impairs ligand-stimulated tyrosine kinase activity, prevents induction of c-MYC and cyclin D1 genes, and blocks CSF-1-dependent progression through the G1 phase of the cell cycle. We devised an unbiased genetic screen to isolate genes that restore the ability of CSF-1 to stimulate growth in cells that express mutant CSF-1R (Y809F). This screen led us to identify a truncated form of the murine type Ibeta phosphatidylinositol 4-phosphate 5-kinase (mPIP5K-Ibeta). This truncated protein lacks residues 1 to 238 of mPIP5K-Ibeta and is catalytically inactive. When we transfected cells expressing CSF-1R (Y809F) with mPIP5K-Ibeta (delta1-238), CSF-1-dependent induction of c-MYC and cyclin D1 was restored and ligand-dependent cell proliferation was sustained. CSF-1 normally triggers the rapid disappearance of CSF-1R (Y809F) from the cell surface; however, transfection of cells with mPIP5K-Ibeta (delta1-238) stabilized CSF-1R (Y809F) expression on the cell surface, resulting in elevated levels of ligand-activated CSF-1R (Y809F). These results suggest a role for PIP5K-Ibeta in receptor endocytosis and that the truncated enzyme compensated for a mitogenically defective CSF-1R by interfering with this process.

  6. Low dose monoethyl phthalate (MEP) exposure triggers proliferation by activating PDX-1 at 1.1B4 human pancreatic beta cells.

    PubMed

    Güven, Celal; Dal, Fulya; Aydoğan Ahbab, Müfide; Taskin, Eylem; Ahbab, Süleyman; Adin Çinar, Suzan; Sırma Ekmekçi, Sema; Güleç, Çağrı; Abacı, Neslihan; Akçakaya, Handan

    2016-07-01

    Phthalate plasticizers used in a wide range of common plastic products are released into the environment and may pose a risk of increased incidence of type 2 diabetes. In this work, we studied the effects of monoethyl phthalate (MEP), the metabolite of diethyl phthalate, exposure on 1.1B4 human pancreatic beta cells at low doses (1-1000 nM). We showed that MEP treatment induced proliferation in 1.1B4 cells. Also PCNA protein expression levels were increased related to proliferation induction. It has been noted that phthalates can exert estrogen mediated response by interacting with ER. In our study 24 h MEP treatment decreased ERα protein expression level conversely it increased the same protein expression level after 72 h treatment. Also MEP treatment decreased ERβ expression after 72 h at 1.1B4 cells. Our results further show that insulin content of 1.1B4 cells were increased with low dose MEP treatment. Along with our insulin content results, PDX- 1 expression levels were also increased at 1.1B4 cells with MEP treatment. These findings suggest that MEP acts as an estrogenic compound and PPARγ agonist at lower concentrations. Also it should be noted that PDX-1 may be a critical regulator of 1.1B4 cells treated with MEP.

  7. Microcomputer Materials from MEP. An Annotated Directory of Packages of Interest to Further Education. An Occasional Paper.

    ERIC Educational Resources Information Center

    Gilbert, Leslie

    Designed to disseminate information to the post-school sector of United Kingdom education, this directory provides information on 50 microcomputer software packages developed by the Microelectronics Education Program (MEP) and available through educational publishers. Subject areas represented include accountancy, biology, business education,…

  8. MEP50/PRMT5 reduces gene expression by histone arginine methylation and this is reversed by PKCδ/p38δ signaling

    PubMed Central

    Saha, Kamalika; Adhikary, Gautam; Eckert, Richard L.

    2016-01-01

    PKCδ and p38δ are key proteins in a cascade that stimulates keratinocyte differentiation. This cascade activates transcription of involucrin (hINV) and other genes associated with differentiation. Protein arginine methyltransferase 5 (PRMT5) is an arginine methyltransferase that symmetrically dimethylates arginine residues. This protein interacts with a cofactor, MEP50, and symmetrically dimethylates arginine eight of histone 3 (H3R8me2s) and arginine three of histone 4 (H4R3me2s) to silence gene expression. We use the involucrin gene as a tool to understand the relationship between PKCδ/p38δ and PRMT5/MEP50 signaling. MEP50 suppresses hINV mRNA level and promoter activity. This is associated with increased arginine dimethylation of hINV gene-associated H3/H4. We further show that the PKCδ/p38δ keratinocyte differentiation cascade reduces PRMT5 and MEP50 expression, association with the hINV gene promoter, and H3R8me2s and H4R2me2s formation. We propose that PRMT5/MEP50-dependent methylation is an epigenetic mechanism that assists in silencing of hINV expression, and that PKCδ signaling activates gene expression by directly activating transcription and by suppressing PRMT5/MEP50 dependent arginine dimethylation of promoter associated histones. This is an example of crosstalk between PKCδ/p38δ signaling and PRMT5/MEP50 epigenetic silencing. PMID:26763441

  9. Evidence that fungal MEP proteins mediate diffusion of the uncharged species NH(3) across the cytoplasmic membrane.

    PubMed

    Soupene, E; Ramirez, R M; Kustu, S

    2001-09-01

    Methylammonium and ammonium (MEP) permeases of Saccharomyces cerevisiae belong to a ubiquitous family of cytoplasmic membrane proteins that transport only ammonium (NH(4)(+) + NH(3)). Transport and accumulation of the ammonium analog [(14)C]methylammonium, a weak base, led to the proposal that members of this family were capable of energy-dependent concentration of the ammonium ion, NH(4)(+). In bacteria, however, ATP-dependent conversion of methylammonium to gamma-N-methylglutamine by glutamine synthetase precludes its use in assessing concentrative transport across the cytoplasmic membrane. We have confirmed that methylammonium is not metabolized in the yeast S. cerevisiae and have shown that it is little metabolized in the filamentous fungus Neurospora crassa. However, its accumulation depends on the energy-dependent acidification of vacuoles. A Deltavph1 mutant of S. cerevisiae and a Deltavma1 mutant, which lack vacuolar H(+)-ATPase activity, had large (fivefold or greater) defects in the accumulation of methylammonium, with little accompanying defect in the initial rate of transport. A vma-1 mutant of N. crassa largely metabolized methylammonium to methylglutamine. Thus, in fungi as in bacteria, subsequent energy-dependent utilization of methylammonium precludes its use in assessing active transport across the cytoplasmic membrane. The requirement for a proton gradient to sequester the charged species CH(3)NH(3)(+) in acidic vacuoles provides evidence that the substrate for MEP proteins is the uncharged species CH(3)NH(2). By inference, their natural substrate is NH(3), a gas. We postulate that MEP proteins facilitate diffusion of NH(3) across the cytoplasmic membrane and speculate that human Rhesus proteins, which lie in the same domain family as MEP proteins, facilitate diffusion of CO(2).

  10. An Efficient Synthetic Strategy for Obtaining 4-Methoxy Carbon Isotope Labeled Combretastatin A-4 Phosphate and Other Z-Combretastatins1

    PubMed Central

    Pettit, George R.; Minardi, Mathew D.; Hogan, Fiona; Price, Pat M.

    2010-01-01

    Human cancer and other clinical trials under development employing combretastatin A-4 phosphate (1b, CA4P) should benefit from the availability of a [11C]-labeled derivative for position emission tomography (PET). In order to obtain a suitable precursor for addition of a [11C]methyl group at the penultimate step, several new synthetic pathways to CA4P were evaluated. Geometrical isomerization (Z to E) proved to be a challenge, but it was overcome by development of a new CA4P synthesis suitable for 4-methoxy isotope labeling. PMID:20028026

  11. A higher number of TMS-elicited MEP from a combined hotspot improves intra- and inter-session reliability of the upper limb muscles in healthy individuals.

    PubMed

    Bastani, Andisheh; Jaberzadeh, Shapour

    2012-01-01

    We aimed to determine, using transcranial magnetic stimulation (TMS), the number of elicited motor evoked potentials (MEPs) that induces the highest intra- and inter-sessions reliability for the extensor carpi radialis (ECR) and first dorsal interosseus (FDI) muscles. Twelve healthy subjects participated in this study on two separate days. Single pulse magnetic stimuli were triggered with Magstim 200(2) to obtain MEPs from the muscles of interest, with the subjects in a relaxed position. Reliability of MEP responses was investigated in three blocks of 5, 10 and 15 trials. The intra- and inter-session reliability of the MEPs' amplitudes and latencies were assessed using intraclass correlation coefficients (ICCs). Repeated measures ANOVA and paired t-tests revealed no significant time effect in the MEP amplitude and latency measurements (P>0.05). The ICCs indicated high intra-session reliability in the MEPs' amplitudes for the ECR and FDI muscles (0.77 to 0.99, 0.90 to 0.99, respectively) and latency (0.80 to 1.00, 0.75 to 0.97, respectively). The MEPs' amplitudes also had high inter-session reliability (0.84 to 0.97, 0.88 to 0.93, respectively), as did their latency (0.80 to 0.90, 0.75 to 0.97, respectively). Highest intra- and inter-session reliability was achieved for blocks of 10 and 15 trials. Our data suggest that intra- and inter-session comparisons should be performed using at least 10 MEPs in "combined hotspot" stimulation technique to ensure highest reliability.

  12. Peptidoglycan-associated outer membrane protein Mep45 of rumen anaerobe Selenomonas ruminantium forms a non-specific diffusion pore via its C-terminal transmembrane domain

    PubMed Central

    Kojima, Seiji; Hayashi, Kanako; Tochigi, Saeko; Kusano, Tomonobu; Kaneko, Jun; Kamio, Yoshiyuki

    2016-01-01

    The major outer membrane protein Mep45 of Selenomonas ruminantium, an anaerobic Gram-negative bacterium, comprises two distinct domains: the N-terminal S-layer homologous (SLH) domain that protrudes into the periplasm and binds to peptidoglycan, and the remaining C-terminal transmembrane domain, whose function has been unknown. Here, we solubilized and purified Mep45 and characterized its function using proteoliposomes reconstituted with Mep45. We found that Mep45 forms a nonspecific diffusion channel via its C-terminal region. The channel was permeable to solutes smaller than a molecular weight of roughly 600, and the estimated pore radius was 0.58 nm. Truncation of the SLH domain did not affect the channel property. On the basis of the fact that Mep45 is the most abundant outer membrane protein in S. ruminantium, we conclude that Mep45 serves as a main pathway through which small solutes diffuse across the outer membrane of this bacterium. PMID:27310312

  13. Investigating the cortical regions involved in MEP modulation in tDCS

    PubMed Central

    Salvador, Ricardo; Wenger, Cornelia; Miranda, Pedro C.

    2015-01-01

    Transcranial magnetic stimulation (TMS) is used in several studies to evaluate cortical excitability changes induced by transcranial direct current stimulation (tDCS) of the primary motor cortex. Interpretation of these results, however, is hindered by the very different spatial distribution of the electric field (E-field) induced by the two techniques and by the different target neurons that they might act upon. In this study we used the finite element method to calculate the E-field distribution induced by TMS and tDCS in a realistically shaped model of a human head. A model of a commercially available figure-8 coil was placed over a position above the identified hand knob (HK) region. We also modeled two configurations of bipolar tDCS montages with one of the electrodes placed over the HK and a return electrode over the contralateral orbital region. The electrodes over the HK were either rectangular in shape, with an area of 35 cm2 or cylindrical with an area of π cm2 (1 cm radius). To compare the E-field distribution in TMS and the two tDCS models, average values of the E-field's magnitude as well as the polar and azimuthal angle were investigated in the HK region and premotor areas. The results show that both techniques induce fields with different magnitudes and directions in the HK: the field in tDCS is predominantly perpendicular to the cortical surface, contrary to what happens in TMS where the field is mostly parallel to it. In the premotor areas, the magnitude of the E-field induced in TMS was well below the accepted threshold for MEP generation, 100 V/m. In tDCS, the magnitude of the field in these areas was comparable to that induced at the HK with a significant component perpendicular to the cortical surface. These results indicate that tDCS and TMS target preferentially different neuronal structures at the HK. Besides, they show that premotor areas may play a role in the tDCS-induced after effects on motor cortex excitability. PMID:26528134

  14. Physiological function of IspE, a plastid MEP pathway gene for isoprenoid biosynthesis, in organelle biogenesis and cell morphogenesis in Nicotiana benthamiana.

    PubMed

    Ahn, Chang Sook; Pai, Hyun-Sook

    2008-03-01

    Isoprenoid biosynthesis in plants occurs by two independent pathways: the cytosolic mevalonate (MVA) pathway and the plastidic methylerythritol phosphate (MEP) pathway. In this study, we investigated the cellular effects of depletion of IspE, a protein involved in the MEP pathway, using virus-induced gene silencing (VIGS). The IspE gene is preferentially expressed in young tissues, and induced by light and methyl jasmonate. The GFP fusion protein of IspE was targeted to chloroplasts. Reduction of IspE expression by VIGS resulted in a severe leaf yellowing phenotype. At the cellular level, depletion of IspE severely affected chloroplast development, dramatically reducing both the number and size of chloroplasts. Interestingly, mitochondrial development was also impaired, suggesting a possibility that the plastidic MEP pathway contributes to mitochondrial isoprenoid biosynthesis in leaves. A deficiency in IspE activity decreased cellular levels of the metabolites produced by the MEP pathway, such as chlorophylls and carotenoids, and stimulated expression of some of the downstream MEP pathway genes, particularly IspF and IspG. Interestingly, the IspE VIGS lines had significantly increased numbers of cells of reduced size in all leaf layers, compared with TRV control and other VIGS lines for the MEP pathway genes. The increased cell division in the IspE VIGS lines was particularly pronounced in the abaxial epidermal layer, in which the over-proliferated cells bulged out of the plane, making the surface uneven. In addition, trichome numbers dramatically increased and the stomata size varied in the affected tissues. Our results show that IspE deficiency causes novel developmental phenotypes distinct from the phenotypes of other MEP pathway mutants, indicating that IspE may have an additional role in plant development besides its role in isoprenoid biosynthesis.

  15. Fair play doesn't matter: MEP modulation as a neurophysiological signature of status quo bias in economic interactions.

    PubMed

    Pisoni, Alberto; Lo Gerfo, Emanuele; Ottone, Stefania; Ponzano, Ferruccio; Zarri, Luca; Vergallito, Alessandra; Romero Lauro, Leonor Josefina

    2014-11-01

    Transcranial magnetic stimulation (TMS) studies show that watching others' movements enhances motor evoked potential (MEPs) amplitude of the muscles involved in the observed action (motor facilitation, MF). MF has been attributed to a mirror neuron system mediated mechanism, causing an excitability increment of primary motor cortex. It is still unclear whether the meaning an action assumes when performed in an interpersonal exchange context could affect MF. This study aims at exploring this issue by measuring MF induced by the observation of the same action coupled with opposite reward values (gain vs loss) in an economic game. Moreover, the interaction frame was manipulated by showing the same actions within different economic games, the Dictator Game (DG) and the Theft Game (TG). Both games involved two players: a Dictator/Thief and a receiver. Experimental participants played the game always as receivers whereas the Dictator/Thief roles were played by our confederates. In each game Dictator/Thief's choices were expressed by showing a grasping action of one of two cylinders, previously associated with fair/unfair choices. In the DG the dictator decides whether to share (gain condition) or not (no-gain condition) a sum of money with the receiver, while in TGs the thief decides whether to steal (loss condition) or not to steal (no-loss condition) it from the participants. While the experimental subjects watched the videos showing these movements, a single TMS pulse was delivered to their motor hand area and a MEP was recorded from the right FDI muscle. Results show that, in the DG, MF was enhanced by the status quo modification, i.e. MEP amplitude increased when the dictator decided to change the receivers' status quo and share his/her money, and this was true when the status quo was more salient. The same was true for the TG, where the reverse happened: MF was higher for trials in which the thief decided to steal the participants' money, thus changing the status

  16. cisMEP: an integrated repository of genomic epigenetic profiles and cis-regulatory modules in Drosophila

    PubMed Central

    2014-01-01

    Background Cis-regulatory modules (CRMs), or the DNA sequences required for regulating gene expression, play the central role in biological researches on transcriptional regulation in metazoan species. Nowadays, the systematic understanding of CRMs still mainly resorts to computational methods due to the time-consuming and small-scale nature of experimental methods. But the accuracy and reliability of different CRM prediction tools are still unclear. Without comparative cross-analysis of the results and combinatorial consideration with extra experimental information, there is no easy way to assess the confidence of the predicted CRMs. This limits the genome-wide understanding of CRMs. Description It is known that transcription factor binding and epigenetic profiles tend to determine functions of CRMs in gene transcriptional regulation. Thus integration of the genome-wide epigenetic profiles with systematically predicted CRMs can greatly help researchers evaluate and decipher the prediction confidence and possible transcriptional regulatory functions of these potential CRMs. However, these data are still fragmentary in the literatures. Here we performed the computational genome-wide screening for potential CRMs using different prediction tools and constructed the pioneer database, cisMEP (cis-regulatory module epigenetic profile database), to integrate these computationally identified CRMs with genomic epigenetic profile data. cisMEP collects the literature-curated TFBS location data and nine genres of epigenetic data for assessing the confidence of these potential CRMs and deciphering the possible CRM functionality. Conclusions cisMEP aims to provide a user-friendly interface for researchers to assess the confidence of different potential CRMs and to understand the functions of CRMs through experimentally-identified epigenetic profiles. The deposited potential CRMs and experimental epigenetic profiles for confidence assessment provide experimentally testable

  17. Fair play doesn't matter: MEP modulation as a neurophysiological signature of status quo bias in economic interactions.

    PubMed

    Pisoni, Alberto; Lo Gerfo, Emanuele; Ottone, Stefania; Ponzano, Ferruccio; Zarri, Luca; Vergallito, Alessandra; Romero Lauro, Leonor Josefina

    2014-11-01

    Transcranial magnetic stimulation (TMS) studies show that watching others' movements enhances motor evoked potential (MEPs) amplitude of the muscles involved in the observed action (motor facilitation, MF). MF has been attributed to a mirror neuron system mediated mechanism, causing an excitability increment of primary motor cortex. It is still unclear whether the meaning an action assumes when performed in an interpersonal exchange context could affect MF. This study aims at exploring this issue by measuring MF induced by the observation of the same action coupled with opposite reward values (gain vs loss) in an economic game. Moreover, the interaction frame was manipulated by showing the same actions within different economic games, the Dictator Game (DG) and the Theft Game (TG). Both games involved two players: a Dictator/Thief and a receiver. Experimental participants played the game always as receivers whereas the Dictator/Thief roles were played by our confederates. In each game Dictator/Thief's choices were expressed by showing a grasping action of one of two cylinders, previously associated with fair/unfair choices. In the DG the dictator decides whether to share (gain condition) or not (no-gain condition) a sum of money with the receiver, while in TGs the thief decides whether to steal (loss condition) or not to steal (no-loss condition) it from the participants. While the experimental subjects watched the videos showing these movements, a single TMS pulse was delivered to their motor hand area and a MEP was recorded from the right FDI muscle. Results show that, in the DG, MF was enhanced by the status quo modification, i.e. MEP amplitude increased when the dictator decided to change the receivers' status quo and share his/her money, and this was true when the status quo was more salient. The same was true for the TG, where the reverse happened: MF was higher for trials in which the thief decided to steal the participants' money, thus changing the status

  18. CO2 as main carbon source for isoprenoid biosynthesis via the mevalonate-independent methylerythritol 4-phosphate route in the marine diatoms Phaeodactylum tricornutum and Nitzschia ovalis.

    PubMed

    Cvejić, J H; Rohmer, M

    2000-01-01

    Isoprenoid biosynthesis was investigated in the two diatoms Phaeodactylum tricornutum and Nitzschia ovalis by labeling experiments performed in mixotrophic growth conditions with sodium [1-(13)C]acetate, 13CO2, [1-(13)C]glucose, sodium [3-(13)C]pyruvate and 1-deoxy-D-[5,5-(2)H2]xylulose. A clear dichotomy was found. Acetate was the preferred carbon source for the formation of the sterols in the cytoplasm via the mevalonate pathway. Carbon dioxide was the main source for phytol biosynthesis in the chloroplasts via the mevalonate-independent methylerythritol 4-phosphate pathway. The two diatoms showed the same compartmentation for isoprenoid biosynthesis as that previously found in higher plants, the red alga Porphyridium cruentum and the Chrysophyte Ochromonas danica.

  19. Studies on the 4-carbon precursor in the biosynthesis of riboflavin. Purification and properties of L-3,4-dihydroxy-2-butanone-4-phosphate synthase.

    PubMed

    Volk, R; Bacher, A

    1990-11-15

    The formation of the riboflavin precursor, 6,7-dimethyl-8-ribityllumazine, from 5-amino-6-ribitylamino-2,4(1H,3H)-pyrimidinedione requires a phosphorylated 4-carbon intermediate which has been designated as Compound X (Neuberger, G., and Bacher, A. (1985) Biochem. Biophys. Res. Commun. 127, 175-181). The enzyme catalyzing the formation of Compound X has been purified about 600-fold from the cell extract of the flavinogenic yeast Candida guilliermondii by chromatographic procedures. The purified protein appeared homogeneous as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and consisted of a single polypeptide of 24 kDa. The committed substrate of the enzyme was identified as D-ribulose 5-phosphate. The enzyme yields two products which were identified as L-3,4-dihydroxy-2-butanone 4-phosphate and formate by NMR and CD spectroscopy. Mg2+ is required for activity. PMID:2246238

  20. Check-Testing of Manufacturer Self Reported Labeling Data& Compliance with MEPS

    SciTech Connect

    Zhou, Nan; Zhou, Nan; Zheng, Nina; Fridley, David; Wang, Ruohong; Egan, Christine

    2008-03-01

    China first adopted minimum energy performance standards (MEPS) in 1989. Today, there are standards for a wide range of domestic, commercial and selected industrial equipment. In 1999, China launched a voluntary endorsement label, which has grown to cover over 40 products including water-saving products. Further, in 2005, China started a mandatory energy information label that initially covered two products and in 2007 was extended to cover four products total including: air conditioners; household refrigerators; clothes washers; and unitary air conditioners. These programs have had an important impact in reducing the energy consumption of appliances in China. China has built up a strong infrastructure to develop and implement standards. Historically, however, the government's primary focus has been on the technical requirements for specifying efficiency performance. Less attention has been paid to monitoring and enforcement with a minimal commitment of resources and little expansion of administrative capacity in this area. Thus, market compliance with both mandatory standard and labeling programs has been questionable. Furthermore, actual energy savings have quite possibly been undermined as a result. The establishment of a regularized monitoring system for tracking compliance with the mandatory standard and energy information label programs in China is a major area for program improvement. Over the years, the Collaborative Labeling and Appliance Standards Program (CLASP) has partnered with several Chinese institutions to promote energy-efficient products in China. CLASP, together with its implementing partner Lawrence Berkeley National Laboratory (LBNL), has assisted China in developing and updating the above-mentioned standards and labeling programs. Because of the increasing need for the development of a monitoring system to track compliance with the standard, CLASP, with support from Japan's Ministry of Economy, Trade and Industry (METI) and the Institute of

  1. Purification, crystallization and X-ray crystallographic studies of a Bacillus cereus MepR-like transcription factor, BC0657.

    PubMed

    Cho, Min Uk; Kim, Meong Il; Hong, Minsun

    2015-06-01

    Transcription factors of the MarR family respond to internal and external changes and regulate a variety of biological functions through ligand association with microorganisms. MepR belongs to the MarR family, and its mutations are associated with the development of multidrug resistance in Staphylococcus aureus, which has caused a growing health problem. In this study, a Bacillus cereus MepR-like transcription regulator, BC0657, was crystallized. The BC0657 crystals diffracted to 2.05 Å resolution and belonged to either space group P6(2)22 or P6(4)22, with unit-cell parameters a = 110.57, b = 110.57, c = 67.29 Å. There was one molecule per asymmetric unit. Future comparative structural studies on BC0657 would extend knowledge of ligand-induced transcriptional regulatory mechanisms in the MarR family and would make a significant contribution to the design of antibiotic drugs against multidrug-resistant bacteria.

  2. FT-IR and FT-Raman spectra, MEP and HOMO-LUMO of 2,5-dichlorobenzonitrile: DFT study.

    PubMed

    Alcolea Palafox, M; Bhat, Daisy; Goyal, Yasha; Ahmad, Shabbir; Hubert Joe, I; Rastogi, V K

    2015-02-01

    The experimental FT-IR and FT-Raman spectra of 2,5-dichlorobenzonitrile molecule were recorded at room temperature, and the results compared with quantum chemical theoretical values using MP2 and DFT methods. Molecular geometry, vibrational wavenumbers and thermodynamic parameters were calculated. With the help of specific scaling procedures for the computed wavenumbers, the experimentally observed FTIR and FT-Raman bands were analyzed and assigned to different normal modes of the molecule. Most of the modes have wavenumbers in the expected range and the error obtained was in general very low. Several general conclusions were deduced. The NBO analysis has been done and Molecular Electrostatic Potential (MEP) has been plotted.

  3. Lenz-Majewski mutations in PTDSS1 affect phosphatidylinositol 4-phosphate metabolism at ER-PM and ER-Golgi junctions.

    PubMed

    Sohn, Mira; Ivanova, Pavlina; Brown, H Alex; Toth, Daniel J; Varnai, Peter; Kim, Yeun Ju; Balla, Tamas

    2016-04-19

    Lenz-Majewski syndrome (LMS) is a rare disease characterized by complex craniofacial, dental, cutaneous, and limb abnormalities combined with intellectual disability. Mutations in thePTDSS1gene coding one of the phosphatidylserine (PS) synthase enzymes, PSS1, were described as causative in LMS patients. Such mutations render PSS1 insensitive to feedback inhibition by PS levels. Here we show that expression of mutant PSS1 enzymes decreased phosphatidylinositol 4-phosphate (PI4P) levels both in the Golgi and the plasma membrane (PM) by activating the Sac1 phosphatase and altered PI4P cycling at the PM. Conversely, inhibitors of PI4KA, the enzyme that makes PI4P in the PM, blocked PS synthesis and reduced PS levels by 50% in normal cells. However, mutant PSS1 enzymes alleviated the PI4P dependence of PS synthesis. Oxysterol-binding protein-related protein 8, which was recently identified as a PI4P-PS exchanger between the ER and PM, showed PI4P-dependent membrane association that was significantly decreased by expression of PSS1 mutant enzymes. Our studies reveal that PS synthesis is tightly coupled to PI4P-dependent PS transport from the ER. Consequently, PSS1 mutations not only affect cellular PS levels and distribution but also lead to a more complex imbalance in lipid homeostasis by disturbing PI4P metabolism. PMID:27044099

  4. Lenz-Majewski mutations in PTDSS1 affect phosphatidylinositol 4-phosphate metabolism at ER-PM and ER-Golgi junctions

    PubMed Central

    Sohn, Mira; Ivanova, Pavlina; Brown, H. Alex; Varnai, Peter; Kim, Yeun Ju; Balla, Tamas

    2016-01-01

    Lenz-Majewski syndrome (LMS) is a rare disease characterized by complex craniofacial, dental, cutaneous, and limb abnormalities combined with intellectual disability. Mutations in the PTDSS1 gene coding one of the phosphatidylserine (PS) synthase enzymes, PSS1, were described as causative in LMS patients. Such mutations render PSS1 insensitive to feedback inhibition by PS levels. Here we show that expression of mutant PSS1 enzymes decreased phosphatidylinositol 4-phosphate (PI4P) levels both in the Golgi and the plasma membrane (PM) by activating the Sac1 phosphatase and altered PI4P cycling at the PM. Conversely, inhibitors of PI4KA, the enzyme that makes PI4P in the PM, blocked PS synthesis and reduced PS levels by 50% in normal cells. However, mutant PSS1 enzymes alleviated the PI4P dependence of PS synthesis. Oxysterol-binding protein–related protein 8, which was recently identified as a PI4P-PS exchanger between the ER and PM, showed PI4P-dependent membrane association that was significantly decreased by expression of PSS1 mutant enzymes. Our studies reveal that PS synthesis is tightly coupled to PI4P-dependent PS transport from the ER. Consequently, PSS1 mutations not only affect cellular PS levels and distribution but also lead to a more complex imbalance in lipid homeostasis by disturbing PI4P metabolism. PMID:27044099

  5. Lenz-Majewski mutations in PTDSS1 affect phosphatidylinositol 4-phosphate metabolism at ER-PM and ER-Golgi junctions.

    PubMed

    Sohn, Mira; Ivanova, Pavlina; Brown, H Alex; Toth, Daniel J; Varnai, Peter; Kim, Yeun Ju; Balla, Tamas

    2016-04-19

    Lenz-Majewski syndrome (LMS) is a rare disease characterized by complex craniofacial, dental, cutaneous, and limb abnormalities combined with intellectual disability. Mutations in thePTDSS1gene coding one of the phosphatidylserine (PS) synthase enzymes, PSS1, were described as causative in LMS patients. Such mutations render PSS1 insensitive to feedback inhibition by PS levels. Here we show that expression of mutant PSS1 enzymes decreased phosphatidylinositol 4-phosphate (PI4P) levels both in the Golgi and the plasma membrane (PM) by activating the Sac1 phosphatase and altered PI4P cycling at the PM. Conversely, inhibitors of PI4KA, the enzyme that makes PI4P in the PM, blocked PS synthesis and reduced PS levels by 50% in normal cells. However, mutant PSS1 enzymes alleviated the PI4P dependence of PS synthesis. Oxysterol-binding protein-related protein 8, which was recently identified as a PI4P-PS exchanger between the ER and PM, showed PI4P-dependent membrane association that was significantly decreased by expression of PSS1 mutant enzymes. Our studies reveal that PS synthesis is tightly coupled to PI4P-dependent PS transport from the ER. Consequently, PSS1 mutations not only affect cellular PS levels and distribution but also lead to a more complex imbalance in lipid homeostasis by disturbing PI4P metabolism.

  6. Protein Kinase D1 regulates focal adhesion dynamics and cell adhesion through Phosphatidylinositol-4-phosphate 5-kinase type-l γ

    PubMed Central

    Durand, Nisha; Bastea, Ligia I.; Long, Jason; Döppler, Heike; Ling, Kun; Storz, Peter

    2016-01-01

    Focal adhesions (FAs) are highly dynamic structures that are assembled and disassembled on a continuous basis. The balance between the two processes mediates various aspects of cell behavior, ranging from cell adhesion and spreading to directed cell migration. The turnover of FAs is regulated at multiple levels and involves a variety of signaling molecules and adaptor proteins. In the present study, we show that in response to integrin engagement, a subcellular pool of Protein Kinase D1 (PKD1) localizes to the FAs. PKD1 affects FAs by decreasing turnover and promoting maturation, resulting in enhanced cell adhesion. The effects of PKD1 are mediated through direct phosphorylation of FA-localized phosphatidylinositol-4-phosphate 5-kinase type-l γ (PIP5Klγ) at serine residue 448. This phosphorylation occurs in response to Fibronectin-RhoA signaling and leads to a decrease in PIP5Klγs’ lipid kinase activity and binding affinity for Talin. Our data reveal a novel function for PKD1 as a regulator of FA dynamics and by identifying PIP5Klγ as a novel PKD1 substrate provide mechanistic insight into this process. PMID:27775029

  7. Development of an image-based screening system for inhibitors of the plastidial MEP pathway and of protein geranylgeranylation

    PubMed Central

    Hartmann, Michael; Gas-Pascual, Elisabet; Hemmerlin, Andrea; Rohmer, Michel; Bach, Thomas J.

    2015-01-01

    In a preceding study we have recently established an in vivo visualization system for the geranylgeranylation of proteins in a stably transformed tobacco BY-2 cell line, which involves expressing a dexamethasone-inducible GFP fused to the prenylable, carboxy-terminal basic domain of the rice calmodulin CaM61, which naturally bears a CaaL geranylgeranylation motif (GFP-BD-CVIL). By using pathway-specific inhibitors it was there demonstrated that inhibition of the methylerythritol phosphate (MEP) pathway with oxoclomazone and fosmidomycin, as well as inhibition of protein geranylgeranyl transferase type 1 (PGGT-1), shifted the localization of the GFP-BD-CVIL protein from the membrane to the nucleus. In contrast, the inhibition of the mevalonate (MVA) pathway with mevinolin did not affect this localization. Furthermore, in this initial study complementation assays with pathway-specific intermediates confirmed that the precursors for the cytosolic isoprenylation of this fusion protein are predominantly provided by the MEP pathway. In order to optimize this visualization system from a more qualitative assay to a statistically trustable medium or a high-throughput screening system, we established now new conditions that permit culture and analysis in 96-well microtiter plates, followed by fluorescence microscopy. For further refinement, the existing GFP-BD-CVIL cell line was transformed with an estradiol-inducible vector driving the expression of a RFP protein, C-terminally fused to a nuclear localization signal (NLS-RFP). We are thus able to quantify the total number of viable cells versus the number of inhibited cells after various treatments. This approach also includes a semi-automatic counting system, based on the freely available image processing software. As a result, the time of image analysis as well as the risk of user-generated bias is reduced to a minimum. Moreover, there is no cross-induction of gene expression by dexamethasone and estradiol, which is an

  8. Type B Phosphatidylinositol-4-Phosphate 5-Kinases Mediate Arabidopsis and Nicotiana tabacum Pollen Tube Growth by Regulating Apical Pectin Secretion[W

    PubMed Central

    Ischebeck, Till; Stenzel, Irene; Heilmann, Ingo

    2008-01-01

    Phosphatidylinositol-4,5-bisphosphate [PtdIns(4,5)P2] occurs in the apical plasma membrane of growing pollen tubes. Because enzymes responsible for PtdIns(4,5)P2 production at that location are uncharacterized, functions of PtdIns(4,5)P2 in pollen tube tip growth are unresolved. Two candidate genes encoding pollen-expressed Arabidopsis thaliana phosphatidylinositol-4-phosphate 5-kinases (PI4P 5-kinases) of Arabidopsis subfamily B were identified (PIP5K4 and PIP5K5), and their recombinant proteins were characterized as being PI4P 5-kinases. Pollen of T-DNA insertion lines deficient in both PIP5K4 and PIP5K5 exhibited reduced pollen germination and defects in pollen tube elongation. Fluorescence-tagged PIP5K4 and PIP5K5 localized to an apical plasma membrane microdomain in Arabidopsis and tobacco (Nicotiana tabacum) pollen tubes, and overexpression of either PIP5K4 or PIP5K5 triggered multiple tip branching events. Further studies using the tobacco system revealed that overexpression caused massive apical pectin deposition accompanied by plasma membrane invaginations. By contrast, callose deposition and cytoskeletal structures were unaltered in the overexpressors. Morphological effects depended on PtdIns(4,5)P2 production, as an inactive enzyme variant did not produce any effects. The data indicate that excessive PtdIns(4,5)P2 production by type B PI4P 5-kinases disturbs the balance of membrane trafficking and apical pectin deposition. Polar tip growth of pollen tubes may thus be modulated by PtdIns(4,5)P2 via regulatory effects on membrane trafficking and/or apical pectin deposition. PMID:19060112

  9. Phosphatidylinositol-4-phosphate 5-Kinase 1α Modulates Ribosomal RNA Gene Silencing through Its Interaction with Histone H3 Lysine 9 Trimethylation and Heterochromatin Protein HP1-α*

    PubMed Central

    Chakrabarti, Rajarshi; Sanyal, Sulagna; Ghosh, Amit; Bhar, Kaushik; Das, Chandrima; Siddhanta, Anirban

    2015-01-01

    Phosphoinositide signaling has been implicated in the regulation of numerous cellular processes including cytoskeletal dynamics, cellular motility, vesicle trafficking, and gene transcription. Studies have also shown that nuclear phosphoinositide(s) regulates processes such as mRNA export, cell cycle progression, gene transcription, and DNA repair. We have shown previously that the nuclear form of phosphatidylinositol-4-phosphate 5-kinase 1α (PIP5K), the enzyme responsible for phosphatidylinositol 4,5-bisphosphate synthesis, is modified by small ubiquitin-like modifier (SUMO)-1. In this study, we have shown that due to the site-specific Lys to Ala mutations of PIP5K at Lys-244 and Lys-490, it is unable to localize in the nucleus and nucleolus, respectively. Furthermore, by using chromatin immunoprecipitation assays, we have observed that PIP5K associates with the chromatin silencing complex constituted of H3K9me3 and heterochromatin protein 1α at multiple ribosomal DNA (rDNA) loci. These interactions followed a definite cyclical pattern of occupancy (mostly G1) and release from the rDNA loci (G1/S) throughout the cell cycle. Moreover, the immunoprecipitation results clearly demonstrate that PIP5K SUMOylated at Lys-490 interacts with components of the chromatin silencing machinery, H3K9me3 and heterochromatin protein 1α. However, PIP5K does not interact with the gene activation signature protein H3K4me3. This study, for the first time, demonstrates that PIP5K, an enzyme actively associated with lipid modification pathway, has additional roles in rDNA silencing. PMID:26157143

  10. In Vivo Near-Infrared Spectroscopy and Magnetic Resonance Imaging Monitoring of Tumor Response to Combretastatin A-4-Phosphate Correlated With Therapeutic Outcome

    SciTech Connect

    Zhao Dawen; Chang Chenghui; Kim, Jae G.; Liu Hanli; Mason, Ralph P.

    2011-06-01

    Purpose: To develop a combination treatment consisting of combretastatin A-4-phosphate (CA4P) with radiation based on tumor oxygenation status. Methods and Materials: In vivo near-infrared spectroscopy (NIRS) and diffusion-weighted (DW) magnetic resonance imaging (MRI) were applied to noninvasively monitor changes in tumor blood oxygenation and necrosis induced by CA4P (30 mg/kg) in rat mammary 13762NF adenocarcinoma, and the evidence was used to optimize combinations of CA4P and radiation treatment (a single dose of 5 Gy). Results: NIRS showed decreasing concentrations of tumor vascular oxyhemoglobin and total hemoglobin during the first 2 h after CA4P treatment, indicating significant reductions in tumor blood oxygenation and perfusion levels (p < 0.001). Twenty-four hours later, in response to oxygen inhalation, significant recovery was observed in tumor vascular and tissue oxygenation according to NIRS and pimonidazole staining results, respectively (p < 0.05). DW MRI revealed significantly increased water diffusion in tumors measured by apparent diffusion coefficient at 24 h (p < 0.05), suggesting that CA4P-induced central necrosis. In concordance with the observed tumor oxygen dynamics, we found that treatment efficacy depended on the timing of the combined therapy. The most significant delay in tumor growth was seen in the group of tumors treated with radiation while the rats breathed oxygen 24 h after CA4P administration. Conclusions: Noninvasive evaluation of tumor oxygen dynamics allowed us to rationally enhance the response of syngeneic rat breast tumors to combined treatment of CA4P with radiation.

  11. Phosphatidylinositol 4-phosphate 5-kinase reduces cell surface expression of the epithelial sodium channel (ENaC) in cultured collecting duct cells.

    PubMed

    Weixel, Kelly M; Edinger, Robert S; Kester, Lauren; Guerriero, Christopher J; Wang, Huamin; Fang, Liang; Kleyman, Thomas R; Welling, Paul A; Weisz, Ora A; Johnson, John P

    2007-12-14

    Ubiquitination of ENaC subunits has been shown to negatively regulate the cell surface expression of ENaC channels. We have previously demonstrated that epsin links ubiquitinated ENaC to clathrin adaptors for clathrin-mediated endocytosis. Epsin is thought to directly modify the curvature of membranes upon binding to phosphatidylinositol 4,5-bisphosphate (PIP2) where it recruits clathrin and stimulates lattice assembly. Murine phosphatidylinositol 4-phosphate 5-kinase alpha (PI5KIalpha) has been shown to enhance endocytosis in a PIP2-dependent manner. We tested the hypothesis that PI5KIalpha-mediated PIP2 production would negatively regulate ENaC current by enhancing epsin-mediated endocytosis of the channel. Expression of PI5KIalpha decreased ENaC currents in Xenopus oocytes by 80%, entirely because of a decrease in cell surface ENaC levels. Catalytically inactive mutants of PI5Kalpha had no effect on ENaC activity. Expression of the PIP2 binding region of epsin increased ENaC current in oocytes, an effect completely reversed by co-expression of PI5KIalpha. Overexpression of epsin reduced amiloride-sensitive current in CCD cells. Overexpression of PI5KIalpha enhanced membrane PIP2 levels and reduced apical surface expression of ENaC in CCD cells, down-regulating amiloride-sensitive current. Knockdown of PI5KIalpha with isoform-specific siRNA resulted in a 4-fold enhancement of ENaC activity. PI5KIalpha localized exclusively to the apical plasma membrane domain when overexpressed in mouse CCD cells, consistent for a role in regulating PIP2 production at the apical plasma membrane. We conclude that membrane turnover events regulating ENaC surface expression and activity in oocytes and CCD cells can be regulated by PI5KIalpha. PMID:17940289

  12. Cadaverine covalently linked to peptidoglycan is required for interaction between the peptidoglycan and the periplasm-exposed S-layer-homologous domain of major outer membrane protein Mep45 in Selenomonas ruminantium.

    PubMed

    Kojima, Seiji; Ko, Kyong-Cheol; Takatsuka, Yumiko; Abe, Naoki; Kaneko, Jun; Itoh, Yoshifumi; Kamio, Yoshiyuki

    2010-11-01

    The peptidoglycan of Selenomonas ruminantium is covalently bound to cadaverine (PG-cadaverine), which likely plays a significant role in maintaining the integrity of the cell surface structure. The outer membrane of this bacterium contains a 45-kDa major protein (Mep45) that is a putative peptidoglycan-associated protein. In this report, we determined the nucleotide sequence of the mep45 gene and investigated the relationship between PG-cadaverine, Mep45, and the cell surface structure. Amino acid sequence analysis showed that Mep45 is comprised of an N-terminal S-layer-homologous (SLH) domain followed by α-helical coiled-coil region and a C-terminal β-strand-rich region. The N-terminal SLH domain was found to be protruding into the periplasmic space and was responsible for binding to peptidoglycan. It was determined that Mep45 binds to the peptidoglycan in a manner dependent on the presence of PG-cadaverine. Electron microscopy revealed that defective PG-cadaverine decreased the structural interactions between peptidoglycan and the outer membrane, consistent with the proposed role for PG-cadaverine. The C-terminal β-strand-rich region of Mep45 was predicted to be a membrane-bound unit of the 14-stranded β-barrel structure. Here we propose that PG-cadaverine possesses functional importance to facilitate the structural linkage between peptidoglycan and the outer membrane via specific interaction with the SLH domain of Mep45.

  13. Histone H2A and H4 N-terminal tails are positioned by the MEP50 WD repeat protein for efficient methylation by the PRMT5 arginine methyltransferase.

    PubMed

    Burgos, Emmanuel S; Wilczek, Carola; Onikubo, Takashi; Bonanno, Jeffrey B; Jansong, Janina; Reimer, Ulf; Shechter, David

    2015-04-10

    The protein arginine methyltransferase PRMT5 is complexed with the WD repeat protein MEP50 (also known as Wdr77 or androgen coactivator p44) in vertebrates in a tetramer of heterodimers. MEP50 is hypothesized to be required for protein substrate recruitment to the catalytic domain of PRMT5. Here we demonstrate that the cross-dimer MEP50 is paired with its cognate PRMT5 molecule to promote histone methylation. We employed qualitative methylation assays and a novel ultrasensitive continuous assay to measure enzyme kinetics. We demonstrate that neither full-length human PRMT5 nor the Xenopus laevis PRMT5 catalytic domain has appreciable protein methyltransferase activity. We show that histones H4 and H3 bind PRMT5-MEP50 more efficiently compared with histone H2A(1-20) and H4(1-20) peptides. Histone binding is mediated through histone fold interactions as determined by competition experiments and by high density histone peptide array interaction studies. Nucleosomes are not a substrate for PRMT5-MEP50, consistent with the primary mode of interaction via the histone fold of H3-H4, obscured by DNA in the nucleosome. Mutation of a conserved arginine (Arg-42) on the MEP50 insertion loop impaired the PRMT5-MEP50 enzymatic efficiency by increasing its histone substrate Km, comparable with that of Caenorhabditis elegans PRMT5. We show that PRMT5-MEP50 prefers unmethylated substrates, consistent with a distributive model for dimethylation and suggesting discrete biological roles for mono- and dimethylarginine-modified proteins. We propose a model in which MEP50 and PRMT5 simultaneously engage the protein substrate, orienting its targeted arginine to the catalytic site.

  14. Effector Vγ9Vδ2 T cells dominate the human fetal γδ T-cell repertoire

    PubMed Central

    Dimova, Tanya; Brouwer, Margreet; Gosselin, Françoise; Tassignon, Joël; Leo, Oberdan; Donner, Catherine; Marchant, Arnaud; Vermijlen, David

    2015-01-01

    γδ T cells are unconventional T cells recognizing antigens via their γδ T-cell receptor (TCR) in a way that is fundamentally different from conventional αβ T cells. γδ T cells usually are divided into subsets according the type of Vγ and/or Vδ chain they express in their TCR. T cells expressing the TCR containing the γ-chain variable region 9 and the δ-chain variable region 2 (Vγ9Vδ2 T cells) are the predominant γδ T-cell subset in human adult peripheral blood. The current thought is that this predominance is the result of the postnatal expansion of cells expressing particular complementary-determining region 3 (CDR3) in response to encounters with microbes, especially those generating phosphoantigens derived from the 2-C-methyl-d-erythritol 4-phosphate pathway of isoprenoid synthesis. However, here we show that, rather than requiring postnatal microbial exposure, Vγ9Vδ2 T cells are the predominant blood subset in the second-trimester fetus, whereas Vδ1+ and Vδ3+ γδ T cells are present only at low frequencies at this gestational time. Fetal blood Vγ9Vδ2 T cells are phosphoantigen responsive and display very limited diversity in the CDR3 of the Vγ9 chain gene, where a germline-encoded sequence accounts for >50% of all sequences, in association with a prototypic CDR3δ2. Furthermore, these fetal blood Vγ9Vδ2 T cells are functionally preprogrammed (e.g., IFN-γ and granzymes-A/K), with properties of rapidly activatable innatelike T cells. Thus, enrichment for phosphoantigen-responsive effector T cells has occurred within the fetus before postnatal microbial exposure. These various characteristics have been linked in the mouse to the action of selecting elements and would establish a much stronger parallel between human and murine γδ T cells than is usually articulated. PMID:25617367

  15. Spared Primary Motor Cortex and The Presence of MEP in Cerebral Palsy Dictate the Responsiveness to tDCS during Gait Training.

    PubMed

    Grecco, Luanda A Collange; Oliveira, Claudia Santos; Galli, Manuela; Cosmo, Camila; Duarte, Natália de Almeida Carvalho; Zanon, Nelci; Edwards, Dylan J; Fregni, Felipe

    2016-01-01

    The current priority of investigations involving transcranial direct current stimulation (tDCS) and neurorehabilitation is to identify biomarkers associated with the positive results of the interventions such that respondent and non-respondent patients can be identified in the early phases of treatment. The aims were to determine whether: (1) present motor evoked potential (MEP); and (2) injuries involving the primary motor cortex, are associated with tDCS-enhancement in functional outcome following gait training in children with cerebral palsy (CP). We reviewed the data from our parallel, randomized, sham-controlled, double-blind studies. Fifty-six children with spastic CP received gait training (either treadmill training or virtual reality training) and tDCS (active or sham). Univariate and multivariate logistic regression analyses were employed to identify clinical, neurophysiologic and neuroanatomic predictors associated with the responsiveness to treatment with tDCS. MEP presence during the initial evaluation and the subcortical injury were associated with positive effects in the functional results. The logistic regression revealed that present MEP was a significant predictor for the six-minute walk test (6MWT; p = 0.003) and gait speed (p = 0.028), whereas the subcortical injury was a significant predictor of gait kinematics (p = 0.013) and gross motor function (p = 0.021). In this preliminary study involving children with CP, two important prediction factors of good responses to anodal tDCS combined with gait training were identified. Apparently, MEP (integrity of the corticospinal tract) and subcortical location of the brain injury exerted different influences on aspects related to gait, such as velocity and kinematics. PMID:27486393

  16. Spared Primary Motor Cortex and The Presence of MEP in Cerebral Palsy Dictate the Responsiveness to tDCS during Gait Training.

    PubMed

    Grecco, Luanda A Collange; Oliveira, Claudia Santos; Galli, Manuela; Cosmo, Camila; Duarte, Natália de Almeida Carvalho; Zanon, Nelci; Edwards, Dylan J; Fregni, Felipe

    2016-01-01

    The current priority of investigations involving transcranial direct current stimulation (tDCS) and neurorehabilitation is to identify biomarkers associated with the positive results of the interventions such that respondent and non-respondent patients can be identified in the early phases of treatment. The aims were to determine whether: (1) present motor evoked potential (MEP); and (2) injuries involving the primary motor cortex, are associated with tDCS-enhancement in functional outcome following gait training in children with cerebral palsy (CP). We reviewed the data from our parallel, randomized, sham-controlled, double-blind studies. Fifty-six children with spastic CP received gait training (either treadmill training or virtual reality training) and tDCS (active or sham). Univariate and multivariate logistic regression analyses were employed to identify clinical, neurophysiologic and neuroanatomic predictors associated with the responsiveness to treatment with tDCS. MEP presence during the initial evaluation and the subcortical injury were associated with positive effects in the functional results. The logistic regression revealed that present MEP was a significant predictor for the six-minute walk test (6MWT; p = 0.003) and gait speed (p = 0.028), whereas the subcortical injury was a significant predictor of gait kinematics (p = 0.013) and gross motor function (p = 0.021). In this preliminary study involving children with CP, two important prediction factors of good responses to anodal tDCS combined with gait training were identified. Apparently, MEP (integrity of the corticospinal tract) and subcortical location of the brain injury exerted different influences on aspects related to gait, such as velocity and kinematics.

  17. Spared Primary Motor Cortex and The Presence of MEP in Cerebral Palsy Dictate the Responsiveness to tDCS during Gait Training

    PubMed Central

    Grecco, Luanda A. Collange; Oliveira, Claudia Santos; Galli, Manuela; Cosmo, Camila; Duarte, Natália de Almeida Carvalho; Zanon, Nelci; Edwards, Dylan J.; Fregni, Felipe

    2016-01-01

    The current priority of investigations involving transcranial direct current stimulation (tDCS) and neurorehabilitation is to identify biomarkers associated with the positive results of the interventions such that respondent and non-respondent patients can be identified in the early phases of treatment. The aims were to determine whether: (1) present motor evoked potential (MEP); and (2) injuries involving the primary motor cortex, are associated with tDCS-enhancement in functional outcome following gait training in children with cerebral palsy (CP). We reviewed the data from our parallel, randomized, sham-controlled, double-blind studies. Fifty-six children with spastic CP received gait training (either treadmill training or virtual reality training) and tDCS (active or sham). Univariate and multivariate logistic regression analyses were employed to identify clinical, neurophysiologic and neuroanatomic predictors associated with the responsiveness to treatment with tDCS. MEP presence during the initial evaluation and the subcortical injury were associated with positive effects in the functional results. The logistic regression revealed that present MEP was a significant predictor for the six-minute walk test (6MWT; p = 0.003) and gait speed (p = 0.028), whereas the subcortical injury was a significant predictor of gait kinematics (p = 0.013) and gross motor function (p = 0.021). In this preliminary study involving children with CP, two important prediction factors of good responses to anodal tDCS combined with gait training were identified. Apparently, MEP (integrity of the corticospinal tract) and subcortical location of the brain injury exerted different influences on aspects related to gait, such as velocity and kinematics. PMID:27486393

  18. Protein Arginine Methyltransferase Prmt5-Mep50 Methylates Histones H2A and H4 and the Histone Chaperone Nucleoplasmin in Xenopus laevis Eggs*

    PubMed Central

    Wilczek, Carola; Chitta, Raghu; Woo, Eileen; Shabanowitz, Jeffrey; Chait, Brian T.; Hunt, Donald F.; Shechter, David

    2011-01-01

    Histone proteins carry information contained in post-translational modifications. Eukaryotic cells utilize this histone code to regulate the usage of the underlying DNA. In the maturing oocytes and eggs of the frog Xenopus laevis, histones are synthesized in bulk in preparation for deposition during the rapid early developmental cell cycles. During this key developmental time frame, embryonic pluripotent chromatin is established. In the egg, non-chromatin-bound histones are complexed with storage chaperone proteins, including nucleoplasmin. Here we describe the identification and characterization of a complex of the protein arginine methyltransferase 5 (Prmt5) and the methylosome protein 50 (Mep50) isolated from Xenopus eggs that specifically methylates predeposition histones H2A/H2A.X-F and H4 and the histone chaperone nucleoplasmin on a conserved motif (GRGXK). We demonstrate that nucleoplasmin (Npm), an exceedingly abundant maternally deposited protein, is a potent substrate for Prmt5-Mep50 and is monomethylated and symmetrically dimethylated at Arg-187. Furthermore, Npm modulates Prmt5-Mep50 activity directed toward histones, consistent with a regulatory role for Npm in vivo. We show that H2A and nucleoplasmin methylation appears late in oogenesis and is most abundant in the laid egg. We hypothesize that these very abundant arginine methylations are constrained to pre-mid blastula transition events in the embryo and therefore may be involved in the global transcriptional repression found in this developmental time frame. PMID:22009756

  19. Protein arginine methyltransferase Prmt5-Mep50 methylates histones H2A and H4 and the histone chaperone nucleoplasmin in Xenopus laevis eggs.

    PubMed

    Wilczek, Carola; Chitta, Raghu; Woo, Eileen; Shabanowitz, Jeffrey; Chait, Brian T; Hunt, Donald F; Shechter, David

    2011-12-01

    Histone proteins carry information contained in post-translational modifications. Eukaryotic cells utilize this histone code to regulate the usage of the underlying DNA. In the maturing oocytes and eggs of the frog Xenopus laevis, histones are synthesized in bulk in preparation for deposition during the rapid early developmental cell cycles. During this key developmental time frame, embryonic pluripotent chromatin is established. In the egg, non-chromatin-bound histones are complexed with storage chaperone proteins, including nucleoplasmin. Here we describe the identification and characterization of a complex of the protein arginine methyltransferase 5 (Prmt5) and the methylosome protein 50 (Mep50) isolated from Xenopus eggs that specifically methylates predeposition histones H2A/H2A.X-F and H4 and the histone chaperone nucleoplasmin on a conserved motif (GRGXK). We demonstrate that nucleoplasmin (Npm), an exceedingly abundant maternally deposited protein, is a potent substrate for Prmt5-Mep50 and is monomethylated and symmetrically dimethylated at Arg-187. Furthermore, Npm modulates Prmt5-Mep50 activity directed toward histones, consistent with a regulatory role for Npm in vivo. We show that H2A and nucleoplasmin methylation appears late in oogenesis and is most abundant in the laid egg. We hypothesize that these very abundant arginine methylations are constrained to pre-mid blastula transition events in the embryo and therefore may be involved in the global transcriptional repression found in this developmental time frame.

  20. Determination of the active stereoisomer of the MEP pathway-targeting antimalarial agent MMV008138, and initial structure-activity studies

    PubMed Central

    Yao, Zhong-Ke; Krai, Priscilla M.; Merino, Emilio F.; Simpson, Morgan E.; Slebodnick, Carla; Cassera, Maria Belen; Carlier, Paul R.

    2015-01-01

    Compounds that target isoprenoid biosynthesis in Plasmodium falciparum could be a welcome addition to malaria chemotherapy, since the methylerythritol phosphate (MEP) pathway used by the parasite is not present in humans. We previously reported that MMV008138 targets the apicoplast of P. falciparum and that its target in the MEP pathway differs from that of Fosmidomycin. In this article, we determine that the active stereoisomer of MMV008138 is 4a, which is (1R,3S)-configured. 2′,4′-Disubstitution of the D ring was also found to be crucial for inhibition of the parasite growth. Limited variation of the C3-carboxylic acid substitutent was carried out, and methylamide derivative 8a was found to be more potent than 4a; other amides, acylhydrazines, and esters were less potent. Finally, lead compounds 4a, 4e, 4f, 4h, 8a, and 8e did not inhibit growth of Escherichia coli, suggesting that protozoan-selective inhibition of the MEP pathway of P. falciparum can be achieved. PMID:25754494

  1. Spectroscopic (infrared, Raman, UV and NMR) analysis, Gaussian hybrid computational investigation (MEP maps/HOMO and LUMO) on cyclohexanone oxime.

    PubMed

    Ramalingam, S; Karabacak, M; Periandy, S; Puviarasan, N; Tanuja, D

    2012-10-01

    In the present analysis, FT-IR/FT-Raman spectra of the cyclohexanone oxime (CHO, C(6)H(11)NO) are recorded. The observed vibrational frequencies are assigned and the computational calculations are carried out by HF and DFT (B3LYP and B3PW91) methods with 6-311++G(d,p) basis set and the corresponding results are tabulated. In order to yield good coherence with observed values, the calculated frequencies are scaled by appropriate scale factors. The complete assignments are performed on the basis of the total energy distribution (TED) of the vibrational modes, calculated with scaled quantum mechanics (SQM) method. The alternation of structure of cyclohexanone due to the substitution of NOH is investigated. The vibrational sequence pattern of the molecule related to the substitutions is analyzed. Comparison of the observed fundamental vibrational frequencies of CHO and calculated results by density functional (B3LYP and B3PW91) and HF methods indicates that B3LYP is superior to the scaled HF and B3PW91 approach for molecular vibrational problems. Moreover, (13)C NMR and (1)H NMR chemical shifts are calculated by using the gauge independent atomic orbital (GIAO) method with HF/B3LYP/B3PW91 methods and the same basis set. A study on the electronic properties; absorption wavelengths, excitation energy, dipole moment and frontier molecular orbital energies, are performed by HF and DFT methods. The calculated HOMO and LUMO energies show that charge transfer occurs within the molecule. Besides frontier molecular orbitals (FMO), molecular electrostatic potential (MEP) was performed. NLO properties and Mulliken charges of the CHO was also calculated and interpreted. The thermodynamic properties (heat capacity, entropy, and enthalpy) of the title compound at different temperatures are calculated in gas phase.

  2. Spectroscopic (infrared, Raman, UV and NMR) analysis, Gaussian hybrid computational investigation (MEP maps/HOMO and LUMO) on cyclohexanone oxime

    NASA Astrophysics Data System (ADS)

    Ramalingam, S.; Karabacak, M.; Periandy, S.; Puviarasan, N.; Tanuja, D.

    2012-10-01

    In the present analysis, FT-IR/FT-Raman spectra of the cyclohexanone oxime (CHO, C6H11NO) are recorded. The observed vibrational frequencies are assigned and the computational calculations are carried out by HF and DFT (B3LYP and B3PW91) methods with 6-311++G(d,p) basis set and the corresponding results are tabulated. In order to yield good coherence with observed values, the calculated frequencies are scaled by appropriate scale factors. The complete assignments are performed on the basis of the total energy distribution (TED) of the vibrational modes, calculated with scaled quantum mechanics (SQM) method. The alternation of structure of cyclohexanone due to the substitution of NOH is investigated. The vibrational sequence pattern of the molecule related to the substitutions is analyzed. Comparison of the observed fundamental vibrational frequencies of CHO and calculated results by density functional (B3LYP and B3PW91) and HF methods indicates that B3LYP is superior to the scaled HF and B3PW91 approach for molecular vibrational problems. Moreover, 13C NMR and 1H NMR chemical shifts are calculated by using the gauge independent atomic orbital (GIAO) method with HF/B3LYP/B3PW91 methods and the same basis set. A study on the electronic properties; absorption wavelengths, excitation energy, dipole moment and frontier molecular orbital energies, are performed by HF and DFT methods. The calculated HOMO and LUMO energies show that charge transfer occurs within the molecule. Besides frontier molecular orbitals (FMO), molecular electrostatic potential (MEP) was performed. NLO properties and Mulliken charges of the CHO was also calculated and interpreted. The thermodynamic properties (heat capacity, entropy, and enthalpy) of the title compound at different temperatures are calculated in gas phase.

  3. Spectroscopic (infrared, Raman, UV and NMR) analysis, Gaussian hybrid computational investigation (MEP maps/HOMO and LUMO) on cyclohexanone oxime.

    PubMed

    Ramalingam, S; Karabacak, M; Periandy, S; Puviarasan, N; Tanuja, D

    2012-10-01

    In the present analysis, FT-IR/FT-Raman spectra of the cyclohexanone oxime (CHO, C(6)H(11)NO) are recorded. The observed vibrational frequencies are assigned and the computational calculations are carried out by HF and DFT (B3LYP and B3PW91) methods with 6-311++G(d,p) basis set and the corresponding results are tabulated. In order to yield good coherence with observed values, the calculated frequencies are scaled by appropriate scale factors. The complete assignments are performed on the basis of the total energy distribution (TED) of the vibrational modes, calculated with scaled quantum mechanics (SQM) method. The alternation of structure of cyclohexanone due to the substitution of NOH is investigated. The vibrational sequence pattern of the molecule related to the substitutions is analyzed. Comparison of the observed fundamental vibrational frequencies of CHO and calculated results by density functional (B3LYP and B3PW91) and HF methods indicates that B3LYP is superior to the scaled HF and B3PW91 approach for molecular vibrational problems. Moreover, (13)C NMR and (1)H NMR chemical shifts are calculated by using the gauge independent atomic orbital (GIAO) method with HF/B3LYP/B3PW91 methods and the same basis set. A study on the electronic properties; absorption wavelengths, excitation energy, dipole moment and frontier molecular orbital energies, are performed by HF and DFT methods. The calculated HOMO and LUMO energies show that charge transfer occurs within the molecule. Besides frontier molecular orbitals (FMO), molecular electrostatic potential (MEP) was performed. NLO properties and Mulliken charges of the CHO was also calculated and interpreted. The thermodynamic properties (heat capacity, entropy, and enthalpy) of the title compound at different temperatures are calculated in gas phase. PMID:22683556

  4. Merging Q-theory and MEP theory to explain some geographical variations seen in Russian soil C inventory data

    NASA Astrophysics Data System (ADS)

    Yurova, Alla

    2016-04-01

    Soils are as critical for understanding the ecosystem carbon cycle as plants are and here I critically evaluate some of the commonly used assumptions embedded into the soil organic matter dynamics process-based models. According to the biochemical concept (e.g. Mindermann, 1968) plant residues can be divided into liable and more recalcitrant fractions, each decomposing with a specific rate (increasing with temperature) and it is remains of recalcitrant compounds that accumulate to form soil organic matter. The application of this theory in regional to global biogeochemical models leads to conclusion that the high latitude soils stores the highest amount of carbon per square meter due to high percentage of recalcitrant compounds and low temperature. This contradicts with the Russian soil inventory data, demonstrating that within the large span of biomes present in Russia that is steepe that has the highest soil C storage. Here I take an alternative, most theoretical, viewpoint, called Q-theory (from q-quality) (Ågren and Bosatta, 1996) considering the changes in the continuous variable-the quality of the organic matter in the soil as a starting point. I then derive the novel equation for the entropy production of humification process and demonstrate how MEP theory works to explain geographical differences in soil C accumulation seen in Russian soil inventory data. Conceptually close to the work presented is a general theory of humification (Orlov, 1995) based on thermodynamic view on decomposition postulating that independently on acting factors and the soil type it is only the most thermodynamically stable components, such as humic substances, that will be produced and stored in the process of organic matter transformation This work was supported by RFBR grants 15-05-01368 A

  5. The effect of MEP pathway and other inhibitors on the intracellular localization of a plasma membrane-targeted, isoprenylable GFP reporter protein in tobacco BY-2 cells

    PubMed Central

    Bach, Thomas J

    2013-01-01

    We have established an in vivo visualization system for the geranylgeranylation of proteins in a stably transformed tobacco BY-2 cell line, based on the expression of a dexamethasone-inducible GFP fused to the carboxy-terminal basic domain of the rice calmodulin CaM61, which naturally bears a CaaL geranylgeranylation motif (GFP-BD-CVIL). By using pathway-specific inhibitors it was demonstrated that inhibition of the methylerythritol phosphate (MEP) pathway with known inhibitors like oxoclomazone and fosmidomycin, as well as inhibition of the protein geranylgeranyltransferase type 1 (PGGT-1), shifted the localization of the GFP-BD-CVIL protein from the membrane to the nucleus. In contrast, the inhibition of the mevalonate (MVA) pathway with mevinolin did not affect the localization. During the present work, this test system has been used to examine the effect of newly designed inhibitors of the MEP pathway and inhibitors of sterol biosynthesis such as squalestatin, terbinafine and Ro48-8071. In addition, we also studied the impact of different post-prenylation inhibitors or those suspected to affect the transport of proteins to the plasma membrane on the localization of the geranylgeranylable fusion protein GFP-BD-CVIL. PMID:24555083

  6. Overexpression of Ipe protein from the coliphage mEp021 induces pleiotropic effects involving haemolysis by HlyE-containing vesicles and cell death.

    PubMed

    Martínez-Peñafiel, Eva; Fernández-Ramírez, Fernando; Ishida, Cecilia; Reyes-Cortés, Ruth; Sepúlveda-Robles, Omar; Guarneros-Peña, Gabriel; Bermúdez-Cruz, Rosa María; Kameyama, Luis

    2012-06-01

    Lysogenic Escherichia coli K-12 harbouring the prophage mEp021 displays haemolytic activity. From a genomic library of mEp021, we identified an open reading frame (ORF 4) that was responsible for the haemolytic activity. However, the ORF 4 sequence contains four initiation codons in the same frame: ORF 4.1-ORF 4.4, coding for 83-a.a., 82-a.a., 77-a.a. and 72-a.a. products, respectively. The expression of the cloned ORF 4.3, or inducer of pleiotropic effects (ipe), reproduced the haemolytic phenotype in a native strain carrying the gene hlyE(+), but not in the mutant hlyE(-) strain. The overexpression of Ipe induced several pleiotropic effects, such as the inhibition of cell growth and the deregulation of cell division, which resulted in a mixture of normal and desiccated-like cells: normal-filamentous, desiccated-like-filamentous bacilli, minicells etc. Other effects included abnormalities of the cell membrane, the production of vesicles containing HlyE, and finally, cell death. These events were analysed at the molecular level by microarray assays. The global transcription profile of E. coli K-12 strain MC4100, which expressed Ipe after 4 h, revealed differential expression of various genes, most of which were related either to cell membrane and murein biosynthesis or to cell division. The up-regulation of some of these transcripts was confirmed by qRT-PCR. Additional research is needed to determine whether these effects are directly related to Ipe activity or are consequences of the cellular responses to putative structural damage induced by Ipe. PMID:22365985

  7. An animal homolog of plant Mep/Amt transporters promotes ammonia excretion by the anal papillae of the disease vector mosquito Aedes aegypti.

    PubMed

    Chasiotis, Helen; Ionescu, Adrian; Misyura, Lidiya; Bui, Phuong; Fazio, Kimberly; Wang, Jason; Patrick, Marjorie; Weihrauch, Dirk; Donini, Andrew

    2016-05-01

    The transcripts of three putative ammonia (NH3/NH4 (+)) transporters, Rhesus-like glycoproteins AeRh50-1, AeRh50-2 and Amt/Mep-like AeAmt1 were detected in the anal papillae of larval Aedes aegypti Quantitative PCR studies revealed 12-fold higher transcript levels of AeAmt1 in anal papillae relative to AeRh50-1, and levels of AeRh50-2 were even lower. Immunoblotting revealed AeAmt1 in anal papillae as a pre-protein with putative monomeric and trimeric forms. AeAmt1 was immunolocalized to the basal side of the anal papillae epithelium where it co-localized with Na(+)/K(+)-ATPase. Ammonium concentration gradients were measured adjacent to anal papillae using the scanning ion-selective electrode technique (SIET) and used to calculate ammonia efflux by the anal papillae. dsRNA-mediated reductions in AeAmt1 decreased ammonia efflux at larval anal papillae and significantly increased ammonia levels in hemolymph, indicating a principal role for AeAmt1 in ammonia excretion. Pharmacological characterization of ammonia transport mechanisms in the anal papillae suggests that, in addition to AeAmt1, the ionomotive pumps V-type H(+)-ATPase and Na(+)/K(+)-ATPase as well as NHE3 are involved in ammonia excretion at the anal papillae.

  8. Molecular structure, vibrational, UV, NMR, HOMO-LUMO, MEP, NLO, NBO analysis of 3,5 di tert butyl 4 hydroxy benzoic acid

    NASA Astrophysics Data System (ADS)

    Mathammal, R.; Sangeetha, K.; Sangeetha, M.; Mekala, R.; Gadheeja, S.

    2016-09-01

    In this study, we report a combined experimental and theoretical study on molecular structure and vibrational spectra of 3,5 di tert butyl 4 hydroxy benzoic acid. The properties of title compound have been evaluated by quantum chemical calculation (DFT) using B3LYP functional and 6-31 + G (d, p) as basis set. IR Spectra has been recorded using Fourier transform infrared spectroscopy (FT-IR) in the region 4000-400 cm-1. The vibrational assignment of the calculated normal modes has been made on the basis set. The isotropic chemical shifts computed by 13C and 1H NMR (Nuclear Magnetic Resonance) analyses also show good agreement with experimental observations. The theoretical UV-Vis spectrum of the compound are used to study the visible absorption maxima (λ max). The structure activity relationship have been interpreted by mapping electrostatic potential surface (MEP), which is valuable information for the quality control of medicines and drug receptor interactions. The Mullikan charges, HOMO (Highest Occupied Molecular Orbital) - LUMO (Lowest Unoccupied Molecular Orbital) energy are analyzed. HOMO-LUMO energy gap and other related molecular properties are also calculated. The Natural Bond Orbital (NBO) analysis is carried out to investigate the various intra and inter molecular interactions of molecular system. The Non-linear optical properties such as dipole moment (μ), polarizability (αtot) and molecular first order hyperpolarizability (β) of the title compound are computed with B3LYP/6-31 + G (d,p) level of theory.

  9. Molecular structure, vibrational spectra, NLO and MEP analysis of bis[2-hydroxy-кO-N-(2-pyridyl)-1-naphthaldiminato-кN]zinc(II)

    NASA Astrophysics Data System (ADS)

    Tanak, Hasan; Toy, Mehmet

    2013-11-01

    The molecular geometry and vibrational frequencies of bis[2-hydroxy-кO-N-(2-pyridyl)-1-naphthaldiminato-кN]zinc(II) in the ground state have been calculated by using the Hartree-Fock (HF) and density functional method (B3LYP) with 6-311G(d,p) basis set. The results of the optimized molecular structure are presented and compared with the experimental X-ray diffraction. The energetic and atomic charge behavior of the title compound in solvent media has been examined by applying the Onsager and the polarizable continuum model. To investigate second order nonlinear optical properties of the title compound, the electric dipole (μ), linear polarizability (α) and first-order hyperpolarizability (β) were computed using the density functional B3LYP and CAM-B3LYP methods with the 6-31+G(d) basis set. According to our calculations, the title compound exhibits nonzero (β) value revealing second order NLO behavior. In addition, DFT calculations of the title compound, molecular electrostatic potential (MEP), frontier molecular orbitals, and thermodynamic properties were performed at B3LYP/6-311G(d,p) level of theory.

  10. Vibrational spectra, HOMO, LUMO, NBO, MEP analysis and molecular docking study of 2,2-diphenyl-4-(piperidin-1-yl)butanamide.

    PubMed

    Mary, Y Sheena; Varghese, Hema Tresa; Panicker, C Yohannan; Girisha, M; Sagar, B K; Yathirajan, H S; Al-Saadi, Abdulaziz A; Van Alsenoy, Christian

    2015-11-01

    The Fourier-Transform Infrared and Fourier-Transform Raman spectra of 2,2-diphenyl-4-(piperidin-1-yl)butanamide were recorded in the region 4000-400 cm(-1) and 4000-0 cm(-1). The vibrational wavenumbers are computed using HF and DFT methods. The complete vibrational assignments were performed on the basis of potential energy distribution using GAR2PED program. The geometrical parameters of the title compound are in agreement with the XRD data. From the MEP study, the negative electrostatic potential regions are mainly localized of carbonyl group and are possible sites for electrophilic attack and the positive regions are localized all the rings, indicating possible sites for nucleophilic attack. Stability of the molecule arising from hyper-conjugative interaction and charge delocalization has been analyzed using natural bond orbital analysis. The calculated HOMO and LUMO energies also confirm that charge transfer occurs within the molecule. PASS analysis of the title compound predicts among other activities, antidyskinetic activity. Molecular docking results draw us to the conclusion that the compound might exhibit inhibitory activity against adenosine A2A and may act as antidyskinetic agent.

  11. Inhibition of green tea and the catechins against 1-deoxy-d-xylulose 5-phosphate reductoisomerase, the key enzyme of the MEP terpenoid biosynthetic pathway.

    PubMed

    Hui, Xian; Liu, Hui; Tian, Fang-Lin; Li, Fei-Fei; Li, Heng; Gao, Wen-Yun

    2016-09-01

    1-Deoxy-d-xylulose 5-phosphate reductoisomerase (DXR) is the first committed enzyme in the MEP terpenoid biosynthetic pathway and also a validated antimicrobial target. Green tea which is rich in polyphenolic components such as the catechins, possesses a plenty of pharmacological activities, in particular an antibacterial effect. To uncover the antibacterial mechanism of green tea and to seek new DXR inhibitors from natural sources, the DXR inhibitory activity of green tea and its main antimicrobial catechins were investigated in this study. The results show that the raw extract of green tea and its ethyl acetate fraction are able to suppress DXR activity explicitly. Further determination of the DXR inhibitory capacity of eight catechin compounds demonstrates that the most active compound is gallocatechin gallate that is able to inhibit around 50% activity of DXR at 25μM. Based on these data, the primary structure-activity relationship of the catechins against DXR is discussed. This study would be very helpful to elucidate the antimicrobial mechanism of green tea and the catechins and also would be very useful to direct the rational utilization of them as food additives. PMID:27439219

  12. FT-IR, molecular structure, first order hyperpolarizability, HOMO and LUMO analysis, MEP and NBO analysis of 2-(4-chlorophenyl)-2-oxoethyl 3-nitrobenzoate.

    PubMed

    Chidan Kumar, C S; Panicker, C Yohannan; Fun, Hoong-Kun; Mary, Y Sheena; Harikumar, B; Chandraju, S; Quah, Ching Kheng; Ooi, Chin Wei

    2014-05-21

    2-(4-Chlorophenyl)-2-oxoethyl 3-nitrobenzoate is synthesized by reacting 4-chlorophenacyl bromide with 3-nitrobenzoic acid using a slight excess of potassium or sodium carbonate in DMF medium at room temperature. The structure of the compound was confirmed by IR and single-crystal X-ray diffraction studies. FT-IR spectrum of 2-(4-chlorophenyl)-2-oxoethyl 3-nitrobenzoate was recorded and analyzed. The crystal structure is also described. The vibrational wavenumbers were computed using HF and DFT methods and are assigned with the help of potential energy distribution method. The first hyperpolarizability and infrared intensities are also reported. The geometrical parameters of the title compound obtained from XRD studies are in agreement with the calculated (DFT) values. The stability of the molecule arising from hyper-conjugative interaction and charge delocalization has been analyzed using NBO analysis. The HOMO and LUMO analysis are used to determine the charge transfer within the molecule. MEP was performed by the DFT method.

  13. Molecular structure, vibrational spectra, NLO and MEP analysis of bis[2-hydroxy-кO-N-(2-pyridyl)-1-naphthaldiminato-кN]zinc(II).

    PubMed

    Tanak, Hasan; Toy, Mehmet

    2013-11-01

    The molecular geometry and vibrational frequencies of bis[2-hydroxy-кO-N-(2-pyridyl)-1-naphthaldiminato-кN]zinc(II) in the ground state have been calculated by using the Hartree-Fock (HF) and density functional method (B3LYP) with 6-311G(d,p) basis set. The results of the optimized molecular structure are presented and compared with the experimental X-ray diffraction. The energetic and atomic charge behavior of the title compound in solvent media has been examined by applying the Onsager and the polarizable continuum model. To investigate second order nonlinear optical properties of the title compound, the electric dipole (μ), linear polarizability (α) and first-order hyperpolarizability (β) were computed using the density functional B3LYP and CAM-B3LYP methods with the 6-31+G(d) basis set. According to our calculations, the title compound exhibits nonzero (β) value revealing second order NLO behavior. In addition, DFT calculations of the title compound, molecular electrostatic potential (MEP), frontier molecular orbitals, and thermodynamic properties were performed at B3LYP/6-311G(d,p) level of theory.

  14. Inhibition of green tea and the catechins against 1-deoxy-d-xylulose 5-phosphate reductoisomerase, the key enzyme of the MEP terpenoid biosynthetic pathway.

    PubMed

    Hui, Xian; Liu, Hui; Tian, Fang-Lin; Li, Fei-Fei; Li, Heng; Gao, Wen-Yun

    2016-09-01

    1-Deoxy-d-xylulose 5-phosphate reductoisomerase (DXR) is the first committed enzyme in the MEP terpenoid biosynthetic pathway and also a validated antimicrobial target. Green tea which is rich in polyphenolic components such as the catechins, possesses a plenty of pharmacological activities, in particular an antibacterial effect. To uncover the antibacterial mechanism of green tea and to seek new DXR inhibitors from natural sources, the DXR inhibitory activity of green tea and its main antimicrobial catechins were investigated in this study. The results show that the raw extract of green tea and its ethyl acetate fraction are able to suppress DXR activity explicitly. Further determination of the DXR inhibitory capacity of eight catechin compounds demonstrates that the most active compound is gallocatechin gallate that is able to inhibit around 50% activity of DXR at 25μM. Based on these data, the primary structure-activity relationship of the catechins against DXR is discussed. This study would be very helpful to elucidate the antimicrobial mechanism of green tea and the catechins and also would be very useful to direct the rational utilization of them as food additives.

  15. Phosphatidylinositol 4-phosphate 5-kinase α facilitates Toll-like receptor 4-mediated microglial inflammation through regulation of the Toll/interleukin-1 receptor domain-containing adaptor protein (TIRAP) location.

    PubMed

    Nguyen, Tu Thi Ngoc; Kim, Yong Min; Kim, T Doohun; Le, Oanh Thi Tu; Kim, Jae Jin; Kang, Ho Chul; Hasegawa, Hiroshi; Kanaho, Yasunori; Jou, Ilo; Lee, Sang Yoon

    2013-02-22

    Phosphatidylinositol (PI) 4,5-bisphosphate (PIP(2)), generated by PI 4-phosphate 5-kinase (PIP5K), regulates many critical cellular events. PIP(2) is also known to mediate plasma membrane localization of the Toll/IL-1 receptor domain-containing adaptor protein (TIRAP), required for the MyD88-dependent Toll-like receptor (TLR) 4 signaling pathway. Microglia are the primary immune competent cells in brain tissue, and TLR4 is important for microglial activation. However, a functional role for PIP5K and PIP(2) in TLR4-dependent microglial activation remains unclear. Here, we knocked down PIP5Kα, a PIP5K isoform, in a BV2 microglial cell line using stable expression of lentiviral shRNA constructs or siRNA transfection. PIP5Kα knockdown significantly suppressed induction of inflammatory mediators, including IL-6, IL-1β, and nitric oxide, by lipopolysaccharide. PIP5Kα knockdown also attenuated signaling events downstream of TLR4 activation, including p38 MAPK and JNK phosphorylation, NF-κB p65 nuclear translocation, and IκB-α degradation. Complementation of the PIP5Kα knockdown cells with wild type but not kinase-dead PIP5Kα effectively restored the LPS-mediated inflammatory response. We found that PIP5Kα and TIRAP colocalized at the cell surface and interacted with each other, whereas kinase-dead PIP5Kα rendered TIRAP soluble. Furthermore, in LPS-stimulated control cells, plasma membrane PIP(2) increased and subsequently declined, and TIRAP underwent bi-directional translocation between the membrane and cytosol, which temporally correlated with the changes in PIP(2). In contrast, PIP5Kα knockdown that reduced PIP(2) levels disrupted TIRAP membrane targeting by LPS. Together, our results suggest that PIP5Kα promotes TLR4-associated microglial inflammation by mediating PIP(2)-dependent recruitment of TIRAP to the plasma membrane.

  16. A new and fast methodology to assess oxidative damage in cardiovascular diseases risk development through eVol-MEPS-UHPLC analysis of four urinary biomarkers.

    PubMed

    Mendes, Berta; Silva, Pedro; Mendonça, Isabel; Pereira, Jorge; Câmara, José S

    2013-11-15

    In this work, a new, fast and reliable methodology using a digitally controlled microextraction by packed sorbent (eVol(®)-MEPS) followed by ultra-high pressure liquid chromatography (UHPLC) analysis with photodiodes (PDA) detection, was developed to establish the urinary profile levels of four putative oxidative stress biomarkers (OSBs) in healthy subjects and patients evidencing cardiovascular diseases (CVDs). This data was used to verify the suitability of the selected OSBs (uric acid-UAc, malondialdehyde-MDA, 5-(hydroxymethyl)uracil-5-HMUra and 8-hydroxy-2'-deoxyguanosine-8-oxodG) as potential biomarkers of CVDs progression. Important parameters affecting the efficiency of the extraction process were optimized, particularly stationary phase selection, pH influence, sample volume, number of extraction cycles and washing and elution volumes. The experimental conditions that allowed the best extraction efficiency, expressed in terms of total area of the target analytes and data reproducibility, includes a 10 times dilution and pH adjustment of the urine samples to 6.0, followed by a gradient elution through the C8 adsorbent with 5 times 50 µL of 0.01% formic acid and 3×50 µL of 20% methanol in 0.01% formic acid. The chromatographic separation of the target analytes was performed with a HSS T3 column (100 mm × 2.1 mm, 1.7 µm in particle size) using 0.01% formic acid 20% methanol at 250 µL min(-1). The methodology was validated in terms of selectivity, linearity, instrumental limit of detection (LOD), method limit of quantification (LOQ), matrix effect, accuracy and precision (intra-and inter-day). Good results were obtained in terms of selectivity and linearity (r(2)>0.9906), as well as the LOD and LOQ, whose values were low, ranging from 0.00005 to 0.72 µg mL(-1) and 0.00023 to 2.31 µg mL(-1), respectively. The recovery results (91.1-123.0%), intra-day (1.0-8.3%), inter-day precision (4.6-6.3%) and the matrix effect (60.1-110.3%) of eVol(®)-MEPS

  17. A High-Yield Co-Expression System for the Purification of an Intact Drs2p-Cdc50p Lipid Flippase Complex, Critically Dependent on and Stabilized by Phosphatidylinositol-4-Phosphate

    PubMed Central

    Azouaoui, Hassina; Montigny, Cédric; Ash, Miriam-Rose; Fijalkowski, Frank; Jacquot, Aurore; Grønberg, Christina; López-Marqués, Rosa L.; Palmgren, Michael G.; Garrigos, Manuel; le Maire, Marc; Decottignies, Paulette; Gourdon, Pontus; Nissen, Poul; Champeil, Philippe; Lenoir, Guillaume

    2014-01-01

    P-type ATPases from the P4 subfamily (P4-ATPases) are energy-dependent transporters, which are thought to establish lipid asymmetry in eukaryotic cell membranes. Together with their Cdc50 accessory subunits, P4-ATPases couple ATP hydrolysis to lipid transport from the exoplasmic to the cytoplasmic leaflet of plasma membranes, late Golgi membranes, and endosomes. To gain insights into the structure and function of these important membrane pumps, robust protocols for expression and purification are required. In this report, we present a procedure for high-yield co-expression of a yeast flippase, the Drs2p-Cdc50p complex. After recovery of yeast membranes expressing both proteins, efficient purification was achieved in a single step by affinity chromatography on streptavidin beads, yielding ∼1–2 mg purified Drs2p-Cdc50p complex per liter of culture. Importantly, the procedure enabled us to recover a fraction that mainly contained a 1∶1 complex, which was assessed by size-exclusion chromatography and mass spectrometry. The functional properties of the purified complex were examined, including the dependence of its catalytic cycle on specific lipids. The dephosphorylation rate was stimulated in the simultaneous presence of the transported substrate, phosphatidylserine (PS), and the regulatory lipid phosphatidylinositol-4-phosphate (PI4P), a phosphoinositide that plays critical roles in membrane trafficking events from the trans-Golgi network (TGN). Likewise, overall ATP hydrolysis by the complex was critically dependent on the simultaneous presence of PI4P and PS. We also identified a prominent role for PI4P in stabilization of the Drs2p-Cdc50p complex towards temperature- or C12E8-induced irreversible inactivation. These results indicate that the Drs2p-Cdc50p complex remains functional after affinity purification and that PI4P as a cofactor tightly controls its stability and catalytic activity. This work offers appealing perspectives for detailed structural and

  18. Vegetable Crop Pests. MEP 311.

    ERIC Educational Resources Information Center

    Kantzes, James G.; And Others

    As part of a cooperative extension service series by the University of Maryland, this publication introduces the identification and control of common agricultural pests of vegetable crops. The first of the five sections defines "pest" and "weed" and generally introduces different kinds of pests in the categories of insects, weeds, and diseases.…

  19. Fruit Crop Pests. MEP 312.

    ERIC Educational Resources Information Center

    Weaver, Leslie O.; And Others

    As part of a cooperative extension service series by the University of Maryland this publication introduces the identification and control of common agricultural pests of fruit crops. The first of the five sections defines "pest" and "weed" and generally introduces different kinds of pests in the categories of insects, weeds, and diseases. Also in…

  20. Is There Life After MEP?

    ERIC Educational Resources Information Center

    Edwardson, Roger

    1982-01-01

    Difficulties in implementing the Microelectronics Education Programme are discussed. Difficulties include too few microcomputers in schools, networking within schools, teachers working in isolation, distribution of software; and the problem of raising the level of awareness of teachers in all subject areas. (Author/JN)

  1. Evaluation of coriander spice as a functional food by using in vitro bioassays.

    PubMed

    Zhang, Chuan-Rui; Dissanayake, Amila A; Kevseroğlu, Kudret; Nair, Muraleedharan G

    2015-01-15

    Coriander leaves and seeds are widely used as a condiment and spice. The use of roasted coriander seeds in food and beverage is very common. In this study, we investigated raw and roasted coriander seeds for their functional food quality using antioxidant, anti-inflammatory and human tumour cell proliferation inhibitory assays. The hexane and methanolic extracts of raw and roasted coriander seeds showed identical chromatographic and bioassay profiles. Chromatographic purification of the roasted seed extracts afforded tripetroselinin as the predominant component. Other isolates were petroselinic acid, 1,3-dipetroselinin, 2-C-methyl-d-erythritol, 2-C-methyl-d-erythritol 4-O-β-d-glucopyranoside and linalool. Hexane and methanolic extracts of both raw and roasted seeds and pure isolates from them showed comparable antioxidant and anti-inflammatory activities to the positive controls used in the assays, and inhibited the growth of human tumour cells AGS (gastric carcinoma), DU-145 and LNCaP (prostate carcinoma), HCT-116 (colon carcinoma), MCF-7 (breast carcinoma) and NCI-H460 (lung carcinoma) by 4-34%, respectively.

  2. A novel strategy to isolate cell-envelope mutants resistant to phage infection: bacteriophage mEp213 requires lipopolysaccharides in addition to FhuA to enter Escherichia coli K-12.

    PubMed

    Reyes-Cortés, Ruth; Martínez-Peñafiel, Eva; Martínez-Pérez, Francisco; de la Garza, Mireya; Kameyama, Luis

    2012-12-01

    We have developed a direct and efficient strategy, based on a three-step method, to select bacterial cell-envelope mutants resistant to bacteriophage infection. Escherichia coli K-12 strain W3110 underwent classical transposon mutagenesis followed by replica plating and selection for mutants resistant to infection by coliphage mEp213. To verify that phage resistance was due to mutations in the cell envelope, we transformed host cells with the viral genome using electroporation and selected those in which virions were subsequently detected in the supernatant. Among the nine mutants resistant to coliphage infection that we selected, six were in the fhuA gene, two were mutated in the waaC gene, and one was mutated in the gmhD gene. The latter two gene products are involved in the synthesis of lipopolysaccharide (LPS). The efficiency of plating and adsorption of phage mEp213 was affected in these mutants. We verified that LPS is required for the efficient infection of phage λ as well. We propose that this mutation-and-selection strategy can be used to find host factors involved in the initial steps of phage infection for any cognate pair of phage and bacteria.

  3. Molecular structure, FT-IR, NBO, HOMO and LUMO, MEP and first order hyperpolarizability of (2E)-1-(2,4-Dichlorophenyl)-3-(3,4,5-trimethoxyphenyl)prop-2-en-1-one by HF and density functional methods.

    PubMed

    Sheena Mary, Y; Yohannan Panicker, C; Anto, P L; Sapnakumari, M; Narayana, B; Sarojini, B K

    2015-01-25

    (2E)-1-(2,4-Dichlorophenyl)-3-(3,4,5-trimethoxyphenyl)prop-2-en-1-one is synthesized by using 2,4-dichloroacetophenone and 3,4,5-trimethoxybenzaldehyde in ethanol. The structure of the compound was confirmed by IR and single crystal X-ray diffraction studies. FT-IR spectrum of (2E)-1-(2,4-dichloro-phenyl)-3-(3,4,5-trimethoxyphenyl)prop-2-en-1-one was recorded and analyzed. The crystal structure is also described. The vibrational wavenumbers were computed using HF and DFT methods and are assigned with the help of potential energy distribution method. The first hyperpolarizability and infrared intensities are also reported. The geometrical parameters of the title compound obtained from XRD studies are in agreement with the calculated (DFT) values. The stability of the molecule arising from hyper-conjugative interaction and charge delocalization has been analyzed using NBO analysis. The HOMO and LUMO analysis are used to determine the charge transfer within the molecule. MEP was performed by the DFT method. From the MEP map of the title molecule, negative region is mainly localized over the electronegative oxygen atoms, in the carbonyl group and the oxygen atom O4 of the methoxy group and the maximum positive region is localized on the phenyl rings.

  4. Spectroscopic investigation (FT-IR and FT-Raman), vibrational assignments, HOMO-LUMO, NBO, MEP analysis and molecular docking study of 2-(4-hydroxyphenyl)-4,5-dimethyl-1H-imidazole 3-oxide

    NASA Astrophysics Data System (ADS)

    Benzon, K. B.; Varghese, Hema Tresa; Panicker, C. Yohannan; Pradhan, Kiran; Tiwary, Bipransh Kumar; Nanda, Ashis Kumar; Alsenoy, C. Van

    2015-07-01

    In this work, the vibrational spectral analysis was carried out using FT-IR and FT-Raman spectroscopy of 2-(4-hydroxyphenyl)-4,5-dimethyl-1H-imidazole 3-oxide. The computations were performed at DFT levels of theory to get the optimized geometry and vibrational frequencies of the normal modes of the title compound using Gaussian09 software. The complete vibrational assignments of frequencies were made on the basis of potential energy distribution. The calculated HOMO and LUMO energies show the chemical activity of the molecule. The stability of the molecule arising from hyper-conjugative interaction and charge delocalization has been analyzed using NBO analysis. The hyperpolarizability values are reported and the first hyperpolarizability of the title compound is 19.61 times that of standard NLO material urea. From the MEP plot, the negative charge covers the nitro group and the positive region is over the hydroxyl group and N-H part of the imidazole ring. The calculated 1H NMR results are in good agreement with experimental data. Molecular docking study is also reported.

  5. Melanin production by Rhizobium meliloti GR4 is linked to nonsymbiotic plasmid pRmeGR4b: cloning, sequencing, and expression of the tyrosinase gene mepA.

    PubMed Central

    Mercado-Blanco, J; García, F; Fernández-López, M; Olivares, J

    1993-01-01

    Melanin production by Rhizobium meliloti GR4 is linked to nonsymbiotic plasmid pRmeGR4b (140 MDa). Transfer of this plasmid to GR4-cured derivatives or to Agrobacterium tumefaciens enables these bacteria to produce melanin. Sequence analysis of a 3.5-kb PstI fragment of plasmid pRmeGR4b has revealed the presence of a open reading frame 1,481-bp that codes for a protein whose sequence shows strong homology to two conserved regions involved in copper binding in tyrosinases and hemocyanins. In vitro-coupled transcription-translation experiments showed that this open reading frame codes for a 55-kDa polypeptide. Melanin production in GR4 is not under the control of the RpoN-NifA regulatory system, unlike that in R. leguminosarum bv. phaseoli 8002. The GR4 tyrosinase gene could be expressed in Escherichia coli under the control of the lacZ promoter. For avoiding confusion with mel genes (for melibiose), a change of the name of the previously reported mel genes of R. leguminosarum bv. phaseoli and other organisms to mep genes (for melanin production) is proposed. Images PMID:8366027

  6. FT-IR, molecular structure, HOMO-LUMO, MEP, NBO analysis and first order hyperpolarizability of Methyl 4,4″-difluoro-5";-methoxy-1,1";:3";,1″-terphenyl-4";-carboxylate

    NASA Astrophysics Data System (ADS)

    Sheena Mary, Y.; Yohannan Panicker, C.; Narayana, B.; Samshuddin, S.; Sarojini, B. K.; Van Alsenoy, C.

    2014-12-01

    Methyl 4,4″-difluoro-5";-methoxy-1,1";:3";,1″-terphenyl-4";-carboxylate was prepared by the aromatization of a cyclohexenone derivative, Methyl 4,6-bis(4-fluorophenyl)-2-oxocyclohex-3-ene-1-carboxylate using iodine and methanol at reflux conditions. The structure of the compound was confirmed by IR and single crystal X-ray diffraction studies. FT-IR spectrum was recorded and analyzed. The crystal structure is also described. The vibrational wavenumbers are calculated using HF and DFT methods and are assigned with the help of potential energy distribution. The geometrical parameters of the title compound obtained from XRD studies are in agreement with the calculated (DFT) values. The stability of the molecule arising from hyper-conjugative interaction and charge delocalization has been analyzed using NBO analysis. According to the MEP analysis, negative region (red) is mainly over the carbonyl group showing large activity. The calculated hyperpolarizability of the title compound is greater than that of the standard NLO material urea and the title compound is an attractive object for future studies of nonlinear optical properties.

  7. FT-IR, HOMO-LUMO, NBO, MEP analysis and molecular docking study of 3-Methyl-4-{(E)-[4-(methylsulfanyl)-benzylidene]amino}1H-1,2,4-triazole-5(4H)-thione.

    PubMed

    Panicker, C Yohannan; Varghese, Hema Tresa; Manjula, P S; Sarojini, B K; Narayana, B; War, Javeed Ahamad; Srivastava, S K; Van Alsenoy, C; Al-Saadi, Abdulaziz A

    2015-01-01

    FT-IR spectrum of 3-Methyl-4-{(E)-[4-(methylsulfanyl)-benzylidene]amino}1H-1,2,4-triazole-5(4H)-thione was recorded and analysed. The vibrational wavenumbers were computed and at HF and DFT levels of theory. The data obtained from wavenumber calculations are used to assign the vibrational bands obtained in the IR spectrum. The NH stretching wavenumber is red shifted in the IR spectrum from the computed value, which indicates the weakening of the NH bond. The geometrical parameters of the title compound are in agreement with the XRD results. NBO analysis, HOMO-LUMO, first and second order hyperpolarizability and molecular electrostatic potential results are also reported. From the MEP map it is evident that the negative regions are localized over the sulphur atoms and N3 atom of triazole ring and the maximum positive region is localized on NH group, indicating a possible site for nucleophilic attack. Prediction of Activity Spectra analysis of the title compound predicts anti-tuberculostic activity with probability to be active value of 0.543. Molecular docking studies reveal that the triazole nitrogen atoms and the thione sulphur atom play vital role in bonding and results draw us to the conclusion that the compound might exhibit anti-tuberculostic activity.

  8. Spectroscopic (FT-IR and FT-Raman) investigation, first order hyperpolarizability, NBO, HOMO-LUMO and MEP analysis of 6-nitrochromone by ab initio and density functional theory calculations

    NASA Astrophysics Data System (ADS)

    Senthil kumar, J.; Jeyavijayan, S.; Arivazhagan, M.

    2015-02-01

    The vibrational spectral analysis is carried out using FT-Raman and FT-IR spectroscopy in the range 3500-50 cm-1 and 4000-400 cm-1, respectively, for 6-nitrochromone (6NC). The molecular structure, fundamental vibrational frequencies and intensity of the vibrational bands are interpreted with the aid of structure optimization and normal coordinates force field calculation based on ab initio HF and DFT gradient calculations employing the HF/6-311++G(d,p) and B3LYP/6-311++G(d,p) basis set. Stability of the molecule has been analyzed using NBO analysis. The calculated HOMO and LUMO energies show that charge transfer occurs within the molecule. Thermodynamic properties like entropy, heat capacity, zero-point energy and Mulliken's charge analysis have been calculated for the 6NC. The complete assignments were performed on the basis of total energy distribution (TED) of the vibrational modes with scaled quantum mechanical (SQM) method. The MEP map shows the negative potential sites are on oxygen atoms as well as the positive potential sites are around the hydrogen atoms.

  9. Spectroscopic (FT-IR and FT-Raman) investigation, first order hyperpolarizability, NBO, HOMO-LUMO and MEP analysis of 6-nitrochromone by ab initio and density functional theory calculations.

    PubMed

    Senthil kumar, J; Jeyavijayan, S; Arivazhagan, M

    2015-02-01

    The vibrational spectral analysis is carried out using FT-Raman and FT-IR spectroscopy in the range 3500-50 cm(-1) and 4000-400 cm(-1), respectively, for 6-nitrochromone (6NC). The molecular structure, fundamental vibrational frequencies and intensity of the vibrational bands are interpreted with the aid of structure optimization and normal coordinates force field calculation based on ab initio HF and DFT gradient calculations employing the HF/6-311++G(d,p) and B3LYP/6-311++G(d,p) basis set. Stability of the molecule has been analyzed using NBO analysis. The calculated HOMO and LUMO energies show that charge transfer occurs within the molecule. Thermodynamic properties like entropy, heat capacity, zero-point energy and Mulliken's charge analysis have been calculated for the 6NC. The complete assignments were performed on the basis of total energy distribution (TED) of the vibrational modes with scaled quantum mechanical (SQM) method. The MEP map shows the negative potential sites are on oxygen atoms as well as the positive potential sites are around the hydrogen atoms. PMID:25448928

  10. Field and Forage Crop Pests. MEP 310.

    ERIC Educational Resources Information Center

    Morgan, Omar, D.; And Others

    As part of a cooperative extension service series by the University of Maryland, this publication introduces the identification and control of common agricultural pests that can be found in field and forage crops. The first of the five sections defines "pest" and "weed" and generally introduces different kinds of pests in the categories of…

  11. Ornamental, Turf and Nursery Pests. MEP 308.

    ERIC Educational Resources Information Center

    Morgan, Omar D.; And Others

    As part of a cooperative extension service series by the University of Maryland, this publication introduces the identification and control of common turf and plant pests that can be found in the urban environment. The first of the five sections defines "pest" and "weed" and generally introduces different kinds of pests such as insects, weeds, and…

  12. Household and Structural Pests. MEP 307.

    ERIC Educational Resources Information Center

    Wood, F. E.

    This pamphlet is a non-technical description of common household arthropod pests in Maryland. Since most of the pests can be found in houses throughout North America, this publication has a wide geographic range of use. General discussions of arthropod structure, growth and development, and metamorphosis are given before the pages on specific…

  13. Vibrational spectroscopic (FT-IR and FT-Raman) studies, HOMO-LUMO, NBO analysis and MEP of 6-methyl-1-({[(2E)-2-methyl-3-phenyl-prop-2-en-1-yl]oxy}methyl)-1,2,3,4-tetra-hydroquinazoline-2,4-dione, a potential chemotherapeutic agent, using density functional methods.

    PubMed

    Sebastian, Sr S H Roseline; Al-Tamimi, Abdul-Malek S; El-Brollosy, Nasser R; El-Emam, Ali A; Yohannan Panicker, C; Van Alsenoy, Christian

    2015-01-01

    6-Methyl-1-({[(2E)-2-methyl-3-phenyl-prop-2-en-1-yl]oxy}methyl)-1,2,3,4-tetra-hydro quinazoline-2,4-dione was prepared via treatment of silylated 6-methylquinazoline-2,4-dione with bis-[(E)-2-methyl-3-phenylallyloxy]methane. FT-IR and FT-Raman spectra were recorded and analyzed. The vibrational wavenumbers were computed using DFT methods and are assigned with the help of potential energy distribution method. The first hyperpolarizability, infrared intensities and Raman activities also reported. The geometrical parameters of the title compound obtained from XRD studies are in agreement with the calculated (B3LYP) values. The stability of the molecule arising from hyper-conjugative interaction and charge delocalization has been analyzed using NBO analysis. The HOMO and LUMO analysis are used to determine the charge transfer within the molecule. MEP was performed by the B3LYP method and from the MEP it is evident that the negative charge covers the CO group and the positive region is over the phenyl ring and NH group.

  14. Structural, spectral, NLO and MEP analysis of the [MgO₂Ti₂(OPr(i)ⁱⁱ)₆], [MgO₂Ti₂(OPr(i)ⁱ)₂(acac)₄] and [MgO₂Ti₂(OPr(i)ⁱ)2(bzac)₄] by DFT method.

    PubMed

    Sayin, Koray; Karakaş, Duran

    2015-06-01

    Quantum chemical calculations are performed on [MgO2Ti2(OPr(i))6] and [MgO2Ti2(OPr(i))2(L)4] complexes. L is acetylacetonate (acac) and benzoylacetonate (bzac) anion. The crystal structures of these complexes have not been obtained as experimentally but optimized structures of these complexes are obtained as theoretically in this study. Universal force field (UFF) and DFT/B3LYP method are used to obtain optimized structures. Theoretical spectral analysis (IR, (1)H and (13)C NMR) is compared with their experimental values. A good agreement is found between experimental and theoretical spectral analysis. These results mean that the optimized structures of mentioned complexes are appropriate. Additionally, the active sites of mentioned complexes are determined by molecular electrostatic potential (MEP) diagrams and non-linear optical (NLO) properties are investigated. PMID:25754394

  15. Structural, spectral, NLO and MEP analysis of the [MgO₂Ti₂(OPr(i)ⁱⁱ)₆], [MgO₂Ti₂(OPr(i)ⁱ)₂(acac)₄] and [MgO₂Ti₂(OPr(i)ⁱ)2(bzac)₄] by DFT method.

    PubMed

    Sayin, Koray; Karakaş, Duran

    2015-06-01

    Quantum chemical calculations are performed on [MgO2Ti2(OPr(i))6] and [MgO2Ti2(OPr(i))2(L)4] complexes. L is acetylacetonate (acac) and benzoylacetonate (bzac) anion. The crystal structures of these complexes have not been obtained as experimentally but optimized structures of these complexes are obtained as theoretically in this study. Universal force field (UFF) and DFT/B3LYP method are used to obtain optimized structures. Theoretical spectral analysis (IR, (1)H and (13)C NMR) is compared with their experimental values. A good agreement is found between experimental and theoretical spectral analysis. These results mean that the optimized structures of mentioned complexes are appropriate. Additionally, the active sites of mentioned complexes are determined by molecular electrostatic potential (MEP) diagrams and non-linear optical (NLO) properties are investigated.

  16. Spectroscopic investigation (FT-IR and FT-Raman), vibrational assignments, HOMO-LUMO, NBO, MEP analysis and molecular docking study of 2-[(4-chlorobenzyl)sulfanyl]-4-(2-methylpropyl)-6-(phenylsulfanyl)-pyrimidine-5-carbonitrile, a potential chemotherapeutic agent

    NASA Astrophysics Data System (ADS)

    Alzoman, Nourah Z.; Mary, Y. Sheena; Panicker, C. Yohannan; Al-Swaidan, Ibrahim A.; El-Emam, Ali A.; Al-Deeb, Omar A.; Al-Saadi, Abdulaziz A.; Van Alsenoy, Christian; War, Javeed Ahmad

    2015-03-01

    Vibrational spectral analysis of 2-[(4-chlorobenzyl)sulfanyl]-4-(2-methylpropyl)-6-(phenylsulfanyl)-pyrimidine-5-carbonitrile was carried out using FT-IR and FT-Raman spectroscopic techniques. The equilibrium geometry and vibrational wave numbers have been computed using density functional B3LYP method with 6-311++G(d,p)(5D,7F) as basis set. Stability of the molecule arising from hyper conjugative interactions, charge delocalization has been analyzed using NBO analysis. The nonlinear optical behavior of the title compound is also theoretically predicted. From the MEP, it is evident that the negative charge covers the Ctbnd N group and the positive region is over the phenyl and the pyrimidine rings. From the potential energy scan it is clear that the lone pairs of the sulfur atom prefer to point away from the pyrimidine ring and the Ctbnd N group resulting with two possible minimum conformations at the N4C8S1C25 angle equal nearly 0° or 150°. Molecular docking results suggest that the compound might exhibit inhibitory activity against GPb and may act as potential anti-diabetic compound.

  17. Momentous discoveries on Mars: science outside MEP pathways

    NASA Technical Reports Server (NTRS)

    Easter, R. W.; Delitsky, M. I.; Lamassoure, E.; Marshall, M. F.; Matthews, J. B.; Palkovic, L. A.; Wilson, T. J.

    2003-01-01

    The Mars Outpost Team seeks further input from the Mars community about other possible momentous discoveries that could be made, as well as ways to respond to them (types of missions, instruments, spacecraft-orbiters, landers, rovers, airplanes, etc.).

  18. Microelectronics and Special Education. CET/MEP Information Sheet.

    ERIC Educational Resources Information Center

    Council for Educational Technology, London (England).

    Used as an additional aid by the teacher, microelectronics can assist mentally and physically handicapped children to meet educational objectives that have been specifically agreed upon for the individual child. Microelectronics can help deaf children develop speech production, communication skills, and grammar and sentence construction;…

  19. The non-mevalonate isoprenoid biosynthesis of plants as a test system for drugs against malaria and pathogenic bacteria.

    PubMed

    Zeidler, J; Schwender, J; Mueller, C; Lichtenthaler, H K

    2000-12-01

    Two plant test systems are presented in the search for new inhibitors of the non-mevalonate isoprenoid pathway. A derivative of clomazone appears to be an inhibitor of the deoxyxylulose 5-phosphate/methylerythritol 4-phosphate (DOXP/MEP) pathway of isoprenoid formation.

  20. Genome Sequence of the Red Pigment-Forming Meiothermus taiwanensis Strain RP Isolated from Paniphala Hot Spring, India.

    PubMed

    Mukherjee, Trinetra; Bose, Sucharita; Sen, Urmimala; Roy, Chayan; Rameez, Moidu Jameela; Ghosh, Wriddhiman; Mukhopadhyay, Subhra Kanti

    2016-01-01

    Here we report the draft genome sequence of Meiothermus taiwanensis strain RP (MCC 2966), isolated from the Paniphala hot spring of India, which contains genes encoding for enzymes of the methyl erythritol 4-phosphate (MEP) pathway of isoprenoid biosynthesis and carotenoid backbone synthesis. PMID:27365353

  1. Genome Sequence of the Red Pigment-Forming Meiothermus taiwanensis Strain RP Isolated from Paniphala Hot Spring, India

    PubMed Central

    Mukherjee, Trinetra; Bose, Sucharita; Sen, Urmimala; Roy, Chayan; Rameez, Moidu Jameela; Ghosh, Wriddhiman

    2016-01-01

    Here we report the draft genome sequence of Meiothermus taiwanensis strain RP (MCC 2966), isolated from the Paniphala hot spring of India, which contains genes encoding for enzymes of the methyl erythritol 4-phosphate (MEP) pathway of isoprenoid biosynthesis and carotenoid backbone synthesis. PMID:27365353

  2. Synthesis of Methylerythritol Phosphate Analogues and Their Evaluation as Alternate Substrates for IspDF and IspE from Agrobacterium tumefaciens

    PubMed Central

    2015-01-01

    The methylerythritol phosphate biosynthetic pathway, found in most Bacteria, some parasitic protists, and plant chloroplasts, converts d-glyceraldehyde phosphate and pyruvate to isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP), where it intersects with the mevalonate pathway found in some Bacteria, Archaea, and Eukarya, including the cytosol of plants. d-3-Methylerythritol-4-phosphate (MEP), the first pathway-specific intermediate in the pathway, is converted to IPP and DMAPP by the consecutive action of the IspD-H proteins. We synthesized five d-MEP analogues—d-erythritol-4-phosphate (EP), d-3-methylthrietol-4-phosphate (MTP), d-3-ethylerythritol-4-phosphate (EEP), d-1-amino-3-methylerythritol-4-phosphate (NMEP), and d-3-methylerythritol-4-thiolophosphate (MESP)—and studied their ability to function as alternative substrates for the reactions catalyzed by the IspDF fusion and IspE proteins from Agrobacterium tumefaciens, which covert MEP to the corresponding eight-membered cyclic diphosphate. All of the analogues, except MTP, and their products were substrates for the three consecutive enzymes. PMID:25184438

  3. Synthesis of methylerythritol phosphate analogues and their evaluation as alternate substrates for IspDF and IspE from Agrobacterium tumefaciens.

    PubMed

    Krasutsky, Sergiy G; Urbansky, Marek; Davis, Chad E; Lherbet, Christian; Coates, Robert M; Poulter, C Dale

    2014-10-01

    The methylerythritol phosphate biosynthetic pathway, found in most Bacteria, some parasitic protists, and plant chloroplasts, converts D-glyceraldehyde phosphate and pyruvate to isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP), where it intersects with the mevalonate pathway found in some Bacteria, Archaea, and Eukarya, including the cytosol of plants. D-3-Methylerythritol-4-phosphate (MEP), the first pathway-specific intermediate in the pathway, is converted to IPP and DMAPP by the consecutive action of the IspD-H proteins. We synthesized five D-MEP analogues-D-erythritol-4-phosphate (EP), D-3-methylthrietol-4-phosphate (MTP), D-3-ethylerythritol-4-phosphate (EEP), D-1-amino-3-methylerythritol-4-phosphate (NMEP), and D-3-methylerythritol-4-thiolophosphate (MESP)-and studied their ability to function as alternative substrates for the reactions catalyzed by the IspDF fusion and IspE proteins from Agrobacterium tumefaciens, which covert MEP to the corresponding eight-membered cyclic diphosphate. All of the analogues, except MTP, and their products were substrates for the three consecutive enzymes.

  4. Atomic-Resolution Structures of Discrete Stages on the Reaction Coordinate of the [Fe4S4] Enzyme IspG (GcpE).

    PubMed

    Quitterer, Felix; Frank, Annika; Wang, Ke; Rao, Guodong; O'Dowd, Bing; Li, Jikun; Guerra, Francisco; Abdel-Azeim, Safwat; Bacher, Adelbert; Eppinger, Jörg; Oldfield, Eric; Groll, Michael

    2015-06-19

    IspG is the penultimate enzyme in non-mevalonate biosynthesis of the universal terpene building blocks isopentenyl diphosphate and dimethylallyl diphosphate. Its mechanism of action has been the subject of numerous studies but remained unresolved due to difficulties in identifying distinct reaction intermediates. Using a moderate reducing agent and an epoxide substrate analogue, we were now able to trap and crystallographically characterize various stages in the IspG-catalyzed conversion of 2-C-methyl-D-erythritol-2,4-cyclo-diphosphate into (E)-1-hydroxy-2-methylbut-2-enyl-4-diphosphate. In addition, the enzyme's structure was determined in complex with several inhibitors. These results, combined with recent electron paramagnetic resonance data, allowed us to deduce a detailed and complete IspG catalytic mechanism, which describes all stages from initial ring opening to formation of (E)-1-hydroxy-2-methylbut-2-enyl-4-diphosphate via discrete radical and carbanion intermediates. The data presented in this article provide a guide for the design of selective drugs against many prokaryotic and eukaryotic pathogens to which the non-mevalonate pathway is essential for survival and virulence.

  5. The plastidial retrograde signal methyl erythritol cyclopyrophosphate is a regulator of salicylic acid and jasmonic acid crosstalk

    PubMed Central

    Lemos, Mark; Xiao, Yanmei; Bjornson, Marta; Wang, Jin-zheng; Hicks, Derrick; de Souza, Amancio; Wang, Chang-Quan; Yang, Panyu; Ma, Shisong; Dinesh-Kumar, Savithramma; Dehesh, Katayoon

    2016-01-01

    The exquisite harmony between hormones and their corresponding signaling pathways is central to prioritizing plant responses to simultaneous and/or successive environmental trepidations. The crosstalk between jasmonic acid (JA) and salicylic acid (SA) is an established effective mechanism that optimizes and tailors plant adaptive responses. However, the underlying regulatory modules of this crosstalk are largely unknown. Global transcriptomic analyses of mutant plants (ceh1) with elevated levels of the stress-induced plastidial retrograde signaling metabolite 2-C-methyl-D-erythritol cyclopyrophosphate (MEcPP) revealed robustly induced JA marker genes, expected to be suppressed by the presence of constitutively high SA levels in the mutant background. Analyses of a range of genotypes with varying SA and MEcPP levels established the selective role of MEcPP-mediated signal(s) in induction of JA-responsive genes in the presence of elevated SA. Metabolic profiling revealed the presence of high levels of the JA precursor 12-oxo-phytodienoic acid (OPDA), but near wild type levels of JA in the ceh1 mutant plants. Analyses of coronatine-insensitive 1 (coi1)/ceh1 double mutant plants confirmed that the MEcPP-mediated induction is JA receptor COI1 dependent, potentially through elevated OPDA. These findings identify MEcPP as a previously unrecognized central regulatory module that induces JA-responsive genes in the presence of high SA, thereby staging a multifaceted plant response within the environmental context. PMID:26733689

  6. Cloning and expression analysis of ten genes associated with picrosides biosynthesis in Picrorhiza kurrooa.

    PubMed

    Singh, Harsharan; Gahlan, Parul; Kumar, Sanjay

    2013-02-25

    Picrorhiza kurrooa Royle ex Benth. is an economically important medicinal plant known to yield picrosides which have high medicinal value. Picroside I and picroside II are major picrosides associated with various bioactivities. The present work analyzed the expression of various genes of the picrosides biosynthesis pathway in different tissues of the plant in relation to the picrosides content. Eight full-length cDNA sequences namely, 1-deoxy-d-xylulose-5-phosphate synthase (2.317 kb), 1-deoxy-d-xylulose-5-phosphate reductoisomerase (1.767 kb), 4-diphosphocytidyl-2-C-methyl-d-erythritol kinase (1.674 kb), 4-hydroxy-3-methylbut-2-enyl diphosphate reductase (1.701 kb), acetyl-CoA acetyltransferase (1.545 kb), 3-hydroxy-3-methylglutaryl coenzyme A reductase (2.241 kb), isopentenyl pyrophosphate isomerase (987 bp) and geranyl diphosphate synthase (1.434 kb), were cloned to full-length followed by expression analysis of ten genes vis-à-vis picrosides content analysis. There is maximum accumulation of picrosides in leaf tissue followed by the rhizome and root, and a similar pattern of expression was found in all the ten genes. The genes responded to the modulators of the picrosides biosynthesis. Picrosides accumulation was enhanced by application of hydrogen peroxide and abscisic acid, whereas methyl jasmonate and salicylic acid treatment decreased the content.

  7. Atmospheric chemistry in stereo: A new look at secondary organic aerosols from isoprene

    NASA Astrophysics Data System (ADS)

    Nozière, Barbara; González, Nélida J. D.; Borg-Karlson, Anna-Karin; Pei, Yuxin; Redeby, Johan Pettersson; Krejci, Radovan; Dommen, Josef; Prevot, Andre S. H.; Anthonsen, Thorleif

    2011-06-01

    Isoprene, a compound emitted by vegetation, could be a major contributor to secondary organic aerosols (SOA) in the atmosphere. The main evidence for this contribution were the 2-methylbutane-1,2,3,4-tetraols, or 2-methyltetrols (2-methylerythritol and 2-methylthreitol) present in ambient aerosols. In this work, the four stereoisomers of these tetraols were analyzed in aerosols from Aspvreten, Sweden. 2-C-methyl-D-erythritol was found in excess over its enantiomer in the Spring/Summer, by up to 29% in July. This clearly indicated some biological origins for this enantiomer, consistent with its well-documented production by plants and other living organisms. In addition, a minimum of 20 to 60% of the mass of racemic tetraols appeared from biological origin. Thus, the SOA mass produced by isoprene in the atmosphere is less than what indicated by the 2-methyltetrols in aerosols. Our results also demonstrate that stereochemical speciation can distinguish primary and secondary organic material in atmospheric aerosols.

  8. Atomic resolution structures of discrete stages on the reaction coordinate of the [Fe4S4] enzyme IspG (GcpE)

    PubMed Central

    Quitterer, Felix; Frank, Annika; Wang, Ke; Rao, Guodong; O'Dowd, Bing; Li, Jikun; Guerra, Francisco; Abdel-Azeim, Safwat; Bacher, Adelbert; Eppinger, Jörg; Oldfield, Eric; Groll, Michael

    2015-01-01

    IspG is the penultimate enzyme in non-mevalonate biosynthesis of the universal terpene building blocks isopentenyl diphosphate and dimethylallyl diphosphate. Its mechanism of action has been the subject of numerous studies but remained unresolved due to difficulties in identifying distinct reaction intermediates. Using a moderate reducing agent as well as an epoxide substrate analogue, we were now able to trap and crystallographically characterize various stages in the IspG catalyzed conversion of 2-C-methyl-D-erythritol-2,4-cyclo-diphosphate (MEcPP) to (E)-1-hydroxy-2-methylbut-2-enyl-4-diphosphate (HMBPP). In addition, the enzyme's structure was determined in complex with several inhibitors. These results, combined with recent electron paramagnetic resonance data, allowed us to deduce a detailed and complete IspG catalytic mechanism which describes all stages from initial ring opening to formation of HMBPP via discrete radical and carbanion intermediates. The data presented in this article provide a guide for the design of selective drugs against many pro- and eukaryotic pathogens to which the non-mevalonate pathway is essential for survival and virulence. PMID:25868383

  9. Leveraging structure determination with fragment screening for infectious disease drug targets: MECP synthase from Burkholderia pseudomallei

    SciTech Connect

    Begley, Darren W.; Hartley, Robert C.; Davies, Douglas R.; Edwards, Thomas E.; Leonard, Jess T.; Abendroth, Jan; Burris, Courtney A.; Bhandari, Janhavi; Myler, Peter J.; Staker, Bart L.; Stewart, Lance J.

    2011-09-28

    As part of the Seattle Structural Genomics Center for Infectious Disease, we seek to enhance structural genomics with ligand-bound structure data which can serve as a blueprint for structure-based drug design. We have adapted fragment-based screening methods to our structural genomics pipeline to generate multiple ligand-bound structures of high priority drug targets from pathogenic organisms. In this study, we report fragment screening methods and structure determination results for 2C-methyl-D-erythritol-2,4-cyclo-diphosphate (MECP) synthase from Burkholderia pseudomallei, the gram-negative bacterium which causes melioidosis. Screening by nuclear magnetic resonance spectroscopy as well as crystal soaking followed by X-ray diffraction led to the identification of several small molecules which bind this enzyme in a critical metabolic pathway. A series of complex structures obtained with screening hits reveal distinct binding pockets and a range of small molecules which form complexes with the target. Additional soaks with these compounds further demonstrate a subset of fragments to only bind the protein when present in specific combinations. This ensemble of fragment-bound complexes illuminates several characteristics of MECP synthase, including a previously unknown binding surface external to the catalytic active site. These ligand-bound structures now serve to guide medicinal chemists and structural biologists in rational design of novel inhibitors for this enzyme.

  10. Phosphatidylinositol 4-Phosphate 5-Kinases in the Regulation of T Cell Activation

    PubMed Central

    Porciello, Nicla; Kunkl, Martina; Viola, Antonella; Tuosto, Loretta

    2016-01-01

    Phosphatidylinositol 4,5-biphosphate kinases (PIP5Ks) are critical regulators of T cell activation being the main enzymes involved in the synthesis of phosphatidylinositol 4,5-biphosphate (PIP2). PIP2 is indeed a pivotal regulator of the actin cytoskeleton, thus controlling T cell polarization and migration, stable adhesion to antigen-presenting cells, spatial organization of the immunological synapse, and co-stimulation. Moreover, PIP2 also serves as a precursor for the second messengers inositol triphosphate, diacylglycerol, and phosphatidylinositol 3,4,5-triphosphate, which are essential for the activation of signaling pathways regulating cytokine production, cell cycle progression, survival, metabolism, and differentiation. Here, we discuss the impact of PIP5Ks on several T lymphocyte functions with a specific focus on the role of CD28 co-stimulation in PIP5K compartimentalization and activation. PMID:27242793

  11. Phosphatidylinositol 4-Phosphate 5-Kinases in the Regulation of T Cell Activation.

    PubMed

    Porciello, Nicla; Kunkl, Martina; Viola, Antonella; Tuosto, Loretta

    2016-01-01

    Phosphatidylinositol 4,5-biphosphate kinases (PIP5Ks) are critical regulators of T cell activation being the main enzymes involved in the synthesis of phosphatidylinositol 4,5-biphosphate (PIP2). PIP2 is indeed a pivotal regulator of the actin cytoskeleton, thus controlling T cell polarization and migration, stable adhesion to antigen-presenting cells, spatial organization of the immunological synapse, and co-stimulation. Moreover, PIP2 also serves as a precursor for the second messengers inositol triphosphate, diacylglycerol, and phosphatidylinositol 3,4,5-triphosphate, which are essential for the activation of signaling pathways regulating cytokine production, cell cycle progression, survival, metabolism, and differentiation. Here, we discuss the impact of PIP5Ks on several T lymphocyte functions with a specific focus on the role of CD28 co-stimulation in PIP5K compartimentalization and activation.

  12. Phosphatidylinositol 4-Phosphate 5-Kinases in the Regulation of T Cell Activation.

    PubMed

    Porciello, Nicla; Kunkl, Martina; Viola, Antonella; Tuosto, Loretta

    2016-01-01

    Phosphatidylinositol 4,5-biphosphate kinases (PIP5Ks) are critical regulators of T cell activation being the main enzymes involved in the synthesis of phosphatidylinositol 4,5-biphosphate (PIP2). PIP2 is indeed a pivotal regulator of the actin cytoskeleton, thus controlling T cell polarization and migration, stable adhesion to antigen-presenting cells, spatial organization of the immunological synapse, and co-stimulation. Moreover, PIP2 also serves as a precursor for the second messengers inositol triphosphate, diacylglycerol, and phosphatidylinositol 3,4,5-triphosphate, which are essential for the activation of signaling pathways regulating cytokine production, cell cycle progression, survival, metabolism, and differentiation. Here, we discuss the impact of PIP5Ks on several T lymphocyte functions with a specific focus on the role of CD28 co-stimulation in PIP5K compartimentalization and activation. PMID:27242793

  13. Effect of Enzyme Inhibitors on Terpene Trilactones Biosynthesis and Gene Expression Profiling in Ginkgo biloba Cultured Cells.

    PubMed

    Chen, Lijia; Tong, Hui; Wang, Mingxuan; Zhu, Jianhua; Zi, Jiachen; Song, Liyan; Yu, Rongmin

    2015-12-01

    The biosynthetic pathway of terpene trilactones of Ginkgo biloba is unclear. In this present study, suspension cultured cells of G. biloba were used to explore the regulation of the mevalonic acid (MVA) and methylerythritol 4-phosphate (MEP) pathways in response to specific enzyme inhibitors (lovastatin and clomazone). The results showed that the biosynthesis of bilobalide was more highly correlated with the MVA pathway, and the biosynthesis of ginkgolides was more highly correlated with the MEP pathway. Meanwhile, according to the results, it could be speculated that bilobalide might be a product of ginkgolide metabolism. PMID:26882658

  14. Effect of Enzyme Inhibitors on Terpene Trilactones Biosynthesis and Gene Expression Profiling in Ginkgo biloba Cultured Cells.

    PubMed

    Chen, Lijia; Tong, Hui; Wang, Mingxuan; Zhu, Jianhua; Zi, Jiachen; Song, Liyan; Yu, Rongmin

    2015-12-01

    The biosynthetic pathway of terpene trilactones of Ginkgo biloba is unclear. In this present study, suspension cultured cells of G. biloba were used to explore the regulation of the mevalonic acid (MVA) and methylerythritol 4-phosphate (MEP) pathways in response to specific enzyme inhibitors (lovastatin and clomazone). The results showed that the biosynthesis of bilobalide was more highly correlated with the MVA pathway, and the biosynthesis of ginkgolides was more highly correlated with the MEP pathway. Meanwhile, according to the results, it could be speculated that bilobalide might be a product of ginkgolide metabolism.

  15. UV-B modulates the interplay between terpenoids and flavonoids in peppermint (Mentha x piperita L.).

    PubMed

    Dolzhenko, Yuliya; Bertea, Cinzia M; Occhipinti, Andrea; Bossi, Simone; Maffei, Massimo E

    2010-08-01

    Modulation of secondary metabolites by UV-B involves changes in gene expression, enzyme activity and accumulation of defence metabolites. After exposing peppermint (Mentha x piperita L.) plants grown in field (FP) and in a growth chamber (GCP) to UV-B irradiation, we analysed by qRT-PCR the expression of genes involved in terpenoid biosynthesis and encoding: 1-deoxy-D-xylulose-5-phosphate synthase (Dxs), 2-C-methyl-D-erythritol-2,4-cyclodiphosphate synthase (Mds), isopentenyl diphosphate isomerase (Ippi), geranyl diphosphate synthase (Gpps), (-)-limonene synthase (Ls), (-)-limonene-3-hydroxylase (L3oh), (+)-pulegone reductase (Pr), (-)-menthone reductase (Mr), (+)-menthofuran synthase (Mfs), farnesyl diphosphate synthase (Fpps) and a putative sesquiterpene synthase (S-TPS). GCP always showed a higher terpenoid content with respect to FP. We found that in both FP and GCP, most of these genes were regulated by the UV-B treatment. The amount of most of the essential oil components, which were analysed by gas chromatography-mass spectrometry (GC-MS), was not correlated to gene expression. The total phenol composition was found to be always increased after UV-B irradiation; however, FP always showed a higher phenol content with respect to GCP. Liquid chromatography-mass spectrometry (LC-ESI-MS/MS) analyses revealed the presence of UV-B absorbing flavonoids such as eriocitrin, hesperidin, and kaempferol 7-O-rutinoside whose content significantly increased in UV-B irradiated FP, when compared to GCP. The results of this work show that UV-B irradiation differentially modulates the expression of genes involved in peppermint essential oil biogenesis and the content of UV-B absorbing flavonoids. Plants grown in field were better adapted to increasing UV-B irradiation than plants cultivated in growth chambers. The interplay between terpenoid and phenylpropanoid metabolism is also discussed.

  16. Functional analysis of the final steps of the 1-deoxy-D-xylulose 5-phosphate (DXP) pathway to isoprenoids in plants using virus-induced gene silencing.

    PubMed

    Page, Jonathan E; Hause, Gerd; Raschke, Maja; Gao, Wenyun; Schmidt, Jürgen; Zenk, Meinhart H; Kutchan, Toni M

    2004-04-01

    Isoprenoid biosynthesis in plant plastids occurs via the 1-deoxy-d-xylulose 5-phosphate (DXP) pathway. We used tobacco rattle virus (TRV) to posttranscriptionally silence the expression of the last two enzymes of this pathway, the IspG-encoded (E)-4-hydroxy-3-methylbut-2-enyl diphosphate synthase (HDS) and the IspH-encoded isopentenyl/dimethylallyl diphosphate synthase (IDDS), as well as isopentenyl/dimethylallyl diphosphate isomerase (IDI), the enzyme that interconverts IPP and DMAPP. TRV-IspG and TRV-IspH infected Nicotiana benthamiana plants had albino leaves that contained less than 4% of the chlorophyll and carotenoid pigments of control leaves. We applied [(13)C]DXP and [(14)C]DXP to silenced leaves and found that 2-C-methyl-d-erythritol 2,4-cyclodiphosphate accumulated in plants blocked at HDS while DXP, (E)-4-hydroxy-3-methylbut-2-enyl phosphate and (E)-2-methylbut-2-ene-1,4-diol accumulated in IDDS-blocked plants. Albino leaves from IspG- and IspH-silenced plants displayed a disorganized palisade mesophyll, reduced cuticle, fewer plastids, and disrupted thylakoid membranes. These findings demonstrate the participation of HDS and IDDS in the DXP pathway in plants, and support the view that plastid isoprenoid biosynthesis is metabolically and physically segregated from the mevalonate pathway. IDI-silenced plants had mottled white-pale green leaves with disrupted tissue and plastid structure, and showed an 80% reduction in pigments compared to controls. IPP pyrophosphatase activity was higher in chloroplasts isolated from IDI-silenced plants than in control plant chloroplasts. We suggest that a low level of isoprenoid biosynthesis via the DXP pathway can occur without IDI but that this enzyme is required for full function of the DXP pathway.

  17. The analysis of chiral methyltetrols in atmospheric aerosols: A new look at Secondary Organic Aerosols from isoprene

    NASA Astrophysics Data System (ADS)

    Gonzalez Cantu, N. J.; Noziere, B.; Borg-Karlsson, A.; Pei, Y.; Petersson, J.; Krejci, R.; Artaxo, P.; Baltensperger, U.; Dommen, J.; Prevot, A. S.; Anthonsen, T.

    2010-12-01

    The quantities of Secondary Organic Aerosols (SOA) produced in the atmosphere by the transformations of organic gases are difficult to estimate, isoprene having possibly and important contribution, but also containing the most uncertainties. One of the main challenges in this topic is the limited information on real atmospheric SOA, as there was, until recently, no method to distinguish unambiguously between secondary and primary organic compounds in atmospheric aerosols. We have developed a new analytical method to make this distinction, based on separating the enantiomers of organic compounds (isomers mirror images of each other) in aerosols. In this work, this method is applied to the 2-methyltetrols, 2-methylerythritol and 2-methylthreitol, present in PM2.5 aerosols collected at Aspvreten, Sweden from May to November 2008. These compounds are currently considered as the main evidence supporting the presence of SOA from isoprene in the atmosphere. The results of our analyses show that these compounds are, at least in part, from biological (or “primary”) origin. In particular, 2-C-methyl-D-erythritol was 15 % in excess of the total mass of 2-methyltetrols in May-July, clearly indicating its biological origin, and consistent with its well-documented production by plants. Furthermore, the concentrations of the remaining racemic diastereoisomers (total 2-methylerythritol and total 2-methylthreitol) did not correlate with each other, implying that at least one of them, or both, were also from biological origin. This was supported by their lack of correlation with ozone at the site. While atmospheric (abiotic) reactions might have contributed, it can not be excluded that these compounds were mostly from biological origin, thus challenging the main evidence for the existence of SOA from isoprene in the atmosphere.

  18. 2D numerical simulation of the MEP energy-transport model with a finite difference scheme

    SciTech Connect

    Romano, V. . E-mail: romano@dmi.unict.it

    2007-02-10

    A finite difference scheme of Scharfetter-Gummel type is used to simulate a consistent energy-transport model for electron transport in semiconductors devices, free of any fitting parameters, formulated on the basis of the maximum entropy principle. Simulations of silicon n{sup +}-n-n{sup +} diodes, 2D-MESFET and 2D-MOSFET and comparisons with the results obtained by a direct simulation of the Boltzmann transport equation and with other energy-transport models, known in the literature, show the validity of the model and the robustness of the numerical scheme.

  19. 75 FR 6355 - Manufacturing Extension Partnership (MEP) Availability of Funds for Three Regions Including the...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2010-02-09

    ... methodology for delivering advanced manufacturing technology to small- and medium-sized manufacturers and... the Federal Register Notice of February 11, 2008 (73 FR 7696), are applicable to this notice. Please refer to http://www.gpoaccess.gov/fr/ . Dun and Bradstreet Data Universal Numbering System: On the...

  20. 77 FR 37653 - Manufacturing Extension Partnership (MEP) Centers for Arizona, Maryland and Rhode Island...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2012-06-22

    ... Mechanisms. Does the proposal clearly and sharply define an effective methodology for delivering advanced manufacturing technology to small- and medium-sized manufacturers and mechanism(s) for accelerating the adoption..., which are contained in the Federal Register notice of February 11, 2008 (73 FR 7696), are applicable...

  1. 34 CFR 200.89 - MEP allocations; Re-interviewing; Eligibility documentation; and Quality control.

    Code of Federal Regulations, 2014 CFR

    2014-07-01

    ... the identified problems. (2) Prospective re-interviewing. As part of the system of quality controls... information on which the determination was based were true and correct; (v) Stop serving any children found... children identified in the State. (d) Responsibilities of an SEA to establish and implement a system...

  2. 34 CFR 200.89 - MEP allocations; Re-interviewing; Eligibility documentation; and Quality control.

    Code of Federal Regulations, 2013 CFR

    2013-07-01

    ... the identified problems. (2) Prospective re-interviewing. As part of the system of quality controls... information on which the determination was based were true and correct; (v) Stop serving any children found... children identified in the State. (d) Responsibilities of an SEA to establish and implement a system...

  3. 78 FR 34346 - Proposed Information Collection; Comment Request; NIST MEP Advanced Manufacturing Jobs and...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2013-06-07

    ... Advanced Manufacturing Jobs and Innovation Accelerator Challenge (AMJIAC) Client Impact Survey AGENCY... information collection. The purpose of the Advanced Manufacturing Jobs and Innovation Accelerator Challenge... to support job creation, encourage economic development, and enhance the competitiveness of...

  4. 75 FR 33766 - Manufacturing Extension Partnership (MEP) Availability of Funds for Projects To Develop Client...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2010-06-15

    ... Cooperative Agreements, which are contained in the Federal Register Notice of February 11, 2008 (73 FR 7696), are applicable to this notice. Please refer to http://www.gpoaccess.gov/fr/ . Dun and Bradstreet Data..., technology acceleration, workforce and continuous improvement. Proposals should include a project plan,...

  5. Deoxyxylulose 5-phosphate reductoisomerase is not a rate-determining enzyme for essential oil production in spike lavender.

    PubMed

    Mendoza-Poudereux, Isabel; Muñoz-Bertomeu, Jesús; Arrillaga, Isabel; Segura, Juan

    2014-11-01

    Spike lavender (Lavandula latifolia) is an economically important aromatic plant producing essential oils, whose components (mostly monoterpenes) are mainly synthesized through the plastidial methylerythritol 4-phosphate (MEP) pathway. 1-Deoxy-D-xylulose-5-phosphate (DXP) synthase (DXS), that catalyzes the first step of the MEP pathway, plays a crucial role in monoterpene precursors biosynthesis in spike lavender. To date, however, it is not known whether the DXP reductoisomerase (DXR), that catalyzes the conversion of DXP into MEP, is also a rate-limiting enzyme for the biosynthesis of monoterpenes in spike lavender. To investigate it, we generated transgenic spike lavender plants constitutively expressing the Arabidopsis thaliana DXR gene. Although two out of the seven transgenic T0 plants analyzed accumulated more essential oils than the controls, this is hardly imputable to the DXR transgene effect since a clear correlation between transcript accumulation and monoterpene production could not be established. Furthermore, these increased essential oil phenotypes were not maintained in their respective T1 progenies. Similar results were obtained when total chlorophyll and carotenoid content in both T0 transgenic plants and their progenies were analyzed. Our results then demonstrate that DXR enzyme does not play a crucial role in the synthesis of plastidial monoterpene precursors, suggesting that the control flux of the MEP pathway in spike lavender is primarily exerted by the DXS enzyme. PMID:25151124

  6. Phosphatidylinositol 4-phosphate 5-kinases 1 and 2 are involved in the regulation of vacuole morphology during Arabidopsis thaliana pollen development.

    PubMed

    Ugalde, José-Manuel; Rodriguez-Furlán, Cecilia; Rycke, Riet De; Norambuena, Lorena; Friml, Jiří; León, Gabriel; Tejos, Ricardo

    2016-09-01

    The pollen grains arise after meiosis of pollen mother cells within the anthers. A series of complex structural changes follows, generating mature pollen grains capable of performing the double fertilization of the female megasporophyte. Several signaling molecules, including hormones and lipids, have been involved in the regulation and appropriate control of pollen development. Phosphatidylinositol 4-phophate 5-kinases (PIP5K), which catalyze the biosynthesis of the phosphoinositide PtdIns(4,5)P2, are important for tip polar growth of root hairs and pollen tubes, embryo development, vegetative plant growth, and responses to the environment. Here, we report a role of PIP5Ks during microgametogenesis. PIP5K1 and PIP5K2 are expressed during early stages of pollen development and their transcriptional activity respond to auxin in pollen grains. Early male gametophytic lethality to certain grade was observed in both pip5k1(-/-) and pip5k2(-/-) single mutants. The number of pip5k mutant alleles is directly related to the frequency of aborted pollen grains suggesting the two genes are involved in the same function. Indeed PIP5K1 and PIP5K2 are functionally redundant since homozygous double mutants did not render viable pollen grains. The loss of function of PIP5K1 and PIP5K2results in defects in vacuole morphology in pollen at the later stages and epidermal root cells. Our results show that PIP5K1, PIP5K2 and phosphoinositide signaling are important cues for early developmental stages and vacuole formation during microgametogenesis. PMID:27457979

  7. ME-PS matching in the simulation of multi-jet production in hadron collisions using a subtraction method

    NASA Astrophysics Data System (ADS)

    Odaka, Shigeru; Watanabe, Norihisa; Kurihara, Yoshimasa

    2015-05-01

    The subtraction method for the matching between the matrix element (ME) and parton shower (PS), which has been developed for combining 0-jet and 1-jet production processes in association with electroweak-boson production in hadron collisions, is extended to multi-jet production. In order to include multi-jet MEs, we have to address the soft-gluon divergence together with the collinear divergence. We introduce an approximation that simultaneously reproduces both divergences in a form suitable for application to our subtraction method. The alteration in the subtraction can be compensated by applying an appropriate correction to the corresponding non-radiative events. We demonstrate that W + 0, 1, and 2 jet production processes can be consistently combined using the developed matching method.

  8. Evolution of the Isoprene Biosynthetic Pathway in Kudzu1[w

    PubMed Central

    Sharkey, Thomas D.; Yeh, Sansun; Wiberley, Amy E.; Falbel, Tanya G.; Gong, Deming; Fernandez, Donna E.

    2005-01-01

    Isoprene synthase converts dimethylallyl diphosphate, derived from the methylerythritol 4-phosphate (MEP) pathway, to isoprene. Isoprene is made by some plants in substantial amounts, which affects atmospheric chemistry, while other plants make no isoprene. As part of our long-term study of isoprene synthesis, the genetics of the isoprene biosynthetic pathway of the isoprene emitter, kudzu (Pueraria montana), was compared with similar genes in Arabidopsis (Arabidopsis thaliana), which does not make isoprene. The MEP pathway genes in kudzu were similar to the corresponding Arabidopsis genes. Isoprene synthase genes of kudzu and aspen (Populus tremuloides) were cloned to compare their divergence with the divergence seen in MEP pathway genes. Phylogenetic analysis of the terpene synthase gene family indicated that isoprene synthases are either within the monoterpene synthase clade or sister to it. In Arabidopsis, the gene most similar to isoprene synthase is a myrcene/ocimene (acyclic monoterpenes) synthase. Two phenylalanine residues found exclusively in isoprene synthases make the active site smaller than other terpene synthase enzymes, possibly conferring specificity for the five-carbon substrate rather than precursors of the larger isoprenoids. Expression of the kudzu isoprene synthase gene in Arabidopsis caused Arabidopsis to emit isoprene, indicating that whether or not a plant emits isoprene depends on whether or not it has a terpene synthase capable of using dimethylallyl diphosphate. PMID:15653811

  9. Differential Subplastidial Localization and Turnover of Enzymes Involved in Isoprenoid Biosynthesis in Chloroplasts.

    PubMed

    Perello, Catalina; Llamas, Ernesto; Burlat, Vincent; Ortiz-Alcaide, Miriam; Phillips, Michael A; Pulido, Pablo; Rodriguez-Concepcion, Manuel

    2016-01-01

    Plastidial isoprenoids are a diverse group of metabolites with roles in photosynthesis, growth regulation, and interaction with the environment. The methylerythritol 4-phosphate (MEP) pathway produces the metabolic precursors of all types of plastidial isoprenoids. Proteomics studies in Arabidopsis thaliana have shown that all the enzymes of the MEP pathway are localized in the plastid stroma. However, immunoblot analysis of chloroplast subfractions showed that the first two enzymes of the pathway, deoxyxylulose 5-phosphate synthase (DXS) and reductoisomerase (DXR), can also be found in non-stromal fractions. Both transient and stable expression of GFP-tagged DXS and DXR proteins confirmed the presence of the fusion proteins in distinct subplastidial compartments. In particular, DXR-GFP was found to accumulate in relatively large vesicles that could eventually be released from chloroplasts, presumably to be degraded by an autophagy-independent process. Together, we propose that protein-specific mechanisms control the localization and turnover of the first two enzymes of the MEP pathway in Arabidopsis chloroplasts. PMID:26919668

  10. Differential Subplastidial Localization and Turnover of Enzymes Involved in Isoprenoid Biosynthesis in Chloroplasts

    PubMed Central

    Perello, Catalina; Llamas, Ernesto; Burlat, Vincent; Ortiz-Alcaide, Miriam; Phillips, Michael A.; Pulido, Pablo; Rodriguez-Concepcion, Manuel

    2016-01-01

    Plastidial isoprenoids are a diverse group of metabolites with roles in photosynthesis, growth regulation, and interaction with the environment. The methylerythritol 4-phosphate (MEP) pathway produces the metabolic precursors of all types of plastidial isoprenoids. Proteomics studies in Arabidopsis thaliana have shown that all the enzymes of the MEP pathway are localized in the plastid stroma. However, immunoblot analysis of chloroplast subfractions showed that the first two enzymes of the pathway, deoxyxylulose 5-phosphate synthase (DXS) and reductoisomerase (DXR), can also be found in non-stromal fractions. Both transient and stable expression of GFP-tagged DXS and DXR proteins confirmed the presence of the fusion proteins in distinct subplastidial compartments. In particular, DXR-GFP was found to accumulate in relatively large vesicles that could eventually be released from chloroplasts, presumably to be degraded by an autophagy-independent process. Together, we propose that protein-specific mechanisms control the localization and turnover of the first two enzymes of the MEP pathway in Arabidopsis chloroplasts. PMID:26919668

  11. Differential Subplastidial Localization and Turnover of Enzymes Involved in Isoprenoid Biosynthesis in Chloroplasts.

    PubMed

    Perello, Catalina; Llamas, Ernesto; Burlat, Vincent; Ortiz-Alcaide, Miriam; Phillips, Michael A; Pulido, Pablo; Rodriguez-Concepcion, Manuel

    2016-01-01

    Plastidial isoprenoids are a diverse group of metabolites with roles in photosynthesis, growth regulation, and interaction with the environment. The methylerythritol 4-phosphate (MEP) pathway produces the metabolic precursors of all types of plastidial isoprenoids. Proteomics studies in Arabidopsis thaliana have shown that all the enzymes of the MEP pathway are localized in the plastid stroma. However, immunoblot analysis of chloroplast subfractions showed that the first two enzymes of the pathway, deoxyxylulose 5-phosphate synthase (DXS) and reductoisomerase (DXR), can also be found in non-stromal fractions. Both transient and stable expression of GFP-tagged DXS and DXR proteins confirmed the presence of the fusion proteins in distinct subplastidial compartments. In particular, DXR-GFP was found to accumulate in relatively large vesicles that could eventually be released from chloroplasts, presumably to be degraded by an autophagy-independent process. Together, we propose that protein-specific mechanisms control the localization and turnover of the first two enzymes of the MEP pathway in Arabidopsis chloroplasts.

  12. Properties and inhibition of the first two enzymes of the non-mevalonate pathway of isoprenoid biosynthesis.

    PubMed

    Mueller, C; Schwender, J; Zeidler, J; Lichtenthaler, H K

    2000-12-01

    Enzymes of the 1-deoxy-D-xylulose 5-phosphate/2-C-methylerythritol 4-phosphate (DOXP/MEP) pathway are targets for new herbicides and antibacterial drugs. Until now, no inhibitors for the DOXP synthase have been known of. We show that one of the breakdown products of the herbicide clomazone affects the DOXP synthase. One inhibitor of the non-mevalonate pathway, fosmidomycin, blocks the DOXP reductoisomerase (DXR) of plants and bacteria. The I(50) values of plants are, however, higher than those found for the DXR of Escherichia coli. The DXR of plants, isolated from barley seedlings, shows a pH optimum of 8.1, which is typical for enzymes active in the chloroplast stroma.

  13. Transcript Quantification of Genes Involved in Steviol Glycoside Biosynthesis in Stevia rebaudiana Bertoni by Real-Time Polymerase Chain Reaction (RT-PCR).

    PubMed

    Modi, Arpan; Kumar, Nitish; Narayanan, Subhash

    2016-01-01

    Stevia (Stevia rebaudiana Bertoni) is a medicinal plant having sweet, diterpenoid glycosides known as steviol glycosides which are 200-300 times sweeter than sucrose (0.4 % solution). They are synthesized mainly in the leaves via plastid localized 2-C-methyl-D-erythrose-4-phosphate pathway (MEP pathway). Fifteen genes are involved in the formation of these glycosides. In the present protocol, a method for the quantification of transcripts of these genes is shown. The work involves RNA extraction and cDNA preparation, and therefore, procedures for the confirmation of DNA-free cDNA preparation have also been illustrated. Moreover, details of plant treatments are not mentioned as this protocol may apply to relative gene expression profile in any medicinal plant with any treatment. The treatments are numbered as T0 (Control), T1, T2, T3, and T4. PMID:27108325

  14. Construction and Evaluation of Desulfovibrio vulgaris Whole-Genome Oligonucleotide Microarrays

    SciTech Connect

    Z. He; Q. He; L. Wu; M.E. Clark; J.D. Wall; Jizhong Zhou; Matthew W. Fields

    2004-03-17

    Desulfovibrio vulgaris Hildenborough has been the focus of biochemical and physiological studies in the laboratory, and the metabolic versatility of this organism has been largely recognized, particularly the reduction of sulfate, fumarate, iron, uranium and chromium. In addition, a Desulfovibrio sp. has been shown to utilize uranium as the sole electron acceptor. D. vulgaris is a d-Proteobacterium with a genome size of 3.6 Mb and 3584 ORFs. The whole-genome microarrays of D. vulgaris have been constructed using 70mer oligonucleotides. All ORFs in the genome were represented with 3471 (97.1%) unique probes and 103 (2.9%) non-specific probes that may have cross-hybridization with other ORFs. In preparation for use of the experimental microarrays, artificial probes and targets were designed to assess specificity and sensitivity and identify optimal hybridization conditions for oligonucleotide microarrays. The results indicated that for 50mer and 70mer oligonucleotide arrays, hybridization at 45 C to 50 C, washing at 37 C and a wash time of 2.5 to 5 minutes obtained specific and strong hybridization signals. In order to evaluate the performance of the experimental microarrays, growth conditions were selected that were expected to give significant hybridization differences for different sets of genes. The initial evaluations were performed using D. vulgaris cells grown at logarithmic and stationary phases. Transcriptional analysis of D. vulgaris cells sampled during logarithmic phase growth indicated that 25% of annotated ORFs were up-regulated and 3% of annotated ORFs were downregulated compared to stationary phase cells. The up-regulated genes included ORFs predicted to be involved with acyl chain biosynthesis, amino acid ABC transporter, translational initiation factors, and ribosomal proteins. In the stationary phase growth cells, the two most up-regulated ORFs (70-fold) were annotated as a carboxynorspermidine decarboxylase and a 2C-methyl-D-erythritol-2

  15. Carbon partitioning to the terpenoid biosynthetic pathway enables heterologous β-phellandrene production in Escherichia coli cultures.

    PubMed

    Formighieri, Cinzia; Melis, Anastasios

    2014-12-01

    Escherichia coli was used as a microbial system for the heterologous synthesis of β-phellandrene, a monoterpene of plant origin with several potential commercial applications. Expression of Lavandula angustifolia β-phellandrene synthase (PHLS), alone or in combination with Picea abies geranyl-diphosphate synthase in E. coli, resulted in no β-phellandrene accumulation, in sharp contrast to observations with PHLS-transformed cyanobacteria. Lack of β-phellandrene biosynthesis in E. coli was attributed to the limited endogenous carbon partitioning through the native 2-C-methylerythritol-4-phosphate (MEP) pathway. Heterologous co-expression of the mevalonic acid pathway, enhancing cellular carbon partitioning and flux toward the universal isoprenoid precursors, isopentenyl-diphosphate and dimethylallyl-diphosphate, was required to confer β-phellandrene production. Differences in endogenous carbon flux toward the synthesis of isoprenoids between photosynthetic (Synechocystis) and non-photosynthetic bacteria (E. coli) are discussed in terms of differences in the regulation of carbon partitioning through the MEP biosynthetic pathway in the two systems. PMID:25116411

  16. Are iridoids in leaf beetle larvae synthesized de novo or derived from plant precursors? A methodological approach.

    PubMed

    Søe, Astrid R B; Bartram, Stefan; Gatto, Nathalie; Boland, Wilhelm

    2004-09-01

    Iridoids, belonging to a group of cyclopentanoid monoterpenoids, are secreted by many species of leaf beetles as a defense against predators. Using chemically modified precursors of iridoid biosynthesis, it has been shown that some leaf beetle larvae can synthesize these iridoids de novo as well as sequester plant-produced molecules. Stable isotope techniques can provide useful methods for studying terpenoid biosynthesis without disturbing the natural conditions much. Two terpenoid biosynthesis pathways (mevalonic acid (MVA) pathway and methylerythritol-4-phosphate (MEP) pathway) may lead to different delta13C signatures of the products. Our results from natural abundance 13C and 13C-labelled iridoid precursors in Gastrophysa viridula and Phaedon cochleariae suggested that the two leaf beetle species use only de novo synthesis of their defensive iridoids. We observed that the isotope signature of the leaf-beetle-produced iridoids (via the MVA pathway) resembled that of the MEP-derived monoterpenoids from plants. Owing to this close similarity in the natural 13C abundances in the plant and insect compounds, a determination of iridoid-origin in leaf beetle secretion may only be possible by use of isotopically labelled compounds.

  17. Environmental and Genetic Factors Associated with Solanesol Accumulation in Potato Leaves

    PubMed Central

    Campbell, Raymond; Freitag, Sabine; Bryan, Glenn J.; Stewart, Derek; Taylor, Mark A.

    2016-01-01

    Solanesol is a high value 45-carbon, unsaturated, all-trans-nonaprenol isoprenoid. Recently solanesol has received particular attention because of its utility, both in its own right and as a precursor in the production of numerous compounds used in the treatment of disease states. Solanesol is found mainly in solanaceous crops such as potato, tomato, tobacco and pepper where it accumulates in the foliage. There is considerable potential to explore the extraction of solanesol from these sources as a valuable co-product. In this study we have characterized the genetic variation in leaf solanesol content in a biparental, segregating diploid potato population. We demonstrate that potato leaf solanesol content is genetically controlled and identify several quantitative trait loci associated with leaf solanesol content. Transient over-expression of genes from the methylerythritol 4-phosphate (MEP) and mevalonic acid (MVA) pathways, either singly or in combination, resulted in enhanced accumulation of solanesol in leaves of Nicotiana benthamiana, providing insights for genetically engineering the pathway. We also demonstrate that in potato, leaf solanesol content is enhanced by up to six-fold on exposure to moderately elevated temperature and show corresponding changes in expression patterns of MEP and MVA genes. Our combined approaches offer new insights into solanesol accumulation and strategies for developing a bio-refinery approach to potato production. PMID:27610114

  18. Synthetic Routes to Methylerythritol Phosphate Pathway Intermediates and Downstream Isoprenoids

    PubMed Central

    Jarchow-Choy, Sarah K; Koppisch, Andrew T; Fox, David T

    2014-01-01

    Isoprenoids constitute the largest class of natural products with greater than 55,000 identified members. They play essential roles in maintaining proper cellular function leading to maintenance of human health, plant defense mechanisms against predators, and are often exploited for their beneficial properties in the pharmaceutical and nutraceutical industries. Most impressively, all known isoprenoids are derived from one of two C5-precursors, isopentenyl diphosphate (IPP) or dimethylallyl diphosphate (DMAPP). In order to study the enzyme transformations leading to the extensive structural diversity found within this class of compounds there must be access to the substrates. Sometimes, intermediates within a biological pathway can be isolated and used directly to study enzyme/pathway function. However, the primary route to most of the isoprenoid intermediates is through chemical catalysis. As such, this review provides the first exhaustive examination of synthetic routes to isoprenoid and isoprenoid precursors with particular emphasis on the syntheses of intermediates found as part of the 2C-methylerythritol 4-phosphate (MEP) pathway. In addition, representative syntheses are presented for the monoterpenes (C10), sesquiterpenes (C15), diterpenes (C20), triterpenes (C30) and tetraterpenes (C40). Finally, in some instances, the synthetic routes to substrate analogs found both within the MEP pathway and downstream isoprenoids are examined. PMID:25009443

  19. Environmental and Genetic Factors Associated with Solanesol Accumulation in Potato Leaves

    PubMed Central

    Campbell, Raymond; Freitag, Sabine; Bryan, Glenn J.; Stewart, Derek; Taylor, Mark A.

    2016-01-01

    Solanesol is a high value 45-carbon, unsaturated, all-trans-nonaprenol isoprenoid. Recently solanesol has received particular attention because of its utility, both in its own right and as a precursor in the production of numerous compounds used in the treatment of disease states. Solanesol is found mainly in solanaceous crops such as potato, tomato, tobacco and pepper where it accumulates in the foliage. There is considerable potential to explore the extraction of solanesol from these sources as a valuable co-product. In this study we have characterized the genetic variation in leaf solanesol content in a biparental, segregating diploid potato population. We demonstrate that potato leaf solanesol content is genetically controlled and identify several quantitative trait loci associated with leaf solanesol content. Transient over-expression of genes from the methylerythritol 4-phosphate (MEP) and mevalonic acid (MVA) pathways, either singly or in combination, resulted in enhanced accumulation of solanesol in leaves of Nicotiana benthamiana, providing insights for genetically engineering the pathway. We also demonstrate that in potato, leaf solanesol content is enhanced by up to six-fold on exposure to moderately elevated temperature and show corresponding changes in expression patterns of MEP and MVA genes. Our combined approaches offer new insights into solanesol accumulation and strategies for developing a bio-refinery approach to potato production.

  20. Environmental and Genetic Factors Associated with Solanesol Accumulation in Potato Leaves.

    PubMed

    Campbell, Raymond; Freitag, Sabine; Bryan, Glenn J; Stewart, Derek; Taylor, Mark A

    2016-01-01

    Solanesol is a high value 45-carbon, unsaturated, all-trans-nonaprenol isoprenoid. Recently solanesol has received particular attention because of its utility, both in its own right and as a precursor in the production of numerous compounds used in the treatment of disease states. Solanesol is found mainly in solanaceous crops such as potato, tomato, tobacco and pepper where it accumulates in the foliage. There is considerable potential to explore the extraction of solanesol from these sources as a valuable co-product. In this study we have characterized the genetic variation in leaf solanesol content in a biparental, segregating diploid potato population. We demonstrate that potato leaf solanesol content is genetically controlled and identify several quantitative trait loci associated with leaf solanesol content. Transient over-expression of genes from the methylerythritol 4-phosphate (MEP) and mevalonic acid (MVA) pathways, either singly or in combination, resulted in enhanced accumulation of solanesol in leaves of Nicotiana benthamiana, providing insights for genetically engineering the pathway. We also demonstrate that in potato, leaf solanesol content is enhanced by up to six-fold on exposure to moderately elevated temperature and show corresponding changes in expression patterns of MEP and MVA genes. Our combined approaches offer new insights into solanesol accumulation and strategies for developing a bio-refinery approach to potato production. PMID:27610114

  1. Molecular structure, vibrational spectra, MEP, HOMO-LUMO and NBO analysis of Hf(SeO3)(SeO4)(H2O)4

    NASA Astrophysics Data System (ADS)

    Yankova, Rumyana; Genieva, Svetlana; Halachev, Nenko; Dimitrova, Ginka

    2016-02-01

    Hf(SeO3)(SeO4)(H2O)4 was obtained with the hydrothermal synthesis. The geometry optimization of this molecule was done by Density Functional Theory (DFT/B3LYP) method with 6-31G(d) basis set and LANL2DZ for Hf. The experimental infrared spectrum was compared with calculated and complete vibrational assignment was provided. The bond orders and the electronic properties of the molecule were calculated. The natural bond orbital analysis (NBO) was performed in order to study the intramolecular bonding interactions among bonds and delocalization of unpaired electrons. The calculated highest occupied molecular orbital (HOMO) and lowest unoccupied molecular orbital (LUMO) with frontier orbital gap were presented. The electrostatic potential was calculated in order to investigate the reaction properties of the molecule. The thermodynamic properties of the studied compound at different temperatures were calculated.

  2. A combination of whey protein and potassium bicarbonate supplements during head-down-tilt bed rest: Presentation of a multidisciplinary randomized controlled trial (MEP study)

    NASA Astrophysics Data System (ADS)

    Buehlmeier, Judith; Mulder, Edwin; Noppe, Alexandra; Frings-Meuthen, Petra; Angerer, Oliver; Rudwill, Floriane; Biolo, Gianni; Smith, Scott M.; Blanc, Stéphane; Heer, Martina

    2014-02-01

    Inactivity, as it appears during space flight and in bed rest, induces reduction of lean body and bone mass, glucose intolerance, and weakening of the cardiovascular system. Increased protein intake, whey protein in particular, has been proposed to counteract some of these effects, but has also been associated with negative effects on bone, likely caused by a correspondingly high ratio of acid to alkali precursors in the diet.

  3. Spectroscopic (FT-IR/FT-Raman) and computational (HF/DFT) investigation and HOMO/LUMO/MEP analysis on 1,1-difluoro-2-vinyl-cyclopropane.

    PubMed

    Senthil Raj, P; Shoba, D; Ramalingam, S; Periandy, S

    2015-08-01

    All the computational calculations were made in the ground state using the HF and DFT (B3LYP) methods with 6-31++G (d,p) and 6-311++G (d,p) basis sets. Making use of the recorded data, the complete vibrational assignments were made and analysis of the observed fundamental bands of molecule was carried out. The shifting of the frequencies in the vibrational pattern of the title molecule due to the substitutions; CHCH₂ and F were deeply investigated by the vibrational analysis. Moreover, (13)C NMR and (1)H NMR chemical shifts were calculated by using the gauge independent atomic orbital (GIAO) method with HF/B3LYP methods with 6-311++G (d,p). A study on the electronic properties, such as HOMO and LUMO energies, were performed by time-dependent DFT (TD-DFT) approach. Mulliken charges of the 1DF2VCP were also calculated and interpreted. The thermodynamic properties (heat capacity, entropy, and enthalpy) of the title compound at different temperatures were calculated in gas phase.

  4. Measuring the absolute disintegration rate of a radioactive gas with a moveable endplate discharge counter (MEP) and theoretical calculation of wall effect

    SciTech Connect

    Jaffey, A.H.; Gray, J.; Bentley, W.C.; Lerner, J.L.

    1987-09-01

    A precision built moveable endplate Geiger-Mueller counter was used to measure the absolute disintegration rate of a beta-emitting radioactive gas. A Geiger-Mueller counter used for measuring gaseous radioactivity has <100% counting efficiency owing to two factors: (1) ''end effect,'' due to decreased and distorted fields at the ends where wire-insulator joints are placed, and (2) ''wall effect,'' due to non-ionization by beta particles emitted near to and heading into the wall. The end effect was evaluated by making one end of the counter movable and measuring counting rates at a number of endplate positions. Much of the wall effect was calculated theoretically, based on known data for primary ionization of electrons as a function of energy and gas composition. Corrections were then made for the ''shakeoff'' effect in beta decay and for backscattering of electrons from the counter wall. Measurements and calculations were made for a sample of /sup 85/Kr (beta energy, 0.67 MeV). The wall effect calculation is readily extendable to other beta energies.

  5. COMPARISON OF THE CORRELATION OF MEP AND THE FREE ENERGIES OF REACTION WITH NITRO GROUP REDUCTION OF NITROTOLUENE CONGENERS. (R825689C011)

    EPA Science Inventory

    The perspectives, information and conclusions conveyed in research project abstracts, progress reports, final reports, journal abstracts and journal publications convey the viewpoints of the principal investigator and may not represent the views and policies of ORD and EPA. Concl...

  6. CHLOROPLAST BIOGENESIS genes act cell and noncell autonomously in early chloroplast development.

    PubMed

    Gutiérrez-Nava, María de la Luz; Gillmor, C Stewart; Jiménez, Luis F; Guevara-García, Arturo; León, Patricia

    2004-05-01

    In order to identify nuclear genes required for early chloroplast development, a collection of photosynthetic pigment mutants of Arabidopsis was assembled and screened for lines with extremely low levels of chlorophyll. Nine chloroplast biogenesis (clb) mutants that affect proplastid growth and thylakoid membrane formation and result in an albino seedling phenotype were identified. These mutations identify six new genes as well as a novel allele of cla1. clb mutants have less than 2% of wild-type chlorophyll levels, and little or no expression of nuclear and plastid-encoded genes required for chloroplast development and function. In all but one mutant, proplastids do not differentiate enough to form elongated stroma thylakoid membranes. Analysis of mutants during embryogenesis allows differentiation between CLB genes that act noncell autonomously, where partial maternal complementation of chloroplast development is observed in embryos, and those that act cell autonomously, where complementation during embryogenesis is not observed. Molecular characterization of the noncell autonomous clb4 mutant established that the CLB4 gene encodes for hydroxy-2-methyl-2-(E)-butenyl 4-diphosphate synthase (HDS), the next to the last enzyme of the methylerythritol 4-phosphate (MEP) pathway for the synthesis of plastidic isoprenoids. The noncell autonomous nature of the clb4 mutant suggests that products of the MEP pathway can travel between tissues, and provides in vivo evidence that some movement of MEP intermediates exists from the cytoplasm to the plastid. The isolation and characterization of clb mutants represents the first systematic study of genes required for early chloroplast development in Arabidopsis.

  7. The isogene 1-deoxy-D-xylulose 5-phosphate synthase 2 controls isoprenoid profiles, precursor pathway allocation, and density of tomato trichomes.

    PubMed

    Paetzold, Heike; Garms, Stefan; Bartram, Stefan; Wieczorek, Jenny; Urós-Gracia, Eva-Maria; Rodríguez-Concepción, Manuel; Boland, Wilhelm; Strack, Dieter; Hause, Bettina; Walter, Michael H

    2010-09-01

    Plant isoprenoids are formed from precursors synthesized by the mevalonate (MVA) pathway in the cytosol or by the methyl-D-erythritol 4-phosphate (MEP) pathway in plastids. Although some exchange of precursors occurs, cytosolic sesquiterpenes are assumed to derive mainly from MVA, while plastidial monoterpenes are produced preferentially from MEP precursors. Additional complexity arises in the first step of the MEP pathway, which is typically catalyzed by two divergent 1-deoxy-D-xylulose 5-phosphate synthase isoforms (DXS1, DXS2). In tomato (Solanum lycopersicum), the SlDXS1 gene is ubiquitously expressed with highest levels during fruit ripening, whereas SlDXS2 transcripts are abundant in only few tissues, including young leaves, petals, and isolated trichomes. Specific down-regulation of SlDXS2 expression was performed by RNA interference in transgenic plants to investigate feedback mechanisms. SlDXS2 down-regulation led to a decrease in the monoterpene β-phellandrene and an increase in two sesquiterpenes in trichomes. Moreover, incorporation of MVA-derived precursors into residual monoterpenes and into sesquiterpenes was elevated as determined by comparison of ¹³C to ¹²C natural isotope ratios. A compensatory up-regulation of SlDXS1 was not observed. Down-regulated lines also exhibited increased trichome density and showed less damage by leaf-feeding Spodoptera littoralis caterpillars. The results reveal novel, non-redundant roles of DXS2 in modulating isoprenoid metabolism and a pronounced plasticity in isoprenoid precursor allocation. PMID:20591838

  8. Evolutionary diversification and characterization of the eubacterial gene family encoding DXR type II, an alternative isoprenoid biosynthetic enzyme

    PubMed Central

    2013-01-01

    Background Isoprenoids constitute a vast family of natural compounds performing diverse and essential functions in all domains of life. In most eubacteria, isoprenoids are synthesized through the methylerythritol 4-phosphate (MEP) pathway. The production of MEP is usually catalyzed by deoxyxylulose 5-phosphate reductoisomerase (DXR-I) but a few organisms use an alternative DXR-like enzyme (DXR-II). Results Searches through 1498 bacterial complete proteomes detected 130 sequences with similarity to DXR-II. Phylogenetic analysis identified three well-resolved clades: the DXR-II family (clustering 53 sequences including eleven experimentally verified as functional enzymes able to produce MEP), and two previously uncharacterized NAD(P)-dependent oxidoreductase families (designated DLO1 and DLO2 for DXR-II-like oxidoreductases 1 and 2). Our analyses identified amino acid changes critical for the acquisition of DXR-II biochemical function through type-I functional divergence, two of them mapping onto key residues for DXR-II activity. DXR-II showed a markedly discontinuous distribution, which was verified at several levels: taxonomic (being predominantly found in Alphaproteobacteria and Firmicutes), metabolic (being mostly found in bacteria with complete functional MEP pathways with or without DXR-I), and phenotypic (as no biological/phenotypic property was found to be preferentially distributed among DXR-II-containing strains, apart from pathogenicity in animals). By performing a thorough comparative sequence analysis of GC content, 3:1 dinucleotide frequencies, codon usage and codon adaptation indexes (CAI) between DXR-II sequences and their corresponding genomes, we examined the role of horizontal gene transfer (HGT), as opposed to an scenario of massive gene loss, in the evolutionary origin and diversification of the DXR-II subfamily in bacteria. Conclusions Our analyses support a single origin of the DXR-II family through functional divergence, in which constitutes

  9. Pheophytinase Knockdown Impacts Carbon Metabolism and Nutraceutical Content Under Normal Growth Conditions in Tomato.

    PubMed

    Lira, Bruno Silvestre; Rosado, Daniele; Almeida, Juliana; de Souza, Amanda Pereira; Buckeridge, Marcos Silveira; Purgatto, Eduardo; Guyer, Luzia; Hörtensteiner, Stefan; Freschi, Luciano; Rossi, Magdalena

    2016-03-01

    Although chlorophyll (Chl) degradation is an essential biochemical pathway for plant physiology, our knowledge regarding this process still has unfilled gaps. Pheophytinase (PPH) was shown to be essential for Chl breakdown in dark-induced senescent leaves. However, the catalyzing enzymes involved in pigment turnover and fruit ripening-associated degreening are still controversial. Chl metabolism is closely linked to the biosynthesis of other isoprenoid-derived compounds, such as carotenoids and tocopherols, which are also components of the photosynthetic machinery. Chls, carotenoids and tocopherols share a common precursor, geranylgeranyl diphosphate, produced by the plastidial methylerythritol 4-phosphate (MEP) pathway. Additionally, the Chl degradation-derived phytol can be incorporated into tocopherol biosynthesis. In this context, tomato turns out to be an interesting model to address isoprenoid-metabolic cross-talk since fruit ripening combines degreening and an intensely active MEP leading to carotenoid accumulation. Here, we investigate the impact of PPH deficiency beyond senescence by the comprehensive phenotyping of SlPPH-knockdown tomato plants. In leaves, photosynthetic parameters indicate altered energy usage of excited Chl. As a mitigatory effect, photosynthesis-associated carotenoids increased while tocopherol content remained constant. Additionally, starch and soluble sugar profiles revealed a distinct pattern of carbon allocation in leaves that suggests enhanced sucrose exportation. The higher levels of carbohydrates in sink organs down-regulated carotenoid biosynthesis. Additionally, the reduction in Chl-derived phytol recycling resulted in decreased tocopherol content in transgenic ripe fruits. Summing up, tocopherol and carotenoid metabolism, together with the antioxidant capacity of the hydrophilic and hydrophobic fractions, were differentially affected in leaves and fruits of the transgenic plants. Thus, in tomato, PPH plays a role beyond

  10. An in silico structural insights into Plasmodium LytB protein and its inhibition.

    PubMed

    Bhuyan, Rajabrata; Nandy, Suman Kumar; Seal, Alpana

    2015-01-01

    In most of the pathogenic organisms including Plasmodium falciparum, isoprenoids are synthesized via MEP (MethylErythritol 4-Phosphate) pathway. LytB is the last enzyme of this pathway which catalyzes the conversion of (E)-4-hydroxy-3-methylbut-2-en-1-yl diphosphate (HMBPP) into the two isoprenoid precursors: isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP). Since the MEP pathway is not used by humans, it represents an attractive target for the development of new anti-malarial compounds or inhibitors. Here a systematic in silico study has been conducted to get an insight into the structure of Plasmodium lytB as well as its affinities towards different inhibitors. We used comparative modeling technique to predict the three-dimensional (3D) structure of Plasmodium LytB taking Escherichia coli LytB protein (PDB ID: 3KE8) as template and the model was subsequently refined through molecular dynamics (MD) simulation. A large ligand data-set containing diphospate group was subjected for virtual screening against the target using GOLD 5.2 program. Considering the mode of binding and affinities, 17 leads were selected on basis of binding energies in comparison to its substrate HMBPP (Gold.Chemscore.DG: -20.9734 kcal/mol). Among them, five were discarded because of their inhibitory activity towards other human enzymes. The rest 12 potential leads carry all the properties of any "drug like" molecule and the knowledge of Plasmodium LytB-inhibitory mechanism which can provide valuable support for the anti-malarial-inhibitor design in future.

  11. Engineering of Recombinant Poplar Deoxy-D-Xylulose-5-Phosphate Synthase (PtDXS) by Site-Directed Mutagenesis Improves Its Activity

    PubMed Central

    Banerjee, Aparajita; Preiser, Alyssa L.

    2016-01-01

    Deoxyxylulose 5-phosphate synthase (DXS), a thiamine diphosphate (ThDP) dependent enzyme, plays a regulatory role in the methylerythritol 4-phosphate (MEP) pathway. Isopentenyl diphosphate (IDP) and dimethylallyl diphosphate (DMADP), the end products of this pathway, inhibit DXS by competing with ThDP. Feedback inhibition of DXS by IDP and DMADP constitutes a significant metabolic regulation of this pathway. The aim of this work was to experimentally test the effect of key residues of recombinant poplar DXS (PtDXS) in binding both ThDP and IDP. This work also described the engineering of PtDXS to improve the enzymatic activity by reducing its inhibition by IDP and DMADP. We have designed and tested modifications of PtDXS in an attempt to reduce inhibition by IDP. This could possibly be valuable by removing a feedback that limits the usefulness of the MEP pathway in biotechnological applications. Both ThDP and IDP use similar interactions for binding at the active site of the enzyme, however, ThDP being a larger molecule has more anchoring sites at the active site of the enzyme as compared to the inhibitors. A predicted enzyme structure was examined to find ligand-enzyme interactions, which are relatively more important for inhibitor-enzyme binding than ThDP-enzyme binding, followed by their modifications so that the binding of the inhibitors can be selectively affected compared to ThDP. Two alanine residues important for binding ThDP and the inhibitors were mutated to glycine. In two of the cases, both the IDP inhibition and the overall activity were increased. In another case, both the IDP inhibition and the overall activity were reduced. This provides proof of concept that it is possible to reduce the feedback from IDP on DXS activity. PMID:27548482

  12. Novel insights into structure–function mechanism and tissue-specific expression profiling of full-length dxr gene from Cymbopogon winterianus

    PubMed Central

    Devi, Kamalakshi; Dehury, Budheswar; Phukon, Munmi; Modi, Mahendra Kumar; Sen, Priyabrata

    2015-01-01

    The 1-deoxy-d-xylulose-5-phosphate reductoisomerase (DXR; EC1.1.1.267), an NADPH-dependent reductase, plays a pivotal role in the methylerythritol 4-phosphate pathway (MEP), in the conversion of 1-deoxy-d-xylulose-5-phosphate (DXP) into MEP. The sheath and leaf of citronella (Cymbopogon winterianus) accumulates large amount of terpenes and sesquiterpenes with proven medicinal value and economic uses. Thus, sequencing of full length dxr gene and its characterization seems to be a valuable resource in metabolic engineering to alter the flux of isoprenoid active ingredients in plants. In this study, full length DXR from citronella was characterized through in silico and tissue-specific expression studies to explain its structure–function mechanism, mode of cofactor recognition and differential expression. The modelled DXR has a three-domain architecture and its active site comprised of a cofactor (NADPH) binding pocket and the substrate-binding pocket. Molecular dynamics simulation studies indicated that DXR model retained most of its secondary structure during 10 ns simulation in aqueous solution. The modelled DXR superimposes well with its closest structural homolog but subtle variations in the charge distribution over the cofactor recognition site were noticed. Molecular docking study revealed critical residues aiding tight anchoring NADPH within the active pocket of DXR. Tissue-specific differential expression analysis using semi-quantitative RT-PCR and qRT-PCR in various tissues of citronella plant revealed distinct differential expression of DXR. To our knowledge, this is the first ever report on DXR from the important medicinal plant citronella and further characterization of this gene will open up better avenues for metabolic engineering of secondary metabolite pathway genes from medicinal plants in the near future. PMID:25941629

  13. Evaluation of combined use of transcranial and direct cortical motor evoked potential monitoring during unruptured aneurysm surgery.

    PubMed

    Motoyama, Yasushi; Kawaguchi, Masahiko; Yamada, Shuichi; Nakagawa, Ichiro; Nishimura, Fumihiko; Hironaka, Yasuo; Park, Young-Su; Hayashi, Hironobu; Abe, Ryuichi; Nakase, Hiroyuki

    2011-01-01

    The feasibility and reliability of combined use of transcranial and direct cortical motor evoked potential (MEP) monitoring during unruptured aneurysm surgery were evaluated. Forty-eight patients with unruptured cerebral aneurysms underwent craniotomy and neck clipping accompanied by muscle MEP monitoring. MEPs were elicited successfully by transcranial electrical stimulation in all patients. Direct cortical stimulation elicited MEPs in 44 patients. Reduction in MEP amplitude to less than 50% of baseline was considered significant. No postoperative motor paresis occurred in 39 patients in whom transcranial and direct MEPs remained unchanged. Four patients in whom direct MEPs could not be recorded had no intraoperative abnormality in transcranial MEPs and no postoperative motor dysfunction. Four of the other 5 patients manifested significant transient direct MEP changes without transcranial MEP changes. The transient MEP changes were observed in 3 patients during temporary clipping of the parent artery and in one patient with inadequate clipping of an middle cerebral artery aneurysm, and were considered due to insufficiency of blood flow. Decrease or disappearance of direct MEP waves recovered immediately after re-application of the clip and release of the temporary clip. Direct MEP waves disappeared and did not recover until the end of microsurgical procedures in one patient, although transcranial MEP amplitude remained at less than 50% of baseline. She developed hemiparesis postoperatively, which recovered within 6 hours. The duration of temporary occlusion in patients with direct MEP changes was significantly longer than that in patients without (p < 0.05). Direct MEP was sensitive in detecting ischemic stress to descending motor pathways during aneurysm surgery. Transcranial MEPs could be elicited in patients in whom direct MEPs could not be obtained, and during periods such as craniotomy or after dural closure, in which direct MEPs could not be recorded. These

  14. FT-IR, NBO, HOMO-LUMO, MEP analysis and molecular docking study of Methyl N-({[2-(2-methoxyacetamido)-4-(phenylsulfanyl)phenyl]amino}[(methoxycarbonyl) imino]methyl)carbamate.

    PubMed

    Panicker, C Yohannan; Varghese, Hema Tresa; Narayana, B; Divya, K; Sarojini, B K; War, Javeed Ahmad; Van Alsenoy, C; Fun, H K

    2015-09-01

    The optimized molecular structure, vibrational frequencies, corresponding vibrational assignments of Methyl N-({[2-(2-methoxyacetamido)-4-(phenylsulfanyl) phenyl]amino} [(methoxycarbonyl)imino]methyl)carbamate have been investigated using HF and DFT levels of calculations. The geometrical parameters are in agreement with XRD data. The stability of the molecule arising from hyper-conjugative interaction and charge delocalization has been analyzed using NBO analysis. The HOMO and LUMO analysis is used to determine the charge transfer within the molecule. Molecular electrostatic potential study was also performed. The first and second hyperpolarizability was calculated in order to find its role in nonlinear optics. Molecular docking studies are also reported. Prediction of Activity Spectra analysis of the title compound predicts anthelmintic and antiparasitic activity as the most probable activity with Pa (probability to be active) value of 0.808 and 0.797, respectively. Molecular docking studies show that both the phenyl groups and the carbonyl oxygens of the molecule are crucial for bonding and these results draw us to the conclusion that the compound might exhibit pteridine reductase inhibitory activity.

  15. Synthesis, growth, structural and HOMO and LUMO, MEP analysis of a new stilbazolium derivative crystal: A enhanced third-order NLO properties with a high laser-induced damage threshold for NLO applications

    NASA Astrophysics Data System (ADS)

    Senthil, K.; Kalainathan, S.; Hamada, F.; Yamada, M.; Aravindan, P. G.

    2015-08-01

    A new organic third-order nonlinear optical crystal from stilbazolium family 2-[2-(4-methoxy-phenyl) vinyl]-1-methyl-pyridinium tetrafluoroborate (4MSTB) has been synthesized and grown by slow evaporation method for the first time. The grown crystal structure was confirmed by single crystal X-ray diffraction analysis, and it is revealed that the grown crystal crystallized in a triclinic crystal system with centrosymmetric space group P 1 bar . The HOMO and LUMO energies were calculated for the grown crystal explains charge transfer takes place within the molecule and confirms the suitability of the title crystal for NLO applications. The presence of various vibration modes of expected functional groups was identified by FT-IR analysis. The transmittance ability of the grown crystal was also analyzed by using UV-Vis-NIR spectral studies and shows that the crystal has no absorption of light in the entire Vis-NIR region. The thermal stability of the title crystal has been investigated by TGA/DTA studies and revealed that the material was thermally stable up to the melting point, 193 °C. The hardness number, Meyer index, yield strength, and elastic stiffness constant has been estimated for the grown 4MSTB crystal using Vickers microhardness tester. Photoluminescence excitation studies showed green emission radiation occurred at 517 nm. The dielectric properties of the grown crystal have been analyzed as a function of temperature over a wide range of frequency (50 Hz-5 MHz) by using LCR meter. The result of ac electrical conductivity of 4MSTB was found to be 5.25 × 10-5 (Ω m)-1. The laser damage threshold (LDT) energy for the grown crystal has been measured by using a Q-switched Nd:YAG laser as a source in single-shot mode (1064 nm, 10 Hz, 420 mJ). The result of LDT indicates that grown title crystal has excellent resistance to laser radiation than those of known some inorganic NLO materials. The chemical etching studies were carried out to assess the perfection of the grown crystals. Its third-order nonlinear optical properties were investigated by Z-scan technique and proved that the 4MSTB crystal possesses two-photon absorptions (TPA) and the self-defocusing effect. The second-order molecular hyperpolarizability (γ) value at 632.8 nm is calculated to be 3.555 × 10-34 esu. The photoconductivity study of 4MSTB reveals that the negative photoconducting nature. The obtained all the results in the present work are making a 4MSTB promising candidate for the possible applications in optical switches, optical power limiter, and non-linear optical applications.

  16. Vibrational assignments, spectroscopic investigation (FT-IR and FT-Raman), NBO, MEP, HOMO‒LUMO analysis and intermolecular hydrogen bonding interactions of 7-fluoroisatin, 7-bromoisatin and 1-methylisatin ‒ A comparative study

    NASA Astrophysics Data System (ADS)

    Polat, Turgay; Bulut, Fatih; Arıcan, Ilknur; Kandemirli, Fatma; Yildirim, Gürcan

    2015-12-01

    In this comprehensive study, theoretical and experimental studies were carried out on 7-fluoroisatin, 7-bromoisatin and 1-methylisatin using FT-Raman and FT-IR spectra. The optimized geometrical parameters and theoretical vibrational frequencies were calculated by means of density functional theory (DFT/B3LYP) with 6-311++G(d,p) basis set based on scaled quantum mechanical (SQM) method for the first time. The relative abundances of the possible tautomers or conformers found were calculated with respect to the Boltzmann distribution. Moreover, the harmonic vibrational frequencies including IR and Raman intensities, thermodynamic and electronic parameters were computed in detail. The effects of substituents -F, ‒Br and -CH3 on the crucial characteristics pertaining to the title compound of isatin were investigated, and the obtained data were compared with each other. Natural bond orbital (NBO) analysis was applied to study the stability arising from charge delocalization along with the compound. The chemical reactivity parameters (chemical hardness and softness, electronegativity, chemical potential and electrophilicity index) were discussed clearly. The HOMO and LUMO energies determined showed that the serious charge transfer occurs in the title molecules studied. Furthermore, the size, shape, charge density distributions and chemical reactivity sites belonging to the molecules were obtained by mapping electron density isosurface with electrostatic potential surfaces (ESP). Additionally, the hydrogen-bonded complexes were simulated to describe the roles of intermolecular hydrogen bonding on the molecular structures and vibrational frequencies.

  17. Prevalence of use study for amphetamine (AMP), methamphetamine (MAMP), 3,4-methylenedioxy-amphetamine (MDA), 3,4-methylenedioxy-methamphetamine (MDMA), and 3,4-methylenedioxy-ethylamphetamine (MDEA) in military entrance processing stations (MEPS) specimens.

    PubMed

    Klette, Kevin L; Kettle, Aaron R; Jamerson, Matthew H

    2006-06-01

    The Roche Abuscreen Onlinetrade mark Amphetamine immunoassay (IA), modified to include sodium periodate, and the Microgenics DRI Ecstasy IA were used to determine the prevalence of amphetamine (AMP), methamphetamine (MAMP), 3,4-methylenedioxyamphetamine (MDA), 3,4-methylenedioxymethamphetamine (MDMA), and 3,4-methylenedioxyethylamphetamine (MDEA) in urine specimens from applicants seeking to join the United States Armed Forces. Over a 4-month period, a total of 85,658 specimens were IA screened using the Department of Defense 500 ng/mL administrative cutoff level for AMP and MDMA. All presumptively positive specimens were confirmed using a solid-phase extraction procedure coupled with simultaneous analysis of AMP, MAMP, MDA, MDMA, and MDEA by fast gas chromatography-mass spectrometry using the same cutoff levels as the IA. The Roche Online Amphetamine IA identified 216 specimens as presumptively positive; of these, 70 specimens confirmed positive for AMP and 87 specimens confirmed positive for AMP and/or MAMP, resulting in a confirmation rate of 73%. The Microgenics DRI Ecstasy IA identified eight specimens as presumptively positive; of these, five specimens confirmed positive for MDMA and/or MDA, resulting in a confirmation rate of 63%. The total use prevalence for AMP, MAMP, MDA, MDMA, and/or MDEA in military entrance processing stations specimens over the testing period was determined to be 0.19%.

  18. Molecular structure, NLO, MEP, NBO analysis and spectroscopic characterization of 2,5-dimethylanilinium dihydrogen phosphate with experimental (FT-IR and FT-Raman) techniques and DFT calculations.

    PubMed

    Guidara, Sameh; Feki, Habib; Abid, Younes

    2014-12-10

    Single crystals of 2,5-dimethylanilinium dihydrogen phosphate were grown by slow evaporation method at room temperature. The synthesized compound was characterized by powder X-ray diffraction analysis to confirm its crystalline nature. The optimized molecular structure, vibrational spectra and the optical properties were calculated by the density functional theory (DFT) method using the B3LYP function with the 6-31G(d,p) basis set. Theoretical simulation of infrared and Raman spectra led to excellent overall agreement with the observed spectral patterns. The complete assignments of the vibrational spectra were carried out with the aid of potential energy distribution (PED). The stability of the molecule arising from hyperconjugative interaction and charge delocalization has been analyzed using natural bond orbital (NBO) analysis leading to high nonlinear optical (NLO) activity. The lowering in the HOMO and LUMO energy gap explains the eventual charge transfer interactions that take place within the molecules.

  19. De Novo Transcriptome and Expression Profile Analysis to Reveal Genes and Pathways Potentially Involved in Cantharidin Biosynthesis in the Blister Beetle Mylabris cichorii

    PubMed Central

    Huang, Yi; Wang, Zhongkang; Zha, Shenfang; Wang, Yu; Jiang, Wei; Liao, Yufeng; Song, Zhangyong; Qi, Zhaoran; Yin, Youping

    2016-01-01

    The dried body of Mylabris cichorii is well-known Chinese traditional medicine. The sesquiterpenoid cantharidin, which is secreted mostly by adult male beetles, has recently been used as an anti-cancer drug. However, little is known about the mechanisms of cantharidin biosynthesis. Furthermore, there is currently no genomic or transcriptomic information for M. cichorii. In this study, we performed de novo assembly transcriptome of M. cichorii using the Illumina Hiseq2000. A single run produced 9.19 Gb of clean nucleotides comprising 29,247 sequences, including 23,739 annotated sequences (about 81%). We also constructed two expression profile libraries (20–25 day-old adult males and 20–25 day-old adult females) and discovered 2,465 significantly differentially-expressed genes. Putative genes and pathways involved in the biosynthesis of cantharidin were then characterized. We also found that cantharidin biosynthesis in M. cichorii might only occur via the mevalonate (MVA) pathway, not via the methylerythritol 4-phosphate/deoxyxylulose 5-phosphate (MEP/DOXP) pathway or a mixture of these. Besides, we considered that cantharidin biosynthesis might be related to the juvenile hormone (JH) biosynthesis or degradation. The results of transcriptome and expression profiling analysis provide a comprehensive sequence resource for M. cichorii that could facilitate the in-depth study of candidate genes and pathways involved in cantharidin biosynthesis, and may thus help to improve our understanding of the mechanisms of cantharidin biosynthesis in blister beetles. PMID:26752526

  20. Carotenoid Derivates in Achiote (Bixa orellana) Seeds: Synthesis and Health Promoting Properties

    PubMed Central

    Rivera-Madrid, Renata; Aguilar-Espinosa, Margarita; Cárdenas-Conejo, Yair; Garza-Caligaris, Luz E.

    2016-01-01

    Bixa orellana (family Bixaceae) is a neotropical fast growing perennial tree of great agro-industrial value because its seeds have a high carotenoid content, mainly bixin. It has been used since pre-colonial times as a culinary colorant and spice, and for healing purposes. It is currently used as a natural pigment in the food, in pharmaceutical, and cosmetic industries, and it is commercially known as annatto. Recently, several studies have addressed the biological and medical properties of this natural pigment, both as potential source of new drugs or because its ingestion as a condiment or diet supplement may protect against several diseases. The most documented properties are anti-oxidative; but its anti-cancer, hypoglucemic, antibiotic and anti-inflammatory properties are also being studied. Bixin’s pathway elucidation and its regulation mechanisms are critical to improve the produce of this important carotenoid. Even though the bixin pathway has been established, the regulation of the genes involved in bixin production remains largely unknown. Our laboratory recently published B. orellana’s transcriptome and we have identified most of its MEP (methyl-D-erythritol 4-phosphate) and carotenoid pathway genes. Annatto is a potential source of new drugs and can be a valuable nutraceutical supplement. However, its nutritional and healing properties require further study. PMID:27708658

  1. De novo characterization of the root transcriptome of a traditional Chinese medicinal plant Polygonum cuspidatum.

    PubMed

    Hao, DaCheng; Ma, Pei; Mu, Jun; Chen, ShiLin; Xiao, PeiGen; Peng, Yong; Huo, Li; Xu, LiJia; Sun, Chao

    2012-05-01

    Various active components have been extracted from the root of Polygonum cuspidatum. However, the genetic basis for their activity is virtually unknown. In this study, 25600002 short reads (2.3 Gb) of P. cuspidatum root transcriptome were obtained via Illumina HiSeq 2000 sequencing. A total of 86418 unigenes were assembled de novo and annotated. Twelve, 18, 60 and 54 unigenes were respectively mapped to the mevalonic acid (MVA), methyl-D-erythritol 4-phosphate (MEP), shikimate and resveratrol biosynthesis pathways, suggesting that they are involved in the biosynthesis of pharmaceutically important anthraquinone and resveratrol. Eighteen potential UDP-glycosyltransferase unigenes were identified as the candidates most likely to be involved in the biosynthesis of glycosides of secondary metabolites. Identification of relevant genes could be important in eventually increasing the yields of the medicinally useful constituents of the P. cuspidatum root. From the previously published transcriptome data of 19 non-model plant taxa, 1127 shared orthologs were identified and characterized. This information will be very useful for future functional, phylogenetic and evolutionary studies of these plants.

  2. De Novo Transcriptome and Expression Profile Analysis to Reveal Genes and Pathways Potentially Involved in Cantharidin Biosynthesis in the Blister Beetle Mylabris cichorii.

    PubMed

    Huang, Yi; Wang, Zhongkang; Zha, Shenfang; Wang, Yu; Jiang, Wei; Liao, Yufeng; Song, Zhangyong; Qi, Zhaoran; Yin, Youping

    2016-01-01

    The dried body of Mylabris cichorii is well-known Chinese traditional medicine. The sesquiterpenoid cantharidin, which is secreted mostly by adult male beetles, has recently been used as an anti-cancer drug. However, little is known about the mechanisms of cantharidin biosynthesis. Furthermore, there is currently no genomic or transcriptomic information for M. cichorii. In this study, we performed de novo assembly transcriptome of M. cichorii using the Illumina Hiseq2000. A single run produced 9.19 Gb of clean nucleotides comprising 29,247 sequences, including 23,739 annotated sequences (about 81%). We also constructed two expression profile libraries (20-25 day-old adult males and 20-25 day-old adult females) and discovered 2,465 significantly differentially-expressed genes. Putative genes and pathways involved in the biosynthesis of cantharidin were then characterized. We also found that cantharidin biosynthesis in M. cichorii might only occur via the mevalonate (MVA) pathway, not via the methylerythritol 4-phosphate/deoxyxylulose 5-phosphate (MEP/DOXP) pathway or a mixture of these. Besides, we considered that cantharidin biosynthesis might be related to the juvenile hormone (JH) biosynthesis or degradation. The results of transcriptome and expression profiling analysis provide a comprehensive sequence resource for M. cichorii that could facilitate the in-depth study of candidate genes and pathways involved in cantharidin biosynthesis, and may thus help to improve our understanding of the mechanisms of cantharidin biosynthesis in blister beetles. PMID:26752526

  3. Metalloprotease genes of Trichophyton mentagrophytes are important for pathogenicity.

    PubMed

    Zhang, Xinke; Wang, Yanchun; Chi, Wanyi; Shi, Yao; Chen, Si; Lin, Degui; Jin, Yipeng

    2014-01-01

    Metalloproteases (Mep) of the M36 family are important virulence factors for the host invasion by the dermatophyte Trichophyton mentagrophytes. Dermatophytes secrete keratinase to degrade human and animal keratin and invade the skin. In previous studies, primers designed from the MEP gene sequences of Aspergillus fumigatus and A. oryzae were used to amplify the MEP genes from T. mentagrophytes, and the five MEP genes (MEP1-MEP5) were expressed. Differences in the expression of these five MEP genes in different dermatophytes were observed in an in vitro protein induction study, indicating their different functions and proteolytic abilities. However, specific pathogenic functions and mechanisms of each of the metalloproteases, as well as differences in their proteolytic activities, remain uncertain. In the current study, Agrobacterium tumefaciens-mediated transformation (ATMT) was used to successfully transform five MEP genes, resulting in five MEP mutant strains. MEP3 showed strongest proteolytic activity, hair biodegradation ability, and animal pathogenicity among the mutant strains. The MEP4 and MEP5 mutants were the least pathogenic through the above tests. Therefore, we hypothesize that the MEP4 and MEP5 genes are most likely to significantly affect the pathogenicity of T. mentagrophytes. PMID:23859078

  4. 15 CFR 292.2 - Training development and deployment projects.

    Code of Federal Regulations, 2010 CFR

    2010-01-01

    ... of distance learning activities, and preparation of self-learning and technical-guideline materials... the MEP system; and demonstrated interest in the training activity especially by MEP extension...

  5. Use of a cloned multidrug resistance gene for coamplification and overproduction of major excreted protein, a transformation-regulated secreted acid protease

    SciTech Connect

    Kane, S.E.; Troen, B.R.; Gal, S.; Ueda, K.; Pastan, I.; Gottesman, M.M.

    1988-08-01

    Malignantly transformed mouse fibroblasts synthesize and secrete large amounts of major excreted protein (MEP), a 39,000-dalton precursor to an acid protease (cathepsin L). To evaluate the possible role of this protease in the transformed phenotype, the authors transfected cloned genes for mouse or human MEP into mouse MIH 3T3 cells with an expression vector for the dominant, selectable human multidrug resistance (MDR1) gene. The cotransfected MEP sequences were efficiently coamplified and transcribed during stepwise selection for multidrug resistance in colchicine. The transfected NIH 3T3 cell lines containing amplified MEP sequences synthesized as much MEP as did Kirsten sarcoma virus-transformed NIH 3T3 cells. The MEP synthesized by cells transfected with the cloned mouse and human MEP genes were also secreted. Elevated synthesis and secretion of MEP by NIH 3T3 cells did not change the nontransformed phenotype of these cells.

  6. Whatever Happened to "Talk and Chalk".

    ERIC Educational Resources Information Center

    Aston, Mike

    1982-01-01

    The Microelectronics Education Programme (MEP) has focused its attentions on the classroom teacher as the agent of educational change by providing computer based learning coordinators to implement teacher training programs to support all aspects of the school curriculum. Discusses computer training before MEP, with MEP involvement, and after…

  7. Bacterial population succession and adaptation affected by insecticide application and soil spraying history

    PubMed Central

    Itoh, Hideomi; Navarro, Ronald; Takeshita, Kazutaka; Tago, Kanako; Hayatsu, Masahito; Hori, Tomoyuki; Kikuchi, Yoshitomo

    2014-01-01

    Although microbial communities have varying degrees of exposure to environmental stresses such as chemical pollution, little is known on how these communities respond to environmental disturbances and how past disturbance history affects these community-level responses. To comprehensively understand the effect of organophosphorus insecticide application on microbiota in soils with or without insecticide-spraying history, we investigated the microbial succession in response to the addition of fenitrothion [O,O-dimethyl O-(3-methyl-p-nitrophenyl) phosphorothioate, abbreviated as MEP] by culture-dependent experiments and deep sequencing of 16S rRNA genes. Despite similar microbial composition at the initial stage, microbial response to MEP application was remarkably different between soils with and without MEP-spraying history. MEP-degrading microbes more rapidly increased in the soils with MEP-spraying history, suggesting that MEP-degrading bacteria might already exist at a certain level and could quickly respond to MEP re-treatment in the soil. Culture-dependent and -independent evaluations revealed that MEP-degrading Burkholderia bacteria are predominant in soils after MEP application, limited members of which might play a pivotal role in MEP-degradation in soils. Notably, deep sequencing also revealed that some methylotrophs dramatically increased after MEP application, strongly suggesting that these bacteria play a role in the consumption and removal of methanol, a harmful derivative from MEP-degradation, for better growth of MEP-degrading bacteria. This comprehensive study demonstrated the succession and adaptation processes of microbial communities under MEP application, which were critically affected by past experience of insecticide-spraying. PMID:25221549

  8. Comparison of false-negative/positive results of intraoperative evoked potential monitoring between no and partial neuromuscular blockade in patients receiving propofol/remifentanil-based anesthesia during cerebral aneurysm clipping surgery: A retrospective analysis of 685 patients.

    PubMed

    Kim, Sung-Hoon; Jin, Seok-Joon; Karm, Myong-Hwan; Moon, Young-Jin; Jeong, Hye-Won; Kim, Jae-Won; Ha, Seung-Il; Kim, Joung-Uk

    2016-08-01

    Although the elicited responses of motor evoked potential (MEP) monitoring are very sensitive to suppression by anesthetic agents and muscle relaxants, the use of neuromuscular blockade (NMB) during MEP monitoring is still controversial because of serious safety concerns and diagnostic accuracy. Here, we evaluated the incidence of unacceptable movement and compared false-negative MEP results between no and partial NMB during cerebral aneurysm clipping surgery. We reviewed patient medical records for demographic data, anesthesia regimen, neurophysiology event logs, MEP results, and clinical outcomes. Patients were divided into 2 groups according to the intraoperative use of NMB: no NMB group (n = 276) and partial NMB group (n = 409). We compared the diagnostic accuracy of MEP results to predict postoperative outcomes between both groups. Additionally, we evaluated unwanted patient movement during MEP monitoring in both groups. Of the 685 patients, 622 (90.8%) manifested no intraoperative changes in MEP and no postoperative motor deficits. Twenty patients showed postoperative neurologic deficits despite preserved intraoperative MEP. False-positive MEP results were 3.6% in the no NMB group and 3.9% in the partial NMB group (P = 1.00). False-negative MEP results were 1.1% in the no NMB group and 4.2% in the partial NMB group (P = 0.02). No spontaneous movement or spontaneous respiration was observed in either group. Propofol/remifentanil-based anesthesia without NMB decreases the stimulation intensity of MEPs, which may reduce the false-negative ratio of MEP monitoring during cerebral aneurysm surgery. Our anesthetic protocol enabled reliable intraoperative MEP recording and patient immobilization during cerebral aneurysm clipping surgery. PMID:27559984

  9. Comparison of false-negative/positive results of intraoperative evoked potential monitoring between no and partial neuromuscular blockade in patients receiving propofol/remifentanil-based anesthesia during cerebral aneurysm clipping surgery: A retrospective analysis of 685 patients.

    PubMed

    Kim, Sung-Hoon; Jin, Seok-Joon; Karm, Myong-Hwan; Moon, Young-Jin; Jeong, Hye-Won; Kim, Jae-Won; Ha, Seung-Il; Kim, Joung-Uk

    2016-08-01

    Although the elicited responses of motor evoked potential (MEP) monitoring are very sensitive to suppression by anesthetic agents and muscle relaxants, the use of neuromuscular blockade (NMB) during MEP monitoring is still controversial because of serious safety concerns and diagnostic accuracy. Here, we evaluated the incidence of unacceptable movement and compared false-negative MEP results between no and partial NMB during cerebral aneurysm clipping surgery. We reviewed patient medical records for demographic data, anesthesia regimen, neurophysiology event logs, MEP results, and clinical outcomes. Patients were divided into 2 groups according to the intraoperative use of NMB: no NMB group (n = 276) and partial NMB group (n = 409). We compared the diagnostic accuracy of MEP results to predict postoperative outcomes between both groups. Additionally, we evaluated unwanted patient movement during MEP monitoring in both groups. Of the 685 patients, 622 (90.8%) manifested no intraoperative changes in MEP and no postoperative motor deficits. Twenty patients showed postoperative neurologic deficits despite preserved intraoperative MEP. False-positive MEP results were 3.6% in the no NMB group and 3.9% in the partial NMB group (P = 1.00). False-negative MEP results were 1.1% in the no NMB group and 4.2% in the partial NMB group (P = 0.02). No spontaneous movement or spontaneous respiration was observed in either group. Propofol/remifentanil-based anesthesia without NMB decreases the stimulation intensity of MEPs, which may reduce the false-negative ratio of MEP monitoring during cerebral aneurysm surgery. Our anesthetic protocol enabled reliable intraoperative MEP recording and patient immobilization during cerebral aneurysm clipping surgery.

  10. Molecular characterization of a family of metalloendopeptidases from the intestinal brush border of Haemonchus contortus.

    PubMed

    Newlands, G F J; Skuce, P J; Nisbet, A J; Redmond, D L; Smith, S K; Pettit, D; Smith, W D

    2006-09-01

    Substantial protection against the economically important parasitic nematode Haemonchus contortus has been achieved by immunizing sheep with a glycoprotein fraction isolated from the intestinal membranes of the worm (H-gal-GP). Previous studies showed that one of the major components of H-gal-GP is a family of at least 4 zinc metalloendopeptidases, designated MEPs 1-4. This paper describes aspects of the molecular architecture of this protease family, including the proteomic analysis of the MEP fraction of the H-gal-GP complex. These enzymes belong to the M13 zinc metalloendopeptidase family (EC 3.4.24.11), also known as neutral endopeptidases or neprilysins. The sequences of MEPs 1 and 3 suggested a typical Type II integral membrane protein structure, whilst MEPs 2 and 4 had putative cleavable signal peptides, typical of secreted proteins. Proteomic analysis of H-gal-GP indicated that the extracellular domain of all 4 MEPs had been cleaved close to the transmembrane region/signal peptide with additional cleavage sites mid-way along the polypeptide. MEP3 was present as a homo-dimer in H-gal-GP, whereas MEP1 or MEP2 formed hetero-dimers with MEP4. It was found that expression of MEP3 was confined to developing 4th-stage larvae and to adult worms, the stages of Haemonchus which feed on blood. MEP-like activity was detected in the H-gal-GP complex over a broad pH range (5-9). Since all 4 MEPs must share a similar microenvironment in the complex, this suggests that each might have a different substrate specificity. PMID:16740178

  11. Screening and determination of drugs in human saliva utilizing microextraction by packed sorbent and liquid chromatography-tandem mass spectrometry.

    PubMed

    Abdel-Rehim, Abbi; Abdel-Rehim, Mohamed

    2013-09-01

    This study presents a new method for collecting and handling saliva samples using an automated analytical microsyringe and microextraction by packed syringe (MEPS). The screening and determination of lidocaine in human saliva samples utilizing MEPS and liquid chromatography-tandem mass spectrometry (LC-MS/MS) were carried out. An exact volume of saliva could be collected. The MEPS C8 -cartridge could be used for 50 extractions before it was discarded. The extraction recovery was about 60%. The pharmacokinetic curve of lidocaine in saliva using MEPS-LC-MS/MS is reported.

  12. Metabolic profiling of the methylerythritol phosphate pathway reveals the source of post-illumination isoprene burst from leaves.

    PubMed

    Li, Ziru; Sharkey, Thomas D

    2013-02-01

    The methylerythritol phosphate (MEP) pathway in plants produces the prenyl precursors for all plastidic isoprenoids, including carotenoids and quinones. The MEP pathway is also responsible for synthesis of approximately 600 Tg of isoprene per year, the largest non-methane hydrocarbon flux into the atmosphere. There have been few studies of the regulation of the MEP pathway in plants under physiological conditions. In this study, we combined gas exchange techniques and high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS-MS) and measured the profile of MEP pathway metabolites under different conditions. We report that in the MEP pathway, metabolites immediately preceding steps requiring reducing power were in high concentration. Inhibition of the MEP pathway by fosmidomycin caused deoxyxylulose phosphate accumulation in leaves as expected. Evidence is presented that accumulation of MEP pathway intermediates, primarily methylerythritol cyclodiphosphate, is responsible for the post-illumination isoprene burst phenomenon. Pools of intermediate metabolites stayed at approximately the same level 10 min after light was turned off, but declined eventually under prolonged darkness. In contrast, a strong inhibition of the second-to-last step of the MEP pathway caused suppression of isoprene emission in pure N(2). Our study suggests that reducing equivalents may be a key regulator of the MEP pathway and therefore isoprene emission from leaves. PMID:22831282

  13. 75 FR 72787 - Submission for OMB Review; Comment Request

    Federal Register 2010, 2011, 2012, 2013, 2014

    2010-11-26

    ... local and national levels, will provide information critical to monitoring and reporting on MEP... revenues, partners and affiliates, strategic plan, operating plans, and client success stories....

  14. Effects of olfactory and gustatory stimuli on neural excitability for swallowing.

    PubMed

    Abdul Wahab, Norsila; Jones, Richard D; Huckabee, Maggie-Lee

    2010-12-01

    This project evaluated the effects of olfactory and gustatory stimuli on the amplitude and latency of motor-evoked potentials (MEPs) from the submental muscles when evoked by transcranial magnetic stimulation (TMS). Sixteen healthy volunteers (8 males; age range 19-43) participated in the study. Lemon concentrate at 100% and diluted in water to 25% were presented separately as odor and tastant stimuli. Tap water was used as control. 15 trials of TMS-evoked MEPs triggered by volitional contraction of the submental muscles and volitional swallowing were measured at baseline, during control condition, during stimulus presentation, and immediately, 30-, 60-, and 90-min poststimulation for each of the four stimulus presentations. Experiments were repeated using the combined odor and tastant concentrations that most influenced the MEP independently. Differences in MEP amplitude measured during swallowing were seen at 30-, 60-, and 90-min poststimulation for simultaneous olfactory and gustatory stimulation as opposed to no differences seen at any point for stimuli presented separately. This study has shown that combined odor and tastant stimulation (i.e., flavor) can increase MEP amplitude during swallowing and that this enhancement of MEP can persist for at least 90min following stimulation. As increased MEP amplitude has been associated with improved swallowing performance, a follow-up study is underway to determine the biomechanical changes produced by altered MEPs to facilitate translation of these data to clinical dysphagia management. PMID:20849867

  15. Monitoring of Motor and Somatosensory Evoked Potentials During Spine Surgery: Intraoperative Changes and Postoperative Outcomes

    PubMed Central

    2016-01-01

    Objective To evaluate whether the combination of muscle motor evoked potentials (mMEPs) and somatosensory evoked potentials (SEPs) measured during spinal surgery can predict immediate and permanent postoperative motor deficits. Methods mMEP and SEP was monitored in patients undergoing spinal surgery between November 2012 and July 2014. mMEPs were elicited by a train of transcranial electrical stimulation over the motor cortex and recorded from the upper/lower limbs. SEPs were recorded by stimulating the tibial and median nerves. Results Combined mMEP/SEP recording was successfully achieved in 190 operations. In 117 of these, mMEPs and SEPs were stable and 73 showed significant changes. In 20 cases, motor deficits in the first 48 postoperative hours were observed and 6 patients manifested permanent neurological deficits. The two potentials were monitored in a number of spinal surgeries. For surgery on spinal deformities, the sensitivity and specificity of combined mMEP/SEP monitoring were 100% and 92.4%, respectively. In the case of spinal cord tumor surgeries, sensitivity was only 50% but SEP changes were observed preceding permanent motor deficits in some cases. Conclusion Intraoperative monitoring is a useful tool in spinal surgery. For spinal deformity surgery, combined mMEP/SEP monitoring showed high sensitivity and specificity; in spinal tumor surgery, only SEP changes predicted permanent motor deficits. Therefore, mMEP, SEP, and joint monitoring may all be appropriate and beneficial for the intraoperative monitoring of spinal surgery. PMID:27446784

  16. Identification of Stimulated Sites Using Artificial Neural Networks Based on Transcranial Magnetic Stimulation-Elicited Motor Evoked Potentials and Finger Forces

    NASA Astrophysics Data System (ADS)

    Fukuda, Hiroshi; Odagaki, Masato; Hiwaki, Osamu

    Motor evoked potentials (MEPs) elicited by transcranial magnetic stimulation (TMS) over the primary motor cortex (M1) vary in their amplitude from trial to trial. To investigate the functions of motor cortex by TMS, it is necessary to confirm the causal relationship between stimulated sites and variable MEPs. We created artificial neural networks to classify sets of variable MEP signals and finger forces into the corresponding stimulated sites. We conducted TMS at three different positions over M1 and measured MEPs of hand and forearm muscles and forces of the index finger in four subjects. We estimated the sites within motor cortex stimulated by TMS based on cortical columnar structure and nerve excitation properties. Finally, we tried to classify the various MEPs and finger forces into three groups using artificial neural networks. MEPs and finger forces varied from trial to trial, even if the stimulating coil was fixed on the subject's head. Our proposed neural network was able to identify the MEPs and finger forces with the corresponding stimulated sites in M1. We proposed the artificial neural networks to confirm the TMS-stimulated sites using various MEPs and evoked finger forces.

  17. Botulinum Toxin Type A Injections in the Psoas Muscle of Children with Cerebral Palsy: Muscle Atrophy after Motor End Plate-Targeted Injections

    ERIC Educational Resources Information Center

    Van Campenhout, Anja; Verhaegen, Ann; Pans, Steven; Molenaers, Guy

    2013-01-01

    MEP targeting during BoNT-A injections has been demonstrated to improve outcome. Two injection techniques of the psoas muscle--proximal MEP targeting versus a widely used more distal injection technique--are compared using muscle volume assessment by digital MRI segmentation as outcome measure. Method: 7 spastic diplegic children received…

  18. Effect of Training Frequency on Maximum Expiratory Pressure

    ERIC Educational Resources Information Center

    Anand, Supraja; El-Bashiti, Nour; Sapienza, Christine

    2012-01-01

    Purpose: To determine the effects of expiratory muscle strength training (EMST) frequency on maximum expiratory pressure (MEP). Method: We assigned 12 healthy participants to 2 groups of training frequency (3 days per week and 5 days per week). They completed a 4-week training program on an EMST trainer (Aspire Products, LLC). MEP was the primary…

  19. Microelectronics in Education--A Changing Future.

    ERIC Educational Resources Information Center

    Humphries, Chris

    1985-01-01

    Describes major program activities of Microelectronics Education Programme (MEP) for England, Wales, and Northern Ireland, which is designed to help schools prepare children for society with microelectronics. The core elements and potential consequences of the Microelectronics Support Unit, a follow-up to MEP which will conclude activities in…

  20. Schools' Computing Policy as State-Directed Innovation.

    ERIC Educational Resources Information Center

    Boyd-Barrett, Oliver

    1990-01-01

    Analyzes government's role in promoting computer use in the United Kingdom educational system from 1980 to 1990. Evaluates the Microelectronics Education Programme (MEP) in initiating policy. Concludes MEP's contribution was ultimately positive. Considers problems of top-down innovation, teacher attitudes and training, and hardware and software…

  1. Information Retrieval in Education - A Worthy Tradition?

    ERIC Educational Resources Information Center

    Irving, Ann

    1982-01-01

    Because information handling applies to all forms of learning, one Microelectronics Education Programme (MEP) INSET strategy is devoted to a discussion of the generation, storage, retrieval, communication, and use of information in all subject areas. Discusses the nature of information; MEP and the information domain; information gathering, use,…

  2. Today's Development for Tomorrow's World - Educating for the Information Society.

    ERIC Educational Resources Information Center

    Longworth, Norman

    1982-01-01

    Discusses new demands in education resulting from rapid advances in information technology and the role of the Microelectronics Education Programme (MEP) in meeting this need. The MEP-funded Consortium Information Project is meeting specific information technology challenges by offering a variety of skills and backgrounds to 9- to 13-year-old…

  3. INSET - A Way to Meet an Urgent Requirement in Teacher Training.

    ERIC Educational Resources Information Center

    Bevis, Graham

    1982-01-01

    The objective of the Microelectronics Education Programme (MEP) Electronics and Control Technology is to enable teachers (under the INSET scheme) to develop the necessary expertise to teach the current electronics component of courses which include this subject. Described are MEP-INSET guidelines, which include six-hour course modules, equipment,…

  4. Prevalence and Costs of Five Chronic Conditions in Children

    ERIC Educational Resources Information Center

    Miller, Gabrielle F.; Coffield, Edward; Leroy, Zanie; Wallin, Robin

    2016-01-01

    The objective is to examine the prevalence and health-care costs associated with asthma, epilepsy, hypertension, food allergies, and diabetes in children aged 0-18 years. Prevalence was calculated using 2005-2012 Medical Expenditure Panel Survey (MEPS) data, a population-based, nationally representative sample. Using MEPS, two-part models…

  5. Minority Engineering Programs: A Case for Institutional Support.

    ERIC Educational Resources Information Center

    Morrison, Catherine; Williams, Lea E.

    1993-01-01

    Minority engineering programs (MEPs) were established in the late 1970s to recruit minority students to engineering education and reduce their dropout rate. MEPs at 20 engineering schools were examined in relation to institutional environment; pre-enrollment activities; and services that encourage community building, provide academic support, and…

  6. Improving the Retention and Graduation of Minorities in Engineering. Handbook.

    ERIC Educational Resources Information Center

    Landis, Raymond B., Ed.

    Procedures for operating undergraduate minority engineering programs (MEPs) are provided in this handbook. The findings and suggestions presented are based on the experiences of MEP directors who have managed support programs, which focus on improving the retention of minority students within engineering. The titles and authors of the handbook's…

  7. Improving the Academic Achievement of the Disadvantaged; Migrant Education Program. Final Rule. Federal Register, Department of Education, 34 CFR Part 200

    ERIC Educational Resources Information Center

    National Archives and Records Administration, 2008

    2008-01-01

    The Secretary amends the regulations governing the Migrant Education Program (MEP) administered under Part C of Title I of the Elementary and Secondary Education Act of 1965, as amended (ESEA). These final regulations adjust the base amounts of the MEP Basic State Formula grant allocations for fiscal year (FY) 2006 and subsequent years (as well…

  8. 75 FR 25221 - Notice Extending the Deadline Date for Transmittal of Applications and the Deadline Date for...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2010-05-07

    ..., we published in the Federal Register (75 FR 16446) a notice inviting applications for new awards for... Intergovernmental Review for the Migrant Education Program (MEP) Consortium Incentive Grants Program for Fiscal Year... Intergovernmental Review for the Migrant Education Program (MEP) Consortium Incentive Grants Program for Fiscal...

  9. State Auditor's Review of the Vermont Migrant Education Program, July 1, 1997 to December 31, 1999.

    ERIC Educational Resources Information Center

    Vermont State Office of the State Auditor, Montpelier.

    Since 1992, the Vermont Department of Education has granted funds to the University of Vermont (UVM) to operate the Migrant Education Program (MEP). This review by the State Auditor found that prior to fiscal year 1999, the Department did not make a concerted effort to oversee the MEP and, as a result, several critical programs components were not…

  10. Multiple Exit Points in Vocational Education for Students with Disabilities.

    ERIC Educational Resources Information Center

    Florida State Dept. of Education, Tallahassee. Bureau of Education for Exceptional Students.

    This technical assistance paper is designed as a general guide to assist school districts in developing and implementing multiple-exit vocational programs for students with disabilities. The following topics are covered in Part 1: the concept of multiple-exit points (MEPs) and its background, purposes and benefits of MEPs, goals and guiding…

  11. A Whole-Cell Phenotypic Screening Platform for Identifying Methylerythritol Phosphate Pathway-Selective Inhibitors as Novel Antibacterial Agents

    PubMed Central

    Johnson, L. Jeffrey

    2012-01-01

    Isoprenoid biosynthesis is essential for survival of all living organisms. More than 50,000 unique isoprenoids occur naturally, with each constructed from two simple five-carbon precursors: isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP). Two pathways for the biosynthesis of IPP and DMAPP are found in nature. Humans exclusively use the mevalonate (MVA) pathway, while most bacteria, including all Gram-negative and many Gram-positive species, use the unrelated methylerythritol phosphate (MEP) pathway. Here we report the development of a novel, whole-cell phenotypic screening platform to identify compounds that selectively inhibit the MEP pathway. Strains of Salmonella enterica serovar Typhimurium were engineered to have separately inducible MEP (native) and MVA (nonnative) pathways. These strains, RMC26 and CT31-7d, were then used to differentiate MVA pathway- and MEP pathway-specific perturbation. Compounds that inhibit MEP pathway-dependent bacterial growth but leave MVA-dependent growth unaffected represent MEP pathway-selective antibacterials. This screening platform offers three significant results. First, the compound is antibacterial and is therefore cell permeant, enabling access to the intracellular target. Second, the compound inhibits one or more MEP pathway enzymes. Third, the MVA pathway is unaffected, suggesting selectivity for targeting the bacterial versus host pathway. The cell lines also display increased sensitivity to two reported MEP pathway-specific inhibitors, further biasing the platform toward inhibitors selective for the MEP pathway. We demonstrate development of a robust, high-throughput screening platform that combines phenotypic and target-based screening that can identify MEP pathway-selective antibacterials simply by monitoring optical density as the readout for cell growth/inhibition. PMID:22777049

  12. Basic study on the influence of inhibition induced by the magnetic stimulation on the peripheral nerve

    NASA Astrophysics Data System (ADS)

    Sato, Aya; Torii, Tetsuya; Iwahashi, Masakuni; Iramina, Keiji

    2015-05-01

    The purpose of this study is to analyze the inhibition mechanism of magnetic stimulation on motor function. A magnetic stimulator with a flat figure-eight coil was used to stimulate the peripheral nerve of the antebrachium. The intensity of magnetic stimulation was 0.8 T, and the stimulation frequency was 1 Hz. The amplitudes of the motor-evoked potentials (MEPs) at the abductor pollicis brevis muscle and first dorsal interosseous muscle were used to evaluate the effects of magnetic stimulation. The effects of magnetic stimulation were evaluated by analyzing the MEP amplitude before and after magnetic stimulation to the primary motor cortex. The results showed that MEP amplitude after magnetic stimulation compared with before magnetic stimulation decreased. Because there were individual differences in MEP amplitude induced by magnetic stimulation, the MEP amplitude after stimulation was normalized by the amplitude of each participant before stimulation. The MEP amplitude after stimulation decreased by approximately 58% (p < 0.01) on average compared with before stimulation. Previous studies suggested that magnetic stimulation to the primary motor cortex induced an increase or a decrease in MEP amplitude. Furthermore, previous studies have shown that the alteration in MEP amplitude was induced by cortical excitability based on magnetic stimulation. The results of this study showed that MEP amplitude decreased following magnetic stimulation to the peripheral nerve. We suggest that the decrease in MEP amplitude found in this study was obtained via the feedback from a peripheral nerve through an afferent nerve to the brain. This study suggests that peripheral excitement by magnetic stimulation of the peripheral nerve may control the central nervous system via afferent feedback.

  13. Usefulness of Transcranial Magnetic Stimulation to Assess Motor Function in Patients With Parkinsonism

    PubMed Central

    Park, Jaechan; Cho, Jin Whan; Youn, Jinyoung; Kim, Yun Kwan; Kim, Sun Woong; Kim, Yun-Hee

    2016-01-01

    Objective To investigate the clinical significance of upper and lower extremity transcranial magnetic stimulation (TMS)-induced motor evoked potentials (MEPs) in patients with parkinsonism. Methods Twenty patients (14 men, 6 women; mean age 70.5±9.1 years) suffering from parkinsonism were included in this study. All participants underwent single-pulse TMS session to assess the corticospinal excitability of the upper and lower extremity motor cortex. The resting motor threshold (RMT) was defined as the lowest stimulus intensity able to evoke MEPs of an at least 50 µV peak-to-peak amplitude in 5 of 10 consecutive trials. Five sweeps of MEPs at 120% of the RMT were performed, and the mean amplitude and latency of the MEPs were calculated. Patients were also assessed using the Unified Parkinson's Disease Rating Scale part III (UPDRS-III) and the 5-meter Timed Up and Go (5m-TUG) test. Results There was a significant positive correlation between the RMTs of MEPs in the upper and lower extremities (r=0.612, p=0.004) and between the amplitude of MEPs in the upper and lower extremities (r=0.579, p=0.007). The RMT of upper extremity MEPs showed a significant negative relationship with the UPDRS-III score (r=–0.516, p=0.020). In addition, RMTs of lower extremity MEPs exhibited a negative relationship with the UPDRS-III score, but the association was not statistically significant (r=–406, p=0.075). Conclusion These results indicated that the RMT of MEPs reflect the severity of motor dysfunction in patients with parkinsonism. MEP is a potential quantitative, electrodiagnostic method to assess motor function in patients with parkinsonism. PMID:26949673

  14. TMS-induced motor evoked potentials in Wilson's disease: a systematic literature review.

    PubMed

    Bembenek, Jan P; Kurczych, Katarzyna; Członkowska, Anna

    2015-04-01

    Wilson's disease (WD) is a metabolic brain disease resulting from improper copper metabolism. Although pyramidal symptoms are rarely observed, subclinical injury is highly possible as copper accumulates in all brain structures. The usefulness of motor evoked potentials (MEPs) in pyramidal tracts damage evaluation still appears to be somehow equivocal. We searched for original papers assessing the value of transcranial magnetic stimulation elicited MEPs with respect to motor function of upper and lower extremity in WD. We searched PubMed for original papers evaluating use of MEPs in WD using key words: "motor evoked potentials Wilson's disease" and "transcranial magnetic stimulation Wilson's disease." We found six articles using the above key words. One additional article and one case report were found while viewing the references lists. Therefore, we included eight studies. Number of patients in studies was low and their clinical characteristic was variable. There were also differences in methodology. Abnormal MEPs were confirmed in 20-70% of study participants. MEPs were not recorded in 7.6-66.7% of patients. Four studies reported significantly increased cortical excitability (up to 70% of patients). Prolonged central motor conduction time was observed in four studies (30-100% of patients). One study reported absent or prolonged central motor latency in 66.7% of patients. Although MEPs may be abnormal in WD, this has not been thoroughly assessed. Hence, further studies are indispensable to evaluate MEPs' usefulness in assessing pyramidal tract damage in WD.

  15. Microextraction in packed sorbent for analysis of antidepressants in human plasma by liquid chromatography and spectrophotometric detection.

    PubMed

    Chaves, Andréa R; Leandro, Fernanda Z; Carris, Juciene A; Queiroz, Maria Eugênia C

    2010-08-01

    A sensitive and reproducible method by microextraction packed sorbent and liquid chromatography with UV detection (MEPS/LC-UV) is described for the determination of new generation antidepressants (sertraline, mirtazapine, fluoxetine, citalopram and paroxetine) in human plasma samples. The MEPS variables, such as sample volume, pH, number of extraction cycles (draw-eject), and desorption conditions (solvent and solvent volume of elution) influenced the MEPS/LC efficiency significantly. Important factors in the optimization of MEPS efficiency, as well as washing steps and carryover effect are discussed. The analyses were carried out using small sample volumes (400 microL), and in a short time period (3 min for the entire sample preparation step). The MEPS/LC-UV method was shown to be linear at concentrations ranging from the limit of quantification (LOQ) to 1000 ng mL(-1). The LOQ values ranged from 10 to 25 ng mL(-1). The inter-day precision of the method presented coefficient of the variation ranging from 1.3% to 8.7%. On the basis of analytical validation, it is shown that the MEPS/LC-UV methodology is adequate for antidepressant analysis, from therapeutic to toxic levels. In order to evaluate the proposed method for clinical use, the MEPS/LC-UV method was applied to analysis of plasma samples from elderly depressed patients. PMID:20630812

  16. Transcranial Motor Evoked Potentials of Lower Limbs Can Prognosticate Ambulation in Hemiplegic Stroke Patients

    PubMed Central

    2016-01-01

    Objective To examine the association between motor evoked potentials (MEPs) in lower limbs and ambulatory outcomes of hemiplegic stroke patients. Methods Medical records of hemiplegic patients with the first ever stroke who received inpatient rehabilitation from January 2013 to May 2014 were reviewed. Patient who had diabetes, quadriplegia, bilateral lesion, brainstem lesion, severe musculoskeletal problem, and old age over 80 years were excluded. MEPs in lower limbs were measured when they were transferred to the Department of Rehabilitation Medicine. Subjects were categorized into three groups (normal, abnormal, and absent response) according to MEPs findings. Berg Balance Scale (BBS) and Functional Ambulation Category (FAC) at initial and discharge were compared among the three groups by one-way analysis of variance (ANOVA). Correlation was determined using a linear regression model. Results Fifty-eight hemiplegic patients were included. BBS and FAC at discharge were significantly (ANOVA, p<0.001) different according to MEPs findings. In linear regression model of BBS and FAC using stepwise selection, patients' age (p<0.01), BBS at admission (p<0.01), and MEPs (p<0.01) remained significant covariates. In regression assumption model of BBS and FAC at admission, MEPs and gender were significant covariates. Conclusion Initial MEPs of lower limbs can prognosticate the ambulatory outcomes of hemiplegic patients. PMID:27446774

  17. Mechanisms of central motor reorganization in pediatric hemiplegic patients.

    PubMed

    Maegaki, Y; Maeoka, Y; Ishii, S; Shiota, M; Takeuchi, A; Yoshino, K; Takeshita, K

    1997-06-01

    Twenty hemiplegic patients were studied with transcranial magnetic stimulation (TMS). Motor evoked potentials (MEPs) of the biceps brachii (BB) and the abductor pollicis brevis muscles (APB) were recorded on both sides simultaneously. TMS was carried out with an 8-shaped coil over different scalp positions in the intact hemisphere. Bilateral MEPs of BB were elicited in patients with later childhood lesions as well as early lesion, but those of APB were only elicited in the latter (up to 2 years). In patients with prenatal or birth lesion on BB and in all patients on APB, cortical maps of MEP amplitude of paretic and non-paretic sides showed similar distributions. There were no remarkable differences in mean latency between both sides, and correlation coefficients of MEP amplitude between both sides were high in these patients. In patients with postnatal lesion on BB, MEP maps of both sides showed different distributions, ipsilateral MEP latencies were delayed and correlation coefficients were low. We suspect that ipsilateral MEPs after early lesion derive from axonal sprouting both in the proximal and the distal muscles. After postnatal lesion, other mechanisms of ipsilateral motor projection take place in the proximal muscles, but not in the distal ones. PMID:9266555

  18. Microextraction by packed sorbent: an emerging, selective and high-throughput extraction technique in bioanalysis.

    PubMed

    Pereira, Jorge; Câmara, José S; Colmsjö, Anders; Abdel-Rehim, Mohamed

    2014-06-01

    Sample preparation is an important analytical step regarding the isolation and concentration of desired components from complex matrices and greatly influences their reliable and accurate analysis and data quality. It is the most labor-intensive and error-prone process in analytical methodology and, therefore, may influence the analytical performance of the target analytes quantification. Many conventional sample preparation methods are relatively complicated, involving time-consuming procedures and requiring large volumes of organic solvents. Recent trends in sample preparation include miniaturization, automation, high-throughput performance, on-line coupling with analytical instruments and low-cost operation through extremely low volume or no solvent consumption. Micro-extraction techniques, such as micro-extraction by packed sorbent (MEPS), have these advantages over the traditional techniques. This paper gives an overview of MEPS technique, including the role of sample preparation in bioanalysis, the MEPS description namely MEPS formats (on- and off-line), sorbents, experimental and protocols, factors that affect the MEPS performance, and the major advantages and limitations of MEPS compared with other sample preparation techniques. We also summarize MEPS recent applications in bioanalysis.

  19. Influence of sodium hypochlorite treatment of electropolished and magnetoelectropolished nitinol surfaces on adhesion and proliferation of MC3T3 pre-osteoblast cells.

    PubMed

    Rokicki, Ryszard; Haider, Waseem; Hryniewicz, Tadeusz

    2012-09-01

    The influence of 6 % sodium hypochlorite (NaClO) treatment on adhesion and proliferation of MC3T3 pre-osteoblast cells seeded on electropolished (EP) and magnetoelectropolished (MEP) nitinol surfaces were investigated. The chemistry, topography, roughness, surface energy, wettability of EP and MEP nitinol surfaces before and after NaClO treatment were studied with X-ray photoelectron spectroscopy (XPS), profilometry, and contact angle meter. In vitro interaction of osteoblast cell and NaClO treated EP and MEP nitinol surfaces were assessed after 3 days of incubation by scanning electron microscopy. The XPS analysis shows that NaClO treatment increases oxygen content especially in subsurface oxide layer of EP and MEP nitinol. The changes of both basic components of nitinol, namely nickel and titanium in oxide layer, were negligible. The NaClO treatment did not influence physico-morphological surface properties of EP and MEP nitinol to a big extent. The osteoblast cells show remarkable adherence and proliferation improvement on NaClO treated EP and MEP nitinol surfaces. After 3 days of incubation they show almost total confluence on both NaClO treated surfaces. The present study shows that NaClO treatment of EP and MEP nitinol surfaces alters oxide layer by enriching it in oxygen and by this improves bone cell-nitinol interaction.

  20. Accuracy of Medicare expenditures in the medical expenditure panel survey.

    PubMed

    Zuvekas, Samuel H; Olin, Gary L

    2009-01-01

    This paper examines underreporting and underrepresentation of high expenditure cases in the Medical Expenditure Panel Survey (MEPS) and their implications for analyses. Our data come from a sample of Medicare beneficiaries in the MEPS who were matched to their Medicare claims and enrollment files, with supplemental data from the Medicare Current Beneficiary Survey (MCBS). Underreporting of expenditures affected all groups of Medicare beneficiaries in the matched sample, but uniformly so that behavioral analyses were largely unaffected. Straightforward adjustments to the MEPS expenditure estimates could align them with aggregate sources, such as the National Health Expenditure Accounts, while preserving underlying relationships between expenditures and key correlates.

  1. A family-oriented therapy program for youths with substance abuse: long-term outcomes related to relapse and academic or social status

    PubMed Central

    Wang, Liang-Jen; Lu, Shing-Fang; Chong, Mian-Yoon; Chou, Wen-Jiun; Hsieh, Yu-Lian; Tsai, Tung-ning; Chen, Ching; Lee, Yi-Hsuan

    2016-01-01

    Objective The abuse of illegal substances by youths in Taiwan has become a major public health issue. This study explores the outcomes (relapse rate and academic or social status) of a family-oriented therapy program conducted for substance-using youths who were referred by a judge to participate in it. Methods The present study includes 121 participants categorized into three groups: 36 youths underwent a weekly ten-session outpatient motivational enhancement psychotherapy (MEP) group program; 41 youths participated in a program that combined the aforementioned MEP program with an additional weekly ten-session parenting skill training (PST) program for their guardians (MEP + PST group); and 44 adolescents who received standard supervision by the court served as the control group. All participants were followed-up for a maximum of 2 years. Results Of the 121 participants (mean age: 16.1±1.1 years), 33.1% relapsed into substance use during the follow-up period. The probability of relapse did not differ significantly between the MEP group (36.1%) and the control group (40.9%), but the youths in the MEP + PST group (22.0%) were at a lower risk of relapse than the control group participants (adjusted hazard ratio =0.48, 95% confidence interval [CI] =0.21–1.09). By the end of the study follow-up period, participants in both the MEP group and the MEP + PST group were more likely to be attending school (MEP group: adjusted odds ratio [aOR] =6.61, 95% CI =1.60–27.35; MEP + PST group: aOR =8.57, 95% CI =1.94–37.82) or employed (MEP group: aOR =7.75, 95% CI =1.95–30.75; MEP + PST group: aOR =7.27, 95% CI =1.76–29.97), when compared to the control group. Conclusion This study revealed that a family-oriented treatment approach may be a more effective option for preventing youths’ relapsing into substance abuse. In comparison to individuals who received standard supervision by the court, those who received MEP experienced a better school attendance or social

  2. 78 FR 41185 - Denial of Exemption Applications; Epilepsy and Seizure Disorders

    Federal Register 2010, 2011, 2012, 2013, 2014

    2013-07-09

    ... driving; by metabolic derangement not likely to recur; and by alcohol or illicit drug withdrawal. Examples... complications from brain surgery with significant brain hemorrhage; brain tumor; or stroke. The MEP...

  3. Master environmental plan for Fort Devens, Massachusetts

    SciTech Connect

    Biang, C.A.; Peters, R.W.; Pearl, R.H.; Tsai, S.Y. . Energy Systems Div.)

    1991-11-01

    Argonne National Laboratory has prepared a master environmental plan (MEP) for Fort Devens, Massachusetts, for the US Army Toxic and Hazardous Materials Agency. The MEP is an assessment based on environmental laws and regulations of both the federal government and the Commonwealth of Massachusetts. The MEP assess the physical and environmental status of 58 potential hazardous waste sites, including 54 study areas (SAs) that pose a potential for releasing contamination into the environment and 4 areas of concern (AOCs) that are known to have substantial contamination. For each SA or AOC, this MEP describes the known history and environment, identifies additional data needs, and proposes possible response actions. Most recommended response actions consist of environmental sampling and monitoring and other characterization studies. 74 refs., 63 figs., 50 tabs.

  4. Achilles tendon vibration-induced changes in plantar flexor corticospinal excitability.

    PubMed

    Lapole, Thomas; Temesi, John; Gimenez, Philippe; Arnal, Pierrick J; Millet, Guillaume Y; Petitjean, Michel

    2015-02-01

    Daily Achilles tendon vibration has been shown to increase muscle force, likely via corticospinal neural adaptations. The aim of the present study was to determine the extent by which corticospinal excitability is influenced during direct Achilles tendon vibration. Motor-evoked potentials (MEPs) were elicited in the soleus (SOL), gastrocnemius medialis (GM) and tibialis anterior (TA) by transcranial magnetic stimulation of the motor cortical area of the leg with and without Achilles tendon vibration at various frequencies (50, 80 and 110 Hz). Contralateral homologues were also investigated. SOL and GM MEP amplitude significantly increased by 226 ± 188 and 66 ± 39%, respectively, during Achilles tendon vibration, without any difference between the tested frequencies. No MEP changes were reported for TA or contralateral homologues. Increased SOL and GM MEP amplitude suggests increased vibration-induced corticospinal excitability independent of vibration frequency.

  5. 75 FR 19941 - Manufacturing Extension Partnership Advisory Board

    Federal Register 2010, 2011, 2012, 2013, 2014

    2010-04-16

    ... National Institute of Standards and Technology Manufacturing Extension Partnership Advisory Board AGENCY... announces that the Manufacturing Extension Partnership (MEP) Advisory Board, National Institute of Standards..., 2010. FOR FURTHER INFORMATION CONTACT: Karen Lellock, Manufacturing Extension Partnership,...

  6. 77 FR 20790 - Manufacturing Extension Partnership Advisory Board

    Federal Register 2010, 2011, 2012, 2013, 2014

    2012-04-06

    ... National Institute of Standards and Technology Manufacturing Extension Partnership Advisory Board AGENCY... announces that the Manufacturing Extension Partnership (MEP) Advisory Board, National Institute of Standards... INFORMATION section below. FOR FURTHER INFORMATION CONTACT: Karen Lellock, Manufacturing Extension...

  7. 76 FR 22674 - Manufacturing Extension Partnership Advisory Board

    Federal Register 2010, 2011, 2012, 2013, 2014

    2011-04-22

    ... National Institute of Standards and Technology Manufacturing Extension Partnership Advisory Board AGENCY... announces that the Manufacturing Extension Partnership (MEP) Advisory Board, National Institute of Standards... than May 2, 2011. FOR FURTHER INFORMATION CONTACT: Karen Lellock, Manufacturing Extension...

  8. 78 FR 41985 - Qualification of Drivers; Exemption Applications; Epilepsy and Seizure Disorders

    Federal Register 2010, 2011, 2012, 2013, 2014

    2013-07-12

    ...). You may review DOT's complete Privacy Act Statement in the Federal Register (73 FR 3316, January 17... field of neurology (the MEP) on May 14-15, 2007, to review 49 CFR 391.41(b)(8) and the advisory...

  9. 34 CFR 200.82 - Use of program funds for unique program function costs.

    Code of Federal Regulations, 2011 CFR

    2011-07-01

    ... recruitment of eligible migratory children; (b) Interstate and intrastate coordination of the State MEP and... instructional and support staff; (g) Establishment and implementation of a State parent advisory council; and...

  10. 34 CFR 200.82 - Use of program funds for unique program function costs.

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ... recruitment of eligible migratory children; (b) Interstate and intrastate coordination of the State MEP and... instructional and support staff; (g) Establishment and implementation of a State parent advisory council; and...

  11. Transcranial magnetic stimulation in amyotrophic and primary lateral sclerosis.

    PubMed

    Cruz Martínez, A; Trejo, J M

    1999-01-01

    Conduction of the central motor pathways after transcranial magnetic stimulation (TMS) was investigated in 7 patients with amyotrophic lateral sclerosis (ALS) and 1 case with primary lateral sclerosis (PLS). Threshold intensity, central motor conduction time (CMCT) and amplitude of the motor evoked potentials (MEPs) were evaluated. Threshold was abnormal in 85% of tested limbs, and CMCT prolonged and amplitude of the MEPs attenuated in 28.5% of patients with ALS. Abnormal CMCT was asymmetric and related to clinical score. MEPs were absent in lower limbs in PLS, with prolonged or attenuated amplitude of the MEPs in upper limbs. EMG showed widespread signs of lower motor neuron involvement in ALS, but not in PLS. Cranial MRI showed frontoparietal cortical atrophy, more marked in pre-central gyrus, and SPECT there was lower tracer uptake in the perirolandic area in the PLS patient. EMG examination, TMS, cranial MRI and SPECT can help in the diagnosis of PLS.

  12. 76 FR 81944 - Agency Information Collection Activities: Proposed Collection; Comment Request

    Federal Register 2010, 2011, 2012, 2013, 2014

    2011-12-29

    ... that he/she complete the SAQ and give it to the interviewer before she leaves the household after completing the MEPS interview. If the cancer SAQ is not collected before the interviewer leaves the...

  13. 32 CFR 728.73 - Applicants for enlistment or reenlistment in the Armed Forces, and applicants for enlistment in...

    Code of Federal Regulations, 2012 CFR

    2012-07-01

    ... Defense Department of Defense (Continued) DEPARTMENT OF THE NAVY PERSONNEL MEDICAL AND DENTAL CARE FOR... at Navy and Marine Corps facilities or MEPS may be furnished emergency medical and dental...

  14. 32 CFR 728.74 - Applicants for appointment in the regular Navy or Marine Corps and reserve components, including...

    Code of Federal Regulations, 2013 CFR

    2013-07-01

    ... NAVY PERSONNEL MEDICAL AND DENTAL CARE FOR ELIGIBLE PERSONS AT NAVY MEDICAL DEPARTMENT FACILITIES Other... and Marine Corps facilities or MEPS may be furnished emergency medical and dental care,...

  15. 32 CFR 728.73 - Applicants for enlistment or reenlistment in the Armed Forces, and applicants for enlistment in...

    Code of Federal Regulations, 2011 CFR

    2011-07-01

    ... Defense Department of Defense (Continued) DEPARTMENT OF THE NAVY PERSONNEL MEDICAL AND DENTAL CARE FOR... at Navy and Marine Corps facilities or MEPS may be furnished emergency medical and dental...

  16. 32 CFR 728.74 - Applicants for appointment in the regular Navy or Marine Corps and reserve components, including...

    Code of Federal Regulations, 2011 CFR

    2011-07-01

    ... NAVY PERSONNEL MEDICAL AND DENTAL CARE FOR ELIGIBLE PERSONS AT NAVY MEDICAL DEPARTMENT FACILITIES Other... and Marine Corps facilities or MEPS may be furnished emergency medical and dental care,...

  17. 32 CFR 728.74 - Applicants for appointment in the regular Navy or Marine Corps and reserve components, including...

    Code of Federal Regulations, 2012 CFR

    2012-07-01

    ... NAVY PERSONNEL MEDICAL AND DENTAL CARE FOR ELIGIBLE PERSONS AT NAVY MEDICAL DEPARTMENT FACILITIES Other... and Marine Corps facilities or MEPS may be furnished emergency medical and dental care,...

  18. 32 CFR 728.73 - Applicants for enlistment or reenlistment in the Armed Forces, and applicants for enlistment in...

    Code of Federal Regulations, 2013 CFR

    2013-07-01

    ... Defense Department of Defense (Continued) DEPARTMENT OF THE NAVY PERSONNEL MEDICAL AND DENTAL CARE FOR... at Navy and Marine Corps facilities or MEPS may be furnished emergency medical and dental...

  19. 75 FR 50749 - Manufacturing Extension Partnership Advisory Board

    Federal Register 2010, 2011, 2012, 2013, 2014

    2010-08-17

    ... provide input and advice on MEP's programs, plans, and policies. For this meeting, discussions will focus... evaluation metrics and (3) a discussion of how to support small manufacturers' commercialization of...

  20. Use and validity of principles of extremum of entropy production in the study of complex systems

    SciTech Connect

    Heitor Reis, A.

    2014-07-15

    It is shown how both the principles of extremum of entropy production, which are often used in the study of complex systems, follow from the maximization of overall system conductivities, under appropriate constraints. In this way, the maximum rate of entropy production (MEP) occurs when all the forces in the system are kept constant. On the other hand, the minimum rate of entropy production (mEP) occurs when all the currents that cross the system are kept constant. A brief discussion on the validity of the application of the mEP and MEP principles in several cases, and in particular to the Earth’s climate is also presented. -- Highlights: •The principles of extremum of entropy production are not first principles. •They result from the maximization of conductivities under appropriate constraints. •The conditions of their validity are set explicitly. •Some long-standing controversies are discussed and clarified.

  1. Processing and lysosomal localization of a glycoprotein whose secretion is transformation stimulated

    PubMed Central

    1985-01-01

    The major excreted protein (MEP) of transformed mouse fibroblasts is a mannose 6-phosphate-containing glycoprotein whose synthesis and secretion are increased in malignantly transformed 3T3 cells and whose synthesis is increased by treatment of 3T3 cells with tumor promoters or growth factors. When pulse-labeled extracts from Kirsten virus- transformed NIH 3T3 (KNIH) cells were immunoprecipitated using an antibody against secreted MEP, one cellular protein was immunoprecipitated that had the same molecular weight and tryptic peptide map as the secreted protein. Pulse-chase labeling experiments showed that 50-60% of this 39,000-mol-wt form was secreted in transformed cells. Of the 40-50% remaining, approximately 5% was processed into two lower molecular weight forms (29,000 and 20,000) which are sequestered within the cell. Similar processing of these proteins was observed in the nontransformed parent NIH 3T3 (NIH) cells. However, in NIH cells, much less of the synthesized MEP was secreted. Measurements of steady-state levels of these three forms of cellular MEP by Western blot immunolocalization revealed approximately fourfold more MEP in KNIH cells than in NIH cells as well as differences in the relative distribution of MEP forms in transformed and nontransformed cells. Subcellular fractionation of KNIH cells on a Percoll gradient demonstrated a distribution of total MEP similar to that of several lysosomal enzymes. The light lysosomal/Golgi peak from these gradients contained both the precursor 39,000-mol-wt form of MEP and the 20,000- mol-wt form, whereas the heavy lysosomal peak was enriched in the 20,000-mol-wt form. The distribution of MEP forms was found to be similar in NIH cells except that the 29,000-mol-wt form was also seen to be enriched in the heavy lysosomal peak. This biochemical localization of MEP was confirmed by immunolocalization with light and electron microscopy. These data support the hypothesis that MEP is a lysosomal protein that is

  2. Induction of cytochrome P-450 1A1 in human hepatoma HepG2 and lung carcinoma NCI-H322 cells by motorcycle exhaust particulate.

    PubMed

    Ueng, T H; Hu, S H; Chen, R M; Wang, H W; Kuo, M L

    2000-05-26

    The effects of motorcycle exhaust particulate (MEP) on human cytochrome P-450 (P-450)-dependent monooxygenases were determined using human hepatoma cell line HepG2 and lung carcinoma cell line NCI-H322 treated with organic extracts of MEP from a two-stroke engine. Gas chromatography and mass spectrometry analysis of MEP extract revealed the presence of carcinogens benzo[a]pyrene, benz[a]anthracene, benzo[b]fluoranthene, benzo[k]fluoranthene, benzo[g,h,i]perylene, chrysene, and indeno[1,2,3-c,d]pyrene in the chemical mixture. Treatment with MEP extract produced concentration- and time-dependent increases of monooxygenase activity in HepG2 cells. Treatment of the cells with 100 microg/ ml MEP extract for 24 h markedly increased benzo[a]pyrene hydroxylation, 7-ethoxycoumarin, and 7-ethoxyresorufin O-deethylation activities in microsomes. Immunoblot analysis of microsomal proteins using mouse monoclonal antibody 1-12-3 against P-450 1A1 revealed that MEP extract induced a P-450-immunorelated protein in the hepatoma cells. RNA blot analysis of cellular total RNA using a human P-450 1A1 3'-end cDNA probe showed that MEP extract increased the level of a hybridizable P-450 mRNA. These P-450 1A1 inductive effects of MEP extract were similar to those from treatment with 10 microM benzo[a]pyrene or 3-methylcholanthrene (3-MC) in HepG2 cells. Treatment of lung carcinoma NCI-H322 cells with 100 microg/ml MEP extract, 10 microM benzo[a]pyrene, or 3-MC resulted in induction of monooxygenase activity, protein, and mRNA of P-450 1A1, similar to the induction observed with the hepatoma cells. The present study demonstrates that MEP extract has the ability to induce human hepatic and pulmonary P-450 1A1 in the liver- and lung-derived cell lines, and the induction involves a pretranslational mechanism. Induction of the human hepatic and pulmonary P-450 1A1 in vitro may provide important information in the assessment of MEP metabolism and toxicity in humans.

  3. Facilitatory conditioning of the supplementary motor area in humans enhances the corticophrenic responsiveness to transcranial magnetic stimulation.

    PubMed

    Raux, Mathieu; Xie, Haiqun; Similowski, Thomas; Koski, Lisa

    2010-01-01

    Inspiratory loading in awake humans is associated with electroencephalographic signs of supplementary motor area (SMA) activation. To provide evidence for a functional connection between SMA and the diaphragm representation in the primary motor cortex (M1(DIA)), we tested the hypothesis that modulating SMA activity using repetitive transcranial magnetic stimulation (rTMS) would alter M1(DIA) excitability. Amplitude and latency of diaphragm motor evoked potentials (MEP(DIA)), evoked through single pulse M1(DIA) stimulation, before and up to 16 min after SMA stimulation, were taken as indicators of M1(DIA) excitability. MEPs from the first dorsal interosseous muscle (FDI, MEP(FDI)) served as a control. Four SMA conditioning sessions were performed in random order at 1-wk intervals. Two aimed at increasing SMA activity (5 and 10 Hz, both at 110% of FDI active motor threshold; referred to as 5Hz and 10Hz, respectively), and two aimed at decreasing it (1 Hz either at 110% of FDI active or resting motor threshold, referred to as aMT or rMT, respectively). The 5Hz protocol increased MEP(DIA) and MEP(FDI) amplitudes with a maximum 11-16 min poststimulation (P = 0.04 and P = 0.02, respectively). The 10Hz protocol increased MEP(FDI) amplitude with a similar time course (P = 0.03) but did not increase MEP(DIA) amplitude (P = 0.32). Both aMT and rMT failed to decrease MEP(DIA) or MEP(FDI) amplitudes (P = 0.23 and P = 0.90, respectively, for diaphragm and P = 0.48 and P = 0.14 for FDI). MEP(DIA) and MEP(FDI) latencies were unaffected by rTMS. These results demonstrate that 5-Hz rTMS over the SMA can increase the excitability of M1(DIA). These observations are consistent with the hypothesis of a functional connection between SMA and M1(DIA).

  4. Modulation of amplitude and latency of motor evoked potential by direction of transcranial magnetic stimulation

    NASA Astrophysics Data System (ADS)

    Sato, Aya; Torii, Tetsuya; Iwahashi, Masakuni; Itoh, Yuji; Iramina, Keiji

    2014-05-01

    The present study analyzed the effects of monophasic magnetic stimulation to the motor cortex. The effects of magnetic stimulation were evaluated by analyzing the motor evoked potentials (MEPs). The amplitude and latency of MEPs on the abductor pollicis brevis muscle were used to evaluate the effects of repetitive magnetic stimulation. A figure eight-shaped flat coil was used to stimulate the region over the primary motor cortex. The intensity of magnetic stimulation was 120% of the resting motor threshold, and the frequency of magnetic stimulation was 0.1 Hz. In addition, the direction of the current in the brain was posterior-anterior (PA) or anterior-posterior (AP). The latency of MEP was compared with PA and AP on initial magnetic stimulation. The results demonstrated that a stimulus in the AP direction increased the latency of the MEP by approximately 2.5 ms. MEP amplitude was also compared with PA and AP during 60 magnetic stimulations. The results showed that a stimulus in the PA direction gradually increased the amplitude of the MEP. However, a stimulus in the AP direction did not modulate the MEP amplitude. The average MEP amplitude induced from every 10 magnetic pulses was normalized by the average amplitude of the first 10 stimuli. These results demonstrated that the normalized MEP amplitude increased up to approximately 150%. In terms of pyramidal neuron indirect waves (I waves), magnetic stimulation inducing current flowing backward to the anterior preferentially elicited an I1 wave, and current flowing forward to the posterior elicited an I3 wave. It has been reported that the latency of the I3 wave is approximately 2.5 ms longer than the I1 wave elicitation, so the resulting difference in latency may be caused by this phenomenon. It has also been reported that there is no alteration of MEP amplitude at a frequency of 0.1 Hz. However, this study suggested that the modulation of MEP amplitude depends on stimulation strength and stimulation direction.

  5. Motorcycle exhaust particles induce airway inflammation and airway hyperresponsiveness in BALB/C mice.

    PubMed

    Lee, Chen-Chen; Liao, Jiunn-Wang; Kang, Jaw-Jou

    2004-06-01

    A number of large studies have reported that environmental pollutants from fossil fuel combustion can cause deleterious effects to the immune system, resulting in an allergic reaction leading to respiratory tract damage. In this study, we investigated the effect of motorcycle exhaust particles (MEP), a major pollutant in the Taiwan urban area, on airway inflammation and airway hyperresponsiveness in laboratory animals. BALB/c mice were instilled intratracheally (i.t.) with 1.2 mg/kg and 12 mg/kg of MEP, which was collected from two-stroke motorcycle engines. The mice were exposed 3 times i.t. with MEP, and various parameters for airway inflammation and hyperresponsiveness were sequentially analyzed. We found that MEP would induce airway and pulmonary inflammation characterized by infiltration of eosinophils, neutrophils, lymphocytes, and macrophages in bronchoalveolar lavage fluid (BALF) and inflammatory cell infiltration in lung. In addition, MEP treatment enhanced BALF interleukin-4 (IL-4), IL-5, and interferon-gamma (IFN-gamma) cytokine levels and serum IgE production. Bronchial response measured by unrestrained plethysmography with methacholine challenge showed that MEP treatment induced airway hyperresponsiveness (AHR) in BALB/c mice. The chemical components in MEP were further fractionated with organic solvents, and we found that the benzene-extracted fraction exerts a similar biological effect as seen with MEP, including airway inflammation, increased BALF IL-4, serum IgE production, and induction of AHR. In conclusion, we present evidence showing that the filter-trapped particles emitted from the unleaded-gasoline-fueled two-stroke motorcycle engine may induce proinflammatory and proallergic response profiles in the absence of exposure to allergen.

  6. Recurrent Rearrangement during Adaptive Evolution in an Interspecific Yeast Hybrid Suggests a Model for Rapid Introgression

    PubMed Central

    Dunn, Barbara; Paulish, Terry; Stanbery, Alison; Piotrowski, Jeff; Koniges, Gregory; Kroll, Evgueny; Louis, Edward J.; Liti, Gianni; Sherlock, Gavin; Rosenzweig, Frank

    2013-01-01

    Genome rearrangements are associated with eukaryotic evolutionary processes ranging from tumorigenesis to speciation. Rearrangements are especially common following interspecific hybridization, and some of these could be expected to have strong selective value. To test this expectation we created de novo interspecific yeast hybrids between two diverged but largely syntenic Saccharomyces species, S. cerevisiae and S. uvarum, then experimentally evolved them under continuous ammonium limitation. We discovered that a characteristic interspecific genome rearrangement arose multiple times in independently evolved populations. We uncovered nine different breakpoints, all occurring in a narrow ∼1-kb region of chromosome 14, and all producing an “interspecific fusion junction” within the MEP2 gene coding sequence, such that the 5′ portion derives from S. cerevisiae and the 3′ portion derives from S. uvarum. In most cases the rearrangements altered both chromosomes, resulting in what can be considered to be an introgression of a several-kb region of S. uvarum into an otherwise intact S. cerevisiae chromosome 14, while the homeologous S. uvarum chromosome 14 experienced an interspecific reciprocal translocation at the same breakpoint within MEP2, yielding a chimaeric chromosome; these events result in the presence in the cell of two MEP2 fusion genes having identical breakpoints. Given that MEP2 encodes for a high-affinity ammonium permease, that MEP2 fusion genes arise repeatedly under ammonium-limitation, and that three independent evolved isolates carrying MEP2 fusion genes are each more fit than their common ancestor, the novel MEP2 fusion genes are very likely adaptive under ammonium limitation. Our results suggest that, when homoploid hybrids form, the admixture of two genomes enables swift and otherwise unavailable evolutionary innovations. Furthermore, the architecture of the MEP2 rearrangement suggests a model for rapid introgression, a phenomenon seen in

  7. Differences in Motor Evoked Potentials Induced in Rats by Transcranial Magnetic Stimulation under Two Separate Anesthetics: Implications for Plasticity Studies

    PubMed Central

    Sykes, Matthew; Matheson, Natalie A.; Brownjohn, Philip W.; Tang, Alexander D.; Rodger, Jennifer; Shemmell, Jonathan B. H.; Reynolds, John N. J.

    2016-01-01

    Repetitive transcranial magnetic stimulation (rTMS) is primarily used in humans to change the state of corticospinal excitability. To assess the efficacy of different rTMS stimulation protocols, motor evoked potentials (MEPs) are used as a readout due to their non-invasive nature. Stimulation of the motor cortex produces a response in a targeted muscle, and the amplitude of this twitch provides an indirect measure of the current state of the cortex. When applied to the motor cortex, rTMS can alter MEP amplitude, however, results are variable between participants and across studies. In addition, the mechanisms underlying any change and its locus are poorly understood. In order to better understand these effects, MEPs have been investigated in vivo in animal models, primarily in rats. One major difference in protocols between rats and humans is the use of general anesthesia in animal experiments. Anesthetics are known to affect plasticity-like mechanisms and so may contaminate the effects of an rTMS protocol. In the present study, we explored the effect of anesthetic on MEP amplitude, recorded before and after intermittent theta burst stimulation (iTBS), a patterned rTMS protocol with reported facilitatory effects. MEPs were assessed in the brachioradialis muscle of the upper forelimb under two anesthetics: a xylazine/zoletil combination and urethane. We found MEPs could be induced under both anesthetics, with no differences in the resting motor threshold or the average baseline amplitudes. However, MEPs were highly variable between animals under both anesthetics, with the xylazine/zoletil combination showing higher variability and most prominently a rise in amplitude across the baseline recording period. Interestingly, application of iTBS did not facilitate MEP amplitude under either anesthetic condition. Although it is important to underpin human application of TMS with mechanistic examination of effects in animals, caution must be taken when selecting an

  8. Estimate of Technical Potential for Minimum Efficiency Performance Standards in 13 Major World Economies

    SciTech Connect

    Letschert, Virginie; Desroches, Louis-Benoit; Ke, Jing; McNeil, Michael

    2012-07-01

    As part of the ongoing effort to estimate the foreseeable impacts of aggressive minimum efficiency performance standards (MEPS) programs in the world’s major economies, Lawrence Berkeley National Laboratory (LBNL) has developed a scenario to analyze the technical potential of MEPS in 13 major economies around the world1 . The “best available technology” (BAT) scenario seeks to determine the maximum potential savings that would result from diffusion of the most efficient available technologies in these major economies.

  9. AKAP150-dependent cooperative TRPV4 channel gating is central to endothelium-dependent vasodilation and is disrupted in hypertension

    PubMed Central

    Sonkusare, Swapnil K.; Dalsgaard, Thomas; Bonev, Adrian D.; Hill-Eubanks, David C.; Kotlikoff, Michael I.; Scott, John D.; Santana, Luis F.; Nelson, Mark T.

    2015-01-01

    Endothelial cell dysfunction, characterized by a diminished response to endothelial cell–dependent vasodilators, is a hallmark of hypertension. TRPV4 channels play a major role in endothelial-dependent vaso-dilation, a function mediated by local Ca2+ influx through clusters of functionally coupled TRPV4 channels rather than by a global increase in endothelial cell Ca2+. We showed that stimulation of muscarinic acetylcholine receptors on endothelial cells of mouse arteries exclusively activated TRPV4 channels that were localized at myoendothelial projections (MEPs), specialized regions of endothelial cells that contact smooth muscle cells. Muscarinic receptor–mediated activation of TRPV4 depended on protein kinase C (PKC) and the PKC-anchoring protein AKAP150, which was concentrated at MEPs. Cooperative opening of clustered TRPV4 channels specifically amplified Ca2+ influx at MEPs. Cooperativity of TRPV4 channels at non-MEP sites was much lower, and cooperativity at MEPs was greatly reduced by chelation of intracellular Ca2+ or AKAP150 knockout, suggesting that Ca2+ entering through adjacent channels underlies the AKAP150-dependent potentiation of TRPV4 activity. In a mouse model of angiotensin II–induced hypertension, MEP localization of AKAP150 was disrupted, muscarinic receptor stimulation did not activate TRPV4 channels, cooperativity among TRPV4 channels at MEPs was weaker, and vasodilation in response to muscarinic receptor stimulation was reduced. Thus, endothelial-dependent dilation of resistance arteries is enabled by MEP-localized AKAP150, which ensures the proximity of PKC to TRPV4 channels and the coupled channel gating necessary for efficient communication from endothelial to smooth muscle cells in arteries. Disruption of this molecular assembly may contribute to altered blood flow in hypertension. PMID:25005230

  10. Ipsilesional motor-evoked potential absence in pediatric hemiparesis impacts tracking accuracy of the less affected hand.

    PubMed

    Cassidy, Jessica M; Carey, James R; Lu, Chiahao; Krach, Linda E; Feyma, Tim; Durfee, William K; Gillick, Bernadette T

    2015-12-01

    This study analyzed the relationship between electrophysiological responses to transcranial magnetic stimulation (TMS), finger tracking accuracy, and volume of neural substrate in children with congenital hemiparesis. Nineteen participants demonstrating an ipsilesional motor-evoked potential (MEP) were compared with eleven participants showing an absent ipsilesional MEP response. Comparisons of finger tracking accuracy from the affected and less affected hands and ipsilesional/contralesional (I/C) volume ratio for the primary motor cortex (M1) and posterior limb of internal capsule (PLIC) were done using two-sample t-tests. Participants showing an ipsilesional MEP response demonstrated superior tracking performance from the less affected hand (p=0.016) and significantly higher I/C volume ratios for M1 (p=0.028) and PLIC (p=0.005) compared to participants without an ipsilesional MEP response. Group differences in finger tracking accuracy from the affected hand were not significant. These results highlight differentiating factors amongst children with congenital hemiparesis showing contrasting MEP responses: less affected hand performance and preserved M1 and PLIC volume. Along with MEP status, these factors pose important clinical implications in pediatric stroke rehabilitation. These findings may also reflect competitive developmental processes associated with the preservation of affected hand function at the expense of some function in the less affected hand. PMID:26426515

  11. Bidirectional variability in motor cortex excitability modulation following 1 mA transcranial direct current stimulation in healthy participants.

    PubMed

    Strube, Wolfgang; Bunse, Tilmann; Nitsche, Michael A; Nikolaeva, Alexandra; Palm, Ulrich; Padberg, Frank; Falkai, Peter; Hasan, Alkomiet

    2016-08-01

    Due to the high interindividual response variability following transcranial direct current stimulation (tDCS), it is apparent that further research of the long-lasting effects of the stimulation technique is required. We aimed to investigate interindividual variability following anodal tDCS and cathodal tDCS in a large-scale prospective cross-over study. Motor cortex physiology measurements were obtained using transcranial magnetic stimulation (TMS) in 59 healthy participants comparing motor-evoked potential (MEP) magnitudes following two tDCS paradigms: 1 mA anodal tDCS for 13 min and 1 mA cathodal tDCS for 9 min. Analysis compared MEP changes over time for both polarities. Additionally, we applied hierarchical cluster analysis to assess the dynamics of poststimulation changes. Overall, anodal tDCS resulted in a significant increase in corticospinal excitability lasting for 40 min poststimulation, whereas cathodal tDCS did not alter corticospinal excitability. Cluster analysis revealed for cathodal tDCS both a cluster showing significant stable MEP reduction and a second cluster displaying MEP increase over time. Two diametrical clusters were also found for anodal tDCS Regardless of polarity, individuals with MEP increase following stimulation showed steeper cortical recruitment curves compared to the clusters with decreased MEP magnitudes. The observed findings confirm a bidirectional modulation of corticospinal excitability following 1 mA tDCS in separate subgroups and the relationship to cortical recruitment. PMID:27495298

  12. Exploring the concept of maximum entropy production for the local atmosphere-glacier system

    NASA Astrophysics Data System (ADS)

    Mölg, Thomas

    2015-06-01

    The concept of maximum entropy production (MEP) is closely linked to the second law of thermodynamics, which explains spontaneous processes in the universe. In geophysics, studies have argued that planetary atmospheres and various subsystems of Earth also operate at maximum dissipation through MEP. One of the debates, however, has concerned the degree of empirical support. This article extends the topic by considering measurements from a high-altitude, cold glacier in the tropical atmosphere and a numerical model, which represents the open and nonequilibrium system of glacier-air exchanges. Results reveal that several sensitive system parameters, which are mainly tied to the shortwave radiation budget, cause MEP states at values that coincide closely with the in situ observations. Parameters that set up the forcing of the whole system, however, do not show this pattern. Empirical support for the detection of MEP states, therefore, is limited to parameters that regulate the internal efficiency of energy flow in the glacier. System constraints are shown to affect the solutions, yet not critically in the case of the two most sensitive parameters. In terms of MEP and geophysical fluids, the results suggest that the local atmosphere-glacier system might be of relevance in the further discussion. For practical purposes, the results hold promise for using MEP in single or multiparameter optimization for process-based mass balance models of glaciers.

  13. Basic Principles and Recent Trends of Transcranial Motor Evoked Potentials in Intraoperative Neurophysiologic Monitoring.

    PubMed

    Tsutsui, Shunji; Yamada, Hiroshi

    2016-08-15

    Transcranial motor evoked potentials (TcMEPs), which are muscle action potentials elicited by transcranial brain stimulation, have been the most popular method for the last decade to monitor the functional integrity of the motor system during surgery. It was originally difficult to record reliable and reproducible potentials under general anesthesia, especially when inhalation-based anesthetic agents that suppressed the firing of anterior horn neurons were used. Advances in anesthesia, including the introduction of intravenous anesthetic agents, and progress in stimulation techniques, including the use of pulse trains, improved the reliability and reproducibility of TcMEP responses. However, TcMEPs are much smaller in amplitude compared with compound muscle action potentials evoked by maximal peripheral nerve stimulation, and vary from one trial to another in clinical practice, suggesting that only a limited number of spinal motor neurons innervating the target muscle are excited in anesthetized patients. Therefore, reliable interpretation of the critical changes in TcMEPs remains difficult and controversial. Additionally, false negative cases have been occasionally encountered. Recently, several facilitative techniques using central or peripheral stimuli, preceding transcranial electrical stimulation, have been employed to achieve sufficient depolarization of motor neurons and augment TcMEP responses. These techniques might have potentials to improve the reliability of intraoperative motor pathway monitoring using TcMEPs. PMID:26935781

  14. Hyperdynamics boost factor achievable with an ideal bias potential

    DOE PAGES

    Huang, Chen; Perez, Danny; Voter, Arthur F.

    2015-08-20

    Hyperdynamics is a powerful method to significantly extend the time scales amenable to molecular dynamics simulation of infrequent events. One outstanding challenge, however, is the development of the so-called bias potential required by the method. In this work, we design a bias potential using information about all minimum energy pathways (MEPs) out of the current state. While this approach is not suitable for use in an actual hyperdynamics simulation, because the pathways are generally not known in advance, it allows us to show that it is possible to come very close to the theoretical boost limit of hyperdynamics while maintainingmore » high accuracy. We demonstrate this by applying this MEP-based hyperdynamics (MEP-HD) to metallic surface diffusion systems. In most cases, MEP-HD gives boost factors that are orders of magnitude larger than the best existing bias potential, indicating that further development of hyperdynamics bias potentials could have a significant payoff. Lastly, we discuss potential practical uses of MEP-HD, including the possibility of developing MEP-HD into a true hyperdynamics.« less

  15. Repeated tongue lift movement induces neuroplasticity in corticomotor control of tongue and jaw muscles in humans.

    PubMed

    Komoda, Yoshihiro; Iida, Takashi; Kothari, Mohit; Komiyama, Osamu; Baad-Hansen, Lene; Kawara, Misao; Sessle, Barry; Svensson, Peter

    2015-11-19

    This study investigated the effect of repeated tongue lift training (TLT) on the excitability of the corticomotor representation of the human tongue and jaw musculature. Sixteen participants performed three series of TLT for 41 min on each of 5 consecutive days. Each TLT series consisted of two pressure levels (5 kPa and 10 kPa). All participants underwent transcranial magnetic stimulation (TMS) and electromyographic (EMG) recordings of motor evoked potentials (MEPs) in four sessions: (1) before TLT on Day 1 (baseline), (2) after TLT on Day 1, (3) before TLT on Day 5, and (4) after TLT on Day 5. EMG recordings from the left and right tongue dorsum and masseter muscles were made at three pressure levels (5 kPa, 10 kPa, 100% tongue lift), and tongue, masseter, and first dorsal interosseous (FDI) MEPs were measured. There were no significant day-to-day differences in the tongue pressure during maximum voluntary contractions. The amplitudes and thresholds of tongue and masseter MEPs after TLT on Day 5 were respectively higher and lower than before TLT on Day 1 (P<0.005), and there was also a significant increase in tongue and masseter MEP areas; no significant changes occurred in MEP onset latencies. FDI MEP parameters (amplitude, threshold, area, latency) were not significantly different between the four sessions. Our findings suggest that repeated TLT can trigger neuroplasticity reflected in increased excitability of the corticomotor representation of not only the tongue muscles but also the masseter muscles.

  16. Contribution of the mevalonate and methylerythritol phosphate pathways to the biosynthesis of gibberellins in Arabidopsis.

    PubMed

    Kasahara, Hiroyuki; Hanada, Atsushi; Kuzuyama, Tomohisa; Takagi, Motoki; Kamiya, Yuji; Yamaguchi, Shinjiro

    2002-11-22

    Gibberellins (GAs) are diterpene plant hormones essential for many developmental processes. Although the GA biosynthesis pathway has been well studied, our knowledge on its early stage is still limited. There are two possible routes for the biosynthesis of isoprenoids leading to GAs, the mevalonate (MVA) pathway in the cytosol and the methylerythritol phosphate (MEP) pathway in plastids. To distinguish these possibilities, metabolites from each isoprenoid pathway were selectively labeled with (13)C in Arabidopsis seedlings. Efficient (13)C-labeling was achieved by blocking the endogenous pathway chemically or genetically during the feed of a (13)C-labeled precursor specific to the MVA or MEP pathways. Gas chromatography-mass spectrometry analyses demonstrated that both MVA and MEP pathways can contribute to the biosyntheses of GAs and campesterol, a cytosolic sterol, in Arabidopsis seedlings. While GAs are predominantly synthesized through the MEP pathway, the MVA pathway plays a major role in the biosynthesis of campesterol. Consistent with some crossover between the two pathways, phenotypic defects caused by the block of the MVA and MEP pathways were partially rescued by exogenous application of the MEP and MVA precursors, respectively. We also provide evidence to suggest that the MVA pathway still contributes to GA biosynthesis when this pathway is limiting.

  17. Hyperdynamics boost factor achievable with an ideal bias potential

    NASA Astrophysics Data System (ADS)

    Huang, Chen; Perez, Danny; Voter, Arthur F.

    2015-08-01

    Hyperdynamics is a powerful method to significantly extend the time scales amenable to molecular dynamics simulation of infrequent events. One outstanding challenge, however, is the development of the so-called bias potential required by the method. In this work, we design a bias potential using information about all minimum energy pathways (MEPs) out of the current state. While this approach is not suitable for use in an actual hyperdynamics simulation, because the pathways are generally not known in advance, it allows us to show that it is possible to come very close to the theoretical boost limit of hyperdynamics while maintaining high accuracy. We demonstrate this by applying this MEP-based hyperdynamics (MEP-HD) to metallic surface diffusion systems. In most cases, MEP-HD gives boost factors that are orders of magnitude larger than the best existing bias potential, indicating that further development of hyperdynamics bias potentials could have a significant payoff. Finally, we discuss potential practical uses of MEP-HD, including the possibility of developing MEP-HD into a true hyperdynamics.

  18. Hyperdynamics boost factor achievable with an ideal bias potential

    SciTech Connect

    Huang, Chen; Perez, Danny; Voter, Arthur F.

    2015-08-20

    Hyperdynamics is a powerful method to significantly extend the time scales amenable to molecular dynamics simulation of infrequent events. One outstanding challenge, however, is the development of the so-called bias potential required by the method. In this work, we design a bias potential using information about all minimum energy pathways (MEPs) out of the current state. While this approach is not suitable for use in an actual hyperdynamics simulation, because the pathways are generally not known in advance, it allows us to show that it is possible to come very close to the theoretical boost limit of hyperdynamics while maintaining high accuracy. We demonstrate this by applying this MEP-based hyperdynamics (MEP-HD) to metallic surface diffusion systems. In most cases, MEP-HD gives boost factors that are orders of magnitude larger than the best existing bias potential, indicating that further development of hyperdynamics bias potentials could have a significant payoff. Lastly, we discuss potential practical uses of MEP-HD, including the possibility of developing MEP-HD into a true hyperdynamics.

  19. GABAergic mechanisms contributing to categorical amygdala responses to chemosensory signals.

    PubMed

    Westberry, Jenne M; Meredith, Michael

    2016-09-01

    Chemosensory stimuli from conspecific and heterospecific animals, elicit categorically different immediate-early gene response-patterns in medial amygdala in male hamsters and mice. We previously showed that conspecific signals activate posterior (MeP) as well as anterior medial amygdala (MeA), and especially relevant heterospecific signals such as chemosensory stimuli from potential predators also activate MeP in mice. Other heterospecific chemosignals activate MeA, but not MeP. Here we show that male hamster amygdala responds significantly differentially to different conspecific signals, by activating different proportions of cells of different phenotype, possibly leading to differential activation of downstream circuits. Heterospecific signals that fail to activate MeP do activate GABA-immunoreactive cells in the adjacent caudal main intercalated nucleus (mICNc) and elicit selective suppression of MeP cells bearing GABA-Receptors, suggesting GABA inhibition in MeP by GABAergic cells in mICNc. Overall, work presented here suggests that medial amygdala may discriminate between important conspecific social signals, distinguish them from the social signals of other species and convey that information to brain circuits eliciting appropriate social behavior. PMID:27329335

  20. Degradation of methylparaben in water by corona plasma coupled with ozonation.

    PubMed

    Dobrin, D; Magureanu, M; Bradu, C; Mandache, N B; Ionita, P; Parvulescu, V I

    2014-11-01

    The degradation of methylparaben (MeP) in water was investigated using a pulsed corona discharge generated in oxygen, above the liquid. A comparison was made between results obtained in semi-batch corona (SBC) configuration (stationary solution, continuous gas flow) and results obtained in a semi-batch corona with recirculation combined with ozonation (SBCR + O3), where the liquid is continuously circulated between a solution reservoir and the plasma reactor and the effluent gas containing ozone is bubbled through the solution in the reservoir. It was found that MeP was completely degraded after 10-15 min of treatment in both configurations. Oxidation by ozone alone, in the absence of plasma, was a slower process. The energy efficiency for MeP removal (Y MeP) and for mineralization (Y TOC) was significantly higher in the SBCR + O3 configuration (Y MeP = 7.1 g/kWh at 90 % MeP removal and Y TOC = 0.41 g/kWh at 50 % total organic carbon (TOC) removal) than in the SBC configuration (Y MeP = 0.6 g/kWh at 90 % MeP removal and Y TOC = 0.11 g/kWh at 50 % TOC removal).

  1. Antinociceptive and neuropharmacological activities of methanol extract of Phoenix sylvestris fruit pulp

    PubMed Central

    Shajib, Md. Shafiullah; Akter, Saleha; Ahmed, Tajnin; Imam, Mohammad Zafar

    2015-01-01

    Fruits of Phoenix sylvestris Roxb. (Arecaceae) are used to treat back pain, toothache, headache, arthritis, nervous debility and as sedative. The aim of this study was to evaluate the antinociceptive and neuropharmacological activities of methanol extract of P. sylvestris fruit pulp (MEPS). The antinociceptive activity of MEPS was evaluated by heat-induced (hot plate, tail immersion test) and chemical-induced pain models (acetic acid-induced writhing, formalin-induced nociception, glutamate-induced nociception and paw edema test). The effect of MEPS on central nervous system (CNS) was studied using hole cross test, open field test, sodium thiopental-induced sleeping time and elevated plus maze test. MEPS showed strong, significant and dose-dependent antinociceptive activity in all heat-induced and chemical-induced pain models at all experimental doses. Involvement of opioid receptor mediated analgesia was evident from the reversal of analgesic effect by naloxone. MEPS also showed reduced locomotor activity in both hole cross and open field tests. The increase in sleeping time in sodium thiopental-induced sleeping test and anxiolytic activity in elevated plus maze test were also significant. So, it is evident that MEPS possesses strong central and peripheral antinociceptive activity as well as CNS depressant, sedative and anxiolytic activity. The results justify the ethnomedicinal use of P. sylvestris fruit in different painful conditions and CNS disorders. PMID:26483687

  2. Utility of magneto-electropolished ternary nitinol alloys for blood contacting applications.

    PubMed

    Pulletikurthi, Chandan; Munroe, Norman; Stewart, Danique; Haider, Waseem; Amruthaluri, Sushma; Rokicki, Ryszard; Dugrot, Manuel; Ramaswamy, Sharan

    2015-10-01

    The thrombogenicity of a biomaterial is mainly dependent on its surface characteristics, which dictates its interactions with blood. Surface properties such as composition, roughness wettability, surface free energy, and morphology will affect an implant material's hemocompatibility. Additionally, in the realm of metallic biomaterials, the specific composition of the alloy and its surface treatment are important factors that will affect the surface properties. The utility of magneto-electropolished (MEP) ternary Nitinol alloys, NiTiTa, and NiTiCr as blood contacting materials was investigated. The hemcompatibility of these alloys were compared to mechanically polished (MP) metallic biomaterial counterparts. In vitro thrombogenicity tests revealed significantly less platelet adherence on ternary MEP Nitinol, especially MEP NiTi10Ta as compared to the MP metals (p < 0.05). The enhanced anti-platelet-adhesive property of MEP NiTi10Ta was in part, attributed to the Ta2 O5 component of the alloy. Furthermore, the formation of a dense and mixed hydrophobic oxide layer during MEP is believed to have inhibited the adhesion of negatively charged platelets. In conclusion, MEP ternary Nitinol alloys can potentially be utilized for blood-contacting devices where, complications resulting from thrombogenicity can be minimized.

  3. Contact-mediated inhibition between oligodendrocyte progenitor cells and motor exit point glia establishes the spinal cord transition zone.

    PubMed

    Smith, Cody J; Morris, Angela D; Welsh, Taylor G; Kucenas, Sarah

    2014-09-01

    Rapid conduction of action potentials along motor axons requires that oligodendrocytes and Schwann cells myelinate distinct central and peripheral nervous system (CNS and PNS) domains along the same axon. Despite the importance of this arrangement for nervous system function, the mechanisms that establish and maintain this precise glial segregation at the motor exit point (MEP) transition zone are unknown. Using in vivo time-lapse imaging in zebrafish, we observed that prior to myelination, oligodendrocyte progenitor cells (OPCs) extend processes into the periphery via the MEP and immediately upon contact with spinal motor root glia retract back into the spinal cord. Characterization of the peripheral cell responsible for repelling OPC processes revealed that it was a novel, CNS-derived population of glia we propose calling MEP glia. Ablation of MEP glia resulted in the absence of myelinating glia along spinal motor root axons and an immediate breach of the MEP by OPCs. Taken together, our results identify a novel population of CNS-derived peripheral glia located at the MEP that selectively restrict the migration of OPCs into the periphery via contact-mediated inhibition.

  4. Scaling of motor cortical excitability during unimanual force generation.

    PubMed

    Perez, Monica A; Cohen, Leonardo G

    2009-10-01

    During performance of a unimanual force generation task primary motor cortices (M1s) experience clear functional changes. Here, we evaluated the way in which M1s interact during parametric increases in right wrist flexion force in healthy volunteers. We measured the amplitude and the slope of motor evoked potentials (MEP) recruitment curves to transcranial magnetic stimulation (TMS) in the left and right flexor carpi radialis (FCR) muscles at rest and during 10%, 30% and 70% of maximal wrist flexion force. At rest, no differences were observed in the amplitude and slope of MEP recruitment curves in the left and right FCR muscles. With increasing right wrist flexion force, MEP amplitudes increased in both FCR muscles, with larger amplitudes in the right FCR. We found a significant correlation between the left and right MEP amplitudes across conditions. The slope of right and left FCR MEP recruitment curve was significantly steeper at 70% of force compared to rest and 10% of force. A significant correlation between the slope of left and right FCR MEP amplitudes was found at 70% of force only. Our results indicate a differential scaling of excitability in the corticospinal system controlling right and left FCR muscles at increasing levels of unimanual force generation. Specifically, these data highlights that at strong levels of unimanual force the increases in motor cortical excitability with increasing TMS stimulus intensities follow a similar pattern in both M1s, while at low levels of force they do not.

  5. Degradation of methylparaben in water by corona plasma coupled with ozonation.

    PubMed

    Dobrin, D; Magureanu, M; Bradu, C; Mandache, N B; Ionita, P; Parvulescu, V I

    2014-11-01

    The degradation of methylparaben (MeP) in water was investigated using a pulsed corona discharge generated in oxygen, above the liquid. A comparison was made between results obtained in semi-batch corona (SBC) configuration (stationary solution, continuous gas flow) and results obtained in a semi-batch corona with recirculation combined with ozonation (SBCR + O3), where the liquid is continuously circulated between a solution reservoir and the plasma reactor and the effluent gas containing ozone is bubbled through the solution in the reservoir. It was found that MeP was completely degraded after 10-15 min of treatment in both configurations. Oxidation by ozone alone, in the absence of plasma, was a slower process. The energy efficiency for MeP removal (Y MeP) and for mineralization (Y TOC) was significantly higher in the SBCR + O3 configuration (Y MeP = 7.1 g/kWh at 90 % MeP removal and Y TOC = 0.41 g/kWh at 50 % total organic carbon (TOC) removal) than in the SBC configuration (Y MeP = 0.6 g/kWh at 90 % MeP removal and Y TOC = 0.11 g/kWh at 50 % TOC removal). PMID:24801291

  6. Comparison of Diffusion Tensor Tractography and Motor Evoked Potentials for the Estimation of Clinical Status in Subacute Stroke

    PubMed Central

    Chun, Kwang-Soo; Lee, Yong-Taek; Park, Jong-Wan; Lee, Joon-Youn; Park, Chul-Hyun

    2016-01-01

    Objective To compare diffusion tensor tractography (DTT) and motor evoked potentials (MEPs) for estimation of clinical status in patients in the subacute stage of stroke. Methods Patients with hemiplegia due to stroke who were evaluated using both DTT and MEPs between May 2012 and April 2015 were recruited. Clinical assessments investigated upper extremity motor and functional status. Motor status was evaluated using Medical Research Council grading and the Fugl-Meyer Assessment of upper limb and hand (FMA-U and FMA-H). Functional status was measured using the Modified Barthel Index (MBI). Patients were classified into subgroups according to DTT findings, MEP presence, fractional anisotropy (FA) value, FA ratio (rFA), and central motor conduction time (CMCT). Correlations of clinical assessments with DTT parameters and MEPs were estimated. Results Fifty-five patients with hemiplegia were recruited. In motor assessments (FMA-U), MEPs had the highest sensitivity and negative predictive value (NPV) as well as the second highest specificity and positive predictive value (PPV). CMCT showed the highest specificity and PPV. Regarding functional status (MBI), FA showed the highest sensitivity and NPV, whereas CMCT had the highest specificity and PPV. Correlation analysis showed that the resting motor threshold (RMT) ratio was strongly associated with motor status of the upper limb, and MEP parameters were not associated with MBI. Conclusion DTT and MEPs could be suitable complementary modalities for analyzing the motor and functional status of patients in the subacute stage of stroke. The RMT ratio was strongly correlated with motor status. PMID:26949679

  7. A microcalorimetric study of molecular interactions between immunoglobulin G and hydrophobic charge-induction ligand.

    PubMed

    Yuan, Xiao-Ming; Lin, Dong-Qiang; Zhang, Qi-Lei; Gao, Dong; Yao, Shan-Jing

    2016-04-22

    Hydrophobic charge-induction chromatography (HCIC) with 4-mercaptoethyl-pyridine (MEP) as the ligand is a novel technology for antibody purification. In this study, isothermal titration calorimetry (ITC) was used to evaluate the molecular interactions between MEP ligand and immunoglobulin G (IgG). Three types of IgG molecules including human IgG (hIgG), bovine IgG (bIgG) and a monoclonal antibody (mAb) were investigated with human serum albumins (HSA) and bovine serum albumin (BSA) as the comparison. The thermodynamic parameters obtained from ITC were compared with the adsorption data. The results indicated that MEP binding to protein at neutral pH was entropy driven and induced by multimodal molecular interactions that was dominated by hydrophobic forces. The interactions between MEP and IgGs were stronger than that of albumins, which resulted in high binding affinity of IgGs. Moreover, the effects of pH and salt addition on MEP-hIgG binding were studied. The change of enthalpy increased obviously with the decrease of pH, which revealed that the electrostatic forces dominated the MEP-hIgG interactions at acidic condition and caused typical charge-induced elution of HCIC. Salt addition influenced both hydrophobic and electrostatic interactions. With the increase of salt concentration, the hydrophobic interactions decreased first and then increased, while the electrostatic interactions showed the opposite trend. This resulted in trade-off between the multimodal interactions, which caused the salt-tolerant property of MEP resin. In general, ITC studies revealed the molecular mechanism of three critical characteristics of HCIC, multimodal interactions, pH-dependent and salt-tolerant properties.

  8. A microcalorimetric study of molecular interactions between immunoglobulin G and hydrophobic charge-induction ligand.

    PubMed

    Yuan, Xiao-Ming; Lin, Dong-Qiang; Zhang, Qi-Lei; Gao, Dong; Yao, Shan-Jing

    2016-04-22

    Hydrophobic charge-induction chromatography (HCIC) with 4-mercaptoethyl-pyridine (MEP) as the ligand is a novel technology for antibody purification. In this study, isothermal titration calorimetry (ITC) was used to evaluate the molecular interactions between MEP ligand and immunoglobulin G (IgG). Three types of IgG molecules including human IgG (hIgG), bovine IgG (bIgG) and a monoclonal antibody (mAb) were investigated with human serum albumins (HSA) and bovine serum albumin (BSA) as the comparison. The thermodynamic parameters obtained from ITC were compared with the adsorption data. The results indicated that MEP binding to protein at neutral pH was entropy driven and induced by multimodal molecular interactions that was dominated by hydrophobic forces. The interactions between MEP and IgGs were stronger than that of albumins, which resulted in high binding affinity of IgGs. Moreover, the effects of pH and salt addition on MEP-hIgG binding were studied. The change of enthalpy increased obviously with the decrease of pH, which revealed that the electrostatic forces dominated the MEP-hIgG interactions at acidic condition and caused typical charge-induced elution of HCIC. Salt addition influenced both hydrophobic and electrostatic interactions. With the increase of salt concentration, the hydrophobic interactions decreased first and then increased, while the electrostatic interactions showed the opposite trend. This resulted in trade-off between the multimodal interactions, which caused the salt-tolerant property of MEP resin. In general, ITC studies revealed the molecular mechanism of three critical characteristics of HCIC, multimodal interactions, pH-dependent and salt-tolerant properties. PMID:27017449

  9. Comparison of Motor Inhibition in Variants of the Instructed-Delay Choice Reaction Time Task.

    PubMed

    Quoilin, Caroline; Lambert, Julien; Jacob, Benvenuto; Klein, Pierre-Alexandre; Duque, Julie

    2016-01-01

    Using instructed-delay choice reaction time (RT) paradigms, many previous studies have shown that the motor system is transiently inhibited during response preparation: motor-evoked potentials (MEPs) elicited by transcranial magnetic stimulation (TMS) over the primary motor cortex are typically suppressed during the delay period. This effect has been observed in both selected and non-selected effectors, although MEP changes in selected effectors have been more inconsistent across task versions. Here, we compared changes in MEP amplitudes in three different variants of an instructed-delay choice RT task. All variants required participants to choose between left and right index finger movements but the responses were either provided "in the air" (Variant 1), on a regular keyboard (Variant 2), or on a response device designed to control from premature responses (Variant 3). The task variants also differed according to the visual layout (more concrete in Variant 3) and depending on whether participants received a feedback of their performance (absent in Variant 1). Behavior was globally comparable between the three variants of the task although the propensity to respond prematurely was highest in Variant 2 and lowest in Variant 3. MEPs elicited in a non-selected hand were similarly suppressed in the three variants of the task. However, significant differences emerged when considering MEPs elicited in the selected hand: these MEPs were suppressed in Variants 1 and 3 whereas they were often facilitated in Variant 2, especially in the right dominant hand. In conclusion, MEPs elicited in selected muscles seem to be more sensitive to small variations to the task design than those recorded in non-selected effectors, probably because they reflect a complex combination of inhibitory and facilitatory influences on the motor output system. Finally, the use of a standard keyboard seems to be particularly inappropriate because it encourages participants to respond promptly with no

  10. Farnesol-mediated shift in the metabolic origin of prenyl groups used for protein prenylation in plants.

    PubMed

    Huchelmann, Alexandre; Brahim, Mathieu Semir; Gerber, Esther; Tritsch, Denis; Bach, Thomas J; Hemmerlin, Andréa

    2016-08-01

    Little is known about how plant cells regulate the exchange of prenyl diphosphates between the two compartmentalized isoprenoid biosynthesis pathways. Prenylation of proteins is a suitable model to study such interactions between the plastidial methylerythritol phosphate (MEP) and the cytosolic mevalonate (MVA) pathways because prenyl moieties used to modify proteins rely on both origins. Tobacco cells expressing a prenylatable GFP were treated with specific MEP and/or MVA pathways inhibitors to block the formation of prenyl diphosphates and therefore the possibility to modify the proteins. Chemical complementation assays using prenyl alcohol precursors restore the prenylation. Indeed, geranylgeraniol (C20 prenyl alcohol) and to a lesser but significant level C15-farnesol restored the prenylation of a protein bearing a geranylgeranylation CaaX motif, which under standard conditions is modified by a MEP-derived prenyl group. However, the restoration takes place in different ways. While geranylgeraniol operates directly as a metabolic precursor, the C15-prenyl alcohol functions indirectly as a signal that leads to shift the metabolic origin of prenyl groups in modified proteins, here from the plastidial MEP pathway in favor of the cytosolic MVA pathway. Furthermore, farnesol interferes negatively with the MEP pathway in an engineered Escherichia coli strain synthesizing isoprenoids either starting from MVA or from MEP. Following the cellular uptake of a fluorescent analog of farnesol, we showed its close interaction with tobacco plastids and modification of plastid homeostasis. As a consequence, in tobacco farnesol supposedly inhibits the plastidial MEP pathway and activates the cytosolic MVA pathway, leading to the shift in the metabolic origin and thereby acts as a potential regulator of crosstalk between the two pathways. Together, those results suggest a new role for farnesol (or a metabolite thereof) as a central molecule for the regulation of isoprenoid

  11. Development of an analytical method coupling cell membrane chromatography with gas chromatography-mass spectrometry via microextraction by packed sorbent and its application in the screening of volatile active compounds in natural products.

    PubMed

    Li, Miao; Wang, Sicen; He, Langchong

    2015-01-01

    Natural products (NPs) are important sources of lead compounds in modern drug discovery. To facilitate the screening of volatile active compounds in NPs, we have developed a new biochromatography method that uses rat vascular smooth muscle cells (VSMC), which are rich in L-type calcium channels (LCC), to prepare the stationary phase. This integrated method, which couples cell membrane chromatography (CMC) with gas chromatography-mass spectrometry (GC-MS) via microextraction by packed sorbent (MEPS) technology, has been termed VSMC/CMC-MEPS-GC-MS. Methodological validation confirmed its specificity, reliability and convenience. Screening results for Radix Angelicae Dahuricae and Fructus Cnidii obtained using VSMC/CMC-MEPS-GC-MS were consistent with those obtained using VSMC/CMC-offline-GC-MS. MEPS connection plays as simplified solid-phase extraction and replaces the uncontrollable evaporation operation in reported offline connections, so our new method is supposed to be more efficient and reliable than the offline ones, especially for compounds that are volatile, thermally unstable or difficult to purify. In application, senkyunolide A and ligustilide were preliminary identified as the volatile active components in Rhizoma Chuanxiong. We have thus confirmed the suitability of VSMC/CMC-MEPS-GC-MS for volatile active compounds screening in NP.

  12. Highlights of Magnetoelectropolishing

    NASA Astrophysics Data System (ADS)

    Hryniewicz, Tadeusz; Rokosz, Krzysztof

    2014-05-01

    The research work has been concerned on the studies and development of electrochemical polishing in the magnetic field (MEP) in comparison with the standard electropolishing methods performed without stirring (EP) and the process with the forced electrolyte mixing (MIX). Advanced techniques were used in the studies, to measure the effects of the surface treatment. They are as follows: scanning electron microscopy (SEM/EDS), X-ray Photoelectron Spectroscopy (XPS), Secondary Ion Mass Spectroscopy (SIMS), surface roughness, and nanoindentation measurements. For the corrosion studies, the electrochemical methods were used, such as: open circuit potential OCP, potentiodynamic curves PC, and electrochemical impedance spectroscopy EIS measurements. The MEP process is advised mainly due to the opportunity to enrich the surface layer with the chromium compounds (austenitic stainless steels), or titanium compounds (CP Ti Grade 2, Nitinol). The surface layers of biomaterials after MEP process contain much lower amount of carcinogenic compounds, like chromium VI oxidation stage (Cr6+) (austenitic stainless steels) and nickel compounds (austenitic stainless steel & Nitinol) versus those ones obtained after EP & MIX treatments. It is interesting that apart from the significant modification of the surface layer obtained after MEP, also mechanical properties, such as nanohardness, modulus of elasticity, and mechanical resistance to bending and torsion, undergo considerable advantageous changes. All they make the MEP process very promising for application in many clean industries, such as medical equipment and devices, electronics, food industry, etc.

  13. Immediate increases in quadriceps corticomotor excitability during an electromyography biofeedback intervention.

    PubMed

    Pietrosimone, Brian; McLeod, Michelle M; Florea, David; Gribble, Phillip A; Tevald, Michael A

    2015-04-01

    The purpose of the study was to determine the effects of EMG-BF on vastus lateralis corticomotor excitability, measured via motor evoked potential (MEP) amplitudes elicited using Transcranial Magnetic Stimulation (TMS) during a maximal voluntary isometric contraction (MVIC). We also determined the effect of EMG-BF on isometric knee extensor strength. Fifteen healthy participants volunteered for this crossover study with two sessions held one-week apart. Participants were randomly assigned to condition order, during which five intervention MVICs were performed with or without EMG-BF. MEP amplitudes were collected with TMS during five knee extension contractions (5% of MVIC) at baseline and again during intervention MVICs within each session. During the control condition, participants were instructed to perform the same number of MVICs without any EMG-BF. Percent change scores were used to calculate the change in peak-to-peak MEP amplitudes that occurred during EMG-BF and Control MVICs compared to the baseline MEPs. Peak knee extension torque was recorded during MVICs prior to TMS for each condition. EMG-BF produced significantly increased MEP change scores and significantly greater torque than the control condition. The results of the current study suggest that EMG-BF may be a viable clinical method for targeting corticomotor excitability.

  14. Stereospecific suppression of active site mutants by methylphosphonate substituted substrates reveals the stereochemical course of site-specific DNA recombination.

    PubMed

    Rowley, Paul A; Kachroo, Aashiq H; Ma, Chien-Hui; Maciaszek, Anna D; Guga, Piotr; Jayaram, Makkuni

    2015-07-13

    Tyrosine site-specific recombinases, which promote one class of biologically important phosphoryl transfer reactions in DNA, exemplify active site mechanisms for stabilizing the phosphate transition state. A highly conserved arginine duo (Arg-I; Arg-II) of the recombinase active site plays a crucial role in this function. Cre and Flp recombinase mutants lacking either arginine can be rescued by compensatory charge neutralization of the scissile phosphate via methylphosphonate (MeP) modification. The chemical chirality of MeP, in conjunction with mutant recombinases, reveals the stereochemical contributions of Arg-I and Arg-II. The SP preference of the native reaction is specified primarily by Arg-I. MeP reaction supported by Arg-II is nearly bias-free or RP-biased, depending on the Arg-I substituent. Positional conservation of the arginines does not translate into strict functional conservation. Charge reversal by glutamic acid substitution at Arg-I or Arg-II has opposite effects on Cre and Flp in MeP reactions. In Flp, the base immediately 5' to the scissile MeP strongly influences the choice between the catalytic tyrosine and water as the nucleophile for strand scission, thus between productive recombination and futile hydrolysis. The recombinase active site embodies the evolutionary optimization of interactions that not only favor the normal reaction but also proscribe antithetical side reactions. PMID:25999343

  15. Development of an analytical method coupling cell membrane chromatography with gas chromatography-mass spectrometry via microextraction by packed sorbent and its application in the screening of volatile active compounds in natural products.

    PubMed

    Li, Miao; Wang, Sicen; He, Langchong

    2015-01-01

    Natural products (NPs) are important sources of lead compounds in modern drug discovery. To facilitate the screening of volatile active compounds in NPs, we have developed a new biochromatography method that uses rat vascular smooth muscle cells (VSMC), which are rich in L-type calcium channels (LCC), to prepare the stationary phase. This integrated method, which couples cell membrane chromatography (CMC) with gas chromatography-mass spectrometry (GC-MS) via microextraction by packed sorbent (MEPS) technology, has been termed VSMC/CMC-MEPS-GC-MS. Methodological validation confirmed its specificity, reliability and convenience. Screening results for Radix Angelicae Dahuricae and Fructus Cnidii obtained using VSMC/CMC-MEPS-GC-MS were consistent with those obtained using VSMC/CMC-offline-GC-MS. MEPS connection plays as simplified solid-phase extraction and replaces the uncontrollable evaporation operation in reported offline connections, so our new method is supposed to be more efficient and reliable than the offline ones, especially for compounds that are volatile, thermally unstable or difficult to purify. In application, senkyunolide A and ligustilide were preliminary identified as the volatile active components in Rhizoma Chuanxiong. We have thus confirmed the suitability of VSMC/CMC-MEPS-GC-MS for volatile active compounds screening in NP. PMID:25463192

  16. Different Stimulation Frequencies Alter Synchronous Fluctuations in Motor Evoked Potential Amplitude of Intrinsic Hand Muscles-a TMS Study.

    PubMed

    Sale, Martin V; Rogasch, Nigel C; Nordstrom, Michael A

    2016-01-01

    The amplitude of motor-evoked potentials (MEPs) elicited with transcranial magnetic stimulation (TMS) varies from trial-to-trial. Synchronous oscillations in cortical neuronal excitability contribute to this variability, however it is not known how different frequencies of stimulation influence MEP variability, and whether these oscillations are rhythmic or aperiodic. We stimulated the motor cortex with TMS at different regular (i.e., rhythmic) rates, and compared this with pseudo-random (aperiodic) timing. In 18 subjects, TMS was applied at three regular frequencies (0.05 Hz, 0.2 Hz, 1 Hz) and one aperiodic frequency (mean 0.2 Hz). MEPs (n = 50) were recorded from three intrinsic hand muscles of the left hand with different functional and anatomical relations. MEP amplitude correlation was highest for the functionally related muscle pair, less for the anatomically related muscle pair and least for the functionally- and anatomically-unrelated muscle pair. MEP correlations were greatest with 1 Hz, and least for stimulation at 0.05 Hz. Corticospinal neuron synchrony is higher with shorter TMS intervals. Further, corticospinal neuron synchrony is similar irrespective of whether the stimulation is periodic or aperiodic. These findings suggest TMS frequency is a crucial consideration for studies using TMS to probe correlated activity between muscle pairs.

  17. Changes of excitability in M1 induced by neuromuscular electrical stimulation differ between presence and absence of voluntary drive.

    PubMed

    Sugawara, Kenichi; Tanabe, Shigeo; Higashi, Toshio; Tsurumi, Takamasa; Kasai, Tatsuya

    2011-06-01

    The aim of this study is to investigate excitability changes in the human motor cortex induced by variable therapeutic electrical stimulations (TESs) with or without voluntary drive. We recorded motor-evoked potentials (MEPs) from extensor and flexor carpi radialis (FCR) muscles at rest and during FCR muscle contraction after the application of TES on FCR. TES application conditions were changed intensities, frequencies, and trains. In addition, to evaluate the contribution of M1 inhibitory circuits to the effects of TES application, we also recorded MEPs using paired-pulse transcranial magnetic stimulation. In resting muscle states, an increase in TES intensity resulted in an increase in MEP ratio in both the muscles. In contrast, when TES was applied to FCR during contraction, MEP ratios of both the muscles decreased with increased number of pulse trains. However, under both the states, MEP ratios decreased induced by paired-pulse transcranial magnetic stimulation in extensor carpi radialis to which TES was not applied. Excitability changes in M1 induced by TES application were reversibly modulated depending on the presence or absence of voluntary drive. This study showed that the therapy and the voluntary drive of the target muscles act together, and complement the effects of each other, which may be beneficial for optimizing the rehabilitation if the therapy accompanies voluntary drive. PMID:21088609

  18. Abnormal surround inhibition does not affect asymptomatic limbs in people with cervical dystonia.

    PubMed

    McDougall, Laura; Kiernan, Dovin; Kiss, Zelma H T; Suchowersky, Oksana; Welsh, Timothy N

    2015-09-14

    Surround inhibition is a neural mechanism hypothesized to facilitate goal-directed action by disinhibiting agonist muscle activity while simultaneously inhibiting antagonist and other uninvolved muscle activity. The present study was designed to investigate if abnormalities in surround inhibition are found in asymptomatic body parts (the hand) of people with focal cervical dystonia (neck). Participants with (n=7) and without (n=17) cervical dystonia completed a protocol in which they abducted their index finger while EMG was recorded from the first dorsal interosseous (agonist) and abductor digiti minimi (uninvolved) muscles. Transcranial magnetic stimulation was delivered over the primary motor cortex at intervals ranging from 0 to 950+ms after the onset of agonist muscle activity. Motor-evoked potential (MEP) amplitudes from both muscles were compared. In control participants, MEPs from the uninvolved muscle were significantly lower than agonist MEPs at intervals from 0 to 480ms. Similarly, in the hands of participants with cervical dystonia - the asymptomatic body part - MEPs from the uninvolved muscle were significantly lower than agonist MEPs from 0 to 175ms. These findings suggest that surround inhibition in people with focal dystonia may be intact in asymptomatic hands. In other words, abnormalities in surround inhibition may be restricted to the dystonic limb.

  19. Reproducibility of transcranial magnetic stimulation metrics in the study of proximal upper limb muscles

    PubMed Central

    Sankarasubramanian, Vishwanath; Roelle, Sarah; Bonnett, Corin E; Janini, Daniel; Varnerin, Nicole; Cunningham, David A; Sharma, Jennifer S; Potter-Baker, Kelsey A; Wang, Xiaofeng; Yue, Guang H; Plow, Ela B

    2015-01-01

    Objective Reproducibility of transcranial magnetic stimulation (TMS) metrics is essential in accurately tracking recovery and disease. However, majority of evidence pertains to reproducibility of metrics for distal upper limb muscles. We investigate for the first time, reliability of corticospinal physiology for a large proximal muscle-the biceps brachii and relate how varying statistical analyses can influence interpretations. Methods 14 young right-handed healthy participants completed two sessions assessing resting motor threshold (RMT), motor evoked potentials (MEPs), motor map and intra-cortical inhibition (ICI) from the left biceps brachii. Analyses included paired t-tests, Pearson's, intra-class (ICC) and concordance correlation coefficients (CCC) and Bland-Altman plots. Results Unlike paired t-tests, ICC, CCC and Pearson's were >0.6 indicating good reliability for RMTs, MEP intensities and locations of map; however values were <0.3 for MEP responses and ICI. Conclusions Corticospinal physiology, defining excitability and output in terms of intensity of the TMS device, and spatial loci are the most reliable metrics for the biceps. MEPs and variables based on MEPs are less reliable since biceps receives fewer cortico-motor-neuronal projections. Statistical tests of agreement and associations are more powerful reliability indices than inferential tests. Significance Reliable metrics of proximal muscles when translated to a larger number of participants would serve to sensitively track and prognosticate function in neurological disorders such as stroke where proximal recovery precedes distal. PMID:26111434

  20. Evidence for the presence of a critical disulfide bond in the mouse EP3γ receptor

    PubMed Central

    Downey, Jason D.; Sanders, Charles R.

    2011-01-01

    To determine the contribution of cysteines to the function of the mouse E-prostanoid subtype 3 gamma (mEP3γ), we tested a series of cysteine-to-alanine mutants. Two of these mutants, C107A and C184A, showed no agonist-dependent activation in a cell-based reporter assay for mEP3γ, whereas none of the other cysteine-to-alanine mutations disrupted mEP3γ signal transduction. Total cell membranes prepared from HEK293 cells transfected with mEP3γ C107A or C184A had no detectable radioligand binding. Other mutant mEP3γ receptors had radioligand affinities and receptor densities similar to wild-type. Cell-surface ELISA against the N-terminal HA-tag of C107A and C184A demonstrated 40 % and 47 % reductions respectively in receptor protein expression at the cell surface, and no radioligand binding was detected as assessed by intact cell radioligand binding experiments. These data suggest a key role for C107 and C184 in both receptor structure/stability and function and is consistent with the presence of a conserved disulfide bond between C107 and C184 in mouse EP3 that is required for normal receptor expression and function. Our results also indicate that if a second disulfide bond is present in the native receptor it is non-essential for receptor assembly or function. PMID:21236356

  1. Effect of Paired-Pulse Electrical Stimulation on the Activity of Cortical Circuits

    PubMed Central

    Saito, Kei; Onishi, Hideaki; Miyaguchi, Shota; Kotan, Shinichi; Fujimoto, Shuhei

    2015-01-01

    Objective: We investigated the transient effect of short-duration paired-pulse electrical stimulation (ppES) on corticospinal excitability and the after-effect of long-duration ppES on excitability, short-latency afferent inhibition (SAI), and afferent facilitation (AF). Methods: A total of 28 healthy subjects participated in two different experiments. In Experiment 1, motor-evoked potentials (MEPs) were measured in the abductor pollicis brevis (APB) and abductor digiti minimi (ADM) muscles before and immediately after short-duration ppES (5 s) at various inter-pulse intervals (2, 3, 4, 5, 6, 7, 10, 15, 20, and 30 ms). In Experiment 2, MEPs, SAI, and AF were measured before, immediately, and 20 and 40 min after long-duration ppES (20 min, inter-pulse interval of 5 and 15 ms) and peripheral electrical stimulation (20 min, 10 and 20 Hz). Results: Short-duration ppES with inter-pulse intervals of 5 and 20 ms significantly increased MEP measured in APB but not in ADM. Long-duration ppES with an inter-pulse interval of 5 ms significantly decreased SAI but not MEPs in APB. In contrast, long-duration ppES did not affect ADM. Conclusion: The afferent inputs induced by ppES-5 ms were effective for transiently increasing MEP and sustaining SAI reduction. PMID:26733847

  2. Stereospecific suppression of active site mutants by methylphosphonate substituted substrates reveals the stereochemical course of site-specific DNA recombination.

    PubMed

    Rowley, Paul A; Kachroo, Aashiq H; Ma, Chien-Hui; Maciaszek, Anna D; Guga, Piotr; Jayaram, Makkuni

    2015-07-13

    Tyrosine site-specific recombinases, which promote one class of biologically important phosphoryl transfer reactions in DNA, exemplify active site mechanisms for stabilizing the phosphate transition state. A highly conserved arginine duo (Arg-I; Arg-II) of the recombinase active site plays a crucial role in this function. Cre and Flp recombinase mutants lacking either arginine can be rescued by compensatory charge neutralization of the scissile phosphate via methylphosphonate (MeP) modification. The chemical chirality of MeP, in conjunction with mutant recombinases, reveals the stereochemical contributions of Arg-I and Arg-II. The SP preference of the native reaction is specified primarily by Arg-I. MeP reaction supported by Arg-II is nearly bias-free or RP-biased, depending on the Arg-I substituent. Positional conservation of the arginines does not translate into strict functional conservation. Charge reversal by glutamic acid substitution at Arg-I or Arg-II has opposite effects on Cre and Flp in MeP reactions. In Flp, the base immediately 5' to the scissile MeP strongly influences the choice between the catalytic tyrosine and water as the nucleophile for strand scission, thus between productive recombination and futile hydrolysis. The recombinase active site embodies the evolutionary optimization of interactions that not only favor the normal reaction but also proscribe antithetical side reactions.

  3. Activation and suppression of the trapezius muscle induced by transcranial magnetic stimulation.

    PubMed

    Strenge, H; Jahns, R

    1998-01-01

    Motor evoked potentials (MEPs) and silent periods (SPs) in the trapezius muscle induced by transcranial magnetic stimulation (TMS) were investigated in 15 healthy subjects. Stimuli were applied with a Novametrix Magnetic stimulator using a 14 cm circular coil 4 cm lateral to the vertex on the biauricular line. Surface electrodes were used for simultaneous bilateral electromyographic recordings of the trapezius. TMS invariably induced contralateral MEPs (latency 10.5 +/- 1.3 ms, mean +/- SD), with ipsilateral responses in 53% of the subjects (latency 11.1 +/- 2.5 ms). The mean duration of the SPs was approximately 90 ms on both sides. There were no significant side differences between any of the MEP or SP parameters. To study the influence of subcortical inhibition phenomena TMS induced responses were assessed following electrical mental nerve stimulation with interstimulus intervals (ISI) of 0-100 ms. MEP latencies significantly increased at ISI of 10-100 ms, whereas MEP amplitudes and SPs did not change. These findings may reflect a trigeminal induced exteroceptive suppression of trapezius muscle activity. PMID:9637939

  4. Activity of human motor system during action observation is modulated by object presence.

    PubMed

    Villiger, Michael; Chandrasekharan, Sanjay; Welsh, Timothy N

    2011-03-01

    Neurons in the monkey mirror neuron system (MNS) become active when actions are observed or executed. Increases in activity are greater when objects are engaged than when the actions are mimed. This modulation occurs even when object manipulation is hidden from view. We examined whether human motor systems are similarly modulated during action observation because such observation-related modulations are potentially mediated by a putative human MNS. Transcranial magnetic stimulation (TMS) was used to elicit motor-evoked potentials (MEPs) of a grasping muscle while participants observed actual or pantomimed grasping movements whose endpoints were sometimes hidden from view. MEP amplitudes were found to be modulated by object presence. Critically, the object-based modulation was found when the participant directly observed object manipulation and when the object manipulation had to be inferred because it was hidden. These findings parallel studies of MNS activity in monkeys and support the hypothesis that the MNS influences motor system activity during action observation. Although the object-based modulation of MEP amplitudes was consistent with the hypotheses, the direction of the modulation was not--MEP amplitudes decreased during action observation in contrast to the increase that has previously been observed. We suggest that the decrease in MEP amplitude on object-present trials resulted from inhibitory mechanisms that were activated to suppress the observation-evoked response codes from generating overt muscle activity.

  5. Immediate increases in quadriceps corticomotor excitability during an electromyography biofeedback intervention.

    PubMed

    Pietrosimone, Brian; McLeod, Michelle M; Florea, David; Gribble, Phillip A; Tevald, Michael A

    2015-04-01

    The purpose of the study was to determine the effects of EMG-BF on vastus lateralis corticomotor excitability, measured via motor evoked potential (MEP) amplitudes elicited using Transcranial Magnetic Stimulation (TMS) during a maximal voluntary isometric contraction (MVIC). We also determined the effect of EMG-BF on isometric knee extensor strength. Fifteen healthy participants volunteered for this crossover study with two sessions held one-week apart. Participants were randomly assigned to condition order, during which five intervention MVICs were performed with or without EMG-BF. MEP amplitudes were collected with TMS during five knee extension contractions (5% of MVIC) at baseline and again during intervention MVICs within each session. During the control condition, participants were instructed to perform the same number of MVICs without any EMG-BF. Percent change scores were used to calculate the change in peak-to-peak MEP amplitudes that occurred during EMG-BF and Control MVICs compared to the baseline MEPs. Peak knee extension torque was recorded during MVICs prior to TMS for each condition. EMG-BF produced significantly increased MEP change scores and significantly greater torque than the control condition. The results of the current study suggest that EMG-BF may be a viable clinical method for targeting corticomotor excitability. PMID:25561075

  6. Microvascular decompression for glossopharyngeal neuralgia using intraoperative neurophysiological monitoring: Technical case report

    PubMed Central

    Motoyama, Yasushi; Nakagawa, Ichiro; Takatani, Tsunenori; Park, Hun-Soo; Kotani, Yukiko; Tanaka, Yoshitaka; Gurung, Pritam; Park, Young-Soo; Nakase, Hiroyuki

    2016-01-01

    Background: Glossopharyngeal neuralgia (GN) is a rare functional disorder representing around 1% of cases of trigeminal neuralgia. Lancinating throat and ear pain while swallowing are the typical manifestations, and are initially treated using anticonvulsants such as carbamazepine. Medically refractory GN is treated surgically. Microvascular decompression (MVD) is reportedly effective against GN, superseding rhizotomy and tractotomy. Methods: We encountered three patients with medically refractory GN who underwent MVD using intraoperative neurophysiological monitoring (IONM). The offending vessels were the posterior inferior cerebellar arteries, which were confirmed intraoperatively via a transcondylar fossa approach to be affecting the root exit zones of the glossopharyngeal and vagus nerves. As IONM, facial motor-evoked potentials (MEPs) and brainstem auditory-evoked potentials were monitored during microsurgery in all three patients. Pharyngeal and vagal MEPs were added for two patients to avoid postoperative dysphagia. Results: GN disappeared immediately after surgery with complete preservation of hearing acuity and facial nerve function. Transient mild swallowing disturbance was observed in 1 patient without pharyngeal or vagal MEPs, whereas the remaining two patients with pharyngeal and vagal MEPs demonstrated no postoperative dysphagia. Conclusion: Although control of severe pain is expected in surgical intervention for GN, lower cranial nerves are easily damaged because of their fragility, even in MVD. IONM including pharyngeal and vagal MEPs appears very useful for avoiding postoperative sequelae during MVD for GN. PMID:26862458

  7. "Severe" wear challenge to 36 mm mechanically enhanced highly crosslinked polyethylene hip liners.

    PubMed

    Bowsher, J G; Williams, P A; Clarke, I C; Green, D D; Donaldson, T K

    2008-07-01

    Our purpose was to compare the wear performance of mechanically enhanced 5Mrad highly crosslinked polyethylene (MEP, ArComXL) hip liners to (control) 3Mrad UHMWPE liners (ArCom) in 36 mm head size. As a more severe synergy of clinically relevant test models, we contrasted wear with custom roughened Co-Cr surfaces (Ra 500 nm) to the standard pristine Co-Cr heads (Ra < 20 nm) using a severe microseparation test mode in our hip simulator. We adopted a previously published model to estimate potential biological activity. On new Co-Cr heads, the MEP liners showed a 47% reduction in volumetric wear a 13% reduction in wear particle size and a 27% reduction in Functional Biological Activity (FBA) compared to our control. On rough Co-Cr heads, the MEP liners showed little advantage in terms of volumetric wear compared with the control. However, the MEP liners overall showed a 38% reduction in FBA compared to the control owing to a larger volume fraction of larger particles. Thus overall the MEP liners appeared to offer advantages in terms of reduced FBA indices.

  8. Differential activation of ammonium transporters during the accumulation of ammonia by Colletotrichum gloeosporioides and its effect on appressoria formation and pathogenicity.

    PubMed

    Shnaiderman, Chen; Miyara, Itay; Kobiler, Ilana; Sherman, Amir; Prusky, Dov

    2013-03-01

    Ammonium secreted by the post-harvest pathogen Colletotrichum gloeosporioides during host colonization accumulates in the host environment due to enhanced fungal nitrogen metabolism. Two types of ammonium transporter-encoding genes, AMET and MEP, are expressed during pathogenicity. Gene disruption of AMET, a gene modulating ammonia secretion, showed twofold reduced ammonia secretion and 45% less colonization on avocado fruit, suggesting a contribution to pathogenicity. MEPB, a gene modulating ammonium transport, is expressed by C. gloeosporioides during pathogenicity and starvation conditions in culture. Gene disruption of MEPB, the most highly expressed gene of the MEP family, resulted in twofold overexpression of MEPA and MEPC but reduced colonization, suggesting MEPB expression's contribution to pathogenicity. Analysis of internal and external ammonia accumulation by ΔmepB strains in mycelia and germinated spores showed rapid uptake and accumulation, and reduced secretion of ammonia in the mutant versus wild-type (WT) strains. Ammonia uptake by the WT germinating spores but not by the ΔmepB strain with compromised ammonium transport activated cAMP and transcription of PKA subunits PKAR and PKA2. ΔmepB mutants showed 75% less appressorium formation and colonization than the WT, which was partially restored by 10 mM exogenous ammonia. Thus, whereas both AMET and MEPB genes modulate ammonia secretion, only MEPB contributes to ammonia accumulation by mycelia and germinating spores that activate the cAMP pathways, inducing the morphogenetic processes contributing to C. gloeosporioides pathogenicity. PMID:23387470

  9. Verification and intercomparison of mesoscale ensemble prediction systems in the Beijing 2008 Olympics Research and Development Project

    NASA Astrophysics Data System (ADS)

    Kunii, Masaru; Saito, Kazuo; Seko, Hiromu; Hara, Masahiro; Hara, Tabito; Yamaguchi, Munehiko; Gong, Jiandong; Charron, Martin; Du, Jun; Wang, Yong; Chen, Dehui

    2011-05-01

    During the period around the Beijing 2008 Olympic Games, the Beijing 2008 Olympics Research and Development Project (B08RDP) was conducted as part of the World Weather Research Program short-range weather forecasting research project. Mesoscale ensemble prediction (MEP) experiments were carried out by six organizations in near-real time, in order to share their experiences in the development of MEP systems. The purpose of this study is to objectively verify these experiments and to clarify the problems associated with the current MEP systems through the same experiences. Verification was performed using the MEP outputs interpolated into a common verification domain with a horizontal resolution of 15 km. For all systems, the ensemble spreads grew as the forecast time increased, and the ensemble mean improved the forecast errors compared with individual control forecasts in the verification against the analysis fields. However, each system exhibited individual characteristics according to the MEP method. Some participants used physical perturbation methods. The significance of these methods was confirmed by the verification. However, the mean error (ME) of the ensemble forecast in some systems was worse than that of the individual control forecast. This result suggests that it is necessary to pay careful attention to physical perturbations.

  10. Determination of parabens in urine samples by microextraction using packed sorbent and ultra-performance liquid chromatography coupled to tandem mass spectrometry.

    PubMed

    Cristina Jardim, Valeria; de Paula Melo, Lidervan; Soares Domingues, Diego; Costa Queiroz, Maria Eugênia

    2015-01-01

    A simple, sensitive, and selective method using ultra-performance liquid chromatography coupled to tandem mass spectrometry (UPLC-MS/MS) was developed and validated for simultaneous determination of parabens [methyl paraben (MeP), ethyl paraben (EtP), propyl paraben (PrP), butyl paraben (BuP), and benzyl paraben (BzP)] in human urine samples. After microextraction by packed sorbent (MEPS) using a C18 phase, the parabens were separated on a Kinetex C18 column (100 mm × 2.1 mm × 1.7 μm) within 4.6 min using isocratic elution. These compounds were detected on a triple quadrupole tandem mass spectrometer using the multiple reactions monitoring (MRM) mode via an electrospray ionization source operating in the negative ionization mode. Important factors that influence MEPS performance were evaluated, such as the sample pH, draw-eject sample volume, clean-up step, and desorption conditions. The proposed MEPS/UPLC-MS/MS method presented a linear range from 0.5 ng mL(-1) (limit of quantification - LOQ) to 50 ng mL(-1), and interassay precision with coefficients of variation lower than 15%, and relative standard error values of the accuracy ranged from -8.8% to 15%. The MEPS/UPLC-MS/MS method was applied successfully to determine parabens in urine samples from 30 postpartum volunteers, enabling assessment of human exposure to these compounds.

  11. Paired Associative Stimulation with High-Frequency Peripheral Component Leads to Enhancement of Corticospinal Transmission at Wide Range of Interstimulus Intervals

    PubMed Central

    Shulga, Anastasia; Zubareva, Aleksandra; Lioumis, Pantelis; Mäkelä, Jyrki P.

    2016-01-01

    Background: In spinal paired associative stimulation (PAS), orthodromic and antidromic volleys elicited by transcranial magnetic stimulation (TMS) and peripheral nerve stimulation (PNS) coincide at corticomotoneuronal synapses at the spinal cord. The interstimulus interval (ISI) between TMS and PNS determines whether PAS leads to motor-evoked potential (MEP) potentiation or depression. PAS applied as a long-term treatment for neurological patients might alter conduction of neural fibers over time. Moreover, measurements of motoneuron conductance for determination of ISIs may be challenging in these patients. Results: We sought to design a PAS protocol to induce MEP potentiation at wide range of ISIs. We tested PAS consisting of high-intensity (100% stimulator output, SO) TMS and high-frequency (50 Hz) PNS in five subjects at five different ISIs. Our protocol induced potentiation of MEP amplitudes in all subjects at all tested intervals. TMS and PNS alone did not result in MEP potentiation. The variant of PAS protocol described here does not require exact adjustment of ISIs in order to achieve effective potentiation of MEPs. Conclusions: This variant of PAS might be feasible as a long-term treatment in rehabilitation of neurological patients. PMID:27721747

  12. Soil Moisture and Vegetation Controls on Surface Energy Balance Using the Maximum Entropy Production Model of Evapotranspiration

    NASA Astrophysics Data System (ADS)

    Wang, J.; Parolari, A.; Huang, S. Y.

    2014-12-01

    The objective of this study is to formulate and test plant water stress parameterizations for the recently proposed maximum entropy production (MEP) model of evapotranspiration (ET) over vegetated surfaces. . The MEP model of ET is a parsimonious alternative to existing land surface parameterizations of surface energy fluxes from net radiation, temperature, humidity, and a small number of parameters. The MEP model was previously tested for vegetated surfaces under well-watered and dry, dormant conditions, when the surface energy balance is relatively insensitive to plant physiological activity. Under water stressed conditions, however, the plant water stress response strongly affects the surface energy balance. This effect occurs through plant physiological adjustments that reduce ET to maintain leaf turgor pressure as soil moisture is depleted during drought. To improve MEP model of ET predictions under water stress conditions, the model was modified to incorporate this plant-mediated feedback between soil moisture and ET. We compare MEP model predictions to observations under a range of field conditions, including bare soil, grassland, and forest. The results indicate a water stress function that combines the soil water potential in the surface soil layer with the atmospheric humidity successfully reproduces observed ET decreases during drought. In addition to its utility as a modeling tool, the calibrated water stress functions also provide a means to infer ecosystem influence on the land surface state. Challenges associated with sampling model input data (i.e., net radiation, surface temperature, and surface humidity) are also discussed.

  13. Effects of high frequency electromagnetic field (EMF) emitted by mobile phones on the human motor cortex.

    PubMed

    Inomata-Terada, Satomi; Okabe, Shingo; Arai, Noritoshi; Hanajima, Ritsuko; Terao, Yasuo; Frubayashi, Toshiaki; Ugawa, Yoshikazu

    2007-10-01

    We investigated whether the pulsed high frequency electromagnetic field (EMF) emitted by a mobile phone has short term effects on the human motor cortex. We measured motor evoked potentials (MEPs) elicited by single pulse transcranial magnetic stimulation (TMS), before and after mobile phone exposure (active and sham) in 10 normal volunteers. Three sites were stimulated (motor cortex (CTX), brainstem (BST) and spinal nerve (Sp)). The short interval intracortical inhibition (SICI) of the motor cortex reflecting GABAergic interneuronal function was also studied by paired pulse TMS method. MEPs to single pulse TMS were also recorded in two patients with multiple sclerosis showing temperature dependent neurological symptoms (hot bath effect). Neither MEPs to single pulse TMS nor the SICI was affected by 30 min of EMF exposure from mobile phones or sham exposure. In two MS patients, mobile phone exposure had no effect on any parameters of MEPs even though conduction block occurred at the corticospinal tracts after taking a bath. As far as available methods are concerned, we did not detect any short-term effects of 30 min mobile phone exposure on the human motor cortical output neurons or interneurons even though we can not exclude the possibility that we failed to detect some mild effects due to a small sample size in the present study. This is the first study of MEPs after electromagnetic exposure from a mobile phone in neurological patients. PMID:17516508

  14. Changes of excitability in M1 induced by neuromuscular electrical stimulation differ between presence and absence of voluntary drive.

    PubMed

    Sugawara, Kenichi; Tanabe, Shigeo; Higashi, Toshio; Tsurumi, Takamasa; Kasai, Tatsuya

    2011-06-01

    The aim of this study is to investigate excitability changes in the human motor cortex induced by variable therapeutic electrical stimulations (TESs) with or without voluntary drive. We recorded motor-evoked potentials (MEPs) from extensor and flexor carpi radialis (FCR) muscles at rest and during FCR muscle contraction after the application of TES on FCR. TES application conditions were changed intensities, frequencies, and trains. In addition, to evaluate the contribution of M1 inhibitory circuits to the effects of TES application, we also recorded MEPs using paired-pulse transcranial magnetic stimulation. In resting muscle states, an increase in TES intensity resulted in an increase in MEP ratio in both the muscles. In contrast, when TES was applied to FCR during contraction, MEP ratios of both the muscles decreased with increased number of pulse trains. However, under both the states, MEP ratios decreased induced by paired-pulse transcranial magnetic stimulation in extensor carpi radialis to which TES was not applied. Excitability changes in M1 induced by TES application were reversibly modulated depending on the presence or absence of voluntary drive. This study showed that the therapy and the voluntary drive of the target muscles act together, and complement the effects of each other, which may be beneficial for optimizing the rehabilitation if the therapy accompanies voluntary drive.

  15. Eating tools in hand activate the brain systems for eating action: a transcranial magnetic stimulation study.

    PubMed

    Yamaguchi, Kaori; Nakamura, Kimihiro; Oga, Tatsuhide; Nakajima, Yasoichi

    2014-07-01

    There is increasing neuroimaging evidence suggesting that visually presented tools automatically activate the human sensorimotor system coding learned motor actions relevant to the visual stimuli. Such crossmodal activation may reflect a general functional property of the human motor memory and thus can be operating in other, non-limb effector organs, such as the orofacial system involved in eating. In the present study, we predicted that somatosensory signals produced by eating tools in hand covertly activate the neuromuscular systems involved in eating action. In Experiments 1 and 2, we measured motor evoked response (MEP) of the masseter muscle in normal humans to examine the possible impact of tools in hand (chopsticks and scissors) on the neuromuscular systems during the observation of food stimuli. We found that eating tools (chopsticks) enhanced the masseter MEPs more greatly than other tools (scissors) during the visual recognition of food, although this covert change in motor excitability was not detectable at the behavioral level. In Experiment 3, we further observed that chopsticks overall increased MEPs more greatly than scissors and this tool-driven increase of MEPs was greater when participants viewed food stimuli than when they viewed non-food stimuli. A joint analysis of the three experiments confirmed a significant impact of eating tools on the masseter MEPs during food recognition. Taken together, these results suggest that eating tools in hand exert a category-specific impact on the neuromuscular system for eating.

  16. The methylerythritol phosphate pathway for isoprenoid biosynthesis in coccidia: presence and sensitivity to fosmidomycin.

    PubMed

    Clastre, Marc; Goubard, Armelle; Prel, Anne; Mincheva, Zoia; Viaud-Massuart, Marie-Claude; Bout, Daniel; Rideau, Marc; Velge-Roussel, Florence; Laurent, Fabrice

    2007-08-01

    The apicoplast is a recently discovered, plastid-like organelle present in most apicomplexa. The methylerythritol phosphate (MEP) pathway involved in isoprenoid biosynthesis is one of the metabolic pathways associated with the apicoplast, and is a new promising therapeutic target in Plasmodium falciparum. Here, we check the presence of isoprenoid genes in four coccidian parasites according to genome database searches. Cryptosporidium parvum and C. hominis, which have no plastid genome, lack the MEP pathway. In contrast, gene expression studies suggest that this metabolic pathway is present in several development stages of Eimeria tenella and in tachyzoites of Toxoplasma gondii. We studied the potential of fosmidomycin, an antimalarial drug blocking the MEP pathway, to inhibit E. tenella and T. gondii growth in vitro. The drug was poorly effective even at high concentrations. Thus, both fosmidomycin sensitivity and isoprenoid metabolism differs substantially between apicomplexan species.

  17. Validation of a protocol to evaluate maximal expiratory pressure using a pressure transducer and a signal conditioner.

    PubMed

    Soares, Viviane; Rodrigues, Fábio B; Vieira, Marcus F; Silva, Maria Sebastiana

    2011-09-01

    The respiratory muscles can present fatigue and even chronic inability to generate force. So, reliable devices are necessary to their evaluation. The aim of this study is to evaluate the MEP (Maximal Expiratory Pressure) values of individuals between 20 and 25 years old and to validate a protocol using a pressure transducer and a signal conditioner comparing it with the digital manometer. We evaluated the MEP of 10 participants. They remained seated and made six respiratory maneuvers from Total Lung Capacity (TLC) to Residual Volume (RV). The results in the study showed no statistically significant differences when compared to values reported in the literature, and that the pressure transducer provides reliable values for MEP. PMID:21969962

  18. Milia En Plaque as a Distinct Follicular Hamartoma With Cystic Trichoepitheliomatous Features

    PubMed Central

    Hashimoto, Akira; Yamasaki, Kenshi; Aiba, Setsuya

    2016-01-01

    Abstract: Milia en plaque (MEP) is an uncommon disorder characterized by an erythematous plaque containing numerous milia. The pathogenesis of MEP is not clear. The authors report a man with an erythematous plaque on the right retroauricular area, containing numerous white-yellow cysts varying in size. Histological examination showed that multiple cystic structures at various levels of the dermis that were lined by stratified squamous epithelium and contained keratinous material—these findings were consistent with the diagnosis of multiple milia. In addition to epidermal cysts, however, the lesion consisted of a branched proliferation of pale-staining keratinocytes lined with basal keratinocytes budding from the overlying epidermis. Moreover, some cysts were formed within the branched epithelial proliferation, had thicker cyst walls than the ordinary milium, or had irregular or branched projections toward the surrounding dermis. From these findings, the authors conclude that MEP is a distinct follicular hamartoma with cystic trichoepitheliomatous features. PMID:26381115

  19. Terpenoids and Their Biosynthesis in Cyanobacteria

    PubMed Central

    Pattanaik, Bagmi; Lindberg, Pia

    2015-01-01

    Terpenoids, or isoprenoids, are a family of compounds with great structural diversity which are essential for all living organisms. In cyanobacteria, they are synthesized from the methylerythritol-phosphate (MEP) pathway, using glyceraldehyde 3-phosphate and pyruvate produced by photosynthesis as substrates. The products of the MEP pathway are the isomeric five-carbon compounds isopentenyl diphosphate and dimethylallyl diphosphate, which in turn form the basic building blocks for formation of all terpenoids. Many terpenoid compounds have useful properties and are of interest in the fields of pharmaceuticals and nutrition, and even potentially as future biofuels. The MEP pathway, its function and regulation, and the subsequent formation of terpenoids have not been fully elucidated in cyanobacteria, despite its relevance for biotechnological applications. In this review, we summarize the present knowledge about cyanobacterial terpenoid biosynthesis, both regarding the native metabolism and regarding metabolic engineering of cyanobacteria for heterologous production of non-native terpenoids. PMID:25615610

  20. Chaotic motif sampler: detecting motifs from biological sequences by using chaotic neurodynamics

    NASA Astrophysics Data System (ADS)

    Matsuura, Takafumi; Ikeguchi, Tohru

    Identification of a region in biological sequences, motif extraction problem (MEP) is solved in bioinformatics. However, the MEP is an NP-hard problem. Therefore, it is almost impossible to obtain an optimal solution within a reasonable time frame. To find near optimal solutions for NP-hard combinatorial optimization problems such as traveling salesman problems, quadratic assignment problems, and vehicle routing problems, chaotic search, which is one of the deterministic approaches, has been proposed and exhibits better performance than stochastic approaches. In this paper, we propose a new alignment method that employs chaotic dynamics to solve the MEPs. It is called the Chaotic Motif Sampler. We show that the performance of the Chaotic Motif Sampler is considerably better than that of the conventional methods such as the Gibbs Site Sampler and the Neighborhood Optimization for Multiple Alignment Discovery.

  1. Effect of transcranial magnetic stimulation on force of finger pinch

    NASA Astrophysics Data System (ADS)

    Odagaki, Masato; Fukuda, Hiroshi; Hiwaki, Osamu

    2009-04-01

    Transcranial magnetic stimulation (TMS) is used to explore many aspects of brain function, and to treat neurological disorders. Cortical motor neuronal activation by TMS over the primary motor cortex (M1) produces efferent signals that pass through the corticospinal tracts. Motor-evoked potentials (MEPs) are observed in muscles innervated by the stimulated motor cortex. TMS can cause a silent period (SP) following MEP in voluntary electromyography (EMG). The present study examined the effects of TMS eliciting MEP and SP on the force of pinching using two fingers. Subjects pinched a wooden block with the thumb and index finger. TMS was applied to M1 during the pinch task. EMG of first dorsal interosseous muscles and pinch forces were measured. Force output increased after the TMS, and then oscillated. The results indicated that the motor control system to keep isotonic forces of the muscles participated in the finger pinch was disrupted by the TMS.

  2. Phase-dependent modulation of corticospinal excitability during the observation of the initial phase of gait.

    PubMed

    Takahashi, Makoto; Uchida, Natsuko; Yoshida, Mami; Liang, Nan; Nakazawa, Kimitaka; Sekikawa, Kiyokazu; Inamizu, Tsutomu; Hamada, Hironobu

    2014-12-01

    This study was undertaken to identify the temporal characteristics of corticospinal excitability of tibialis anterior muscle during the observation of the initial phase of gait. For this purpose, using transcranial magnetic stimulation, we recorded motor evoked potentials (MEPs) during the observation of the second step of an actor's first three steps of gait initiation with (complex gait) or without (normal gait) an obstacle and unstable surface. The results demonstrate that (1) MEPs during the observation of the initial phase of normal gait were significantly increased only at early swing phase, but not other phases (mid-swing, heel contact, mid-stance, and heel off) and (2) MEPs during the observation of the initial phase of complex gait were significantly increased at early swing and also at mid-swing and heel contact phases. These findings provide the first evidence that corticospinal excitability during the observation of gait, especially the initial phase, is modulated in phase- and motor-demanded-dependent manners.

  3. Time-dependent changes in motor cortical excitability by electrical stimulation combined with voluntary drive.

    PubMed

    Sugawara, Kenichi; Yamaguchi, Tomofumi; Tanabe, Shigeo; Suzuki, Tomotaka; Saito, Kei; Higashi, Toshio

    2014-04-16

    Prolonged changes in primary motor cortex excitability in response to combined neuromuscular electrical stimulation (NMES) and voluntary contraction with motor evoked potentials (MEPs) were investigated by transcranial magnetic stimulation and recorded by mechanomyography. Participants included 22 healthy individuals. NMES was applied to the extensor carpi radialis (ECR) by voluntary ECR contraction with 20% maximum voluntary contraction (MVC) of wrist extension. MEPs were recorded from the flexor carpi radialis (FCR) and ECR at rest with NMES, at 20% MVC with NMES (combined), and at 20% MVC alone. Significant conditional effects were revealed in ECR and FCR. In the combined condition, MEPs showed gradual enhancement, and those in FCR were more inhibited than those in the control condition. Voluntary contraction with NMES increased primary motor cortex excitability in the agonist muscle, whereas the antagonist muscle might affect reciprocal modulation in the combined condition. PMID:24356108

  4. Evolution of Electrogenic Ammonium Transporters (AMTs).

    PubMed

    McDonald, Tami R; Ward, John M

    2016-01-01

    The ammonium transporter gene family consists of three main clades, AMT, MEP, and Rh. The evolutionary history of the AMT/MEP/Rh gene family is characterized by multiple horizontal gene transfer events, gene family expansion and contraction, and gene loss; thus the gene tree for this family of transporters is unlike the organismal tree. The genomes of angiosperms contain genes for both electrogenic and electroneutral ammonium transporters, but it is not clear how far back in the land plant lineage electrogenic ammonium transporters occur. Here, we place Marchantia polymorpha ammonium transporters in the AMT/MEP/Rh phylogeny and we show that AMTs from the liverwort M. polymorpha are electrogenic. This information suggests that electrogenic ammonium transport evolved at least as early as the divergence of bryophytes in the land plant lineage. PMID:27066024

  5. Evolution of Electrogenic Ammonium Transporters (AMTs)

    PubMed Central

    McDonald, Tami R.; Ward, John M.

    2016-01-01

    The ammonium transporter gene family consists of three main clades, AMT, MEP, and Rh. The evolutionary history of the AMT/MEP/Rh gene family is characterized by multiple horizontal gene transfer events, gene family expansion and contraction, and gene loss; thus the gene tree for this family of transporters is unlike the organismal tree. The genomes of angiosperms contain genes for both electrogenic and electroneutral ammonium transporters, but it is not clear how far back in the land plant lineage electrogenic ammonium transporters occur. Here, we place Marchantia polymorpha ammonium transporters in the AMT/MEP/Rh phylogeny and we show that AMTs from the liverwort M. polymorpha are electrogenic. This information suggests that electrogenic ammonium transport evolved at least as early as the divergence of bryophytes in the land plant lineage. PMID:27066024

  6. Removing External Degrees of Freedom from Transition-State Search Methods using Quaternions.

    PubMed

    Melander, Marko; Laasonen, Kari; Jónsson, Hannes

    2015-03-10

    In finite systems, such as nanoparticles and gas-phase molecules, calculations of minimum energy paths (MEPs) connecting initial and final states of transitions as well as searches for saddle points are complicated by the presence of external degrees of freedom, such as overall translation and rotation. A method based on quaternion algebra for removing the external degrees of freedom is described here and applied in calculations using two commonly used methods: the nudged elastic band (NEB) method for MEPs and the DIMER method for finding the minimum mode in minimum mode following searches of first-order saddle points. With the quaternion approach, fewer images in the NEB are needed to represent MEPs accurately. In both NEB and DIMER calculations of finite systems, the number of iterations required to reach convergence is significantly reduced. The algorithms have been implemented in the Atomic Simulation Environment (ASE) open source software. PMID:26579757

  7. The Thermodynamics of Marine Biogeochemical Cycles: Lotka Revisited

    NASA Astrophysics Data System (ADS)

    Vallino, Joseph J.; Algar, Christopher K.

    2016-01-01

    Nearly 100 years ago, Alfred Lotka published two short but insightful papers describing how ecosystems may organize. Principally, Lotka argued that ecosystems will grow in size and that their cycles will spin faster via predation and nutrient recycling so as to capture all available energy, and that evolution and natural selection are the mechanisms by which this occurs and progresses. Lotka's ideas have often been associated with the maximum power principle, but they are more consistent with recent developments in nonequilibrium thermodynamics, which assert that complex systems will organize toward maximum entropy production (MEP). In this review, we explore Lotka's hypothesis within the context of the MEP principle, as well as how this principle can be used to improve marine biogeochemistry models. We need to develop the equivalent of a climate model, as opposed to a weather model, to understand marine biogeochemistry on longer timescales, and adoption of the MEP principle can help create such models.

  8. Molecular basis of the low activity of antitumor anthracenediones, mitoxantrone and ametantrone, in oxygen radical generation catalyzed by NADH dehydrogenase. Enzymatic and molecular modelling studies.

    PubMed

    Tarasiuk, Jolanta; Mazerski, Jan; Tkaczyk-Gobis, Katarzyna; Borowski, Edward

    2005-04-01

    Synthetic antitumor anthracenedione drugs, in contrast to anthracycline antibiotics, are ineffective in free radical formation in NADH dehydrogenase system. Our results have indicated that neither the reduction potential nor the side chain conformation and the energies of border orbitals (HOMO and LUMO) determine the ability of anthracenediones to stimulate reactive oxygen species formation in NADH dehydrogenase system. It was shown that the distribution of the molecular electrostatic potential (MEP), around the quinone system was crucial for this ability. We have found for non-stimulating anthracenediones that the clouds of positive MEP cover the quinone carbon atoms while for agents effective in stimulating reactive oxygen species formation the clouds of negative MEP cover continuously the aromatic core together with the quinone system.

  9. Methylerythritol Phosphate Pathway of Isoprenoid Biosynthesis

    PubMed Central

    Zhao, Lishan; Chang, Wei-chen; Xiao, Youli; Liu, Hung-wen; Liu, Pinghua

    2016-01-01

    Isoprenoids are a class of natural products with more than 50,000 members. All isoprenoids are constructed from two precursors, isopentenyl diphosphate (IPP) and its isomer dimethylallyl diphosphate (DMAPP). Two of the most important discoveries in isoprenoid biosynthetic studies in recent years are the elucidation of a second isoprenoid biosynthetic pathway (the methylerythritol phosphate (MEP) pathway) and a modified mevalonate (MVA) pathway. In this review, mechanistic insights on the MEP pathway enzymes are summarized. Since many isoprenoids have important biological activities, the need to produce them in sufficient quantities for downstream research efforts or commercial application is apparent. Recent advances in both the MVA and MEP pathway-based synthetic biology efforts are also illustrated by reviewing the landmark work of artemisinic acid and taxadien-5α-ol production through microbial fermentations. PMID:23746261

  10. Selective suppression of the incorrect response implementation in choice behavior assessed by transcranial magnetic stimulation.

    PubMed

    Tandonnet, Christophe; Garry, Michael I; Summers, Jeffery J

    2011-04-01

    Selecting the adequate alternative in choice situations may involve an inhibition process. Here we assessed response implementation during the reaction time of a between-hand choice task with single- or paired-pulse (3 or 15 ms interstimulus intervals [ISIs]) transcranial magnetic stimulation of the motor cortex. The amplitude of the single-pulse motor evoked potential (MEP) initially increased for both hands. At around 130 ms, the single-pulse MEP kept increasing for the responding hand and decreased for the nonresponding hand. The paired-pulse MEP revealed a similar pattern for both ISIs with no effect on short intracortical inhibition and intracortical facilitation measures. The results suggest that the incorrect response implementation was selectively suppressed before execution of the correct response, preventing errors in choice context. The results favor models assuming that decision making involves an inhibition process.

  11. Emotional stimuli modulate readiness for action: a transcranial magnetic stimulation study.

    PubMed

    van Loon, Anouk M; van den Wildenberg, Wery P M; van Stegeren, Anda H; Hajcak, Greg; Ridderinkhof, K Richard

    2010-05-01

    Emotional stimuli may prime the motor system and facilitate action readiness. Direct evidence for this effect has been shown by recent studies using transcranial magnetic stimulation (TMS). When administered over the primary motor cortex involved in responding, TMS pulses elicit motor-evoked potentials (MEPs) in the represented muscles. The amplitudes of these MEPs reflect the state of corticospinal excitability. Here, we investigated the dynamic effects of induced emotions on action readiness, as reflected by corticospinal excitability. Subjects performed a choice task while viewing task-irrelevant emotional and neutral pictures. The pattern of MEP amplitudes showed a typical increase as the TMS pulse was presented closer in time to the imminent response. This dynamic pattern was amplified by both pleasant and unpleasant emotional stimuli, but more so when unpleasant pictures were viewed. These patterns present novel evidence in support of the notion that emotional stimuli modulate action readiness.

  12. Development and Characterization of Healable Carbon Fiber Composites with a Reversibly Cross Linked Polymer

    SciTech Connect

    Ghezzo, Fabrizia; Smith, David R.; Starr, Tatiana N.; Perram, Timothy; Starr, Anthony F.; Darlington, Thomas K.; Baldwin, Richard K.; Oldenburg, Steven J.

    2010-10-18

    Carbon fiber reinforced polymer (CFRP) laminates with remendable cross-linked polymeric matrices were fabricated using a modified resin transfer mold (RTM) technique. The healable composite resin, bis-maleimide tetrafuran (2MEP4F), was synthesized by mixing two monomers, furan (4F) and maleimide (2MEP), at elevated temperatures. The fast kinetic rate of the reaction of polymer constituents requires a fast injection of the healable resin into the carbon fiber preform. The polymer viscosity as a function of time and temperature was experimentally quantified in order to optimize the fabrication of the composite material and to guarantee a uniform flow of the resin through the reinforcement. The method was validated by characterizing the thermo-mechanical properties of the polymerized 2MEP4F. Additionally, the thermo-mechanical properties of the remendable CFRP material were studied.

  13. Nudged-elastic band method with two climbing images: Finding transition states in complex energy landscapes

    DOE PAGES

    Zarkevich, Nikolai A.; Johnson, Duane D.

    2015-01-09

    The nudged-elastic band (NEB) method is modified with concomitant two climbing images (C2-NEB) to find a transition state (TS) in complex energy landscapes, such as those with a serpentine minimal energy path (MEP). If a single climbing image (C1-NEB) successfully finds the TS, then C2-NEB finds it too. Improved stability of C2-NEB makes it suitable for more complex cases, where C1-NEB misses the TS because the MEP and NEB directions near the saddle point are different. Generally, C2-NEB not only finds the TS, but guarantees, by construction, that the climbing images approach it from the opposite sides along the MEP.more » In addition, C2-NEB provides an accuracy estimate from the three images: the highest-energy one and its climbing neighbors. C2-NEB is suitable for fixed-cell NEB and the generalized solid-state NEB.« less

  14. Nudged-elastic band method with two climbing images: Finding transition states in complex energy landscapes

    SciTech Connect

    Zarkevich, Nikolai A.; Johnson, Duane D.

    2015-01-09

    The nudged-elastic band (NEB) method is modified with concomitant two climbing images (C2-NEB) to find a transition state (TS) in complex energy landscapes, such as those with a serpentine minimal energy path (MEP). If a single climbing image (C1-NEB) successfully finds the TS, then C2-NEB finds it too. Improved stability of C2-NEB makes it suitable for more complex cases, where C1-NEB misses the TS because the MEP and NEB directions near the saddle point are different. Generally, C2-NEB not only finds the TS, but guarantees, by construction, that the climbing images approach it from the opposite sides along the MEP. In addition, C2-NEB provides an accuracy estimate from the three images: the highest-energy one and its climbing neighbors. C2-NEB is suitable for fixed-cell NEB and the generalized solid-state NEB.

  15. Influence of mevinolin on chloroplast terpenoids in Cannabis sativa.

    PubMed

    Mansouri, Hakimeh; Salari, Fatemeh

    2014-04-01

    Plants synthesize a myriad of isoprenoid products that are required both for essential constitutive processes and for adaptive responses to the environment. Two independent pathways for the biosynthesis of isoprenoid precursors coexist within the plant cell: the cytosolic mevalonic acid (MVA) pathway and the plastidial methylerythritol phosphate (MEP) pathway. In this study, we investigated the inhibitory effect of the MVA pathway on isoprenoid biosynthesized by the MEP pathway in Cannabis sativa by treatment with mevinolin. The amount of chlorophyll a, b, and total showed to be significantly enhanced in treated plants in comparison with control plants. Also, mevinolin induced the accumulation of carotenoids and α-tocopherol in treated plants. Mevinolin caused a significant decrease in tetrahydrocannabinol (THC) content. This result show that the inhibition of the MVA pathway stimulates MEP pathway but none for all metabolites.

  16. The Thermodynamics of Marine Biogeochemical Cycles: Lotka Revisited.

    PubMed

    Vallino, Joseph J; Algar, Christopher K

    2016-01-01

    Nearly 100 years ago, Alfred Lotka published two short but insightful papers describing how ecosystems may organize. Principally, Lotka argued that ecosystems will grow in size and that their cycles will spin faster via predation and nutrient recycling so as to capture all available energy, and that evolution and natural selection are the mechanisms by which this occurs and progresses. Lotka's ideas have often been associated with the maximum power principle, but they are more consistent with recent developments in nonequilibrium thermodynamics, which assert that complex systems will organize toward maximum entropy production (MEP). In this review, we explore Lotka's hypothesis within the context of the MEP principle, as well as how this principle can be used to improve marine biogeochemistry models. We need to develop the equivalent of a climate model, as opposed to a weather model, to understand marine biogeochemistry on longer timescales, and adoption of the MEP principle can help create such models.

  17. Phosphatidylinositol 4,5-bisphosphate formation in rabbit skeletal and heart muscle membranes.

    PubMed

    Varsányi, M; Messer, M; Brandt, N R; Heilmeyer, L M

    1986-08-14

    Incubation of rabbit skeletal muscle microsomes or isolated triads with gamma 32P-ATP/Mg2+ in the absence and in the presence of added phosphatidylinositol resulted in the formation of phosphatidylinositol 4-phosphate catalyzed by phosphatidylinositol kinase. When phosphatidylinositol 4-phosphate was added as exogenous substrate, phosphatidylinositol 4,5-bisphosphate was also formed demonstrating the presence of a membrane bound phosphatidylinositol 4-phosphate kinase. Triads were broken mechanically in a French press and separated on a continuous sucrose gradient. Incubation of these fractions with gamma 32P-ATP/Mg2+ resulted in a rapid labeling of phospholipid in a membrane fraction banding between transverse tubules and the terminal cisternae. Partial triad breakage and triad reformation experiments indicated that this phosphatidylinositol kinase was associated with T-tubules. When exogenous phosphatidylinositol 4-phosphate was employed as substrate phosphatidylinositol 4,5-bisphosphate and phosphatidic acid were formed, indicating the presence of all the enzymes of the polyphosphoinositide signaling system in this special membrane fraction. In contrast, heart muscle microsomes or plasma membranes can catalyze this reaction sequence from endogenous formed phosphatidylinositol 4-phosphate.

  18. Investigation of electrical responses to acupuncture stimulation: the effect of electrical grounding and insulation conditions.

    PubMed

    Lee, Yong-Heum; Ryu, Yeon-Hang; Jung, Byungjo

    2009-03-01

    Acupuncture in Oriental medicine has been widely used as a core therapeutic method due to its minimal side-effects and therapeutic efficacy. However, the electrical response to acupuncture stimulation (ERAS) has not been clearly studied under acupuncture conditions that might affect the efficacy of acupuncture therapy. In this study, the ERAS was objectively investigated by measuring meridian electric potentials (MEPs) when the electrical grounding conditions of the operator and subject were varied, and when the insulation conditions of acupuncture needle were varied. MEPs between Sang-geoheo (ST37) and Ha-geoheo (ST39) of the Stomach Meridian (ST) were measured by stimulating Jok-samni (ST36) with an acupuncture needle. For non-insulated acupuncture stimulation (NIAS), the average MEP peak was 148.6 +/- 20.6 when neither the operator nor the subject were electrically grounded, 23.1 +/- 8.8 when the subject only was electrically grounded, 348 +/- 76.8 when the operator only was electrically grounded, and 19.9 +/- 4.7 when both the operator and the subject were electrically grounded. The MEPs presented various magnitudes and patterns depending on the electrical grounding conditions. The MEP pattern was very similar to that of the charge and discharge of a capacitor. For insulated acupuncture stimulation (IAS), the average MEP peak was 20 +/- 4 in all electrical grounding conditions, which is not a significant electric response for acupuncture stimulation. In terms of electricity, this study verified that acupuncture therapy might be affected by acupuncture conditions such as (1) the electrical grounding condition of the operator and the subject and (2) the insulation condition of the acupuncture needle.

  19. Maggot excretion products from the blowfly Lucilia sericata contain contact phase/intrinsic pathway-like proteases with procoagulant functions.

    PubMed

    Kahl, M; Gökçen, A; Fischer, S; Bäumer, M; Wiesner, J; Lochnit, G; Wygrecka, M; Vilcinskas, A; Preissner, K T

    2015-08-01

    For centuries, maggots have been used for the treatment of wounds by a variety of ancient cultures, as part of their traditional medicine. With increasing appearance of antimicrobial resistance and in association with diabetic ulcers, maggot therapy was revisited in the 1980s. Three mechanisms by which sterile maggots of the green bottle fly Lucilia sericata may improve healing of chronic wounds have been proposed: Biosurgical debridement, disinfecting properties, and stimulation of the wound healing process. However, the influence of maggot excretion products (MEP) on blood coagulation as part of the wound healing process has not been studied in detail. Here, we demonstrate that specific MEP-derived serine proteases from Lucilia sericata induce clotting of human plasma and whole blood, particularly by activating contact phase proteins factor XII and kininogen as well as factor IX, thereby providing kallikrein-bypassing and factor XIa-like activities, both in plasma and in isolated systems. In plasma samples deficient in contact phase proteins, MEP restored full clotting activity, whereas in plasma deficient in either factor VII, IX, X or II no effect was seen. The observed procoagulant/intrinsic pathway-like activity was mediated by (chymo-) trypsin-like proteases in total MEP, which were significantly blocked by C1-esterase inhibitor or other contact phase-specific protease inhibitors. No significant influence of MEP on platelet activation or fibrinolysis was noted. Together, MEP provides contact phase bypassing procoagulant activity and thereby induces blood clotting in the context of wound healing. Further characterisation of the active serine protease(s) may offer new perspectives for biosurgical treatment of chronic wounds. PMID:25948398

  20. Maximum Entropy Production Modeling of Evapotranspiration Partitioning on Heterogeneous Terrain and Canopy Cover: advantages and limitations.

    NASA Astrophysics Data System (ADS)

    Gutierrez-Jurado, H. A.; Guan, H.; Wang, J.; Wang, H.; Bras, R. L.; Simmons, C. T.

    2015-12-01

    Quantification of evapotranspiration (ET) and its partition over regions of heterogeneous topography and canopy poses a challenge using traditional approaches. In this study, we report the results of a novel field experiment design guided by the Maximum Entropy Production model of ET (MEP-ET), formulated for estimating evaporation and transpiration from homogeneous soil and canopy. A catchment with complex terrain and patchy vegetation in South Australia was instrumented to measure temperature, humidity and net radiation at soil and canopy surfaces. Performance of the MEP-ET model to quantify transpiration and soil evaporation was evaluated during wet and dry conditions with independently and directly measured transpiration from sapflow and soil evaporation using the Bowen Ratio Energy Balance (BREB). MEP-ET transpiration shows remarkable agreement with that obtained through sapflow measurements during wet conditions, but consistently overestimates the flux during dry periods. However, an additional term introduced to the original MEP-ET model accounting for higher stomatal regulation during dry spells, based on differences between leaf and air vapor pressure deficits and temperatures, significantly improves the model performance. On the other hand, MEP-ET soil evaporation is in good agreement with that from BREB regardless of moisture conditions. The experimental design allows a plot and tree scale quantification of evaporation and transpiration respectively. This study confirms for the first time that the MEP-ET originally developed for homogeneous open bare soil and closed canopy can be used for modeling ET over heterogeneous land surfaces. Furthermore, we show that with the addition of an empirical function simulating the plants ability to regulate transpiration, and based on the same measurements of temperature and humidity, the method can produce reliable estimates of ET during both wet and dry conditions without compromising its parsimony.

  1. Corticomotor excitability of back muscles is affected by intervertebral disc lesion in pigs.

    PubMed

    Hodges, Paul W; Galea, Mary P; Holm, Sten; Holm, Allison Kaigle

    2009-04-01

    Morphological and behavioural changes in back muscles are common in back pain and injury. Recent data indicate a rapid reduction in the size of the multifidus, a deep back muscle, within 3 days of experimental intervertebral disc (IVD) injury in pigs. A reduced neural drive may contribute to this. We investigated changes in corticomotor excitability following IVD lesion by evaluation of the response of back muscles to electrical stimulation of the motor cortex. Motor evoked potentials (MEPs) were studied in 12 Swedish landrace pigs before injury, immediately after abdominal incision, immediately after L3-4 IVD lesion with a scalpel, and 15 min later. In two animals, responses were also evoked by descending volleys excited at the level of the mastoid processes (cervicomedullary evoked potentials) without motor cortex activation. In five animals, a sham procedure was followed without IVD lesion. MEPs were recorded in short (deep) and long (superficial) fibres of the multifidus at L3-5 on the lesioned side and at L4 contralaterally with intramuscular wire electrodes. Although the MEP amplitude increased in several muscles after incision, at 15 min after IVD lesion only the MEP amplitude of the deep L4 multifidus on the lesioned side was increased [36% (SD 15%), P < 0.05]. There were no changes in MEP amplitude after 15 min at adjacent or contralateral levels. The response to cervicomedullary stimulation reduced slightly. This suggests that the increased MEP amplitude was due to changes in cortical excitability. These data indicate that IVD lesion induces localized increases, and not decreases, in the excitability of cortical inputs to the deep paraspinal muscles that cross a lesioned disc. PMID:19519631

  2. Urinary excretion and daily intake rates of diethyl phthalate in the general Canadian population.

    PubMed

    Saravanabhavan, Gurusankar; Walker, Mike; Guay, Mireille; Aylward, Lesa

    2014-12-01

    We have analyzed the trends in the body-weight-adjusted urinary monoethyl phthalate (MEP) concentrations and the diethyl ethyl phthalate (DEP) daily intake estimates in the general Canadian population (aged 6-49 years) using the Canadian Health Measures Survey 2007-2009 dataset. The creatinine correction approach, as well as the urine volume approach in a simple one compartment model were used to calculate the daily urinary MEP excretion rates and DEP intake rates in individual survey participants. Using multiple regression models, we have estimated least square geometric means (LSGMs) of body-weight-adjusted MEP concentration, daily excretion and intake rates among different age groups and sex. We observed that body weight affects the trends in the MEP concentrations significantly among children (aged 6-11 years), adolescents (aged 12-19 years) and adults (aged 20-49 years). The body-weight-adjusted MEP concentrations in children were significantly higher than those in adults. On the other hand the DEP daily intakes in children were significantly lower than those in adults. We did not observe any differences in the DEP daily intake rates between males and females. Although the urinary MEP concentrations are correlated well with DEP daily intake estimates in the overall population, one should be cautious when directly using the urinary concentrations to compare the intake trends in the sub-populations (e.g. children vs. adults) as these trends are governed by additional physiological factors. The DEP daily intake calculated using the creatinine approach and that using the urine volume approach were similar to each other. The estimated geometric mean and 95th percentile of DEP daily intake in the general Canadian population are 2 and 20 μg/kg-bw/day, respectively. These daily intake estimates are significantly lower than the US Environmental Protection Agency's oral reference dose of 800 μg/kg-bw/day. PMID:25217994

  3. Abnormal motor cortex excitability during linguistic tasks in adductor-type spasmodic dysphonia.

    PubMed

    Suppa, A; Marsili, L; Giovannelli, F; Di Stasio, F; Rocchi, L; Upadhyay, N; Ruoppolo, G; Cincotta, M; Berardelli, A

    2015-08-01

    In healthy subjects (HS), transcranial magnetic stimulation (TMS) applied during 'linguistic' tasks discloses excitability changes in the dominant hemisphere primary motor cortex (M1). We investigated 'linguistic' task-related cortical excitability modulation in patients with adductor-type spasmodic dysphonia (ASD), a speech-related focal dystonia. We studied 10 ASD patients and 10 HS. Speech examination included voice cepstral analysis. We investigated the dominant/non-dominant M1 excitability at baseline, during 'linguistic' (reading aloud/silent reading/producing simple phonation) and 'non-linguistic' tasks (looking at non-letter strings/producing oral movements). Motor evoked potentials (MEPs) were recorded from the contralateral hand muscles. We measured the cortical silent period (CSP) length and tested MEPs in HS and patients performing the 'linguistic' tasks with different voice intensities. We also examined MEPs in HS and ASD during hand-related 'action-verb' observation. Patients were studied under and not-under botulinum neurotoxin-type A (BoNT-A). In HS, TMS over the dominant M1 elicited larger MEPs during 'reading aloud' than during the other 'linguistic'/'non-linguistic' tasks. Conversely, in ASD, TMS over the dominant M1 elicited increased-amplitude MEPs during 'reading aloud' and 'syllabic phonation' tasks. CSP length was shorter in ASD than in HS and remained unchanged in both groups performing 'linguistic'/'non-linguistic' tasks. In HS and ASD, 'linguistic' task-related excitability changes were present regardless of the different voice intensities. During hand-related 'action-verb' observation, MEPs decreased in HS, whereas in ASD they increased. In ASD, BoNT-A improved speech, as demonstrated by cepstral analysis and restored the TMS abnormalities. ASD reflects dominant hemisphere excitability changes related to 'linguistic' tasks; BoNT-A returns these excitability changes to normal.

  4. Differences in Supraspinal and Spinal Excitability during Various Force Outputs of the Biceps Brachii in Chronic- and Non-Resistance Trained Individuals

    PubMed Central

    Pearcey, Gregory E. P.; Power, Kevin E.; Button, Duane C.

    2014-01-01

    Motor evoked potentials (MEP) and cervicomedullary evoked potentials (CMEP) may help determine the corticospinal adaptations underlying chronic resistance training-induced increases in voluntary force production. The purpose of the study was to determine the effect of chronic resistance training on corticospinal excitability (CE) of the biceps brachii during elbow flexion contractions at various intensities and the CNS site (i.e. supraspinal or spinal) predominantly responsible for any training-induced differences in CE. Fifteen male subjects were divided into two groups: 1) chronic resistance-trained (RT), (n = 8) and 2) non-RT, (n = 7). Each group performed four sets of ∼5 s elbow flexion contractions of the dominant arm at 10 target forces (from 10%–100% MVC). During each contraction, subjects received 1) transcranial magnetic stimulation, 2) transmastoid electrical stimulation and 3) brachial plexus electrical stimulation, to determine MEP, CMEP and compound muscle action potential (Mmax) amplitudes, respectively, of the biceps brachii. All MEP and CMEP amplitudes were normalized to Mmax. MEP amplitudes were similar in both groups up to 50% MVC, however, beyond 50% MVC, MEP amplitudes were lower in the chronic RT group (p<0.05). CMEP amplitudes recorded from 10–100% MVC were similar for both groups. The ratio of MEP amplitude/absolute force and CMEP amplitude/absolute force were reduced (p<0.012) at all contraction intensities from 10–100% MVC in the chronic-RT compared to the non-RT group. In conclusion, chronic resistance training alters supraspinal and spinal excitability. However, adaptations in the spinal cord (i.e. motoneurone) seem to have a greater influence on the altered CE. PMID:24875495

  5. Astacin proteases cleave dentin sialophosphoprotein (Dspp) to generate dentin phosphoprotein (Dpp).

    PubMed

    Tsuchiya, Shuhei; Simmer, James P; Hu, Jan C-C; Richardson, Amelia S; Yamakoshi, Fumiko; Yamakoshi, Yasuo

    2011-01-01

    Dentin sialophosphoprotein (Dspp) is critical for proper dentin biomineralization because genetic defects in DSPP cause dentin dysplasia type II and dentinogenesis imperfecta types II and III. Dspp is processed by proteases into smaller subunits; the initial cleavage releases dentin phosphoprotein (Dpp). We incubated fluorescence resonance energy transfer (FRET) peptides containing the amino acid context of the Dpp cleavage site (YEFDGKSMQGDDPN, designated Dspp-FRET) or a mutant version of that context (YEFDGKSIEGDDPN, designated mutDspp-FRET) with BMP-1, MEP1A, MEP1B, MMP-2, MMP-8, MMP-9, MT1-MMP, MT3-MMP, Klk4, MMP-20, plasmin, or porcine Dpp and characterized the peptide cleavage products. Only BMP-1, MEP1A, and MEP1B cleaved Dspp-FRET at the G-D peptide bond that releases Dpp from Dspp in vivo. We isolated Dspp proteoglycan from dentin power and incubated it with the three enzymes that cleaved Dspp-FRET at the G-D bond. In each case, the released Dpp domain was isolated, and its N-terminus was characterized by Edman degradation. BMP-1 and MEP1A both cleaved native Dspp at the correct site to generate Dpp, making both these enzymes prime candidates for the protease that cleaves Dspp in vivo. MEP1B was able to degrade Dpp when the Dpp was at sufficiently high concentration to deplete free calcium ion concentration. Immunohistochemistry of developing porcine molars demonstrated that astacins are expressed by odontoblasts, a result that is consistent with RT-PCR analyses. We conclude that during odontogenesis, astacins in the predentin matrix cleave Dspp before the DDPN sequence at the N-terminus of Dpp to release Dpp from the parent Dspp protein.

  6. Maggot excretion products from the blowfly Lucilia sericata contain contact phase/intrinsic pathway-like proteases with procoagulant functions.

    PubMed

    Kahl, M; Gökçen, A; Fischer, S; Bäumer, M; Wiesner, J; Lochnit, G; Wygrecka, M; Vilcinskas, A; Preissner, K T

    2015-08-01

    For centuries, maggots have been used for the treatment of wounds by a variety of ancient cultures, as part of their traditional medicine. With increasing appearance of antimicrobial resistance and in association with diabetic ulcers, maggot therapy was revisited in the 1980s. Three mechanisms by which sterile maggots of the green bottle fly Lucilia sericata may improve healing of chronic wounds have been proposed: Biosurgical debridement, disinfecting properties, and stimulation of the wound healing process. However, the influence of maggot excretion products (MEP) on blood coagulation as part of the wound healing process has not been studied in detail. Here, we demonstrate that specific MEP-derived serine proteases from Lucilia sericata induce clotting of human plasma and whole blood, particularly by activating contact phase proteins factor XII and kininogen as well as factor IX, thereby providing kallikrein-bypassing and factor XIa-like activities, both in plasma and in isolated systems. In plasma samples deficient in contact phase proteins, MEP restored full clotting activity, whereas in plasma deficient in either factor VII, IX, X or II no effect was seen. The observed procoagulant/intrinsic pathway-like activity was mediated by (chymo-) trypsin-like proteases in total MEP, which were significantly blocked by C1-esterase inhibitor or other contact phase-specific protease inhibitors. No significant influence of MEP on platelet activation or fibrinolysis was noted. Together, MEP provides contact phase bypassing procoagulant activity and thereby induces blood clotting in the context of wound healing. Further characterisation of the active serine protease(s) may offer new perspectives for biosurgical treatment of chronic wounds.

  7. Modulation of soleus corticospinal excitability during Achilles tendon vibration.

    PubMed

    Lapole, Thomas; Temesi, John; Arnal, Pierrick J; Gimenez, Philippe; Petitjean, Michel; Millet, Guillaume Y

    2015-09-01

    Soleus (SOL) corticospinal excitability has been reported to increase during Achilles tendon vibration. The aim of the present study was to further investigate SOL corticospinal excitability and elucidate the changes to intracortical mechanisms during Achilles tendon vibration. Motor-evoked potentials (MEPs) were elicited in the SOL by transcranial magnetic stimulation (TMS) of the corresponding motor cortical area of the leg with and without 50-Hz Achilles tendon vibration. SOL input-output curves were determined. Paired-pulse protocols were also performed to investigate short-interval intracortical inhibition (SICI) and intracortical facilitation (ICF) by conditioning test TMS pulses with sub-threshold TMS pulses at inter-stimulus intervals of 3 and 13 ms, respectively. During Achilles tendon vibration, motor threshold was lower than in the control condition (43 ± 13 vs. 49 ± 11 % of maximal stimulator output; p = 0.008). Input-output curves were also influenced by vibration, i.e. there was increased maximal MEP amplitude (0.694 ± 0.347 vs. 0.268 ± 0.167 mV; p < 0.001), decreased TMS intensity to elicit a MEP of half the maximal MEP amplitude (100 ± 13 vs. 109 ± 9 % motor threshold; p = 0.009) and a strong tendency for decreased slope constant (0.076 ± 0.04 vs. 0.117 ± 0.04; p = 0.068). Vibration reduced ICF (98 ± 61 vs. 170 ± 105 % of test MEP amplitude; p = 0.05), but had no effect on SICI (53 ± 26 vs. 48 ± 22 % of test MEP amplitude; p = 0.68). The present results further document the increased vibration-induced corticospinal excitability in the soleus muscle and suggest that this increase is not mediated by changes in SICI or ICF.

  8. Acute effects of muscle vibration on sensorimotor integration.

    PubMed

    Lapole, Thomas; Tindel, Jérémy

    2015-02-01

    Projections from the somesthetic cortex are believed to be involved in the modulation of motor cortical excitability by muscle vibration. The aim of the present pilot study was to analyse the effects of a vibration intervention on short-latency afferent inhibition (SAI), long-latency afferent inhibition (LAI), and afferent facilitation (AF), three intracortical mechanisms reflecting sensorimotor integration. Abductor pollicis brevis (APB) SAI, AF and LAI were investigated on 10 subjects by conditioning test transcranial magnetic stimulation pulses with median nerve electrical stimulation at inter-stimuli intervals in the range 15-25 ms, 25-60 ms, and 100-200 ms, respectively. Test motor evoked potentials (MEPs) were compared to unconditioned MEPs. Measurements were performed before and just after 15 min of vibration applied to the muscle belly of APB at a frequency of 80 Hz. SAI and LAI responses were significantly reduced compared to unconditioned test MEPs (P=0.039 and P<0.001, respectively). AF MEP amplitude was greater than SAI and LAI one (P=0.009 and P=0.004, respectively), but not different from test MEP (P=0.511). There was no significant main effect of vibration (P=0.905). However, 4 subjects were clearly identified as responders. Their mean vibration-induced increase was 324 ± 195% in APB SAI MEP amplitude, and 158 ± 53% and 319 ± 80% in AF and LAI, respectively. Significant differences in SAI, AF and LAI vibration-induced changes were found for responders when compared to non-responders (P=0.019, P=0.038, and P=0.01, respectively). A single session of APB vibration may increase sensorimotor integration, via decreased inhibition and increased facilitation. However, such results were not observed for all subjects, suggesting that other factors (such as attention to the sensory inputs) may have played a role.

  9. Pre-stimulus Alpha Oscillations and Inter-subject Variability of Motor Evoked Potentials in Single- and Paired-Pulse TMS Paradigms

    PubMed Central

    Iscan, Zafer; Nazarova, Maria; Fedele, Tommaso; Blagovechtchenski, Evgeny; Nikulin, Vadim V.

    2016-01-01

    Inter- and intra-subject variability of the motor evoked potentials (MEPs) to TMS is a well-known phenomenon. Although a possible link between this variability and ongoing brain oscillations was demonstrated, the results of the studies are not consistent with each other. Exploring this topic further is important since the modulation of MEPs provides unique possibility to relate oscillatory cortical phenomena to the state of the motor cortex probed with TMS. Given that alpha oscillations were shown to reflect cortical excitability, we hypothesized that their power and variability might explain the modulation of subject-specific MEPs to single- and paired-pulse TMS (spTMS, ppTMS, respectively). Neuronal activity was recorded with multichannel electroencephalogram. We used spTMS and two ppTMS conditions: intracortical facilitation (ICF) and short-interval intracortical inhibition (SICI). Spearman correlations were calculated within and across subjects between MEPs and the pre-stimulus power of alpha oscillations in low (8–10 Hz) and high (10–12 Hz) frequency bands. Coefficient of quartile variation was used to measure variability. Across-subject analysis revealed no difference in the pre-stimulus alpha power among the TMS conditions. However, the variability of high-alpha power in spTMS condition was larger than in the SICI condition. In ICF condition pre-stimulus high-alpha power variability correlated positively with MEP amplitude variability. No correlation has been observed between the pre-stimulus alpha power and MEP responses in any of the conditions. Our results show that the variability of the alpha oscillations can be more predictive of TMS effects than the commonly used power of oscillations and we provide further support for the dissociation of high and low-alpha bands in predicting responses produced by the stimulation of the motor cortex. PMID:27774060

  10. Brain-robot interface driven plasticity: Distributed modulation of corticospinal excitability.

    PubMed

    Kraus, Dominic; Naros, Georgios; Bauer, Robert; Leão, Maria Teresa; Ziemann, Ulf; Gharabaghi, Alireza

    2016-01-15

    Brain-robot interfaces (BRI) are studied as novel interventions to facilitate functional restoration in patients with severe and persistent motor deficits following stroke. They bridge the impaired connection in the sensorimotor loop by providing brain-state dependent proprioceptive feedback with orthotic devices attached to the hand or arm of the patients. The underlying neurophysiology of this BRI neuromodulation is still largely unknown. We investigated changes of corticospinal excitability with transcranial magnetic stimulation in thirteen right-handed healthy subjects who performed 40min of kinesthetic motor imagery receiving proprioceptive feedback with a robotic orthosis attached to the left hand contingent to event-related desynchronization of the right sensorimotor cortex in the β-band (16-22Hz). Neural correlates of this BRI intervention were probed by acquiring the stimulus-response curve (SRC) of both motor evoked potential (MEP) peak-to-peak amplitudes and areas under the curve. In addition, a motor mapping was obtained. The specificity of the effects was studied by comparing two neighboring hand muscles, one BRI-trained and one control muscle. Robust changes of MEP amplitude but not MEP area occurred following the BRI intervention, but only in the BRI-trained muscle. The steep part of the SRC showed an MEP increase, while the plateau of the SRC showed an MEP decrease. MEP mapping revealed a distributed pattern with a decrease of excitability in the hand area of the primary motor cortex, which controlled the BRI, but an increase of excitability in the surrounding somatosensory and premotor cortex. In conclusion, the BRI intervention induced a complex pattern of modulated corticospinal excitability, which may boost subsequent motor learning during physiotherapy.

  11. Effects of 4-aminopyridine on motor evoked potentials in patients with spinal cord injury: a double-blinded, placebo-controlled crossover trial.

    PubMed

    Wolfe, D L; Hayes, K C; Hsieh, J T; Potter, P J

    2001-08-01

    4-Aminopyridine (4-AP) is a potassium (K+) channel blocking agent that has been shown to reduce the latency and increase the amplitude of motor evoked potentials (MEPs) elicited with transcranial magnetic stimulation (TMS) in patients with chronic spinal cord injury (SCI). These effects on MEPs are thought to reflect enhanced conduction in long tract axons brought about by overcoming conduction deficits due to focal demyelination and/or by enhancing neuroneuronal transmission at one or more sites of the neuraxis. The present study was designed to obtain further evidence of reduced central motor conduction time (CMCT) and to determine whether MEPs could be recorded from paretic muscles in which they were not normally elicited. MEPs were elicited with TMS being delivered to subjects (n = 25) pre- and post-administration of 4-AP (10 mg capsule) or placebo. The principal finding was that 4-AP lowered the stimulation threshold, increased the amplitude and reduced the latency of MEPs in all muscles tested, including those that were unimpaired, but did not alter measures of the peripheral nervous system (i.e., M-wave, H-reflex, F-wave). These 4-AP-induced changes in MEPs were significantly greater than those seen with placebo (p < 0.05). The primary implication of these results is that a low dose of 4-AP (immediate-release formulation) appears to improve the impaired central motor conduction of some patients with incomplete SCI. This is most likely attributable to overcoming conduction deficits at the site of injury but may also involve an increase in cortical excitability.

  12. Evaluation of optimal electrode configurations for epidural spinal cord stimulation in cervical spinal cord injured rats

    PubMed Central

    Alam, Monzurul; Garcia-Alias, Guillermo; Shah, Prithvi K.; Gerasimenko, Yury; Zhong, Hui; Roy, Roland R.; Edgerton, V. Reggie

    2015-01-01

    Background Epidural spinal cord stimulation is a promising technique for modulating the level of excitability and reactivation of dormant spinal neuronal circuits after spinal cord injury (SCI). We examined the ability of chronically implanted epidural stimulation electrodes within the cervical spinal cord to (1) directly elicit spinal motor evoked potentials (sMEPs) in forelimb muscles and (2) determine whether these sMEPs can serve as a biomarker of forelimb motor function after SCI. New method We implanted EMG electrodes in forelimb muscles and epidural stimulation electrodes at C6 and C8 in adult rats. After recovering from a dorsal funiculi crush (C4), rats were tested with different stimulation configurations and current intensities to elicit sMEPs and determined forelimb grip strength. Results: sMEPs were evoked in all muscles tested and their characteristics were dependent on electrode configurations and current intensities. C6(−) stimulation elicited more robust sMEPs than stimulation at C8(−). Stimulating C6 and C8 simultaneously produced better muscle recruitment and higher grip strengths than stimulation at one site. Comparison with existing method(s) Classical method to select the most optimal stimulation configuration is to empirically test each combination individually for every subject and relate to functional improvements. This approach is impractical, requiring extensively long experimental time to determine the more effective stimulation parameters. Our proposed method is fast and physiologically sound. Conclusions Results suggest that sMEPs from forelimb muscles can be useful biomarkers for identifying optimal parameters for epidural stimulation of the cervical spinal cord after SCI. PMID:25791014

  13. Hemocompatibility Improvement of Chromium-Bearing Bare-Metal Stent Platform After Magnetoelectropolishing

    NASA Astrophysics Data System (ADS)

    Rokicki, Ryszard; Haider, Waseem; Maffi, Shivani Kaushal

    2015-01-01

    Research was undertaken to determine the influence of the increased content of chromium in the outermost passive layer of magneto-electrochemically refined Co-Cr alloy L-605 surface on its hemocompatibility. The chemistry, roughness, surface energy, and wettability of conventionally electropolished (EP) and magnetoelectropolished (MEP) samples were studied with x-ray photoelectron spectroscopy (XPS), open circuit potential, atomic force microscopy, and contact angle meter. In vitro hemocompatibility of tested material surfaces was assessed using two important indicators of vascular responses to biomaterial, namely endothelialization and platelets adhesion. The endothelialization was assessed by seeding and incubating samples with human umbilical vein endothelial cells (HUVEC) for 3 days before counting and observing them under a fluorescent microscope. The platelet (rich plasma blood) adhesion and activation test on EP and MEP L-605 alloy surfaces was assessed using a laser scanning confocal microscope. The XPS analysis of MEP samples showed significant enrichment of the passive layer with Cr and O when compared with the EP one. The amount of other elements in the passive layer did not show a significant difference between EP and MEP treatments. The adhesion of HUVEC cells shows remarkable affinity to surfaces enriched in Cr (MEP) with almost 100% confluency. In addition, the number of platelets that adhered to standard EP surfaces was higher compared to the MEP surface. The present study shows that the chromium-enriched surface of cobalt-chromium alloy L-605 by the magnetoelectropolishing process tremendously improves surface hemocompatibility with regard to stent functionality by enhanced endothelialization and lower platelet adhesion and should be taken under consideration as an alternative surface of biodegradable polymer drug-eluting stents, polymer-free drug-eluting stents as well as bare-metal stents.

  14. Theory of using magnetic deflections to combine charged particle beams