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

  1. 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

  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 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

  3. 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.

  4. 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.

  5. 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

  6. 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.

  7. 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.

  8. 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

  9. 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.

  10. 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.

  11. 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...

  12. 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.

  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. [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.

  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. 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.

  17. 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

  18. 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.

  19. 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.

  20. 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

  1. 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.

  2. 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.

  3. 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.

  4. 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.

  5. 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.

  6. 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.

  7. 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.

  8. 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.

  9. 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.

  10. 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.

  11. 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.

  12. 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

  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. 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

  15. 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.

  16. [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.

  17. 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

  18. 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

  19. 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

  20. 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.

  1. 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.

  2. 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

  3. 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

  4. 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...

  5. 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.

  6. 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.

  7. 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

  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. 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.

  10. 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.

  11. 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.

  12. 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.

  13. 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.

  14. 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.

  15. 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

  16. 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.

  17. 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

  18. 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.

  19. 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.

  20. 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

  1. 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

  2. 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

  3. 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

  4. 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

  5. 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.

  6. 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.

  7. 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.

  8. 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

  9. 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

  10. 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.

  11. 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

  12. 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

  13. 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.

  14. 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.

  15. 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.

  16. 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.

  17. 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.

  18. 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.

  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. 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.

  1. 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.

  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. 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.

  4. 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.

  5. 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

  6. 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

  7. 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.

  8. 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

  9. 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

  10. 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

  11. 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.

  12. 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

  13. 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

  14. 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

  15. 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.

  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. 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

  10. 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

  11. 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.

  12. 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.

  13. 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.

  14. 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

  15. 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.

  16. 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.

  17. 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

  18. 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.

  19. 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.

  20. 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

  1. [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

  2. 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.

  3. 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.

  4. 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.

  5. 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.

  6. 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.

  7. 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.

  8. 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.

  9. 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.

  10. 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.

  11. 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.

  12. 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

  13. 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

  14. 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.

  15. 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

  16. 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

  17. 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.

  18. 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

  19. 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

  20. 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

  1. 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.

  2. 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

  3. 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

  4. 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.

  5. 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

  6. 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.

  7. 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.

  8. 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.

  9. 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.

  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. 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.

  12. 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

  13. 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

  14. 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.

  15. 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

  16. 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.

  17. 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

  18. 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

  19. 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

  20. 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.

  1. 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

  2. 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.

  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

    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

  4. 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

  5. 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

  6. Alternative security

    SciTech Connect

    Weston, B.H. )

    1990-01-01

    This book contains the following chapters: The Military and Alternative Security: New Missions for Stable Conventional Security; Technology and Alternative Security: A Cherished Myth Expires; Law and Alternative Security: Toward a Just World Peace; Politics and Alternative Security: Toward a More Democratic, Therefore More Peaceful, World; Economics and Alternative Security: Toward a Peacekeeping International Economy; Psychology and Alternative Security: Needs, Perceptions, and Misperceptions; Religion and Alternative Security: A Prophetic Vision; and Toward Post-Nuclear Global Security: An Overview.

  7. 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

  8. 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.

  9. Alternate Alternates: A Medley of Alternate Assessments.

    ERIC Educational Resources Information Center

    Burdette, Paula J.; Olsen, Ken

    This paper highlights eight states that have implemented alternate assessments for children with disabilities who cannot participate in their state and district-wide assessment programs. The alternate assessment systems in Delaware, Florida, Georgia, Indiana, Minnesota, North Dakota, Utah, and West Virginia are briefly described, along with their…

  10. Alternative Therapies

    MedlinePlus

    ... Late Effects of Poliomyelitis for Physicians and Survivors © Alternative Therapies Alternative therapies, also called complementary, can support ... of motion, pain, and fatigue are often reported. Energy work includes acupuncture and acupressure, traditional Chinese medicine ...

  11. Alternative strategies: a better alternative.

    PubMed

    Doody, Dennis

    2010-05-01

    Alternatives can be defined as being any financial asset other than traditional stocks and bonds. They include marketable alternatives, private capital, and equity real estate. There are two primary reasons for investing in alternatives: the potential for greater return and the opportunity to diversify a portfolio. Although alternatives were challenged in the highly volatile environment that existed in 2008 and early 2009, they generally lived up to expectations.

  12. Alternative Schools.

    ERIC Educational Resources Information Center

    Pritchett, Stanley; Kimsey, Steve

    2002-01-01

    Describes the design of the DeKalb Alternative School in Atlanta, Georgia, located in a renovated shopping center. Purchasing commercial land and renovating the existing building saved the school system time and money. (EV)

  13. [Alternative medicine].

    PubMed

    Mitello, L

    2001-01-01

    In a critical situation of world official medicine, we can find different alternatives therapies: natural therapy traditional and complementary, survival sometimes, of antique stiles and conditions of life. New sciences presented for them empiricism to the margin of official science. Doctors and sorcerer do the best to defeat the horrible virus that contribute to build symbols categories of sick. The alternatives put dangerously in game the scientific myth of experiment and exhume, if they got lost, antique remedy, almost preserved like cultural wreck very efficient where the medicine is impotent. Besides alternatives and complementary therapies, that are remedies not recognized conventional from official medicine, there are the homeopathic, phytotherapy, pranotherapy, nutritional therapy, the ayurveda, the yoga, ecc. Italians and internationals research show a composite picture of persons that apply that therapies. Object of this work is to understand and know the way that sick lighten their sufferings and role that have o that can assume the nurses to assist this sick. PMID:12146072

  14. Cosmic alternatives?

    NASA Astrophysics Data System (ADS)

    Gregory, Ruth

    2009-04-01

    "Cosmologists are often in error but never in doubt." This pithy characterization by the Soviet physicist Lev Landau sums up the raison d'être of Facts and Speculations in Cosmology. Authors Jayant Narlikar and Geoffrey Burbidge are proponents of a "steady state" theory of cosmology, and they argue that the cosmological community has become fixated on a "Big Bang" dogma, suppressing alternative viewpoints. This book very much does what it says on the tin: it sets out what is known in cosmology, and puts forward the authors' point of view on an alternative to the Big Bang.

  15. Alternative Conceptualizations.

    ERIC Educational Resources Information Center

    Borman, Kathryn M., Ed.; O'Reilly, Patricia, Ed.

    1992-01-01

    This theme issue of the serial "Educational Foundations" contains five articles devoted to the topic of "Alternative Conceptualizations" of the foundations of education. In "The Concept of Place in the New Sociology of Education," Paul Theobald examines the notion of place in educational theory and practice. Janice Jipson and Nicholas Paley, in…

  16. Magnetostrictive Alternator

    NASA Technical Reports Server (NTRS)

    Dyson, Rodger; Bruder, Geoffrey

    2013-01-01

    This innovation replaces the linear alternator presently used in Stirling engines with a continuous-gradient, impedance-matched, oscillating magnetostrictive transducer that eliminates all moving parts via compression, maintains high efficiency, costs less to manufacture, reduces mass, and eliminates the need for a bearing system. The key components of this new technology are the use of stacked magnetostrictive materials, such as Terfenol-D, under a biased magnetic and stress-induced compression, continuous-gradient impedance-matching material, coils, force-focusing metallic structure, and supports. The acoustic energy from the engine travels through an impedancematching layer that is physically connected to the magnetostrictive mass. Compression bolts keep the structure under compressive strain, allowing for the micron-scale compression of the magnetostrictive material and eliminating the need for bearings. The relatively large millimeter displacement of the pressure side of the impedance-matching material is reduced to micron motion, and undergoes stress amplification at the magnetostrictive interface. The alternating compression and expansion of the magnetostrictive material creates an alternating magnetic field that then induces an electric current in a coil that is wound around the stack. This produces electrical power from the acoustic pressure wave and, if the resonant frequency is tuned to match the engine, can replace the linear alternator that is commonly used.

  17. Alternative Thinking.

    ERIC Educational Resources Information Center

    Herman, Dan

    1999-01-01

    Explains how advances in diesel and alternative fuels has caused schools to reconsider their use for their bus fleets. Reductions in air pollution emissions, cost-savings developments, and the economies experienced from less downtime and maintenance requirements are explored. (GR)

  18. 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.

  19. Alternative fuels

    NASA Technical Reports Server (NTRS)

    Grobman, J. S.; Butze, H. F.; Friedman, R.; Antoine, A. C.; Reynolds, T. W.

    1977-01-01

    Potential problems related to the use of alternative aviation turbine fuels are discussed and both ongoing and required research into these fuels is described. This discussion is limited to aviation turbine fuels composed of liquid hydrocarbons. The advantages and disadvantages of the various solutions to the problems are summarized. The first solution is to continue to develop the necessary technology at the refinery to produce specification jet fuels regardless of the crude source. The second solution is to minimize energy consumption at the refinery and keep fuel costs down by relaxing specifications.

  20. 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

  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. Alternative Splice in Alternative Lice

    PubMed Central

    Tovar-Corona, Jaime M.; Castillo-Morales, Atahualpa; Chen, Lu; Olds, Brett P.; Clark, John M.; Reynolds, Stuart E.; Pittendrigh, Barry R.; Feil, Edward J.; Urrutia, Araxi O.

    2015-01-01

    Genomic and transcriptomics analyses have revealed human head and body lice to be almost genetically identical; although con-specific, they nevertheless occupy distinct ecological niches and have differing feeding patterns. Most importantly, while head lice are not known to be vector competent, body lice can transmit three serious bacterial diseases; epidemictyphus, trench fever, and relapsing fever. In order to gain insights into the molecular bases for these differences, we analyzed alternative splicing (AS) using next-generation sequencing data for one strain of head lice and one strain of body lice. We identified a total of 3,598 AS events which were head or body lice specific. Exon skipping AS events were overrepresented among both head and body lice, whereas intron retention events were underrepresented in both. However, both the enrichment of exon skipping and the underrepresentation of intron retention are significantly stronger in body lice compared with head lice. Genes containing body louse-specific AS events were found to be significantly enriched for functions associated with development of the nervous system, salivary gland, trachea, and ovarian follicle cells, as well as regulation of transcription. In contrast, no functional categories were overrepresented among genes with head louse-specific AS events. Together, our results constitute the first evidence for transcript pool differences in head and body lice, providing insights into molecular adaptations that enabled human lice to adapt to clothing, and representing a powerful illustration of the pivotal role AS can play in functional adaptation. PMID:26169943

  3. Alternative Splice in Alternative Lice.

    PubMed

    Tovar-Corona, Jaime M; Castillo-Morales, Atahualpa; Chen, Lu; Olds, Brett P; Clark, John M; Reynolds, Stuart E; Pittendrigh, Barry R; Feil, Edward J; Urrutia, Araxi O

    2015-10-01

    Genomic and transcriptomics analyses have revealed human head and body lice to be almost genetically identical; although con-specific, they nevertheless occupy distinct ecological niches and have differing feeding patterns. Most importantly, while head lice are not known to be vector competent, body lice can transmit three serious bacterial diseases; epidemictyphus, trench fever, and relapsing fever. In order to gain insights into the molecular bases for these differences, we analyzed alternative splicing (AS) using next-generation sequencing data for one strain of head lice and one strain of body lice. We identified a total of 3,598 AS events which were head or body lice specific. Exon skipping AS events were overrepresented among both head and body lice, whereas intron retention events were underrepresented in both. However, both the enrichment of exon skipping and the underrepresentation of intron retention are significantly stronger in body lice compared with head lice. Genes containing body louse-specific AS events were found to be significantly enriched for functions associated with development of the nervous system, salivary gland, trachea, and ovarian follicle cells, as well as regulation of transcription. In contrast, no functional categories were overrepresented among genes with head louse-specific AS events. Together, our results constitute the first evidence for transcript pool differences in head and body lice, providing insights into molecular adaptations that enabled human lice to adapt to clothing, and representing a powerful illustration of the pivotal role AS can play in functional adaptation.

  4. Complementary and Alternative Medicine

    MedlinePlus

    ... Help a Friend Who Cuts? Complementary and Alternative Medicine KidsHealth > For Teens > Complementary and Alternative Medicine Print ... replacement. continue How Is CAM Different From Conventional Medicine? Conventional medicine is based on scientific knowledge of ...

  5. Assessment "Honest Alternatives".

    ERIC Educational Resources Information Center

    Mandel, Susan Glazer

    1995-01-01

    Addresses the challenge of finding or creating alternatives to tests and traditional grading systems. Reflects on and describes the experience of creating an assessment tool and cautions against choosing alternatives that merely camouflage the grades. Encourages educators to find authentic alternatives to describe children's growth. (BAC)

  6. Complementary and Alternative Therapies

    PubMed Central

    Moore, Mary Lou

    2002-01-01

    Complementary and alternative therapies are increasingly used by many pregnant women in the United States; however, limited research is available on many therapies. The number of studies should increase with the establishment of the National Center for Complementary and Alternative Medicine by the National Institutes of Health. This column reviews recent studies of both herbal medicines and alternative therapies used in pregnancy. PMID:17273285

  7. Alternative Teacher Certification.

    ERIC Educational Resources Information Center

    Newman, Carol; Thomas, Kay

    This paper examines issues related to alternative teacher certification, discussing teacher certification in Texas and noting that most researchers agree that both traditional and alternative routes to teacher preparation need improvement. For over a decade, alternative certification has become increasingly available in Texas. This paper…

  8. Alternative Certification Isn't Alternative

    ERIC Educational Resources Information Center

    Walsh, Kate; Jacobs, Sandi

    2007-01-01

    While nearly all states now have something on their books labeled "alternate route to certification," these programs defy standard definition due to their enormous variability. States differ in the types of candidates allowed to apply (e.g., career changers or recent college graduates) and in the academic backgrounds these individuals must…

  9. Alternative Solar Indices

    SciTech Connect

    Lantz, L.J.

    1980-07-01

    Possible alternative Solar Indices which could either be a perturbation from the currently defined Solar Index or possible indices based on current technologies for other media markets are discussed. An overview is given of the current project, including the logic that was utilized in defining its current structure and then alternative indices and definitions are presented and finally, recommendations are made for adopting alternative indices.

  10. Alternative solar indices

    NASA Astrophysics Data System (ADS)

    Lantz, L. J.

    1980-07-01

    Possible alternative Solar Indices which could either be a perturbation from the currently defined Solar Index or possible indices based on current technologies for other media markets are discussed. An overview is given of the current project, including the logic that was utilized in defining its current structure and then alternative indices and definitions are presented and finally, recommendations are made for adopting alternative indices.

  11. Alternate drop pulse polarography

    USGS Publications Warehouse

    Christie, J.H.; Jackson, L.L.; Osteryoung, R.A.

    1976-01-01

    The new technique of alternate drop pulse polarography is presented. An experimental evaluation of alternate drop pulse polarography shows complete compensation of the capacitative background due to drop expansion. The capillary response phenomenon was studied in the absence of faradaic reaction and the capillary response current was found to depend on the pulse width to the -0.72 power. Increased signal-to-noise ratios were obtained using alternate drop pulse polarography at shorter drop times.

  12. Piston engine configuration alternatives

    SciTech Connect

    Wyczalek, F.A.

    1989-01-01

    This paper provides a technological assessment of alternate engine component configuration and material alternatives. It includes a comparative analysis of key characteristics of Gasoline, Diesel and Gas Turbine engines built by Daihatsu, Honda, Isuzu, Mazda, Mitsubishi, Nissan, Suburu, Suzuki and Toyota. The piston engines range from two to ten cylinders with inline, vee and opposed configurations. Furthermore, additional special features and alternative choices include variable compression ratio, ceramic structural components, supercharger, turbocharger, twin turbocharger, supercharger-turbocharger combined and the regenerative gas turbine.

  13. Alternator insulation evaluation tests

    NASA Technical Reports Server (NTRS)

    Penn, W. B.; Schaefer, R. F.; Balke, R. L.

    1972-01-01

    Tests were conducted to predict the remaining electrical insulation life of a 60 KW homopolar inductor alternator following completion of NASA turbo-alternator endurance tests for SNAP-8 space electrical power systems application. The insulation quality was established for two alternators following completion of these tests. A step-temperature aging test procedure was developed for insulation life prediction and applied to one of the two alternators. Armature winding insulation life of over 80,000 hours for an average winding temperature of 248 degrees C was predicted using the developed procedure.

  14. System Alternatives Project

    ERIC Educational Resources Information Center

    Petrait, James A.

    1977-01-01

    The Systems Alternatives Project is an attempt to develop open classroom alternatives within a modular scheduling system. Biology students are given both action and test objectives that emphasize individualization. Structure of the project is detailed and an attempt to analyze the project evaluation data statistically is included. (MA)

  15. Alternative Fuels Data Center

    SciTech Connect

    2013-06-01

    Fact sheet describes the Alternative Fuels Data Center, which provides information, data, and tools to help fleets and other transportation decision makers find ways to reduce petroleum consumption through the use of alternative and renewable fuels, advanced vehicles, and other fuel-saving measures.

  16. Alternative Schools, Mainstream Education

    ERIC Educational Resources Information Center

    McKee, Jan; Conner, Evguenia

    2007-01-01

    Alternative education has its own history. Having emerged in the sixties as a response to the social crisis, its goal was primarily to fight increasing bureaucracy and the depersonalization of public education by giving students more freedom and minimal adult supervision. In the eighties, the understanding of "alternative education" narrowed to…

  17. Alternatives: Project Description.

    ERIC Educational Resources Information Center

    Brier, Norman

    Alternatives is a project designed for youngsters, ages 11-15, who display serious conduct problems and severe learning deficiencies. The primary goal of the project is to prevent the development of a chronic antisocial orientation among youngsters who are at high risk for such an outcome. The interventions employed at Alternatives are based on…

  18. Alternative Automobile Engines

    ERIC Educational Resources Information Center

    Wilson, David Gordon

    1978-01-01

    Requirements for cleaner and more efficient engines have stimulated a search for alternatives to the conventional spark-ignition engine. So far, the defects of the alternative engines are clearer than the virtues. The following engines are compared: spark ignition, diesel, vapor-cycle, Stirling, and gas turbine. (Author/MA)

  19. Alternative Work Programs

    ERIC Educational Resources Information Center

    Kuehn, Kerri L.

    2004-01-01

    Employers are feeling the strain of needing to offer alternative work arrangements to retain and recruit employees. Due to a change in demographics, dual-career couples and increased technology; people are demanding a transformation in the workplace environment. Two alternatives, which are being offered by employers, are flextime and…

  20. Alternative fuel information: Alternative fuel vehicle outlook

    SciTech Connect

    Not Available

    1994-06-01

    Major automobile manufacturers continue to examine a variety of alternative fuel vehicle (AFV) options in an effort to provide vehicles that meet the fleet requirements of the Clean Air Act Amendments of 1990 (CAAA) and the Energy Policy Act of 1992 (EPACT). The current generation of AFVs available to consumers is somewhat limited as the auto industry attempts to respond to the presently uncertain market. At the same time, however, the automobile industry must anticipate future demand and is therefore engaged in research, development, and production programs on a wide range of alternative fuels. The ultimate composition of the AFV fleet may be determined by state and local regulations which will have the effect of determining demand. Many state and regional groups may require vehicles to meet emission standards more stringent than those required by the federal government. Therefore, a significant impact on the market could occur if emission classifications begin serving as the benchmark for vehicles, rather than simply certifying a vehicle as capable of operating on an ``alternative`` to gasoline. Vehicles classified as Zero-Emissions, or even Inherently Low-Emissions, could most likely be met only by electricity or natural gas, thereby dictating that multi-fuel vehicles would be unable to participate in some clean air markets. In the near-term, the Clinton Administration desires to accelerate the use of alternative fuels as evidenced by an executive order directing the federal government to increase the rate of conversion of the federal fleet beyond that called for in EPACT. The Administration has expressed particular interest in using more compressed natural gas (CNG) as a motor fuel, which has resulted in the auto industry`s strong response of concentrating short-term efforts on CNG vehicles. For the 1994 model year, a number of CNG cars and trucks will be available from major automobile manufacturers.

  1. Alternative fuel transit buses

    SciTech Connect

    Motta, R.; Norton, P.; Kelly, K.

    1996-10-01

    The National Renewable Energy Laboratory (NREL) is a U.S. Department of Energy (DOE) national laboratory; this project was funded by DOE. One of NREL`s missions is to objectively evaluate the performance, emissions, and operating costs of alternative fuel vehicles so fleet managers can make informed decisions when purchasing them. Alternative fuels have made greater inroads into the transit bus market than into any other. Each year, the American Public Transit Association (APTA) surveys its members on their inventory and buying plans. The latest APTA data show that about 4% of the 50,000 transit buses in its survey run on an alternative fuel. Furthermore, 1 in 5 of the new transit buses that members have on order are alternative fuel buses. This program was designed to comprehensively and objectively evaluate the alternative fuels in use in the industry.

  2. Complementary and Alternative Medicine.

    PubMed

    Quezada, Sandra M; Briscoe, Jessica; Cross, Raymond K

    2016-06-01

    Inflammatory bowel disease is a complex, chronic, multifactorial inflammatory disorder of the digestive tract. Standard therapies include immunosuppressive and biological treatments, but there is increasing interest in the potential benefit of complementary and alternative medicine for the treatment of inflammatory bowel disease. Given the high prevalence of use of complementary and alternative medicine among inflammatory bowel disease patients, gastroenterologists must remain knowledgeable regarding the risks and benefits of these treatment options. This article reviews the updated scientific data on the use of biologically based complementary and alternative therapies for the treatment of inflammatory bowel disease.

  3. Evaluation of Expenditure Alternates

    ERIC Educational Resources Information Center

    Poehlein, Gary W.; And Others

    1973-01-01

    Illustrates a system of calculating dollar expenditures over periods of time in terms of present value. The system enables planners, school boards, and administrators to compare expenditure alternatives as a decisionmaking factor. (Author)

  4. GLOBAL ALTERNATIVE FUTURE SCENARIOS

    EPA Science Inventory

    One way to examine possible future outcomes for environmental protection is through the development and analysis of alternative future scenarios. This type of assessment postulates two or more different paths that social and environmental development might take, using correspond...

  5. Alternative disinfectant water treatments

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Alternative disinfestant water treatments are disinfestants not as commonly used by the horticultural industry. Chlorine products that produce hypochlorous acid are the main disinfestants used for treating irrigation water. Chlorine dioxide will be the primary disinfestant discussed as an alternativ...

  6. Alternative and Complementary Therapies

    MedlinePlus

    ... always designed to treat a particular illness: Some alternative therapies treat the whole person, not an illness. They might restore harmony, balance, or normal energy flow. Acupuncturists, for example, use the pulse to ...

  7. SOLVENT WASTE REDUCTION ALTERNATIVES

    EPA Science Inventory

    This publication contains edited versions of presentations on this subject made at five Technology Transfer seminars in 1988. Chapters are included on land disposal regulations and requirements; waste solvent disposal alternatives from various industries such as process equipment...

  8. Alternatives to Nursing Homes

    MedlinePlus

    ... this website may not be available. Alternatives to nursing homes Before you make any decisions about long ... live and what help you may need. A nursing home may not be your only choice. Discharge ...

  9. Alternative fuel information sources

    SciTech Connect

    Not Available

    1994-06-01

    This short document contains a list of more than 200 US sources of information (Name, address, phone number, and sometimes contact) related to the use of alternative fuels in automobiles and trucks. Electric-powered cars are also included.

  10. Seal design alternatives study

    SciTech Connect

    Van Sambeek, L.L.

    1993-06-01

    This report presents the results from a study of various sealing alternatives for the WIPP sealing system. Overall, the sealing system has the purpose of reducing to the extent possible the potential for fluids (either gas or liquid) from entering or leaving the repository. The sealing system is divided into three subsystems: drift and panel seals within the repository horizon, shaft seals in each of the four shafts, and borehole seals. Alternatives to the baseline configuration for the WIPP seal system design included evaluating different geometries and schedules for seal component installations and the use of different materials for seal components. Order-of-magnitude costs for the various alternatives were prepared as part of the study. Firm recommendations are not presented, but the advantages and disadvantages of the alternatives are discussed. Technical information deficiencies are identified and studies are outlined which can provide required information.

  11. Alternate policies for alternate energy sources

    SciTech Connect

    Hall, F.F.

    1985-09-01

    Some ''alternates within alternates'' are studied and possible improvement of our energy policies are explored. The viability of a hydrogen fuel economy is reviewed. Methanol, ethanol or ammonia versus hydrogen is one area of interest. Others include liquid hydrogen versus jet fuels, the use of geothermal, solar, wind or water energy for production of hydrogen gas versus development of deep earth supplies of natural gas is another. Energy enhancement as opposed to energy conservation is investigated with regard to polar climate and what might be done to improve natural energy balances, particularly in the northern hemisphere. Pumping Arctic Ocean water out into the Pacific Ocean via the Bering Strait would be an energy debit as opposed to energy gains such as biomass conversion of future plant growth throughout the Siberian and Canadian tundra regions and presently very arid desert regions, improved access to northern region fuel, metal ore and mineral resources, year-round shipping and fishing fleet operations in the Arctic Ocean and development of the tremendous Greenland hydro-electric power potential.

  12. Alternative drugs of abuse.

    PubMed

    Sutter, M E; Chenoweth, J; Albertson, T E

    2014-02-01

    The incidence of drug abuse with alternative agents is increasing. The term "alternative drugs of abuse" is a catch-all term for abused chemicals that do not fit into one of the classic categories of drugs of abuse. The most common age group abusing these agents range from 17 to 25 years old and are often associated with group settings. Due to their diverse pharmacological nature, legislative efforts to classify these chemicals as a schedule I drug have lagged behind the development of new alternative agents. The potential reason for abuse of these agents is their hallucinogenic, dissociative, stimulant, anti-muscarinic, or sedative properties. Some of these drugs are easily obtainable such as Datura stramonium (Jimson Weed) or Lophophora williamsii (Peyote) because they are natural plants indigenous to certain regions. The diverse pharmacology and clinical effects of these agents are so broad that they do not produce a universal constellation of signs and symptoms. Detailed physical exams are essential for identifying clues leading one to suspect an alternative drug of abuse. Testing for the presence of these agents is often limited, and even when available, the results do not return in a timely fashion. Intoxications from these agents pose unique challenges for health care providers. Physician knowledge of the physiological effects of these alternative agents and the local patterns of drug of abuse are important for the accurate diagnosis and optimal care of poisoned patients. This review summarizes the current knowledge of alternative drugs of abuse and highlights their clinical presentations. PMID:23636733

  13. Alternative drugs of abuse.

    PubMed

    Sutter, M E; Chenoweth, J; Albertson, T E

    2014-02-01

    The incidence of drug abuse with alternative agents is increasing. The term "alternative drugs of abuse" is a catch-all term for abused chemicals that do not fit into one of the classic categories of drugs of abuse. The most common age group abusing these agents range from 17 to 25 years old and are often associated with group settings. Due to their diverse pharmacological nature, legislative efforts to classify these chemicals as a schedule I drug have lagged behind the development of new alternative agents. The potential reason for abuse of these agents is their hallucinogenic, dissociative, stimulant, anti-muscarinic, or sedative properties. Some of these drugs are easily obtainable such as Datura stramonium (Jimson Weed) or Lophophora williamsii (Peyote) because they are natural plants indigenous to certain regions. The diverse pharmacology and clinical effects of these agents are so broad that they do not produce a universal constellation of signs and symptoms. Detailed physical exams are essential for identifying clues leading one to suspect an alternative drug of abuse. Testing for the presence of these agents is often limited, and even when available, the results do not return in a timely fashion. Intoxications from these agents pose unique challenges for health care providers. Physician knowledge of the physiological effects of these alternative agents and the local patterns of drug of abuse are important for the accurate diagnosis and optimal care of poisoned patients. This review summarizes the current knowledge of alternative drugs of abuse and highlights their clinical presentations.

  14. Chromatin and alternative splicing.

    PubMed

    Alló, M; Schor, I E; Muñoz, M J; de la Mata, M; Agirre, E; Valcárcel, J; Eyras, E; Kornblihtt, A R

    2010-01-01

    Alternative splicing affects more than 90% of human genes. Coupling between transcription and splicing has become crucial in the complex network underlying alternative splicing regulation. Because chromatin is the real template for nuclear transcription, changes in its structure, but also in the "reading" and "writing" of the histone code, could modulate splicing choices. Here, we discuss the evidence supporting these ideas, from the first proposal of chromatin affecting alternative splicing, performed 20 years ago, to the latest findings including genome-wide evidence that nucleosomes are preferentially positioned in exons. We focus on two recent reports from our laboratories that add new evidence to this field. The first report shows that a physiological stimulus such as neuron depolarization promotes intragenic histone acetylation (H3K9ac) and chromatin relaxation, causing the skipping of exon 18 of the neural cell adhesion molecule gene. In the second report, we show how specific histone modifications can be created at targeted gene regions as a way to affect alternative splicing: Using small interfering RNAs (siRNAs), we increased the levels of H3K9me2 and H3K27me3 in the proximity of alternative exon 33 of the human fibronectin gene, favoring its inclusion into mature messenger RNA (mRNA) through a mechanism that recalls RNA-mediated transcriptional gene silencing.

  15. Biodiesel: an alternative fuel.

    PubMed

    Manzanera, Maximino; Molina-Muñoz, Maria L; González-López, Jesús

    2008-01-01

    Biodiesel is an alternative energy source and could be a substitute for petroleum-based diesel fuel. To be a viable alternative, a biofuel should provide a net energy gain, have environmental benefits, be economically competitive, and be producible in large quantities without reducing food supplies. Most of the sources, methods and apparatus to produce biodiesel are reviewed here. Some of the patents propose the use of oils and fats of animal or vegetal origin and other kind of sources. Many others focus on the methods for the production or oxidation stability of the biofuel in order to make its production economically competitive. Several apparatus comprising reactors and refineries are also presented. This review article summarizes recent and important patents relating to the production of biodiesel to make its production a viable alternative.

  16. Alternative Green Solvents Project

    NASA Technical Reports Server (NTRS)

    Maloney, Phillip R.

    2012-01-01

    Necessary for safe and proper functioning of equipment. Mainly halogenated solvents. Tetrachloride, Trichloroethylene (TCE), CFC-113. No longer used due to regulatory/safety concerns. Precision Cleaning at KSC: Small % of total parts. Used for liquid oxygen (LOX) systems. Dual solvent process. Vertrel MCA (decafluoropentane (DFP) and trons-dichloroethylene) HFE-7100. DFP has long term environmental concerns. Project Goals: a) Identify potential replacements. b) 22 wet chemical processes. c) 3 alternative processes. d) Develop test procedures. e) Contamination and cleaning. f) Analysis. g) Use results to recommend alternative processes. Conclusions: a) No alternative matched Vertrel in this study. b) No clear second place solvent. c) Hydrocarbons- easy; Fluorinated greases- difficult. d) Fluorinated component may be needed in replacement solvent. e) Process may need to make up for shortcoming of the solvent. f) Plasma and SCC02 warrant further testing.

  17. The energy cane alternative

    SciTech Connect

    Alexander, A.G.

    1985-01-01

    This book reviews the conceptual and theoretical background of Saccharum botany, which underlies the growing of cane as a total growth commodity. Management details are provided for energy cane planting, cultivation, harvest, and postharvest operations. Chapters on energy cane utilization stress new developments in lignocellulose conversion plus alternative options for fermentable solids usage. Chapters are also included for the management of alternative grasses to supplement energy cane, and the breeding of new hybrid canes with high biomass attributes at the intergeneric and interspecific levels.

  18. Solvent alternatives guide

    SciTech Connect

    Elion, J.M.; Monroe, K.R.; Hill, E.A.

    1996-06-01

    It is no longer legal to manufacture or import chlorofluorocarbon 113 or methyl chloroform solvents, and companies that currently clean their parts with either material are now required to implement environmentally safe substitutes. To help find alternative methods, Research Triangle Institute`s Surface Cleaning Technology Program has designed a Solvent Alternatives Guide (SAGE), an online tool that enables access to practical information and recommendations for acceptable solvents. Developed in partnership with the US Environmental Protection Agency, SAGE is available free of charge on the Internet`s World Wide Web.

  19. PEAT: an energy alternative

    SciTech Connect

    Schora, F. C.; Punwani, D. V.

    1980-01-01

    Even though peat is a low-heating value and low-bulk density fossil fuel which in its natural state contains over 80 percent moisture, it can be an economical alternative to coal, and fuel oil, as is the case in Iceland and Finland for direct combustion applications. This is because of the relative ease with which peat can be harvested, and the generally low sulfur and ash content of peat. Recent studies show that peat also has very favorable characteristics for conversion to synthetic fuels. Tests show that on the basis of chemistry and kinetics, peat is a better raw material than coal for production of synthetic fuels. Recent estimates also show that conversion of peat to high-Btu gas (>950 Btu/scf) is competitive with other alternatives of synthetic high-Btu gas. Therefore, peat can be an economical energy alternative depending upon location of peat deposits, region of energy need, scale of operation and cost of other energy alternatives.

  20. Making room for alternatives.

    PubMed

    Edelberg, D

    1994-07-01

    Alternative healing is an idea whose time has come, and 1993 was the critical year for that recognition. So believes internist David Edelberg, founder of the Chicago Holistic Center. There patients can see one of four allopathic physicians as well as practitioners in 37 additional therapies, including acupuncture, infant massage, homeopathy, nutrition counseling, and Ayurvedic medicine. PMID:10136509

  1. Alternative Energy Busing

    ERIC Educational Resources Information Center

    LaFee, Scott

    2012-01-01

    In recent years, school districts have converted portions of their bus fleets to cleaner-burning, sometimes cheaper, alternative fossil fuels, such as compressed natural gas or propane. Others have adopted biodiesel, which combines regular diesel with fuel derived from organic sources, usually vegetable oils or animal fats. The number of biodiesel…

  2. Publishing: Alternatives and Economics.

    ERIC Educational Resources Information Center

    Penchansky, Mimi; And Others

    The Library Association of the City University of New York presents an annotated bibliography on the subject of small and alternative publishing. In the first section directories, indexes, catalogs, and reviews are briefly described. Book distributors for small publishers are listed next. The major portion of the bibliography is a listing of books…

  3. Alternatives to Traditional Notation.

    ERIC Educational Resources Information Center

    Gaare, Mark

    1997-01-01

    Provides a introduction and overview to alternative music notation systems. Describes guitar tablature, accordion tablature, klavarskribo (a keyboard notational system developed by Cornelius Pot, a Dutch engineer), and the digital piano roll. Briefly discusses the history of notation reform and current efforts. Includes examples from scores. (MJP)

  4. Alternative Approaches to Negotiating.

    ERIC Educational Resources Information Center

    Ramming, Thomas M.

    1997-01-01

    The wait-and-react and laundry-list approaches to combating teachers' collective-bargaining demands are ineffective. An alternative goals-setting approach requires management and the district negotiations team to identify important needs and objectives. West Seneca Central School District ended contentious negotiations by presenting unions with…

  5. Alternatives in solar energy

    NASA Technical Reports Server (NTRS)

    Schueler, D. G.

    1978-01-01

    Although solar energy has the potential of providing a significant source of clean and renewable energy for a variety of applications, it is expected to penetrate the nation's energy economy very slowly. The alternative solar energy technologies which employ direct collection and conversion of solar radiation as briefly described.

  6. Finding alternatives to antibiotics

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The spread of antibiotic-resistant pathogens requires new treatments. The availability of new antibiotics has severely declined, and so alternatives to antibiotics need to be considered in both animal agriculture and human medicine. Products for disease prevention are different than products for d...

  7. Energy conversion alternatives study

    NASA Technical Reports Server (NTRS)

    Shure, L. T.

    1979-01-01

    Comparison of coal based energy systems is given. Study identifies and compares various advanced energy conversion systems using coal or coal derived fuels for baselaoad electric power generation. Energy Conversion Alternatives Study (ECAS) reports provede government, industry, and general public with technically consistent basis for comparison of system's options of interest for fossilfired electric-utility application.

  8. Alternatives in Education.

    ERIC Educational Resources Information Center

    Rusk, Bruce, Ed.

    These six lectures explore alternative approaches to education both within and outside the educational system. The contributions and their authors include: "Telling It Like It Ain't: An Examination of the Language of Education," by Neil Postman; "The Psychology of J. Piaget and Its Relevance to Education," by Vinh Bang (presented also in French);…

  9. Augmentative & Alternative Communication

    ERIC Educational Resources Information Center

    Murphy, Patti

    2007-01-01

    There is no definitive recipe for augmentative and alternative communication (AAC) success, but its universal ingredients can be found at home. The main ones are: (1) Understanding that all children need to express themselves, however outgoing or shy they may be; (2) Willingness to embrace the technology that may help your child regardless of your…

  10. Alternatives for Struggling Learners

    ERIC Educational Resources Information Center

    DeBlois, Robert; Place, Patti

    2007-01-01

    This article talks about two alternative schools that have adapted to be a better fit for at-risk students than a comprehensive middle or high school might be. It is easy to suggest that an increase in the dropout rate is merely a natural result of the recent preoccupation with test scores. Whatever truth there may be to this, educators must still…

  11. Alternatives to Traditional Lecturing.

    ERIC Educational Resources Information Center

    Brooks, David W.

    1984-01-01

    Alternatives to traditional, large-class lecturing are discussed. They include using canned lectures, demonstrations and lecture experiments, computer simulations, problem-solving strategies, breaks during lectures, and movies. Moving out of large classrooms to laboratories and resource rooms (or giving an examination) is also suggested. (JN)

  12. Compensated pulsed alternator

    DOEpatents

    Weldon, William F.; Driga, Mircea D.; Woodson, Herbert H.

    1980-01-01

    This invention relates to an electromechanical energy converter with inertial energy storage. The device, a single phase, two or multi-pole alternator with stationary field coils, and a rotating armature is provided. The rotor itself may be of laminated steel for slower pulses or for faster pulses should be nonmagnetic and electrically nonconductive in order to allow rapid penetration of the field as the armature coil rotates. The armature coil comprises a plurality of power generating conductors mounted on the rotor. The alternator may also include a stationary or counterrotating compensating coil to increase the output voltage thereof and to reduce the internal impedance of the alternator at the moment of peak outout. As the machine voltage rises sinusoidally, an external trigger switch is adapted to be closed at the appropriate time to create the desired output current from said alternator to an external load circuit, and as the output current passes through zero a self-commutating effect is provided to allow the switch to disconnect the generator from the external circuit.

  13. Alternative Break Service Trips

    ERIC Educational Resources Information Center

    DuPre, Carrie

    2010-01-01

    Even as educators understand how their millennial students learn in such different ways than previous generations (watching how-to videos downloaded from YouTube or engaging in experiential learning adventures), colleges still rely heavily on in-the-classroom learning. The author can't offer an alternative to this classroom format, but she…

  14. Alternative Education Options.

    ERIC Educational Resources Information Center

    Little (Arthur D.), Inc., Washington, DC.

    This guide deals with various areas of alternative education programs, including current practices and different options available to school and community personnel. Steps are outlined to assess present educational settings, design new programs, select the participants, and implement and evaluate the new program. The first appendix contains…

  15. The SOLAR Alternative.

    ERIC Educational Resources Information Center

    Warren, E. H., Jr.; Walton, A. L.

    1984-01-01

    Only when the sun's energy can be captured at a comparable or lower opportunity cost than that of competing sources will solar energy systems become viable alternatives. Economic issues of solar energy are discussed. The legitimate role of government is also examined. (RM)

  16. Alternatives to Violence.

    ERIC Educational Resources Information Center

    Children Today, 1994

    1994-01-01

    Notes that our capacity to diffuse conflict rests in our ability to recognize and verbalize feelings, develop empathy, and think of alternatives to violence. Explores the influence of role models and culture on violence and how the media can use violent images effectively in helping us confront a culture of violence. (HTH)

  17. Alternatives to Evolutionism.

    ERIC Educational Resources Information Center

    Harper, G. H.

    1979-01-01

    Discusses alternative theories to evolutionism. Five theories are mentioned, but most attention is given to the steady state theory of species which the author considers the most suitable partner for Darwinism in O- and A-level biology courses in the United Kingdom. (HM)

  18. Alternatives; A Filmography.

    ERIC Educational Resources Information Center

    Covert, Nadine, Comp.; And Others

    Over 120 films are listed which deal with alternatives in education, lifestyles, work, religion, crafts, and politics. Among topics covered are open classrooms, communes, changing family structures, day care, and racial problems. Each film is summarized; length, copyrights, date, producer, and distributor are listed. A brief bibliography, subject…

  19. [Alternatives to animal testing].

    PubMed

    Fabre, Isabelle

    2009-11-01

    The use of alternative methods to animal testing are an integral part of the 3Rs concept (refine, reduce, replace) defined by Russel & Burch in 1959. These approaches include in silico methods (databases and computer models), in vitro physicochemical analysis, biological methods using bacteria or isolated cells, reconstructed enzyme systems, and reconstructed tissues. Emerging "omic" methods used in integrated approaches further help to reduce animal use, while stem cells offer promising approaches to toxicologic and pathophysiologic studies, along with organotypic cultures and bio-artificial organs. Only a few alternative methods can so far be used in stand-alone tests as substitutes for animal testing. The best way to use these methods is to integrate them in tiered testing strategies (ITS), in which animals are only used as a last resort.

  20. Alternative population futures.

    PubMed

    1980-01-01

    Alternative population scenarios to the year 2000 are presented and policy implications of the various scenarios are discussed. Population models are described. Projections are made based on different sets of assumptions regarding changes in birth, death, marriage and migration rates. A "high" series, "medium" series and "low" series of projections are made for total population. Projections are also made regarding urban and rural population, families and households, and the labor force.

  1. Outlook for alternative transportation fuels

    SciTech Connect

    Gushee, D.E.

    1996-12-31

    This presentation provides a brief review of regulatory issues and Federal programs regarding alternative fuel use in automobiles. A number of U.S. DOE initiatives and studies aimed at increasing alternative fuels are outlined, and tax incentives in effect at the state and Federal levels are discussed. Data on alternative fuel consumption and alternative fuel vehicle use are also presented. Despite mandates, tax incentives, and programs, it is concluded alternative fuels will have minimal market penetration. 7 refs., 5 tabs.

  2. State alternative route designations

    SciTech Connect

    Not Available

    1989-07-01

    Pursuant to the Hazardous Materials Transportation Act (HMTA), the Department of Transportation (DOT) has promulgated a comprehensive set of regulations regarding the highway transportation of high-level radioactive materials. These regulations, under HM-164 and HM-164A, establish interstate highways as the preferred routes for the transportation of radioactive materials within and through the states. The regulations also provide a methodology by which a state may select alternative routes. First,the state must establish a state routing agency,'' defined as an entity authorized to use the state legal process to impose routing requirements on carriers of radioactive material (49 CFR 171.8). Once identified, the state routing agency must select routes in accordance with Large Quantity Shipments of Radioactive Materials or an equivalent routing analysis. Adjoining states and localities should be consulted on the impact of proposed alternative routes as a prerequisite of final route selection. Lastly, the states must provide written notice of DOT of any alternative route designation before the routes are deemed effective.

  3. State alternative route designations

    SciTech Connect

    Not Available

    1989-07-01

    Pursuant to the Hazardous Materials Transportation Act (HMTA), the Department of Transportation (DOT) has promulgated a comprehensive set of regulations regarding the highway transportation of high-level radioactive materials. These regulations, under HM-164 and HM-164A, establish interstate highways as the preferred routes for the transportation of radioactive materials within and through the states. The regulations also provide a methodology by which a state may select alternative routes. First,the state must establish a ``state routing agency,`` defined as an entity authorized to use the state legal process to impose routing requirements on carriers of radioactive material (49 CFR 171.8). Once identified, the state routing agency must select routes in accordance with Large Quantity Shipments of Radioactive Materials or an equivalent routing analysis. Adjoining states and localities should be consulted on the impact of proposed alternative routes as a prerequisite of final route selection. Lastly, the states must provide written notice of DOT of any alternative route designation before the routes are deemed effective.

  4. MANOVA versus alternative methods

    NASA Astrophysics Data System (ADS)

    Yatim, Bidin; Ismail, Suzilah

    2014-12-01

    Multivariate analysis of variance (MANOVA) is a powerful tool in analysing multivariate data of multi-factorial experiments. However one of the assumptions in MANOVA requires the data to be normally distributed. This study concerned with the violation of this assumption, particularly when the data are either moderately non-normal or extremely non-normal. Possible alternative methods of handling such data are (i) permutational MANOVA (PMANOVA) or (ii) analysis of distance (AoD). Both of these alternative methods were compared with MANOVA via Monte Carlo experiments using the power of tests. The experiments focussed on testing interaction effects by incorporating different data types (i.e. having multivariate normal distribution, moderately non-normal and extremely non-normal), three level of inter-variable correlations (low: 0.25, medium: 0.5 and high: 0.75), two designs (small: 3×3 and large: 7×7) and two sample sizes (2 and 5 replicates). Overall, the results revealed that irrespective of the data types and the level of inter-variable correlations MANOVA performed satisfactorily in situations having larger sample size (5 replicates). In these situations, no alternative method is necessary. However, in small design with high inter-variable correlations PMANOVA performed slightly better. In small samples (2 replicates), AoD outperformed both MANOVA and PMANOVA. This is especially true in situation having small sample (2 replicates), large design and highly correlated inter-variables.

  5. Alternate fusion fuels workshop

    SciTech Connect

    Not Available

    1981-06-01

    The workshop was organized to focus on a specific confinement scheme: the tokamak. The workshop was divided into two parts: systems and physics. The topics discussed in the systems session were narrowly focused on systems and engineering considerations in the tokamak geometry. The workshop participants reviewed the status of system studies, trade-offs between d-t and d-d based reactors and engineering problems associated with the design of a high-temperature, high-field reactor utilizing advanced fuels. In the physics session issues were discussed dealing with high-beta stability, synchrotron losses and transport in alternate fuel systems. The agenda for the workshop is attached.

  6. Potential alternate life biochemistries

    NASA Astrophysics Data System (ADS)

    Konesky, Gregory

    2013-09-01

    While life on Earth continues to be discovered in unlikely environments, the underlying biochemistry is all very similar, based on the element carbon, and requiring liquid water. We consider alternate biochemistries based on elements other than carbon, including other group IVA elements, such as silicon and germanium, and solvents other than water. Terminal electron acceptors other than oxygen are also discussed. A fundamental issue is raised related to the detection of, and even the definition of life, whether it is carbon or non-carbon based. An extreme example of this issue would be in consideration of speculative life based on electrically charged dusty plasmas, which may have no physical body.

  7. THE ALTERNATIVE FOR SURVIVAL*

    PubMed Central

    Krishnakumar, P.R.

    1985-01-01

    The need of the time is to develop the traditional health care systems as a people oriented health care system. The factors that dominated the health care structure of our nation has been systematically pushing the traditional systems of medicine away from the people. In this background, an attempt has been made to reflect upon one's own experience, to understand the pattern of domination and to probe and suggest further on what could be the possible alternative development strategy and suggest specific tasks that would help define the development problematique in a meaningful manner in order to progress further in a self-determined manner PMID:22557469

  8. Alternate transportation system

    NASA Technical Reports Server (NTRS)

    Zertuche, Tony; Mckinnie, James

    1988-01-01

    Three missions have been identified by NASA for a Space Shuttle-supplementing Alternate Transportation System (ATS) encompassing combinations of booster vehicles, crew modules, and service modules: (1) to achieve manned access to orbit for Space Station crew rotation every 90 days, (2) the lofting of a logistics module resupplying the Space Station every 180 days, and (3) the simultaneous launch of both crews and logistics to the Space Station. A reentry glider is considered, in conjunction with the Space Shuttle's unmanned cargo version and the Apollo manned capsule, as an important ATS element. The Titan IV/NUS is used as a booster.

  9. Alternative nanostructures for thermophones.

    PubMed

    Aliev, Ali E; Mayo, Nathanael K; Jung de Andrade, Monica; Robles, Raquel O; Fang, Shaoli; Baughman, Ray H; Zhang, Mei; Chen, Yongsheng; Lee, Jae Ah; Kim, Seon Jeong

    2015-05-26

    Thermophones are highly promising for applications such as high-power SONAR arrays, flexible loudspeakers, and noise cancellation devices. So far, freestanding carbon nanotube aerogel sheets provide the most attractive performance as a thermoacoustic heat source. However, the limited accessibility of large-size freestanding carbon nanotube aerogel sheets and other even more exotic materials recently investigated hampers the field. We describe alternative materials for a thermoacoustic heat source with high-energy conversion efficiency, additional functionalities, environmentally friendly, and cost-effective production technologies. We discuss the thermoacoustic performance of alternative nanostructured materials and compare their spectral and power dependencies of sound pressure in air. We demonstrate that the heat capacity of aerogel-like nanostructures can be extracted by a thorough analysis of the sound pressure spectra. The study presented here focuses on engineering thermal gradients in the vicinity of nanostructures and subsequent heat dissipation processes from the interior of encapsulated thermoacoustic projectors. Applications of thermoacoustic projectors for high-power SONAR arrays, sound cancellation, and optimal thermal design, regarding enhanced energy conversion efficiency, are discussed.

  10. Alternative Medicine and Your Child

    MedlinePlus

    ... to Know About Zika & Pregnancy Complementary and Alternative Medicine KidsHealth > For Parents > Complementary and Alternative Medicine Print ... works. previous continue How CAM Differs From Traditional Medicine CAM is frequently distinguished by its holistic methods, ...

  11. Alternatives for Jet Engine Control

    NASA Technical Reports Server (NTRS)

    Leake, R. J.; Sain, M. K.

    1976-01-01

    Approaches are developed as alternatives to current design methods which rely heavily on linear quadratic and Riccati equation methods. The main alternatives are discussed in two broad categories, local multivariable frequency domain methods and global nonlinear optimal methods.

  12. Extending the Alternating Series Test

    ERIC Educational Resources Information Center

    Katsuura, Hidefumi

    2012-01-01

    Alternating series have the simplest of sign patterns. What about series with more complicated patterns? By inspecting the alternating series test closely, we find a theorem that applies to more complicated sign patterns, and beyond.

  13. Alternative Schools. Trends and Issues.

    ERIC Educational Resources Information Center

    Hadderman, Margaret

    This article examines so-called "alternative schools," taking care to define precisely what is meant by this designation. There are many types of alternative education available to the public, including those programs intended for special-education students, advanced-placement students, and home-schooled children. The first alternative schools…

  14. Alternative aircraft fuels

    NASA Technical Reports Server (NTRS)

    Longwell, J. P.; Grobman, J.

    1978-01-01

    In connection with the anticipated impossibility to provide on a long-term basis liquid fuels derived from petroleum, an investigation has been conducted with the objective to assess the suitability of jet fuels made from oil shale and coal and to develop a data base which will allow optimization of future fuel characteristics, taking energy efficiency of manufacture and the tradeoffs in aircraft and engine design into account. The properties of future aviation fuels are examined and proposed solutions to problems of alternative fuels are discussed. Attention is given to the refining of jet fuel to current specifications, the control of fuel thermal stability, and combustor technology for use of broad specification fuels. The first solution is to continue to develop the necessary technology at the refinery to produce specification jet fuels regardless of the crude source.

  15. Alternative energy sources

    NASA Astrophysics Data System (ADS)

    Todd, R. W.

    1982-04-01

    Renewable energy sources and their potential contribution for solving energy needs are presented. Centralized supply technologies include those alternative fuels derived from biomass using solar energy, (supplying 57% of the energy supply in some countries), and those using directly collected solar energy to manufacture a fuel. Fuel utilization effects can be doubled by using combined heat and power stations, and other major sources include wind, wave, tidal, and solar. In terms of local supply technology, wood burning appliances are becoming more popular, and methane is being used for heating and to fuel spark ignition engines. Geothermal low temperature heating exists worldwide at a capacity of 7.2 GW, supplying heat, particularly in Hungary, parts of the U.S.S.R., and Iceland, and a geothermal research program has been established in the United States. Sweden has a potential hydroelectric capacity of 600 MW, and the United States has a 100 GW capacity. Many of these technologies are already cost effective.

  16. Electrically conductive alternating copolymers

    DOEpatents

    Aldissi, M.; Jorgensen, B.S.

    1987-08-31

    Polymers which are soluble in common organic solvents and are electrically conductive, but which also may be synthesized in such a manner that they become nonconductive. Negative ions from the electrolyte used in the electrochemical synthesis of a polymer are incorporated into the polymer during the synthesis and serve as a dopant. A further electrochemical step may be utilized to cause the polymer to be conductive. The monomer repeat unit is comprised of two rings, a pyrrole molecule joined to a thienyl group, or a furyl group, or a phenyl group. The individual groups of the polymers are arranged in an alternating manner. For example, the backbone arrangement of poly(furylpyrrole) is -furan-pyrrole-furan-pyrrole- furan-pyrrole. An alkyl group or phenyl group may be substituted for either or both of the hydrogen atoms of the pyrrole ring.

  17. Alternative Fuels Research Laboratory

    NASA Technical Reports Server (NTRS)

    Surgenor, Angela D.; Klettlinger, Jennifer L.; Nakley, Leah M.; Yen, Chia H.

    2012-01-01

    NASA Glenn has invested over $1.5 million in engineering, and infrastructure upgrades to renovate an existing test facility at the NASA Glenn Research Center (GRC), which is now being used as an Alternative Fuels Laboratory. Facility systems have demonstrated reliability and consistency for continuous and safe operations in Fischer-Tropsch (F-T) synthesis and thermal stability testing. This effort is supported by the NASA Fundamental Aeronautics Subsonic Fixed Wing project. The purpose of this test facility is to conduct bench scale F-T catalyst screening experiments. These experiments require the use of a synthesis gas feedstock, which will enable the investigation of F-T reaction kinetics, product yields and hydrocarbon distributions. Currently the facility has the capability of performing three simultaneous reactor screening tests, along with a fourth fixed-bed reactor for catalyst activation studies. Product gas composition and performance data can be continuously obtained with an automated gas sampling system, which directly connects the reactors to a micro-gas chromatograph (micro GC). Liquid and molten product samples are collected intermittently and are analyzed by injecting as a diluted sample into designated gas chromatograph units. The test facility also has the capability of performing thermal stability experiments of alternative aviation fuels with the use of a Hot Liquid Process Simulator (HLPS) (Ref. 1) in accordance to ASTM D 3241 "Thermal Oxidation Stability of Aviation Fuels" (JFTOT method) (Ref. 2). An Ellipsometer will be used to study fuel fouling thicknesses on heated tubes from the HLPS experiments. A detailed overview of the test facility systems and capabilities are described in this paper.

  18. Diesel Engine Alternatives

    SciTech Connect

    Ryan, T

    2003-08-24

    There are basically three different modes of combustion possible for use in reciprocating engines. These include, diffusion burning, as occurs in current diesel engines, flame propagation combustion such as used in conventional SI engines, and homogeneous combustion such as is used in the SwRI HCCI engine. Diesel engines currently offer significant fuel consumption benefits relative to other powerplants for on and off road applications; however, costs and efficiency may become problems as the emissions standards become even more stringent. This presentation presents a discussion of the potentials of HCCI and flame propagation engines as alternatives to the diesel engines. It is suggested that as the emissions standards become more and more stringent, the advantages of the diesel may disappear. The potential for HCCI is limited by the availability of the appropriate fuel. The potential of flame propagation engines is limited by several factors including knock, EGR tolerance, high BMEP operation, and throttling. These limitations are discussed in the context of potential for improvement of the efficiency of the flame propagation engine.

  19. Hospital diversification: evaluating alternatives.

    PubMed

    Hammer, L

    1987-05-01

    The appropriateness of diversification as a growth strategy for hospitals is discussed, and planning for diversification is described. Because new forms of health-care delivery are now in direct competition with hospitals, many hospitals are confronting environmental pressures and preparing for future survival through diversification. To explore the potential risks and benefits of diversification, the hospital must identify opportunities for new business ventures. Diversification can be "related," through an expansion of the primary product line (health care), or "unrelated," into areas not directly associated with health care. The hospital must establish specific criteria for evaluating each diversification alternative, and the two or three most attractive options should be analyzed further through a financial feasibility study. The hospital should also seek legal advice to determine the implications of diversification for maintenance of tax status, antitrust limitations, and applicability of certificate of need. Although diversification may not be appropriate for every institution, hospitals should consider it as a strategy for increasing their revenue base, confronting environmental pressures, and securing future survival. PMID:3300300

  20. Enhanced Design Alternative IV

    SciTech Connect

    N. E. Kramer

    1999-05-18

    This report evaluates Enhanced Design Alternative (EDA) IV as part of the second phase of the License Application Design Selection (LADS) effort. The EDA IV concept was compared to the VA reference design using criteria from the ''Design Input Request for LADS Phase II EDA Evaluations'' (CRWMS M&O 1999b) and (CRWMS M&O 1999f). Briefly, the EDA IV concept arranges the waste packages close together in an emplacement configuration known as ''line load''. Continuous pre-closure ventilation keeps the waste packages from exceeding the 350 C cladding and 200 C (4.3.13) drift wall temperature limits. This EDA concept keeps relatively high, uniform emplacement drift temperatures (post-closure) to drive water away from the repository and thus dry out the pillars between emplacement drifts. The waste package is shielded to permit human access to emplacement drifts and includes an integral filler inside the package to reduce the amount of water that can contact the waste form. Closure of the repository is desired 50 years after first waste is emplaced. Both backfill and a drip shields will be emplaced at closure to improve post-closure performance.

  1. Alternative nuclear technologies

    NASA Astrophysics Data System (ADS)

    Schubert, E.

    1981-10-01

    The lead times required to develop a select group of nuclear fission reactor types and fuel cycles to the point of readiness for full commercialization are compared. Along with lead times, fuel material requirements and comparative costs of producing electric power were estimated. A conservative approach and consistent criteria for all systems were used in estimates of the steps required and the times involved in developing each technology. The impact of the inevitable exhaustion of the low- or reasonable-cost uranium reserves in the United States on the desirability of completing the breeder reactor program, with its favorable long-term result on fission fuel supplies, is discussed. The long times projected to bring the most advanced alternative converter reactor technologies the heavy water reactor and the high-temperature gas-cooled reactor into commercial deployment when compared to the time projected to bring the breeder reactor into equivalent status suggest that the country's best choice is to develop the breeder. The perceived diversion-proliferation problems with the uranium plutonium fuel cycle have workable solutions that can be developed which will enable the use of those materials at substantially reduced levels of diversion risk.

  2. A new quenching alternative

    SciTech Connect

    Brennan, R.J.; Faulkner, C.H.

    1996-12-31

    The quenching of ferrous alloys implies the controlled extraction of heat from a part at a rate sufficient to harden the part and still control the desired dimensional limitations. Quenchants in common use today are: molten metals, molten salts, petroleum oils, polymer solutions, water, and salt/water solutions. Each type of quenchant has its benefits and limitations. With current waste legislation and the trends toward environmentally friendlier industrial working fluids, many of these quenching products are coming under close scrutiny by the users and legislators. The most widely used quenchant is petroleum oil due to its favorable heat extraction characteristics. The dependence upon imports, price vulnerability, and contamination potential have caused suppliers and users to look into alternative products. Research into renewable resource, non-petroleum, vegetable oils has been going on globally for several years. The drawbacks encountered with many vegetable oils were widely known and only years of research enabled them to be overcome. The presently formulated product not only performs as well as petroleum oil but shows some characteristics better than those of the petroleum products, especially in the biodegradability and ecological aspects of the products. Stability and reproducible quenching properties have been proven with over two and one half years of field testing.

  3. Hospital diversification: evaluating alternatives.

    PubMed

    Hammer, L

    1987-05-01

    The appropriateness of diversification as a growth strategy for hospitals is discussed, and planning for diversification is described. Because new forms of health-care delivery are now in direct competition with hospitals, many hospitals are confronting environmental pressures and preparing for future survival through diversification. To explore the potential risks and benefits of diversification, the hospital must identify opportunities for new business ventures. Diversification can be "related," through an expansion of the primary product line (health care), or "unrelated," into areas not directly associated with health care. The hospital must establish specific criteria for evaluating each diversification alternative, and the two or three most attractive options should be analyzed further through a financial feasibility study. The hospital should also seek legal advice to determine the implications of diversification for maintenance of tax status, antitrust limitations, and applicability of certificate of need. Although diversification may not be appropriate for every institution, hospitals should consider it as a strategy for increasing their revenue base, confronting environmental pressures, and securing future survival.

  4. Detecting alternative graph clusterings.

    PubMed

    Mandala, Supreet; Kumara, Soundar; Yao, Tao

    2012-07-01

    The problem of graph clustering or community detection has enjoyed a lot of attention in complex networks literature. A quality function, modularity, quantifies the strength of clustering and on maximization yields sensible partitions. However, in most real world networks, there are an exponentially large number of near-optimal partitions with some being very different from each other. Therefore, picking an optimal clustering among the alternatives does not provide complete information about network topology. To tackle this problem, we propose a graph perturbation scheme which can be used to identify an ensemble of near-optimal and diverse clusterings. We establish analytical properties of modularity function under the perturbation which ensures diversity. Our approach is algorithm independent and therefore can leverage any of the existing modularity maximizing algorithms. We numerically show that our methodology can systematically identify very different partitions on several existing data sets. The knowledge of diverse partitions sheds more light into the topological organization and helps gain a more complete understanding of the underlying complex network.

  5. Alternating hemiplegia of childhood.

    PubMed

    Kansagra, Sujay; Mikati, Mohamad A; Vigevano, Federico

    2013-01-01

    Alternating hemiplegia of childhood (AHC) is a very rare disease characterized by recurrent attacks of loss of muscular tone resulting in hypomobility of one side of the body. The etiology of the disease due to ATP1A3 gene mutations in the majority of patients. Few familial cases have been described. AHC has an onset in the first few months of life. Hemiplegic episodes are often accompanied by other paroxysmal manifestations, such as lateral eyes and head deviation toward the hemiplegic side and a very peculiar monocular nystagmus. As the attack progresses, hemiplegia can shift to the other side of the body. Sometimes the attack can provoke bilateral paralysis, and these patients may have severe clinical impairment, with difficulty in swallowing and breathing. Hemiplegic attacks may be triggered by different stimuli, like bath in warm water, motor activity, or emotion. The frequency of attacks is high, usually several in a month or in a week. The duration is variable from a few minutes to several hours or even days. Sleep can stop the attack. Movement disorders such as dystonia and abnormal movements are frequent. Cognitive delay of variable degree is a common feature. Epilepsy has been reported in 50% of the cases, but seizure onset is usually during the third or fourth year of life. Many drugs have been used in AHC with very few results. Flunarizine has the most supportive anecdotal evidence regarding efficacy. PMID:23622289

  6. Alternative Compression Garments

    NASA Technical Reports Server (NTRS)

    Stenger, M. B.; Lee, S. M. C.; Ribeiro, L. C.; Brown, A. K.; Westby, C. M.; Platts, S. H.

    2011-01-01

    Orthostatic intolerance after spaceflight is still an issue for astronauts as no in-flight countermeasure has been 100% effective. Future anti-gravity suits (AGS) may be similar to the Shuttle era inflatable AGS or may be a mechanical compression device like the Russian Kentavr. We have evaluated the above garments as well as elastic, gradient compression garments of varying magnitude and determined that breast-high elastic compression garments may be a suitable replacement to the current AGS. This new garment should be more comfortable than the AGS, easy to don and doff, and as effective a countermeasure to orthostatic intolerance. Furthermore, these new compression garments could be worn for several days after space flight as necessary if symptoms persisted. We conducted two studies to evaluate elastic, gradient compression garments. The purpose of these studies was to evaluate the comfort and efficacy of an alternative compression garment (ACG) immediately after actual space flight and 6 degree head-down tilt bed rest as a model of space flight, and to determine if they would impact recovery if worn for up to three days after bed rest.

  7. [Alternative oxidase in industrial fungi].

    PubMed

    Gu, Shuai; Liu, Qiang; He, Hao; Li, Shuang

    2015-01-01

    Filamentous fungi have been used in industrial fermentation extensively. Based on non-phosphorylating electron transport process, alternative respiration pathway (ARP) acts as an energy overflow, which can balance carbon metabolism and electron transport, allow the continuance of tricarboxylic acid cycle without the formation of ATP, and permit the turnover of carbon skeletons. Alternative respiration pathway also plays an important role in the stress response of fungi and the physiological function of conditioned pathogen. Alternative oxidase (AOX) is the terminal oxidase responsible for the activity of alternative respiration pathway, which exists widely in higher plants, parts of fungi and algae. Owing to the property that alternative oxidase (AOX) is sensitive to salicylhydroxamic acid (SHAM) and insensitive to conventional inhibitors of cytochrome respiration, alternative respiration pathway by AOX is also named as cyanide-resistant respiration (CRR). In recent years, the study of the alternative respiration pathway and alternative oxidase has been a hot topic in the area involving cellular respiration metabolism. In this review we summarized the latest research advances about the functions of alternative respiration pathway and alternative oxidase in industrial fungi.

  8. Bacterial alternative nitrogen fixation systems.

    PubMed

    Joerger, R D; Bishop, P E

    1988-01-01

    The introduction briefly reviews some of the salient features of the well-characterized conventional molybdo-enzyme system for N2 fixation. This is followed by a brief account of the discovery of an alternative N2 fixation system that does not require molybdenum in the N2-fixing bacterum Azotobacter vinelandii. The next section cites observations from the early literature on N2 fixation suggesting may not always require molybdenum. Next, recent evidence for an alternative N2 fixation system in A. vinelandii is discussed. A brief description of our discovery of an alternative nitrogenase which is not a molybdenum or vanadium enzyme is presented, followed by a summary of recent papers describing an alternative vanadium-containing nitrogenase. Available information on the genetics and regulation of alternative N2 fixation systems is discussed. Finally, the possible/probable presence of alternative N2 fixation systems in bacteria other than Azotobacter species is covered.

  9. Helping Families Explore Parenthood Alternatives

    PubMed Central

    Goodbody, Sandra Toll

    1979-01-01

    As many more couples are choosing childlessness as a lifestyle, physicians are more frequently being consulted about alternatives. Changing cultural values as well as changing attitudes to women have opened up these alternatives; the physician must ensure that he or she does not steer patients towards an alternative that may not be for them. Examples of six couples illustrate how childfree couples cope with the implications of their decision. PMID:21297712

  10. Alternative display and interaction devices

    NASA Technical Reports Server (NTRS)

    Bolas, M. T.; McDowall, I. E.; Mead, R. X.; Lorimer, E. R.; Hackbush, J. E.; Greuel, C.

    1995-01-01

    While virtual environment systems are typically thought to consist of a head mounted display and a flex-sensing glove, alternative peripheral devices are beginning to be developed in response to application requirements. Three such alternatives are discussed: fingertip sensing gloves, fixed stereoscopic viewers, and counterbalanced head mounted displays. A subset of commercial examples that highlight each alternative is presented as well as a brief discussion of interesting engineering and implementation issues.

  11. Private Housing or Alternative Financing?

    ERIC Educational Resources Information Center

    Bruno, Nick

    1999-01-01

    Explores the history of privatizing university housing and some current financing options, including use of developer and private foundations. Examples of successful alternative financing methods are highlighted. (GR)

  12. Alternative fuelds in urban fleets

    SciTech Connect

    Lindsay, T.

    1994-12-31

    In this presentation the author addresses four main objectives. They are to: discuss programs that are driving the introduction of alternative fuels into fleet operations in urban areas around the country; define alternative fuels; quantify the present use and future projections on alternative fuel vehicles (AVFs) in the Chicago metropolitan statistical area; and discuss benefits of increased use of alternative fuels in urban areas. Factors which touch on these points include: present domestic dependence on petroleum for autos, with usage exceeding production; the large populations in urban areas which do not meet Clean Air Standards; recent legislative initiatives which give guidance and aid in the adoption of such strategies.

  13. Normetex Pump Alternatives Study

    SciTech Connect

    Clark, Elliot A.

    2013-04-25

    A mainstay pump for tritium systems, the Normetex scroll pump, is currently unavailable because the Normetex company went out of business. This pump was an all-metal scroll pump that served tritium processing facilities very well. Current tritium system operators are evaluating replacement pumps for the Normetex pump and for general used in tritium service. An all-metal equivalent alternative to the Normetex pump has not yet been identified. 1. The ideal replacement tritium pump would be hermetically sealed and contain no polymer components or oils. Polymers and oils degrade over time when they contact ionizing radiation. 2. Halogenated polymers (containing fluorine, chlorine, or both) and oils are commonly found in pumps. These materials have many properties that surpass those of hydrocarbon-based polymers and oils, including thermal stability (higher operating temperature) and better chemical resistance. Unfortunately, they are less resistant to degradation from ionizing radiation than hydrocarbon-based materials (in general). 3. Polymers and oils can form gaseous, condensable (HF, TF), liquid, and solid species when exposed to ionizing radiation. For example, halogenated polymers form HF and HCl, which are extremely corrosive upon reaction with water. If a pump containing polymers or oils must be used in a tritium system, the system must be designed to be able to process the unwanted by-products. Design features to mitigate degradation products include filters and chemical or physical traps (eg. cold traps, oil traps). 4. Polymer components can work in tritium systems, but must be replaced regularly. Polymer components performance should be monitored or be regularly tested, and regular replacement of components should be viewed as an expected normal event. A radioactive waste stream must be established to dispose of used polymer components and oil with an approved disposal plan developed based on the facility location and its regulators. Polymers have varying

  14. Acceptability of Alternatives to Traditional Emergency Care: Patient Characteristics, Alternate Transport Modes, and Alternate Destinations.

    PubMed

    Jones, Courtney Marie Cora; Wasserman, Erin B; Li, Timmy; Shah, Manish N

    2015-01-01

    To determine the acceptability of alternatives to traditional emergency care, we assessed the proportion of subjects willing to consider alternative modes of transportation and alternative destinations. We further identified patient characteristics associated with willingness to consider these alternatives. We conducted a cross-sectional survey study in the emergency department (ED) of an academic medical center. Research assistants screened all non-critically ill ED patients for eligibility and willingness to participate and administered an interview-based survey that included questions on demographic and clinical characteristics, perceived illness severity, and acceptability of alternatives to traditional emergency care for acute illness and injuries. We calculated the proportions and 95% confidence intervals for subjects who found alternative transport modes and destinations acceptable and developed a log-binomial regression model to identify patient characteristics associated with acceptability of alternative modes of transport and alternative destinations. Complete data were available on 1,058 subjects. Forty-two percent of the study sample arrived to the ED via emergency medical services (EMS). Over two-thirds of the study sample (68.2%) was willing to consider transport via either taxi or medical transport van and 69.0% was willing to consider either transportation to an urgent care center or their primary care physician's office. Other alternatives, including delayed EMS response time, were less frequently endorsed as acceptable alternatives. Subject characteristics associated with willingness to accept alternative modes of transportation included younger age, chief complaint, previous ED use, and place of residence (p < 0.05). Subject characteristics associated with willingness to accept alternative destinations included younger age, non-white race, lower patient acuity, and lower self-perceived illness severity (p < 0.05). In our ED, some patients found

  15. Orion Project: Alternate Attitude Study

    NASA Technical Reports Server (NTRS)

    Alvarez-Hernandez, A.; Miller, Stephen W.

    2009-01-01

    This slide presentation reviews the study done on alternate attitudes for the Orion project's crew exploration vehicle. The analysis focused on the thermal performance of the vehicle with the alternate attitudes. The pressure vessel heater power, other vehicle heaters and radiator sink temperatures were included in the analysis.

  16. Saving Money with Menu Alternatives.

    ERIC Educational Resources Information Center

    Rubin, David

    1998-01-01

    Menu alternatives are substitute meals, whereas menu additions are dishes that complement the main meal. Both should be vegetarian dishes that are less expensive than the main offering and attractive to 20-40% of the camp population. By offering alternatives and additions, one can eliminate complaints, save money, and change eating patterns.…

  17. Alternate Semester 1972. An Evaluation.

    ERIC Educational Resources Information Center

    Copp, Barry D.; And Others

    Five students from a Boston high school and 35 from Lincoln-Sudbury took part in an alternate semester consisting of 5 units: Outward Bound, environmental issues, urban, rural, and river/building. Students had to be juniors or seniors, have parental permission, and meet graduation requirements. The alternate semester began with Outward Bound, a…

  18. An Alternative in Engineering Education.

    ERIC Educational Resources Information Center

    Garrett, Rick L.

    1996-01-01

    To meet increased technical demands, alternative engineering and technical degree programs are needed, such as four-year programs in engineering technology. A technical college and university in Florence, SC, are collaborating on such a 1+2+1 alternative degree program. (SK)

  19. The Alternative Press in Microform.

    ERIC Educational Resources Information Center

    Tsang, Daniel C.

    Presented is an analysis of what has been done regarding the preservation of alternative publications in microfilm. The alternative press was defined, for the purposes of this study, as "nonstandard, nonestablishment publications." Two projects undertaken to microfilm such publications, one by the Microphoto Division of Bell and Howell and the…

  20. Curricular Implications of Alternate Assessments.

    ERIC Educational Resources Information Center

    Browder, Diane; Flowers, Claudia; Ahlgrim-Delzell, Lynn; Karvonen, Meagan; Spooner, Fred; Algozzine, Robert

    The 1997 amendments to the Individuals with Disabilities Education Act (IDEA) required that states provide alternate assessments for students unable to participate in the statewide assessment systems (e.g., those with severe disabilities). IDEA mandated that alternate assessments access the general curriculum. This study examined the curricular…

  1. Difficulties of Alternatively Certified Teachers

    ERIC Educational Resources Information Center

    Schonfeld, Irvin Sam; Feinman, Samantha J.

    2012-01-01

    This daily diary study followed, over a 2-week period, 252 beginning New York City public school teachers. Seventy percent were alternatively certified (New York City Teaching Fellows) and the rest, traditionally certified teachers. Alternatively certified teachers were more likely to experience stressors such as violent incidents and classroom…

  2. How did alternative splicing evolve?

    PubMed

    Ast, Gil

    2004-10-01

    Alternative splicing creates transcriptome diversification, possibly leading to speciation. A large fraction of the protein-coding genes of multicellular organisms are alternatively spliced, although no regulated splicing has been detected in unicellular eukaryotes such as yeasts. A comparative analysis of unicellular and multicellular eukaryotic 5' splice sites has revealed important differences - the plasticity of the 5' splice sites of multicellular eukaryotes means that these sites can be used in both constitutive and alternative splicing, and for the regulation of the inclusion/skipping ratio in alternative splicing. So, alternative splicing might have originated as a result of relaxation of the 5' splice site recognition in organisms that originally could support only constitutive splicing. PMID:15510168

  3. Our favourite alternative splice site.

    PubMed

    Lerivray, Hubert; Méreau, Agnès; Osborne, H Beverley

    2006-05-01

    Alternative splicing is a widespread mechanism in mammals that generates several mRNAs from one gene, thereby creating genetic diversity of the genome. Variant splice patterns are often specific to different stages of development or particular tissues, and alternative splicing defects are being more frequently detected in genetic diseases and cancers. The increasingly important role of alternative splicing in the function and the regulation of cellular process makes it critical to have an easy-to-use data repository for the biological and medical research communities. We have compared web resources that give access to information on alternatively spliced genes, and the FAST DB (Friendly Alternative Splicing and Transcripts DataBase) site came out as our favourite.

  4. NASA Alternative Aviation Fuel Research

    NASA Astrophysics Data System (ADS)

    Anderson, B. E.; Beyersdorf, A. J.; Thornhill, K. L., II; Moore, R.; Shook, M.; Winstead, E.; Ziemba, L. D.; Crumeyrolle, S.

    2015-12-01

    We present an overview of research conducted by NASA Aeronautics Research Mission Directorate to evaluate the performance and emissions of "drop-in" alternative jet fuels, highlighting experiment design and results from the Alternative Aviation Fuel Experiments (AAFEX-I & -II) and Alternative Fuel-Effects on Contrails and Cruise Emissions flight series (ACCESS-I & II). These projects included almost 100 hours of sampling exhaust emissions from the NASA DC-8 aircraft in both ground and airborne operation and at idle to takeoff thrust settings. Tested fuels included Fischer-Tropsch (FT) synthetic kerosenes manufactured from coal and natural-gas feedstocks; Hydro-treated Esters and Fatty-Acids (HEFA) fuels made from beef-tallow and camelina-plant oil; and 50:50 blends of these alternative fuels with Jet A. Experiments were also conducted with FT and Jet A fuels doped with tetrahydrothiophene to examine the effects of fuel sulfur on volatile aerosol and contrail formation and microphysical properties. Results indicate that although the absence of aromatic compounds in the alternative fuels caused DC-8 fuel-system leaks, the fuels did not compromise engine performance or combustion efficiency. And whereas the alternative fuels produced only slightly different gas-phase emissions, dramatic reductions in non-volatile particulate matter (nvPM) emissions were observed when burning the pure alternative fuels, particularly at low thrust settings where particle number and mass emissions were an order of magnitude lower than measured from standard jet fuel combustion; 50:50 blends of Jet A and alternative fuels typically reduced nvPM emissions by ~50% across all thrust settings. Alternative fuels with the highest hydrogen content produced the greatest nvPM reductions. For Jet A and fuel blends, nvPM emissions were positively correlated with fuel aromatic and naphthalene content. Fuel sulfur content regulated nucleation mode aerosol number and mass concentrations within aging

  5. Alternative medicine among college students.

    PubMed

    Gaedeke, R M; Tootelian, D H; Holst, C

    1999-01-01

    The use of "alternative" medicine has become increasingly popular in the United States. Books devoted to alternative medicine, e.g., Spontaneous Healing and 8 Weeks to Optimum Health, have become best sellers. Nevertheless, relatively few research studies have focused on the subject. This study examined the role of alternative medicine among college students. Issues addressed included students' familiarity with, use of, and perceptions regarding unconventional health therapies. The study substantiated a phenomenon health care providers across the country are discovering: a growing number of Americans with interest and financial resources support the development of "mainstream" alternative medicine programs. Results of the study also suggest that well-respected traditional health care organizations would not suffer reputation damage if they were to offer such programs.

  6. Disturbance maintains alternative biome states.

    PubMed

    Dantas, Vinícius de L; Hirota, Marina; Oliveira, Rafael S; Pausas, Juli G

    2016-01-01

    Understanding the mechanisms controlling the distribution of biomes remains a challenge. Although tropical biome distribution has traditionally been explained by climate and soil, contrasting vegetation types often occur as mosaics with sharp boundaries under very similar environmental conditions. While evidence suggests that these biomes are alternative states, empirical broad-scale support to this hypothesis is still lacking. Using community-level field data and a novel resource-niche overlap approach, we show that, for a wide range of environmental conditions, fire feedbacks maintain savannas and forests as alternative biome states in both the Neotropics and the Afrotropics. In addition, wooded grasslands and savannas occurred as alternative grassy states in the Afrotropics, depending on the relative importance of fire and herbivory feedbacks. These results are consistent with landscape scale evidence and suggest that disturbance is a general factor driving and maintaining alternative biome states and vegetation mosaics in the tropics.

  7. Alternative splicing and muscular dystrophy

    PubMed Central

    Pistoni, Mariaelena; Ghigna, Claudia; Gabellini, Davide

    2013-01-01

    Alternative splicing of pre-mRNAs is a major contributor to proteomic diversity and to the control of gene expression in higher eukaryotic cells. For this reasons, alternative splicing is tightly regulated in different tissues and developmental stages and its disruption can lead to a wide range of human disorders. The aim of this review is to focus on the relevance of alternative splicing for muscle function and muscle disease. We begin by giving a brief overview of alternative splicing, muscle-specific gene expression and muscular dystrophy. Next, to illustrate these concepts we focus on two muscular dystrophy, myotonic muscular dystrophy and facioscapulohumeral muscular dystrophy, both associated to disruption of splicing regulation in muscle. PMID:20603608

  8. Peat as an energy alternative

    SciTech Connect

    Punwani, D.V.

    1980-07-01

    The importance of developing alternative energy sources to augment supplies of fossil fuels is growing all over the world. Coal, oil shale, tar sands, biomass, solar, geothermal, nuclear, and hydroelectric power have received considerable attention as alternative energy sources. One large energy resource, however, has received little attention until recently. That resource is peat. Although peat is used as an energy source in some countries such as Russia, Ireland, and Finland, it is virtually unexploited in many countries including the United States. This paper provides an understanding of peat: its varieties, abundance, and distribution; its value as an energy alternative; its current and future role as an energy alternative; and the environmental and socioeconomic impacts of large-scale peat utilization.

  9. Alternative food safety intervention technologies

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Alternative nonthermal and thermal food safety interventions are gaining acceptance by the food processing industry and consumers. These technologies include high pressure processing, ultraviolet and pulsed light, ionizing radiation, pulsed and radiofrequency electric fields, cold atmospheric plasm...

  10. Alternative approaches to plasma confinement

    NASA Technical Reports Server (NTRS)

    Roth, J. R.

    1977-01-01

    The potential applications of fusion reactors, the desirable properties of reactors intended for various applications, and the limitations of the Tokamak concept are discussed. The principles and characteristics of 20 distinct alternative confinement concepts are described, each of which may be an alternative to the Tokamak. The devices are classed as Tokamak-like, stellarator-like, mirror machines, bumpy tori, electrostatically assisted, migma concept, and wall-confined plasma.

  11. State Energy Alternatives: Alternative Energy Resources by State

    DOE Data Explorer

    This U.S. map provides state by state information on incentives and laws related to alternative fuels and advanced vehicles. Discover what's available in each state for innovation grants, infrastructure grants, and production grants and who to contact. Find out how many alternative refueling stations are available in each state and where they are. Tennessee, for example, in 2009, has 114 alternative refueling stations: 36 biodiesel, 1 electrical, 29 ethanol, 4 natural gas, and 44 propane. There are also 5 Truck Stop Electrification (TSE) sites in Tennessee. Users can also find out from this map interface the contacts for Clean Cities in a state, information about renewable energy projects and activities in each state, fuel prices across a state, and biomass potential resources and current production in each state.

  12. Community assembly: alternative stable states or alternative transient states?

    PubMed Central

    Fukami, Tadashi; Nakajima, Mifuyu

    2011-01-01

    The concept of alternative stable states has long been a dominant framework for studying the influence of historical contingency in community assembly. This concept focuses on stable states, yet many real communities are kept in a transient state by disturbance, and the utility of predictions for stable states in explaining transient states remains unclear. Using a simple model of plant community assembly, we show that the conditions under which historical contingency affects community assembly can differ greatly for stable versus transient states. Differences arise because the contribution of such factors as mortality rate, environmental heterogeneity and plant-soil feedback to historical contingency changes as community assembly proceeds. We also show that transient states can last for a long time relative to immigration rate and generation time. These results argue for a conceptual shift of focus from alternative stable states to alternative transient states for understanding historical contingency in community assembly. PMID:21790934

  13. Alternative fuels for maritime use

    SciTech Connect

    Not Available

    1980-01-01

    The objectives of this study were to review the potential fuels which may be available to the marine industry from the present to the year 2000 and to define the economic, technical, and environmental/social impacts of these alternative fuels on marine power plants. Thus, this study is aimed at the fuels/prime mover combination. This study should help to guide the industry in choosing the proper power plant for the future - recognizing that a new power plant installed in 1980 will still be in service in the 21st century. The importance of the marine transportation industry and the need to consider alternate fuels are examined. An overview of potential alternate fuels for marine applications is presented, and power plant/fuel interaction is discussed. An in-depth discussion is presented on the impact of the most likely alternate fuels from the viewpoint of maintenance, retrofit capability, safety, and air-quality impacts. Two nonfossil-fuel alternatives sailing ships and nuclear-powered vessels, are discussed. It is concluded that: there is a high probability of using synfuels from tar sands shale, or coal liquids in both existing and future ships; coal and coal/oil slurries have a high probability of use in future ships and medium probability in existing ships; nuclear and sail-power future ships have a medium probability of commercial development; and is a low probability of commercial maritime use of alcohol fuels, methane, or coal/methanol combinations. (LCL)

  14. Advances in alternative cementitious binders

    SciTech Connect

    Juenger, M.C.G.; Winnefeld, F.; Provis, J.L.; Ideker, J.H.

    2011-12-15

    There is a burgeoning interest in the development, characterization, and implementation of alternatives to Portland cement as a binder in concrete. The construction materials industry is under increasing pressure to reduce the energy used in production of Portland cement clinker and the associated greenhouse gas emissions. Further, Portland cement is not the ideal binder for all construction applications, as it suffers from durability problems in particularly aggressive environments. Several alternative binders have been available for almost as long as Portland cement, yet have not been extensively used, and new ones are being developed. In this paper, four promising binders available as alternatives to Portland cement are discussed, namely calcium aluminate cement, calcium sulfoaluminate cement, alkali-activated binders, and supersulfated cements. The history of the binders, their compositions and reaction mechanisms, benefits and drawbacks, unanswered questions, and primary challenges are described.

  15. Alternative treatments for the menopause.

    PubMed

    Rees, Margaret

    2009-02-01

    Concerns about the safety of oestrogen-based hormone replacement therapy after publication of the Women's Health Initiative study and Million Women Study has led to women turning to alternative therapies, erroneously believing that they are safer and 'more natural'. Evidence from randomized trials that alternative and complementary therapies improve menopausal symptoms or have the same benefits as conventional pharmacopoeia is poor. There are no recognized international criteria for the design of clinical trials of alternative therapies as there are for standard medicines and medical devices for endpoints of treatment and safety evaluations. Studies may have limitations such as design, sample size and duration. There is a wide range of different preparations, making comparison difficult. The evidence regarding botanicals, homeopathy, steroids, vitamin supplements, dietary changes and functional foods, and physical interventions are discussed in this chapter. Standard pharmacopoeia such as clonidine, selective serotonin re-uptake inhibitors and progestogens are also examined.

  16. Experiences in mainstreaming alternative energy

    SciTech Connect

    Cabraal, A.

    1997-12-01

    The author discusses efforts by the Asia Alternative Energy Unit (ASTAE) of the World Bank in supporting alternative energy source projects in Asia. Energy growth rates have been as high as 18% per year, with power capacity doubling each decade in the 1960`s, 70`s and 80`s. Much of this has come from fossil fuel projects coupled with major hydroelectric projects. One consequence is developing air pollution loads originating in Asia. ASTAE has been supporting pilot programs in applying alternative energy sources. The goal has been to mainstream renewable energy sources in World Bank operations, by working with managers from different countries to: include renewable energy in country assistance strategies and sectorial development plans; provide assistance to renewable energy initiatives; expand initiatives to new countries, sectors and technologies.

  17. [Alternative treatment methods in ENT].

    PubMed

    Friese, K H

    1997-08-01

    In this review, the most important complementary und alternative therapies are discussed, focusing particularly on their use in otorhinolaryngology. These therapies include balneology, Kneipp therapy, microbiological therapy, fasting, excretion therapy, different oxygen therapies, hydro-colon therapy, urine therapy, own-blood therapy, Bach therapy, orthomolecular therapy, order therapy, environmental medicine, phytotherapy, homeopathy, complex homeopathy, anthroposophy, neural therapy, electroaccupuncture according to Voll and similar therapies, nasal reflex therapy, reflex-zone massage, manual therapy, massage, lymph drainage, aroma therapy, thermotherapy, bioresonance, kinesiology, hopi candles, and dietetics. Some of these methods and regimens can be recommended, but others should be rejected. In universities, these methods are only represented to a minor extend, but are more accepted by otorhinolaryngologists in practice. This paper provides a guide to which alternative therapies are sensible and possible in otorhinolaryngology. The aim is to stimulate interest in these methods. It is necessary to discuss these alternative methods reasonably and credibly with patients. PMID:9378666

  18. Gasohol - Analysis and biomass alternatives

    NASA Astrophysics Data System (ADS)

    1980-11-01

    The economics of fermentation ethanol as a near-term alternative to liquid hydrocarbon fuels are analyzed and alternatives to grain-fermented ethanol are examined. Based on estimates of raw material and production costs and energy consumption, it is shown that net production costs for alcohol fuel from corn amount to $2.14/gallon, with no significant net consumption or gain in energy. It is also pointed out that the use of grain for alcohol production will influence quantities available for livestock production and export, and that land available for grain production is limited. Consideration is then given to the economic potential of using cellulosic biomass from agricultural and forest residues in the production of ethanol fuels and coal gasification for methanol production, and it is pointed out that these alternatives offer economic, energy and oil-savings advantages over ethanol production from grains.

  19. Revisiting the solar hydrogen alternative

    SciTech Connect

    Tomkiewicz, M.

    1996-09-01

    Research aimed at the development of technology to advance the solar-hydrogen alternative is per definition mission oriented. The priority that society puts on such research rise and fall with the priorities that we associate with the mission. The mission that we associate with the hydrogen economy is to provide a technological option for an indefinitely sustainable energy and material economies in which society is in equilibrium with its environment. In this paper we try to examine some global aspects of the hydrogen alternative and recommend formulation of a {open_quotes}rational{close_quotes} tax and regulatory system that is based on efforts needed to restore the ecological balance. Such a system, once entered into the price structure of the alternative energy schemes, will be used as a standard to compare energy systems that in turn will serve as a base for prioritization of publicly supported research and development.

  20. FMDP reactor alternative summary report: Volume 4, Evolutionary LWR alternative

    SciTech Connect

    1996-09-01

    Significant quantities of weapons-usable fissile materials [primarily plutonium and highly enriched uranium (HEU)] have become surplus to national defense needs both in the United States and Russia. These stocks of fissile materials pose significant dangers to national and international security. The dangers exist not only in the potential proliferation of nuclear weapons but also in the potential for environmental, safety, and health (ES&H) consequences if surplus fissile materials are not properly managed. The purpose of this report is to provide schedule, cost, and technical information that will be used to support the Record of Process (ROD). Following the screening process, DOE/MD via its national laboratories initiated a more detailed analysis activity to further evaluate each of the ten plutonium disposition alternatives that survived the screening process. Three ``Alternative Teams,`` chartered by DOE and comprised of technical experts from across the DOE national laboratory complex, conducted these analyses. One team was chartered for each of the major disposition classes (borehole, immobilization, and reactors). During the last year and a half, the Fissile Materials Disposition Program (FMDP) Reactor Alternative Team (RxAT) has conducted extensive analyses of the cost, schedule, technical maturity, S&S, and other characteristics of reactor-based plutonium disposition. The results of the RxAT`s analyses of the existing LWR, CANDU, and partially complete LWR alternatives are documented in Volumes 1-3 of this report. This document (Volume 4) summarizes the results of these analyses for the ELWR-based plutonium disposition option.

  1. Alternative general-aircraft engines

    NASA Technical Reports Server (NTRS)

    Tomazic, W. A.

    1976-01-01

    The most promising alternative engine (or engines) for application to general aircraft in the post-1985 time period was defined, and the level of technology was cited to the point where confident development of a new engine can begin early in the 1980's. Low emissions, multifuel capability, and fuel economy were emphasized. Six alternative propulsion concepts were considered to be viable candidates for future general-aircraft application: the advanced spark-ignition piston, rotary combustion, two- and four-stroke diesel, Stirling, and gas turbine engines.

  2. Multipebble Simulations for Alternating Automata

    NASA Astrophysics Data System (ADS)

    Clemente, Lorenzo; Mayr, Richard

    We study generalized simulation relations for alternating Büchi automata (ABA), as well as alternating finite automata. Having multiple pebbles allows the Duplicator to "hedge her bets" and delay decisions in the simulation game, thus yielding a coarser simulation relation. We define (k 1,k 2)-simulations, with k 1/k 2 pebbles on the left/right, respectively. This generalizes previous work on ordinary simulation (i.e., (1,1)-simulation) for nondeterministic Büchi automata (NBA)[4] in and ABA in [5], and (1,k)-simulation for NBA in [3].

  3. Spent-fuel-storage alternatives

    SciTech Connect

    Not Available

    1980-01-01

    The Spent Fuel Storage Alternatives meeting was a technical forum in which 37 experts from 12 states discussed storage alternatives that are available or are under development. The subject matter was divided into the following five areas: techniques for increasing fuel storage density; dry storage of spent fuel; fuel characterization and conditioning; fuel storage operating experience; and storage and transport economics. Nineteen of the 21 papers which were presented at this meeting are included in this Proceedings. These have been abstracted and indexed. (ATT)

  4. Two alternative versions of strangeness

    PubMed Central

    Nishijima, Kazuhikoa

    2008-01-01

    The concept of strangeness emerged from the low energy phenomenology before the entry of quarks in particle physics. The connection between strangeness and isospin is rather accidental and loose and we recognize later that the definition of strangeness is model-dependent. Indeed, in Gell-Mann’s triplet quark model we realize that there is a simple alternative representation of strangeness. When the concept of generations is incorporated into the quark model we find that only the second alternative version of strangeness remains meaningful, whereas the original one does no longer keep its significance. PMID:18997448

  5. The neurogenetics of alternative splicing

    PubMed Central

    Vuong, Celine K.; Black, Douglas L.; Zheng, Sika

    2016-01-01

    Alternative precursor-mRNA splicing is a key mechanism for regulating gene expression in mammals and is controlled by specialized RNA-binding proteins. The misregulation of splicing is implicated in multiple neurological disorders. We describe recent mouse genetic studies of alternative splicing that reveal its critical role in both neuronal development and the function of mature neurons. We discuss the challenges in understanding the extensive genetic programmes controlled by proteins that regulate splicing, both during development and in the adult brain. PMID:27094079

  6. Integrative Medicine and Complementary and Alternative Therapies

    MedlinePlus

    ... 000 this month to find cures. Loading... Integrative Medicine and Complementary and Alternative Therapies Integrative Medicine and Complementary and Alternative Therapies SHARE: Print Glossary ...

  7. Alternatives To Dissection. Second Edition.

    ERIC Educational Resources Information Center

    DeRosa, Bill, Ed.; Winiarskyj, Lesia, Ed.

    This packet attempts to provide educationally sound alternatives to dissection in the classroom, thereby making it possible for teachers to eliminate dissection from the curriculum. This packet can also be used by educators who include dissection in their curricula but consider it important to respect the expression of students' ethical, moral, or…

  8. An Alternative to Process Recording

    ERIC Educational Resources Information Center

    Baker, Joan; And Others

    1976-01-01

    Some disadvantages in the use of process recordings as an assessment and teaching tool for evaluating the communication skills of the student in nurse-client interactions are discussed. A more useful alternative process requires actual observation and subsequent participation by the instructor during student-client interviews. (EC)

  9. Looking for alternative energy sources.

    PubMed

    Gross, Michael

    2012-02-21

    With unrest in oil-exporting countries, backlashes against biofuels and photovoltaics, and a nuclear incident in Japan, the year 2011 rattled confidence in future energy supplies. The search for alternatives is all the more urgent, but some of the solutions investigated hark back to fossil fuels that we can't afford to burn.

  10. Mobile Alternative Fueling Station Locator

    SciTech Connect

    Not Available

    2009-04-01

    The Department of Energy's Alternative Fueling Station Locator is available on-the-go via cell phones, BlackBerrys, or other personal handheld devices. The mobile locator allows users to find the five closest biodiesel, electricity, E85, hydrogen, natural gas, and propane fueling sites using Google technology.

  11. Providers issue brief: alternative providers.

    PubMed

    Rothouse, M

    1999-06-29

    Access by managed care plan enrollees, scope of practice issues and fee reimbursement by Medicaid and third parties such as insurance carriers is the engine that drives legislation recognizing alternative health care providers--chiropractors, acupuncturists, physical therapists, naturopaths, massage therapists, homeopaths, and dietitians and nutritionists. PMID:11073386

  12. "Partial Vision" in Alternative Education.

    ERIC Educational Resources Information Center

    Miller, Ron

    1997-01-01

    A comparison of the philosophies and pedagogies of Waldorf and Montessori schools points out that no single educational perspective can encompass all possibilities of human growth. Educators should attend to the "seed qualities" of alternative educational philosophies rather than their dogmas. A caring relationship between teachers and students is…

  13. The Alternate Route to Teaching.

    ERIC Educational Resources Information Center

    Penning, Nick

    1990-01-01

    Alternative certification programs are needed to address the projected teacher shortage by 2000. Only two-thirds of prepared graduates enter the profession, half who do enter leave after five years, and teacher salaries are 25 percent lower than the salaries of college graduates in other fields. Afternoon, evening, and weekend preparation programs…

  14. Alternative Models for Higher Education.

    ERIC Educational Resources Information Center

    Caffrey, John G.

    Higher education is beset from all sides by criticism, fear, doubt, uncertainty, and prophecies of doom. While the young call for change, the faculty often resist anything that might reduce their privileges and prerogatives. Before alternative models to the present system of higher education can be considered, it is useful to question present…

  15. Alternatives for jet engine control

    NASA Technical Reports Server (NTRS)

    Sain, M. K.

    1984-01-01

    The technical progress of researches on alternatives for jet engine control is reported. Extensive numerical testing is included. It is indicated that optimal inputs contribute significantly to the process of calculating tensor approximations for nonlinear systems, and that the resulting approximations may be order-reduced in a systematic way.

  16. Alternatives for jet engine control

    NASA Technical Reports Server (NTRS)

    Sain, M. K.

    1984-01-01

    The technical progress of researches Alternatives for Jet Engine Control is reported. A numerical study employing feedback tensors for optimal control of nonlinear systems was completed. It is believed that these studies are the first of their kind. State regulation, with a decrease in control power is demonstrated. A detailed treatment follows.

  17. Alternatives for jet engine control

    NASA Technical Reports Server (NTRS)

    Sain, M. K.

    1983-01-01

    The technical progress of researches on alternatives for jet engine control, is reported. The principal new activities involved the initial testing of an input design method for choosing the inputs to a non-linear system to aid the approximation of its tensor parameters, and the beginning of order reduction studies designed to remove unnecessary monomials from tensor models.

  18. An Overview of Alternative Education

    ERIC Educational Resources Information Center

    Aron, Laudan Y.

    2006-01-01

    This publication represents the first in a series of papers on alternative education for the U.S. Department of Labor. The main focus of this review is community- or district-based programs that have as their primary focus the re-engagement of out-of-school youth in learning in order to better prepare these youth to successfully enter high growth…

  19. Student Loans: Alternatives for Reauthorization.

    ERIC Educational Resources Information Center

    National Association of Student Financial Aid Administrators, Washington, DC.

    The papers presented at the symposium include: Student Loans: Some Practical Radical Alternatives (D. Bruce Johnstone); Solving the Student Loan Problem by Supplementing Government Loan Policy--An Expanded Role for Institutional Aid Administrators in the 1980's (Kurt L. Kendis); Estimating Manageable Educational Loan Limits for Graduate and…

  20. Not Just Another Alternative School

    ERIC Educational Resources Information Center

    Gulson, Kalervo N.; Webb, P. Taylor

    2016-01-01

    In this article, we problematize the establishment of an Africentric Alternative School in Toronto, Canada. We argue that policy, and race and racializations cannot be understood outside of, or immune to, neoliberalism. We contend that policy is a form of racial biopolitics, and race is now produced through neoliberal markets, in conjunction with…

  1. The Centre for Alternative Technology.

    ERIC Educational Resources Information Center

    Randle, Joan; Randle, Damian

    1983-01-01

    Describes operation and activities of The National Center for Alternative Technology (NCAT), built on the site of a former quarry. A major center function involves encouraging and assisting students toward a holistic approach to their environment. Also describes and includes a map of major center exhibits. (JN)

  2. Alternative Certification: Effectively Preparing Teachers

    ERIC Educational Resources Information Center

    Foster, Christopher; Bishop, Carolyn A.; Hernandez, David A.

    2008-01-01

    This study addresses questions related to teacher preparation through online supported learning in an alternative certification program, CalStateTEACH. Specifically, this study analyzes and reports survey responses of 130 teachers and 84 supervisors related to preparedness of program completers to teach in a multiple subject classroom. The…

  3. Renewable Energy Alternatives in Maryland.

    ERIC Educational Resources Information Center

    Welsh, Greg E.; McClellan, Deborah A. S.

    This handbook discusses the renewable energy resources suitable for use in Maryland. It follows a question and answer format with sections about the following alternative renewable energy sources; solar, wind, wood, water, bio-gas/methane, and geothermal. Each section includes a list of recommended readings, appropriate agencies or organizations,…

  4. Alternatives to Afrocentrism. Second Edition.

    ERIC Educational Resources Information Center

    Miller, John J., Ed.

    The essays in this collection place the current Afrocentric movement in its historical context and offer alternative suggestions about how to teach African American students about their history. The first section deals with the roots of Afrocentrism, analyzes the content of Afrocentric books and curricula, and discusses the impact of Afrocentrism…

  5. An Alternative to Thought Suppression?

    ERIC Educational Resources Information Center

    Boice, Robert

    2012-01-01

    Comments on the original article, "Setting free the bears: Escape from thought suppression," by D. M. Wegner (see record 2011-25622-008). While Wegner supposed that we might have to learn to live with bad thoughts, the present author discusses the use of imagination and guided imagery as an alternative to forced thought suppression.

  6. A Simple Alternative to Grading

    ERIC Educational Resources Information Center

    Potts, Glenda

    2010-01-01

    In this article, the author investigates whether an alternative grading system (contract grading) would yield the same final grades as traditional grading (letter grading), and whether or not it would be accepted by students. The author states that this study demonstrated that contract grading was widely, and for the most part, enthusiastically…

  7. Lecture Alternatives in Teaching English.

    ERIC Educational Resources Information Center

    Judy, Stephen, Ed.

    The five sections of the document are: General Discussion; Classroom Experiences; Evaluation and Non-Lecture Teaching; A Closing Note; and Appendix. The ten papers presented are as follows: "Lecture Alternatives and the English Class" by Stephen Judy; "Let's See How it Goes: A View of the Teacher as Manager of Student-Initiated Activities" by…

  8. Science Literacy and Alternative Futures.

    ERIC Educational Resources Information Center

    O'Hearn, George T.

    Presented is an operational definition of science literacy including knowledge of science, the processes of science, the nature of science and the social implications of science. Future-oriented supplemental teaching materials are proposed as the most viable alternative available for achieving literacy in science within the present school…

  9. Alternative Careers in Physical Education.

    ERIC Educational Resources Information Center

    Lambert, Charlotte

    Several alternate definitions of "physical education" can be presented to illustrate the fact that changes in name or definition open the way to new opportunities and new ways of thinking of career options. Traditional definitions of physical education have limited it to a profession of teaching in the traditional school system normal children…

  10. Alternative Education: The Cutting Edge?

    ERIC Educational Resources Information Center

    Byrne, Jay

    2004-01-01

    Miami Valley Career Technology Youth Connections, an alternative high school located in Dayton, Ohio, has an enrollment of 160 students and was created as a joint venture among local politicians to address the high dropout rates in Montgomery County, Ohio. To achieve this, the Montgomery County commissioners created a task force to develop…

  11. An Alternative School Conceptual Framework.

    ERIC Educational Resources Information Center

    Morris, Lee A.

    This model is designed to serve as a conceptual framework for alternative programs with primary emphasis on provisioning (the way in which time, space, material, equipment, and activities are used in the learning environment). The model's concern is to assess concrete examples or tendencies of teachers, students, and the education program. The…

  12. Plain Talk about Alternative Assessment.

    ERIC Educational Resources Information Center

    Cheek, Dennis W.

    1993-01-01

    Testing is a technology, a socially mediated, value-laden endeavor. Technologies embody trade-offs between what is desired and real-world constraints of cost, time, thought, and energy. Although alternative assessments offer exciting prospects for middle schools, these new forms must not be misused or naively accepted as defensible, consummate…

  13. An Alternative Triangle Area Strategy

    ERIC Educational Resources Information Center

    Shockey, T. L.; Zhang, P.; Brosnan, P.

    2016-01-01

    We explore the mathematics engagement of a group of mathematics coaches, working in k-12 mathematics education. The incenter of a triangle is used to derive an alternative formula for the area of a triangle inspired by Usiskin, Peressini, Marhisotto, and Stanley (2002).

  14. Academic Programs in Alternative Education: An Overview

    ERIC Educational Resources Information Center

    Ruzzi, Betsy Brown; Kraemer, Jacqueline

    2006-01-01

    This paper, second in a series of papers on alternative education, examines the academic programming in alternative education programs by reviewing the literature specifically focused on the academic programs in alternative education and summarizing a survey of fifteen alternative education programs. It suggests options for further research on…

  15. Alternatives for jet engine control

    NASA Technical Reports Server (NTRS)

    Sain, M. K.; Schafer, R. M.

    1980-01-01

    Alternatives to linear quadratic regulator theory in the linear case are examined along with nonlinear modelling and optimization approaches for global control. Context for the studies has been set by the DYNGEN digital simulator and by models generated for various phases of the F100 Multivariable Control Synthesis Program. With respect to the linear alternatives, the multivariable frequency domain is stressed. Progress is reported in both the direct algebraic approach to exact model matching, by means of stimulating work on the basic computational issues, and in the indirect generalized Nyquist approach. With respect to nonlinear modelling and optimization, the emphasis is twofold: the development of analytical nonlinear models of the jet engine and the use of these models in conjunction with techniques of mathematical programming in order to study global control over nonincremental portions of the flight envelope. The possibility of using tensor methods is explored.

  16. Potential alternative approaches to xenotransplantation.

    PubMed

    Mou, Lisha; Chen, Fengjiao; Dai, Yifan; Cai, Zhiming; Cooper, David K C

    2015-11-01

    There is an increasing worldwide shortage of organs and cells for transplantation in patients with end-stage organ failure or cellular dysfunction. This shortage could be resolved by the transplantation of organs or cells from pigs into humans. What competing approaches might provide support for the patient with end-stage organ or cell failure? Four main approaches are receiving increasing attention - (i) implantable mechanical devices, although these are currently limited almost entirely to devices aimed at supporting or replacing the heart, (ii) stem cell technology, at present directed mainly to replace absent or failing cells, but which is also fundamental to progress in (iii) tissue engineering and regenerative medicine, in which the ultimate aim is to replace an entire organ. A final novel potential approach is (iv) blastocyst complementation. These potential alternative approaches are briefly reviewed, and comments added on their current status and whether they are now (or will soon become) realistic alternative therapies to xenotransplantation.

  17. Alternative Splicing in Plant Immunity

    PubMed Central

    Yang, Shengming; Tang, Fang; Zhu, Hongyan

    2014-01-01

    Alternative splicing (AS) occurs widely in plants and can provide the main source of transcriptome and proteome diversity in an organism. AS functions in a range of physiological processes, including plant disease resistance, but its biological roles and functional mechanisms remain poorly understood. Many plant disease resistance (R) genes undergo AS, and several R genes require alternatively spliced transcripts to produce R proteins that can specifically recognize pathogen invasion. In the finely-tuned process of R protein activation, the truncated isoforms generated by AS may participate in plant disease resistance either by suppressing the negative regulation of initiation of immunity, or by directly engaging in effector-triggered signaling. Although emerging research has shown the functional significance of AS in plant biotic stress responses, many aspects of this topic remain to be understood. Several interesting issues surrounding the AS of R genes, especially regarding its functional roles and regulation, will require innovative techniques and additional research to unravel. PMID:24918296

  18. Wastewater privatization: A beneficial alternative

    SciTech Connect

    Wakeman, R.F.; Drewry, W.A.

    1999-07-01

    Municipalities with wastewater operations face increasing requirements to maximize efficiency, implement capital improvements, and ensure environmental compliance. Privatization is a relatively unused alternative offering benefits in the areas of cost-effective operations, flexible financing, technology access, and compliance assurance. Recent executive direction and tax code changes have opened new doors for mutually beneficial public-private partnerships. Wastewater privatization has historically consisted of short-term contract agreements for treatment operations, but looming infrastructure recapitalization and development requirements have catalyzed an exploration of non-traditional alternatives that include private sector financing, development, and operation of entire wastewater systems, The purpose of this paper is to show why privatization must be considered, evaluate the different levels available, and generate an analytical aid for communities taking their first look at privatization opportunities.

  19. Ambitious Philippine alternative energy plans

    SciTech Connect

    Not Available

    1981-09-07

    The Philippines is to spend $5.4 billion over the next ten years for the development of alternative sources of energy. These would include the development of fuel woods and other biomass, and the commercialization of a coconut/diesel-oil fuel. It is hoped that the Philippines' dependence on imported oil will be reduced from about 80% today to around 50% by the end of the decade.

  20. Alternative scenarios utilizing nonterrestrial resources

    NASA Astrophysics Data System (ADS)

    Eldred, Charles H.; Roberts, Barney B.

    A collection of alternative scenarios that are enabled or substantially enhanced by the utilization of nonterrestrial resources is provided. We take a generalized approach to scenario building so that our report will have value in the context of whatever goals are eventually chosen. Some of the topics covered include the following: lunar materials processing; asteroid mining; lunar resources; construction of a large solar power station; solar dynamic power for the space station; reduced gravity; mission characteristics and options; and tourism.

  1. Alternative models in developmental toxicology.

    PubMed

    Lee, Hyung-yul; Inselman, Amy L; Kanungo, Jyotshnabala; Hansen, Deborah K

    2012-02-01

    In light of various pressures, toxicologists have been searching for alternative methods for safety testing of chemicals. According to a recent policy in the European Union (Regulation, Evaluation Authorisation and Restriction of Chemicals, REACH), it has been estimated that over the next twelve to fifteen years, approximately 30,000 chemicals may need to be tested for safety, and under current guidelines such testing would require the use of approximately 7.2 million laboratory animals [ Hofer et al. 2004 ]. It has also been estimated that over 80% of all animals used for safety testing under REACH legislation would be used for examining reproductive and developmental toxicity [Hofer et al., 2004]. In addition to REACH initiatives, it has been estimated that out of 5,000 to 10,000 new drug entities that a pharmaceutical company may start with, only one is finally approved by the Food and Drug Administration at a cost of over one billion dollars [ Garg et al. 2011 ]. A large portion of this cost is due to animal testing. Therefore, both the pharmaceutical and chemical industries are interested in using alternative models and in vitro tests for safety testing. This review will examine the current state of three alternative models - whole embryo culture (WEC), the mouse embryonic stem cell test (mEST), and zebrafish. Each of these alternatives will be reviewed, and advantages and disadvantages of each model will be discussed. These models were chosen because they are the models most commonly used and would appear to have the greatest potential for future applications in developmental toxicity screening and testing.

  2. Alternative scenarios utilizing nonterrestrial resources

    NASA Technical Reports Server (NTRS)

    Eldred, Charles H.; Roberts, Barney B.

    1992-01-01

    A collection of alternative scenarios that are enabled or substantially enhanced by the utilization of nonterrestrial resources is provided. We take a generalized approach to scenario building so that our report will have value in the context of whatever goals are eventually chosen. Some of the topics covered include the following: lunar materials processing; asteroid mining; lunar resources; construction of a large solar power station; solar dynamic power for the space station; reduced gravity; mission characteristics and options; and tourism.

  3. Alternative to Nitric Acid Passivation

    NASA Technical Reports Server (NTRS)

    Kessel, Kurt R.

    2016-01-01

    Corrosion is an extensive problem that affects the National Aeronautics and Space Administration (NASA) and European Space Agency (ESA). The deleterious effects of corrosion result in steep costs, asset downtime affecting mission readiness, and safety risks to personnel. It is vital to reduce corrosion costs and risks in a sustainable manner. The primary objective of this effort is to qualify citric acid as an environmentally-preferable alternative to nitric acid for passivation of stainless steel alloys.

  4. Alternative Aviation Fuel Experiment (AAFEX)

    NASA Technical Reports Server (NTRS)

    Anderson, B. E.; Beyersdorf, A. J.; Hudgins, C. H.; Plant, J. V.; Thornhill, K. L.; Winstead, E. L.; Ziemba, L. D.; Howard, R.; Corporan, E.; Miake-Lye, R. C.; Herndon, S. C.; Timko, M.; Woods, E.; Dodds, W.; Lee, B.; Santoni, G.; Whitefield, P.; Hagen, D.; Lobo, P.; Knighton, W. B.; Bulzan, D.; Tacina, K.; Wey, C.; VanderWal, R.; Bhargava, A.

    2011-01-01

    The rising cost of oil coupled with the need to reduce pollution and dependence on foreign suppliers has spurred great interest and activity in developing alternative aviation fuels. Although a variety of fuels have been produced that have similar properties to standard Jet A, detailed studies are required to ascertain the exact impacts of the fuels on engine operation and exhaust composition. In response to this need, NASA acquired and burned a variety of alternative aviation fuel mixtures in the Dryden Flight Research Center DC-8 to assess changes in the aircraft s CFM-56 engine performance and emission parameters relative to operation with standard JP-8. This Alternative Aviation Fuel Experiment, or AAFEX, was conducted at NASA Dryden s Aircraft Operations Facility (DAOF) in Palmdale, California, from January 19 to February 3, 2009 and specifically sought to establish fuel matrix effects on: 1) engine and exhaust gas temperatures and compressor speeds; 2) engine and auxiliary power unit (APU) gas phase and particle emissions and characteristics; and 3) volatile aerosol formation in aging exhaust plumes

  5. FMDP reactor alternative summary report. Volume 1 - existing LWR alternative

    SciTech Connect

    Greene, S.R.; Bevard, B.B.

    1996-10-07

    Significant quantities of weapons-usable fissile materials [primarily plutonium and highly enriched uranium (HEU)] are becoming surplus to national defense needs in both the United States and Russia. These stocks of fissile materials pose significant dangers to national and international security. The dangers exist not only in the potential proliferation of nuclear weapons but also in the potential for environmental, safety, and health (ES&H) consequences if surplus fissile materials are not properly managed. This document summarizes the results of analysis concerned with existing light water reactor plutonium disposition alternatives.

  6. Alternating copolymers and alternative device geometries for organic photovoltaics.

    PubMed

    Inganäs, Olle; Zhang, Fengling; Andersson, Mats R

    2012-01-01

    The efficiency of conversion of light to electrical energy with the help of conjugated polymers and molecules is rapidly improving. The optical absorption properties of these materials can be designed, and implemented via molecular engineering. Full coverage of the solar spectrum is thus feasible. Narrow absorption spectra allow construction of tandem solar cells. The poor transport properties of these materials require thin devices, which limits optical absorption. Alternative device geometries for these flexible materials compensate for the optical absorption by light trapping, and allow tandem cells. PMID:22434441

  7. [Alternative medicine: really an alternative to academic medicine?].

    PubMed

    Happle, R

    2000-06-01

    Numerous courses on alternative medicine are regularly advertised in Deutsches Arzteblatt, the organ of the German Medical Association. The present German legislation likewise supports this form of medicine, and this explains why Iscador, an extract of the mistletoe, is found in the Rote Liste, a directory of commercially available medical drugs, under the heading "cytostatic and antimetastatic drugs" although such beneficial effect is unproven. To give another example, a German health insurance fund was sentenced to pay for acupuncture as a treatment for hepatic failure. This judgement is characteristic of the present German judicial system and represents a victory of "oracling irrationalism" (Popper). The astonishing popularity of alternative medicine can be explained by a revival of romanticism. An intellectually fair opposite position has been delineated by Karl Popper in the form of critical rationalism. It is important to realize, however, that our decision to adhere to rational thinking is made in the innermost depth of our heart but not on the basis of rational arguing. Rather, the decision in favor of reason has a moral dimension. PMID:10907162

  8. Thermoelectric converters for alternating current standards

    NASA Astrophysics Data System (ADS)

    Anatychuk, L. I.; Taschuk, D. D.

    2012-06-01

    Thermoelectric converters of alternating current remain priority instruments when creating standard equipment. This work presents the results of design and manufacture of alternating current converter for a military standard of alternating current in Ukraine. Results of simulation of temperature distribution in converter elements, ways of optimization to improve the accuracy of alternating current signal reproduction are presented. Results of metrological trials are given. The quality of thermoelectric material specially created for alternating current metrology is verified. The converter was used in alternating current standard for the frequency range from 10 Hz to 30 MHz. The efficiency of using thermoelectric signal converters in measuring instruments is confirmed.

  9. Bayesian Alternation during Tactile Augmentation

    PubMed Central

    Goeke, Caspar M.; Planera, Serena; Finger, Holger; König, Peter

    2016-01-01

    A large number of studies suggest that the integration of multisensory signals by humans is well-described by Bayesian principles. However, there are very few reports about cue combination between a native and an augmented sense. In particular, we asked the question whether adult participants are able to integrate an augmented sensory cue with existing native sensory information. Hence for the purpose of this study, we build a tactile augmentation device. Consequently, we compared different hypotheses of how untrained adult participants combine information from a native and an augmented sense. In a two-interval forced choice (2 IFC) task, while subjects were blindfolded and seated on a rotating platform, our sensory augmentation device translated information on whole body yaw rotation to tactile stimulation. Three conditions were realized: tactile stimulation only (augmented condition), rotation only (native condition), and both augmented and native information (bimodal condition). Participants had to choose one out of two consecutive rotations with higher angular rotation. For the analysis, we fitted the participants' responses with a probit model and calculated the just notable difference (JND). Then, we compared several models for predicting bimodal from unimodal responses. An objective Bayesian alternation model yielded a better prediction (χred2 = 1.67) than the Bayesian integration model (χred2 = 4.34). Slightly higher accuracy showed a non-Bayesian winner takes all (WTA) model (χred2 = 1.64), which either used only native or only augmented values per subject for prediction. However, the performance of the Bayesian alternation model could be substantially improved (χred2 = 1.09) utilizing subjective weights obtained by a questionnaire. As a result, the subjective Bayesian alternation model predicted bimodal performance most accurately among all tested models. These results suggest that information from augmented and existing sensory modalities in

  10. Synergistic epistasis and alternative hypotheses.

    PubMed

    Trouve, S; Ding, A; Goudet, J

    2004-11-01

    Inbreeding generally results in deleterious shifts in mean fitness. If the fitness response to increasing inbreeding coefficient is non-linear, this suggests a contribution of epistasis to inbreeding depression. In a cross-breeding experiment, Salathe & Ebert (2003. J. Evol. Biol. 16: 976-985) tested and found the presence of this non-linearity in Daphnia magna. They argue that epistatic interactions cause this non-linearity. We argue here that their experimental protocol does not allow disentangling the effect of synergistic epistasis from two alternative hypotheses, namely hybrid vigour and statistical non-independence of data.

  11. Alternative approaches to epilepsy treatment.

    PubMed

    McElroy-Cox, Caitlin

    2009-07-01

    Complementary and alternative medicine (CAM) is a diverse group of health care practices and products that fall outside the realm of traditional Western medical theory and practice and that are used to complement or replace conventional medical therapies. The use of CAM has increased over the past two decades, and surveys have shown that up to 44% of patients with epilepsy are using some form of CAM treatment. This article reviews the CAM modalities of meditation, yoga, relaxation techniques, biofeedback, nutritional and herbal supplements, dietary measures, chiropractic care, acupuncture, Reiki, and homeopathy and what is known about their potential efficacy in patients with epilepsy.

  12. Machine translation project alternatives analysis

    NASA Technical Reports Server (NTRS)

    Bajis, Catherine J.; Bedford, Denise A. D.

    1993-01-01

    The Machine Translation Project consists of several components, two of which, the Project Plan and the Requirements Analysis, have already been delivered. The Project Plan details the overall rationale, objectives and time-table for the project as a whole. The Requirements Analysis compares a number of available machine translation systems, their capabilities, possible configurations, and costs. The Alternatives Analysis has resulted in a number of conclusions and recommendations to the NASA STI program concerning the acquisition of specific MT systems and related hardware and software.

  13. Towards an alternative gravitational theory

    NASA Astrophysics Data System (ADS)

    Nieto, J. A.; Beltrán, L. A.

    2015-03-01

    In 1680, Cassini proposed oval curves as alternative trajectories for the visible planets around the sun. The Cassini ovals were of course overshadowed by the Kepler's first law (1609), namely the planets move around the sun describing conic orbits. Here, we describe the possibility that the Cassini's idea works at larger or smaller scales. Indeed, we consider the Spiric curves (which are a generalization of the Cassini oval) and present the first steps towards a Spiric gravitational theory. We show that from our formalism an ellipse associated with a planet can be obtained as a particular case.

  14. Antibodies: an alternative for antibiotics?

    PubMed

    Berghman, L R; Abi-Ghanem, D; Waghela, S D; Ricke, S C

    2005-04-01

    In 1967, the success of vaccination programs, combined with the seemingly unstoppable triumph of antibiotics, prompted the US Surgeon General to declare that "it was time to close the books on infectious diseases." We now know that the prediction was overly optimistic and that the fight against infectious diseases is here to stay. During the last 20 yr, infectious diseases have indeed made a staggering comeback for a variety of reasons, including resistance against existing antibiotics. As a consequence, several alternatives to antibiotics are currently being considered or reconsidered. Passive immunization (i.e., the administration of more or less pathogen-specific antibodies to the patient) prior to or after exposure to the disease-causing agent is one of those alternative strategies that was almost entirely abandoned with the introduction of chemical antibiotics but that is now gaining interest again. This review will discuss the early successes and limitations of passive immunization, formerly referred to as "serum therapy," the current use of antibody administration for prophylaxis or treatment of infectious diseases in agriculture, and, finally, recent developments in the field of antibody engineering and "molecular farming" of antibodies in various expression systems. Especially the potential of producing therapeutic antibodies in crops that are routine dietary components of farm animals, such as corn and soy beans, seems to hold promise for future application in the fight against infectious diseases. PMID:15844826

  15. Alternative therapies for postmenopausal women.

    PubMed

    Speroff, Leon

    2005-01-01

    Alternative therapies are being used by postmenopausal women in attempts to treat all of the complaints and medical conditions of the menopause. One-fifth of those who take prescription drugs for these indications also take herbal remedies and/or high-dose vitamins, most often without disclosing the fact to the physician. Although studies of alternative therapies are short-term and rarely focused on safety--let alone efficacy--in the long-term, there are many studies spread over the large number of substances involved. More than 130 studies, including meta-analyses, are reviewed in this article under the headings of phytoestrogens, especially from soy; therapies for hot flushes; and preventives for cardiovascular disease, osteoporosis, and breast cancer. Special attention is given to the recently recognized daidzein metabolite equol, and for the sake of completeness there are reviews of the unconventional, but not botanical, treatments estriol, transdermal progesterone, and dehydroepiandrosterone. The total picture produced by conscientious review of the studies is bleak overall, but there seems to be good reason to pursue the possibilities inherent in soy protein with phytoestrogens in populations of women who endogenously produce equol.

  16. Alternate Energy for National Security.

    NASA Astrophysics Data System (ADS)

    Rath, Bhakta

    2010-02-01

    Recent price fluctuations at the gas pump have brought our attention to the phenomenal increase of global energy consumption in recent years. It is now evident that we have almost reached a peak in global oil production. Several projections indicate that total world consumption of oil will rise by nearly 60 per cent between 1999 and 2020. In 1999 consumption was equivalent to 86 million barrels of oil per day, which has reached a peak of production extracted from most known oil reserves. These projections, if accurate, will present an unprecedented crisis to the global economy and industry. As an example, in the US, nearly 40 per cent of energy usage is provided by petroleum, of which nearly a third is used in transportation. The US Department of Defense (DOD) is the single largest buyer of fuel, amounting to, on the average, 13 million gallons per day. Additionally, these fuels have to meet different requirements that prevent use of ethanol additives and biodiesel. An aggressive search for alternate energy sources, both renewable and nonrenewable, is vital. The presentation will review national and DOD perspectives on the exploration of alternate energy with a focus on energy derivable from the ocean. )

  17. Alternative cosmology from cusp geometries

    NASA Astrophysics Data System (ADS)

    Rosa, Reinaldo; Herbin Stalder Díaz, Diego

    We study an alternative geometrical approach on the problem of classical cosmological singularity. It is based on a generalized function f(x,y)=x(2+y^2=(1-z)z^n) which consists of a cusped projected coupled isosurface. Such a projected geometry is computed and analized into the context of Friedmann singularity-free cosmology where a pre-big bang scenario is considered. Assuming that the mechanism of cusp formation is described by non-linear oscillations of a pre- big bang extended very high energy density field (>3x10^{94} kg/m^3$), we show that the action under the gravitational field follows a tautochrone of revolution, understood here as the primary projected geometry that alternatively replaces the Friedmann singularity in the standard big bang theory. As shown here this new approach allows us to interpret the nature of both matter and dark energy from first geometric principles [1]. [1] Rosa et al. DOI: 10.1063/1.4756991

  18. Mitigation alternatives for L Lake

    SciTech Connect

    Moore, D.B.

    1988-11-03

    The current condition of L Lake/Steel Creek was summarized in a report to SCDHEC in June 1988 which reported that the L Lake and Steel Creek ecosystems were adequately developing towards balanced biological communities. If mitigation for L Lake inputs, specifically temperature and nutrients, are required, several viable alternatives are available. A report prepared by Spencer in 1986 discusses the various options available for cooling L-Reactor discharges. In effect, a small cooling tower is the only realistic solution to reducing effluent temperatures. Nutrient mitigation can take several approaches including upstream sewage treatment, hypolimnetic withdrawal, dilution of input water by Par Pond water, precipitation of nutrients, and sediment oxidation. None of these systems would influence the thermal regime, but would significantly reduce nutrient input into the system. One beneficial use of L-Lake thermal effluents is algaculture, the production of useful algae. A document prepared in 1988 concludes that algaculture is a technically and economically feasible mitigation alternative for L Lake and could allow L Lake to be handled under Section 318 of the Clean Water Act.

  19. Alternate nozzle ablative materials program

    NASA Technical Reports Server (NTRS)

    Kimmel, N. A.

    1984-01-01

    Four subscale solid rocket motor tests were conducted successfully to evaluate alternate nozzle liner, insulation, and exit cone structural overwrap components for possible application to the Space Shuttle Solid Rocket Motor (SRM) nozzle asasembly. The 10,000 lb propellant motor tests were simulated, as close as practical, the configuration and operational environment of the full scale SRM. Fifteen PAN based and three pitch based materials had no filler in the phenolic resin, four PAN based materials had carbon microballoons in the resin, and the rest of the materials had carbon powder in the resin. Three nozzle insulation materials were evaluated; an aluminum oxide silicon oxide ceramic fiber mat phenolic material with no resin filler and two E-glass fiber mat phenolic materials with no resin filler. It was concluded by MTI/WD (the fabricator and evaluator of the test nozzles) and NASA-MSFC that it was possible to design an alternate material full scale SRM nozzle assembly, which could provide an estimated 360 lb increased payload capability for Space Shuttle launches over that obtainable with the current qualified SRM design.

  20. Complementary and Alternative Methods and Cancer

    MedlinePlus

    ... My Saved Articles » My ACS » Complementary and Alternative Methods and Cancer Download Printable Version [PDF] » ( En español ) ... with cancer here. What are complementary and alternative methods? How are complementary methods used to manage cancer? ...

  1. 7 CFR 982.36 - Alternates.

    Code of Federal Regulations, 2012 CFR

    2012-01-01

    ... and Orders; Fruits, Vegetables, Nuts), DEPARTMENT OF AGRICULTURE HAZELNUTS GROWN IN OREGON AND WASHINGTON Order Regulating Handling Hazelnut Control Board § 982.36 Alternates. An alternate for a member...

  2. 7 CFR 982.36 - Alternates.

    Code of Federal Regulations, 2014 CFR

    2014-01-01

    ... AND ORDERS; FRUITS, VEGETABLES, NUTS), DEPARTMENT OF AGRICULTURE HAZELNUTS GROWN IN OREGON AND WASHINGTON Order Regulating Handling Hazelnut Control Board § 982.36 Alternates. An alternate for a member...

  3. 7 CFR 982.36 - Alternates.

    Code of Federal Regulations, 2011 CFR

    2011-01-01

    ... and Orders; Fruits, Vegetables, Nuts), DEPARTMENT OF AGRICULTURE HAZELNUTS GROWN IN OREGON AND WASHINGTON Order Regulating Handling Hazelnut Control Board § 982.36 Alternates. An alternate for a member...

  4. 7 CFR 982.36 - Alternates.

    Code of Federal Regulations, 2013 CFR

    2013-01-01

    ... AND ORDERS; FRUITS, VEGETABLES, NUTS), DEPARTMENT OF AGRICULTURE HAZELNUTS GROWN IN OREGON AND WASHINGTON Order Regulating Handling Hazelnut Control Board § 982.36 Alternates. An alternate for a member...

  5. 7 CFR 982.36 - Alternates.

    Code of Federal Regulations, 2010 CFR

    2010-01-01

    ... and Orders; Fruits, Vegetables, Nuts), DEPARTMENT OF AGRICULTURE HAZELNUTS GROWN IN OREGON AND WASHINGTON Order Regulating Handling Hazelnut Control Board § 982.36 Alternates. An alternate for a member...

  6. Complementary and Alternative Medicine in Cancer Treatment

    MedlinePlus

    ... Ask about Your Treatment Research Complementary and Alternative Medicine for Patients Complementary and alternative medicine (CAM) is ... based on scientific evidence from research studies. Complementary medicine refers to treatments that are used with standard ...

  7. IBD and Complementary and Alternative Medicine (CAM)

    MedlinePlus

    ... Alternative Medicine (CAM) Go Back Complementary and Alternative Medicine (CAM) Email Print + Share Crohn’s disease and ulcerative ... Energy Medicine, and Biologically-Based Practices. Mind-Body Medicine Mind-body medicine is a set of interventions ...

  8. 40 CFR 152.43 - Alternate formulations.

    Code of Federal Regulations, 2011 CFR

    2011-07-01

    ... PESTICIDE REGISTRATION AND CLASSIFICATION PROCEDURES Registration Procedures § 152.43 Alternate formulations... label text of the alternate formulation product must be identical to that of the basic formulation....

  9. 40 CFR 152.43 - Alternate formulations.

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ... PESTICIDE REGISTRATION AND CLASSIFICATION PROCEDURES Registration Procedures § 152.43 Alternate formulations... label text of the alternate formulation product must be identical to that of the basic formulation....

  10. An alternative to India ink stain.

    PubMed

    Ibembe, Isaac Nicholas; Wiggin, Timothy Roger

    2015-07-01

    Accessing India ink in rural Uganda is difficult and costly. An alternative stain was sought to assist in microbiological diagnoses of cryptococcal infections in immunosuppressed patients with meningitis. Mascara proved to be an excellent and cheap alternative.

  11. 7 CFR 1208.44 - Alternate members.

    Code of Federal Regulations, 2014 CFR

    2014-01-01

    ... AGREEMENTS AND ORDERS; MISCELLANEOUS COMMODITIES), DEPARTMENT OF AGRICULTURE PROCESSED RASPBERRY PROMOTION, RESEARCH, AND INFORMATION ORDER Processed Raspberry Promotion, Research, and Information Order National Processed Raspberry Council § 1208.44 Alternate members. An alternate member of the Council, during...

  12. 7 CFR 1208.44 - Alternate members.

    Code of Federal Regulations, 2013 CFR

    2013-01-01

    ... AGREEMENTS AND ORDERS; MISCELLANEOUS COMMODITIES), DEPARTMENT OF AGRICULTURE PROCESSED RASPBERRY PROMOTION, RESEARCH, AND INFORMATION ORDER Processed Raspberry Promotion, Research, and Information Order National Processed Raspberry Council § 1208.44 Alternate members. An alternate member of the Council, during...

  13. Office of Cancer Complementary and Alternative Medicine

    MedlinePlus

    ... Information FAQs about OCCAM Talking about Complementary and Alternative Medicine with Health Care Providers: A Workbook and Tips ... your health care provider(s) about your complementary and alternative medicine (CAM) use during and after your cancer care. ...

  14. Are There Feasible Alternatives to Laboratory Animals?

    ERIC Educational Resources Information Center

    Rowan, A. N.

    1976-01-01

    Discusses several alternatives to the use of laboratory animals in investigating biomedical problems. Alternatives include tissue culture, use of plant and bacterial material, redesigning experiments, and construction of mathematical and computer models. (CS)

  15. The Pressing Need for Alternative Technology

    ERIC Educational Resources Information Center

    Clarke, Robin

    1973-01-01

    Examines the nature of technology in use today, suggests alternative forms of technology, and looks at the future relationships likely to evolve between alternative technology and developing and developed countries throughout the world. (JR)

  16. An alternative to India ink stain.

    PubMed

    Ibembe, Isaac Nicholas; Wiggin, Timothy Roger

    2015-07-01

    Accessing India ink in rural Uganda is difficult and costly. An alternative stain was sought to assist in microbiological diagnoses of cryptococcal infections in immunosuppressed patients with meningitis. Mascara proved to be an excellent and cheap alternative. PMID:25999353

  17. 33 CFR 127.017 - Alternatives.

    Code of Federal Regulations, 2012 CFR

    2012-07-01

    ... written request for the alternative at least 30 days before facility operations under the alternative... same degree of safety provided by the regulations in this part. (b) The COTP approves or...

  18. 33 CFR 127.017 - Alternatives.

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ... written request for the alternative at least 30 days before facility operations under the alternative... same degree of safety provided by the regulations in this part. (b) The COTP approves or...

  19. 7 CFR 981.35 - Alternates.

    Code of Federal Regulations, 2014 CFR

    2014-01-01

    ... AND ORDERS; FRUITS, VEGETABLES, NUTS), DEPARTMENT OF AGRICULTURE ALMONDS GROWN IN CALIFORNIA Order Regulating Handling Almond Board of California § 981.35 Alternates. An alternate for a member for the...

  20. 7 CFR 981.35 - Alternates.

    Code of Federal Regulations, 2011 CFR

    2011-01-01

    ... and Orders; Fruits, Vegetables, Nuts), DEPARTMENT OF AGRICULTURE ALMONDS GROWN IN CALIFORNIA Order Regulating Handling Almond Board of California § 981.35 Alternates. An alternate for a member for the...

  1. 7 CFR 981.35 - Alternates.

    Code of Federal Regulations, 2013 CFR

    2013-01-01

    ... AND ORDERS; FRUITS, VEGETABLES, NUTS), DEPARTMENT OF AGRICULTURE ALMONDS GROWN IN CALIFORNIA Order Regulating Handling Almond Board of California § 981.35 Alternates. An alternate for a member for the...

  2. 7 CFR 981.35 - Alternates.

    Code of Federal Regulations, 2012 CFR

    2012-01-01

    ... and Orders; Fruits, Vegetables, Nuts), DEPARTMENT OF AGRICULTURE ALMONDS GROWN IN CALIFORNIA Order Regulating Handling Almond Board of California § 981.35 Alternates. An alternate for a member for the...

  3. 7 CFR 981.35 - Alternates.

    Code of Federal Regulations, 2010 CFR

    2010-01-01

    ... and Orders; Fruits, Vegetables, Nuts), DEPARTMENT OF AGRICULTURE ALMONDS GROWN IN CALIFORNIA Order Regulating Handling Almond Board of California § 981.35 Alternates. An alternate for a member for the...

  4. Understanding Conic Sections Using Alternate Graph Paper

    ERIC Educational Resources Information Center

    Brown, Elizabeth M.; Jones, Elizabeth

    2006-01-01

    This article describes two alternative coordinate systems and their use in graphing conic sections. This alternative graph paper helps students explore the idea of eccentricity using the definitions of the conic sections.

  5. LIFE CYCLE ASSESSMENT OF ELECTRICITY GENERATION ALTERNATIVES

    EPA Science Inventory

    This presentation summarizes various electricity and electricity/steam cogeneration alternatives. Among these alternatives, are fossil fuel and biomass power generation plants. These plants have different designs due to the need in fossil fuel (coal) plants to include process u...

  6. 7 CFR 932.30 - Alternates.

    Code of Federal Regulations, 2012 CFR

    2012-01-01

    ... and Orders; Fruits, Vegetables, Nuts), DEPARTMENT OF AGRICULTURE OLIVES GROWN IN CALIFORNIA Order Regulating Handling Olive Administrative Committee § 932.30 Alternates. An alternate for a member of...

  7. 7 CFR 932.30 - Alternates.

    Code of Federal Regulations, 2014 CFR

    2014-01-01

    ... AND ORDERS; FRUITS, VEGETABLES, NUTS), DEPARTMENT OF AGRICULTURE OLIVES GROWN IN CALIFORNIA Order Regulating Handling Olive Administrative Committee § 932.30 Alternates. An alternate for a member of...

  8. 7 CFR 932.30 - Alternates.

    Code of Federal Regulations, 2013 CFR

    2013-01-01

    ... AND ORDERS; FRUITS, VEGETABLES, NUTS), DEPARTMENT OF AGRICULTURE OLIVES GROWN IN CALIFORNIA Order Regulating Handling Olive Administrative Committee § 932.30 Alternates. An alternate for a member of...

  9. 7 CFR 932.30 - Alternates.

    Code of Federal Regulations, 2010 CFR

    2010-01-01

    ... and Orders; Fruits, Vegetables, Nuts), DEPARTMENT OF AGRICULTURE OLIVES GROWN IN CALIFORNIA Order Regulating Handling Olive Administrative Committee § 932.30 Alternates. An alternate for a member of...

  10. 7 CFR 932.30 - Alternates.

    Code of Federal Regulations, 2011 CFR

    2011-01-01

    ... and Orders; Fruits, Vegetables, Nuts), DEPARTMENT OF AGRICULTURE OLIVES GROWN IN CALIFORNIA Order Regulating Handling Olive Administrative Committee § 932.30 Alternates. An alternate for a member of...

  11. 33 CFR 101.120 - Alternatives.

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ....120 Navigation and Navigable Waters COAST GUARD, DEPARTMENT OF HOMELAND SECURITY MARITIME SECURITY MARITIME SECURITY: GENERAL General § 101.120 Alternatives. (a) Alternative Security Agreements. (1) The U.S... Alternative Security Arrangements covering short international voyages on fixed routes between...

  12. 40 CFR 191.04 - Alternative standards.

    Code of Federal Regulations, 2013 CFR

    2013-07-01

    ... 40 Protection of Environment 26 2013-07-01 2013-07-01 false Alternative standards. 191.04 Section... PROGRAMS ENVIRONMENTAL RADIATION PROTECTION STANDARDS FOR MANAGEMENT AND DISPOSAL OF SPENT NUCLEAR FUEL....04 Alternative standards. (a) The Administrator may issue alternative standards from those...

  13. 40 CFR 191.04 - Alternative standards.

    Code of Federal Regulations, 2012 CFR

    2012-07-01

    ... 40 Protection of Environment 26 2012-07-01 2011-07-01 true Alternative standards. 191.04 Section... PROGRAMS ENVIRONMENTAL RADIATION PROTECTION STANDARDS FOR MANAGEMENT AND DISPOSAL OF SPENT NUCLEAR FUEL....04 Alternative standards. (a) The Administrator may issue alternative standards from those...

  14. Alternative Schools: A Practical Manual. Revised.

    ERIC Educational Resources Information Center

    Ascheim, Skip; And Others

    This manual is a practical guide based on school experiences and legal requirements in Massachusetts. It is intended to help alternative schools get off the ground and to stay out of trouble with State and local authorities. Some brief sketches of four alternative schools are presented to indicate alternatives that already exist. Sections…

  15. Alternative School Development: A Guide for Practitioners.

    ERIC Educational Resources Information Center

    DeTurk, Philip; Mackin, Robert

    There is a tendency to simply view alternative schools as against tests, grades, bells, seats in rows, and dress regulations. This obscures the need for alternative schools to have stable organizational structures if they are to play an important role in education. There are five stages in the development of an alternative school. They include (a)…

  16. An alternative to Zoe's Law.

    PubMed

    Dalmau, James

    2015-03-01

    Under the criminal law of New South Wales, the destruction of a foetus (other than in the course of a medical procedure) constitutes grievous bodily harm to the pregnant woman. Charges can be laid for offences against the woman, but not against the foetus. Many are dissatisfied with this. The Crimes Amendment (Zoe's Law) Bill 2013 (No 2) (NSW) aimed to change this by providing that a foetus is taken to be a living person for the purposes of certain offences. The Bill was strongly opposed on the basis that according personhood to a foetus in this way will have undesirable consequences that could erode the reproductive rights of women. The public debate over how the criminal law addresses the destruction of a foetus has centred on Zoe's Law. This article proposes an alternative amendment. that aims, to accommodate the concerns of the Bill's supporters and its detractors.

  17. Basic sciences: an alternative career?

    PubMed

    Khatri, R

    2013-01-01

    Career selection is a crucial and a complex process which is also true for the medical profession. In the context of our country, due to the limited opportunity and proper guidance, migration of medical graduates to foreign countries is increasing. Though, clinical subjects have a huge attraction, basic science field has failed to impress our medical graduates. In current scenario, basic science field seems to be a dumping site for the incompetent as the candidates who have failed trying their luck elsewhere stumble upon basic science careers though it is not true for all. Moreover, a very few medical graduates are interested in developing their career as a basic scientist. Therefore, to motivate today's young medical graduates, there is a need of a good mentor along with a proper career guidance which can help them to understand the basic science field as an alternative career. PMID:23774420

  18. An alternative to Zoe's Law.

    PubMed

    Dalmau, James

    2015-03-01

    Under the criminal law of New South Wales, the destruction of a foetus (other than in the course of a medical procedure) constitutes grievous bodily harm to the pregnant woman. Charges can be laid for offences against the woman, but not against the foetus. Many are dissatisfied with this. The Crimes Amendment (Zoe's Law) Bill 2013 (No 2) (NSW) aimed to change this by providing that a foetus is taken to be a living person for the purposes of certain offences. The Bill was strongly opposed on the basis that according personhood to a foetus in this way will have undesirable consequences that could erode the reproductive rights of women. The public debate over how the criminal law addresses the destruction of a foetus has centred on Zoe's Law. This article proposes an alternative amendment. that aims, to accommodate the concerns of the Bill's supporters and its detractors. PMID:25980199

  19. Alternative Fuels for Cancer Cells

    PubMed Central

    Keenan, Melissa; Chi, Jen-Tsan

    2015-01-01

    Tumor metabolism is significantly altered to support the various metabolic needs of tumor cells. The most prominent change is the increased tumor glycolysis that leads to increased glucose uptake and utilization. However, it has become obvious that many non-glucose nutrients, such as amino acids, lactate, acetate and macromolecules, can serve as alternative fuels for cancer cells. This knowledge reveals an unexpected flexibility and evolutionarily-conserved model in which cancer cells uptake nutrients from their external environment to fulfill their necessary energetic needs. It is possible that tumor cells have evolved the ability to utilize different carbon sources due to the limited supply of nutrient that can be driven by oncogenic mutations and tumor microenvironmental stresses. In certain cases, these factors permanently alter the tumor cells’ metabolism, causing certain nutrients to become indispensable and thus creating opportunities for therapeutic intervention to eradicate tumors by their metabolic vulnerabilities. PMID:25815843

  20. Alternative models of OPEC behavior

    SciTech Connect

    Al-Sultan, A.M.

    1993-12-31

    Since the 1973 oil price jump there has been considerable interest in the Organization of the Petroleum Exporting Countries (OPEC) and its role in the international oil market. However, most of the literature on this subject is mainly concerned with either a time-control problem in which the optimality and implication of certain market behavior is analyzed or a simulation of the oil market assuming a particular market behavior by OPEC members. Our objective in this preliminary research is to present a unified framework in which we construct models of viable alternative market behaviors for OPEC members assuming profit-maximization behavior. Each model will be specified as a system of nonlinear simultaneous equations, and for a particular functional forms specification, we present the estimates of the first two models considered.

  1. Towards an alternative evolution model.

    PubMed

    van Waesberghe, H

    1982-01-01

    Lamarck and Darwin agreed on the inconstancy of species and on the exclusive gradualism of evolution (nature does not jump). Darwinism, revived as neo-Darwinism, was almost generally accepted from about 1930 till 1960. In the sixties the evolutionary importance of selection has been called in question by the neutralists. The traditional conception of the gene is disarranged by recent molecular-biological findings. Owing to the increasing confusion about the concept of genotype, this concept is reconsidered. The idea of the genotype as a cluster of genes is replaced by a cybernetical interpretation of the genotype. As nature does jump, exclusive gradualism is dismissed. Saltatory evolution is a natural phenomenon, provided by a sudden collapse of the thresholds which resist against evolution. The fossil record and the taxonomic system call for a macromutational interpretation. As Lamarck and Darwin overlooked the resistance of evolutionary thresholds, an alternative evolution model is needed, the first to be constructed on a palaeontological and taxonomic basis.

  2. Alternator control for battery charging

    SciTech Connect

    Brunstetter, Craig A.; Jaye, John R.; Tallarek, Glen E.; Adams, Joseph B.

    2015-07-14

    In accordance with an aspect of the present disclosure, an electrical system for an automotive vehicle has an electrical generating machine and a battery. A set point voltage, which sets an output voltage of the electrical generating machine, is set by an electronic control unit (ECU). The ECU selects one of a plurality of control modes for controlling the alternator based on an operating state of the vehicle as determined from vehicle operating parameters. The ECU selects a range for the set point voltage based on the selected control mode and then sets the set point voltage within the range based on feedback parameters for that control mode. In an aspect, the control modes include a trickle charge mode and battery charge current is the feedback parameter and the ECU controls the set point voltage within the range to maintain a predetermined battery charge current.

  3. Alternative approaches to plasma confinement

    NASA Technical Reports Server (NTRS)

    Roth, J. R.

    1978-01-01

    The paper discusses 20 plasma confinement schemes each representing an alternative to the tokamak fusion reactor. Attention is given to: (1) tokamak-like devices (TORMAC, Topolotron, and the Extrap concept), (2) stellarator-like devices (Torsatron and twisted-coil stellarators), (3) mirror machines (Astron and reversed-field devices, the 2XII B experiment, laser-heated solenoids, the LITE experiment, the Kaktus-Surmac concept), (4) bumpy tori (hot electron bumpy torus, toroidal minimum-B configurations), (5) electrostatically assisted confinement (electrostatically stuffed cusps and mirrors, electrostatically assisted toroidal confinement), (6) the Migma concept, and (7) wall-confined plasmas. The plasma parameters of the devices are presented and the advantages and disadvantages of each are listed.

  4. Alternative treatment of gallbladder disease.

    PubMed

    Moga, M M

    2003-01-01

    Major risk factors for gallbladder disease include a sedentary lifestyle and a diet rich in refined sugars. In genetically prone individuals, these two factors lead to an abnormal bile composition, altered gut microflora, and hyperinsulinemia, with resulting gallstone formation. As a large percentage of gallbladder patients have continued digestive complaints following cholecystectomy, the author examines complementary and alternative medicine (CAM) treatments to counteract gallstone formation. Herbal medicine such as turmeric, oregon grape, bupleurum, and coin grass may reduce gallbladder inflammation and relieve liver congestion. Elimination of offending foods, not necessarily 'fatty' foods, is often successful and recommended by many holistic physicians. Regular aerobic exercise has a beneficial effect on hyperinsulinemia, which is often associated with gallbladder disease. Dietary changes that lower plasma insulin levels, such as a change in dietary fats and substitution of unrefined carbohydrates for refined carbohydrates, may also be helpful.

  5. Alternate alloying for environmental resistance

    SciTech Connect

    Smolik, G.R.; Banerji, S.K.

    1986-01-01

    The 35 papers contained in this collection detail efforts being made toward achieving environmental resistance of alloys and conserved usage of strategic and critical materials. An in-depth look is taken at the roles played by various alloying elements in providing desired microstructures, properties, and influences upon environmental attack. Also presented are applications and performances of alternate alloys in aqueous and high temperature gaseous and molten salt environments. The book is broken down into five key sections covering: 1) philosophies and status of programs designing alloys for resistance to various environmental and microstructural stability of some of these alloys systems, 2) applications in hot corrosion and sulfidizing environments, 3) applications in oxidizing conditions, 4) corrosion resistance in aqueous environments, and 5) other properties, such as physical and mechanical, which are necessary to evaluate overall alloy performance.

  6. Alternative calcination development status report

    SciTech Connect

    Boardman, R.D.

    1997-12-01

    The Programmatic Spent Nuclear Fuel and (INEEL) Environmental Restoration and Waste Management Programs Environmental Impact Statement Record of Decision, dated June 1, 1995, specifies that high-level waste stored in the underground tanks at the ICPP continue to be calcined while other options to treat the waste are studied. Therefore, the High-Level Waste Program has funded a program to develop new flowsheets to increase the liquid waste processing rate. Simultaneously, a radionuclide separation process, as well as other options, are also being developed, which will be compared to the calcination treatment option. Two alternatives emerged as viable candidates; (1) elevated temperature calcination (also referred to as high temperature calcination), and (2) sugar-additive calcination. Both alternatives were determined to be viable through testing performed in a lab-scale calcination mockup. Subsequently, 10-cm Calciner Pilot Plant scoping tests were successfully completed for both flowsheets. The results were compared to the standard 500 C, high-ANN flow sheet (baseline flowsheet). The product and effluent streams were characterized to help elucidate the process chemistry and to investigate potential environmental permitting issues. Several supplementary tests were conducted to gain a better understanding of fine-particles generation, calcine hydration, scrub foaming, feed makeup procedures, sugar/organic elimination, and safety-related issues. Many of the experiments are only considered to be scoping tests, and follow-up experiments will be required to establish a more definitive understanding of the flowsheets. However, the combined results support the general conclusion that flowsheet improvements for the NWCF are technically viable.

  7. Quantifying asymmetry: ratios and alternatives.

    PubMed

    Franks, Erin M; Cabo, Luis L

    2014-08-01

    Traditionally, the study of metric skeletal asymmetry has relied largely on univariate analyses, utilizing ratio transformations when the goal is comparing asymmetries in skeletal elements or populations of dissimilar dimensions. Under this approach, raw asymmetries are divided by a size marker, such as a bilateral average, in an attempt to produce size-free asymmetry indices. Henceforth, this will be referred to as "controlling for size" (see Smith: Curr Anthropol 46 (2005) 249-273). Ratios obtained in this manner often require further transformations to interpret the meaning and sources of asymmetry. This model frequently ignores the fundamental assumption of ratios: the relationship between the variables entered in the ratio must be isometric. Violations of this assumption can obscure existing asymmetries and render spurious results. In this study, we examined the performance of the classic indices in detecting and portraying the asymmetry patterns in four human appendicular bones and explored potential methodological alternatives. Examination of the ratio model revealed that it does not fulfill its intended goals in the bones examined, as the numerator and denominator are independent in all cases. The ratios also introduced strong biases in the comparisons between different elements and variables, generating spurious asymmetry patterns. Multivariate analyses strongly suggest that any transformation to control for overall size or variable range must be conducted before, rather than after, calculating the asymmetries. A combination of exploratory multivariate techniques, such as Principal Components Analysis, and confirmatory linear methods, such as regression and analysis of covariance, appear as a promising and powerful alternative to the use of ratios. PMID:24842694

  8. COMMUNICATION: Alternative splicing and genomic stability

    NASA Astrophysics Data System (ADS)

    Cahill, Kevin

    2004-06-01

    Alternative splicing allows an organism to make different proteins in different cells at different times, all from the same gene. In a cell that uses alternative splicing, the total length of all the exons is much shorter than in a cell that encodes the same set of proteins without alternative splicing. This economical use of exons makes genes more stable during reproduction and development because a genome with a shorter exon length is more resistant to harmful mutations. Genomic stability may be the reason why higher vertebrates splice alternatively. For a broad class of alternatively spliced genes, a formula is given for the increase in their stability.

  9. Expression of alternative oxidase in tomato

    SciTech Connect

    Kakefuda, M.; McIntosh, L. )

    1990-05-01

    Tomato fruit ripening is characterized by an increase in ethylene biosynthesis, a burst in respiration (i.e. the climacteric), fruit softening and pigmentation. As whole tomatoes ripened from mature green to red, there was an increase in the alternative oxidase capacity. Aging pink tomato slices for 24 and 48 hrs also showed an increase of alternative oxidase and cytochrome oxidase capacities. Monoclonal antibodies prepared to the Sauromatum guttatum alternative oxidase were used to follow the appearance of alternative oxidase in tomato fruits. There is a corresponding increase in a 36kDa protein with an increase in alternative oxidase capacity. Effects of ethylene and norbornadiene on alternative oxidase capacity were also studied. We are using an alternative oxidase cDNA clone from potato to study the expression of mRNA in ripening and wounded tomatoes to determine if the gene is transcriptionally regulated.

  10. Boom Rendezvous Alternative Docking Approach

    NASA Technical Reports Server (NTRS)

    Bonometti, Joseph A.

    2006-01-01

    Space rendezvous and docking has always been attempted with primarily one philosophic methodology. The slow matching of one vehicle's orbit by a second vehicle and then a final closing sequence that ends in matching the orbits with perfect precision and with near zero relative velocities. The task is time consuming, propellant intensive, risk inherent (plume impingement, collisions, fuel depletion, etc.) and requires substantial hardware mass. The historical background and rationale as to why this approach is used is discussed in terms of the path-not-taken and in light of an alternate methodology. Rendezvous and docking by boom extension is suggested to have inherent advantages that today s technology can readily exploit. Extension from the primary spacecraft, beyond its inherent large inertia, allows low inertia connections to be made rapidly and safely. Plume contamination issues are eliminated as well as the extra propellant mass and risk required for the final thruster (docking) operations. Space vehicle connection hardware can be significantly lightened. Also, docking sensors and controls require less fidelity; allowing them to be more robust and less sensitive. It is the potential safety advantage and mission risk reduction that makes this approach attractive, besides the prospect of nominal time and mass savings.

  11. HFIR spent fuel management alternatives

    SciTech Connect

    Begovich, J.M.; Green, V.M.; Shappert, L.B.; Lotts, A.L.

    1992-10-15

    The High Flux Isotope Reactor (HFIR) at Martin Marietta Energy Systems` Oak Ridge National Laboratory (ORNL) has been unable to ship its spent fuel to Savannah River Site (SRS) for reprocessing since 1985. The HFIR storage pools are expected to fill up in the February 1994 to February 1995 time frame. If a management altemative to existing HFIR pool storage is not identified and implemented before the HFIR pools are full, the HFIR will be forced to shut down. This study investigated several alternatives for managing the HFIR spent fuel, attempting to identify options that could be implemented before the HFIR pools are full. The options investigated were: installing a dedicated dry cask storage facility at ORNL, increasing HFIR pool storage capacity by clearing the HFIR pools of debris and either close-packing or stacking the spent fuel elements, storing the spent fuel at another ORNL pool, storing the spent fuel in one or more hot cells at ORNL, and shipping the spent fuel offsite for reprocessing or storage elsewhere.

  12. HFIR spent fuel management alternatives

    SciTech Connect

    Begovich, J.M.; Green, V.M.; Shappert, L.B.; Lotts, A.L.

    1992-10-15

    The High Flux Isotope Reactor (HFIR) at Martin Marietta Energy Systems' Oak Ridge National Laboratory (ORNL) has been unable to ship its spent fuel to Savannah River Site (SRS) for reprocessing since 1985. The HFIR storage pools are expected to fill up in the February 1994 to February 1995 time frame. If a management altemative to existing HFIR pool storage is not identified and implemented before the HFIR pools are full, the HFIR will be forced to shut down. This study investigated several alternatives for managing the HFIR spent fuel, attempting to identify options that could be implemented before the HFIR pools are full. The options investigated were: installing a dedicated dry cask storage facility at ORNL, increasing HFIR pool storage capacity by clearing the HFIR pools of debris and either close-packing or stacking the spent fuel elements, storing the spent fuel at another ORNL pool, storing the spent fuel in one or more hot cells at ORNL, and shipping the spent fuel offsite for reprocessing or storage elsewhere.

  13. Nonsurgical Alternatives for Uterine Fibroids.

    PubMed

    Zupi, Errico; Centini, Gabriele; Sabbioni, Lorenzo; Lazzeri, Lucia; Argay, István Máté; Petraglia, Felice

    2016-07-01

    Uterine leiomyomata are the direct cause of a significant health-care burden for women, their families, and society as a whole. Because of the long experience with the mode of treatment, surgical myomectomy remains the gold standard for treating reproductive-age women; however, in the recent years, the wide evolution of less invasive approaches led to a change in the options used by the clinician to treat symptomatic fibroids. Minimally invasive procedures such as uterine artery embolization (UAE) are increasingly used to treat symptomatic fibroids. Other alternative treatments are becoming more diffuse, such as magnetic resonance-guided high-frequency focused ultrasound surgery (MRgFUS), cryomyolysis, vaginal occlusion, and laparoscopic closure of the uterine arteries. Both advantages and limitations of these techniques under development must be taken into account, but this wider range of choices is being increasingly considered for a tailored treatment. This article aims to enable health-care providers with the tools to provide the latest evidence-based care in the minimally invasive or noninvasive management of this common problem. PMID:26711881

  14. An Alternative to Laser Cooling

    NASA Astrophysics Data System (ADS)

    Raizen, Mark

    2015-03-01

    Laser cooling has been the standard approach for over thirty years for cooling the translational motion of atoms. While laser cooling is an extremely successful method, it has been limited to a small set of elements in the periodic table. The performance of laser cooling for those elements has saturated in terms of flux of ultra-cold atoms, density, and phase-space density. I report our progress towards the development of an alternative to laser cooling. Our approach relies on magnetic stopping of supersonic beams, an atomic coilgun. A recent advance is the experimental realization of an adiabatic coilgun which preserves phase-space density. Further cooling was demonstrated with a one-way wall, realizing the historic thought experiment of Maxwell's Demon. More recently, we showed how to apply this method to compress atomic phase space with almost no loss of atom number. Our approach is fundamentally different than laser cooling as it does not rely on the momentum of the photon, but rather the photon entropy. I will report on our experimental progress towards this goal, and describe future experiments that will be enabled by this work.

  15. Economics of alternative energy sources.

    PubMed

    Ryle, M

    1977-05-12

    An important part of the oil and natural gas at present consumed in the UK is used for the heating of buildings, a demand which shows large diurnal, day-to-day and annual fluctuations. The replacement of this energy by nuclear-generated electricity, as at present envisaged, would require the construction of some 250 GW of additional capacity by the end of the century, a progamme which does not seem feasible. By incorporating relatively cheap, short term storage in the form of low-grade heat, the generating capacity required to fulfil peak demand could be reduced by more than 50%. As soon as such storage is provided, however, other sources of energy become viable and attractive alternatives, and the UK is well situated to make use of wind, wave, and tidal power. It seems likely that the value of North Sea oil/gas reserves as feedstock to the chemical industry will rise sufficiently to make an early reduction in their consumption as fuel of great economic importance.

  16. Canonical and alternative MAPK signaling.

    PubMed

    Pimienta, Genaro; Pascual, Jaime

    2007-11-01

    The archetype of MAPK cascade activation is somewhat challenged by the most recent discovery of unexpected phosphorylation patterns, alternative activation mechanisms and sub-cellular localization, in various members of this protein kinase family. In particular, activation by autophosphorylation pathways has now been described for the three best understood MAPK subgroups: ERK1/2; JNK1/2 and p38 alpha/beta. Also, a form of dosage compensation between homologs has been shown to occur in the case of ERK1/2 and JNK1/2. In this paper we summarize the MAPK activation pathway, with an emphasis on non-canonical examples. We use this information to propose a model for MAPK signal transduction that considers a cross-talk between MAPKs with different activation loop sequence motifs and unique C-terminal extensions. We highlight the occurrence of non-canonical substrate specificity during MAPK auto-activation, in strong connection with MAPK homo- and hetero-dimerization events.

  17. Alternative Water Processor Test Development

    NASA Technical Reports Server (NTRS)

    Pickering, Karen D.; Mitchell, Julie L.; Adam, Niklas M.; Barta, Daniel; Meyer, Caitlin E.; Pensinger, Stuart; Vega, Leticia M.; Callahan, Michael R.; Flynn, Michael; Wheeler, Ray; Birmele, Michele; Lunn, Griffin; Jackson, Andrew

    2013-01-01

    The Next Generation Life Support Project is developing an Alternative Water Processor (AWP) as a candidate water recovery system for long duration exploration missions. The AWP consists of biological water processor (BWP) integrated with a forward osmosis secondary treatment system (FOST). The basis of the BWP is a membrane aerated biological reactor (MABR), developed in concert with Texas Tech University. Bacteria located within the MABR metabolize organic material in wastewater, converting approximately 90% of the total organic carbon to carbon dioxide. In addition, bacteria convert a portion of the ammonia-nitrogen present in the wastewater to nitrogen gas, through a combination of nitrification and denitrification. The effluent from the BWP system is low in organic contaminants, but high in total dissolved solids. The FOST system, integrated downstream of the BWP, removes dissolved solids through a combination of concentration-driven forward osmosis and pressure driven reverse osmosis. The integrated system is expected to produce water with a total organic carbon less than 50 mg/l and dissolved solids that meet potable water requirements for spaceflight. This paper describes the test definition, the design of the BWP and FOST subsystems, and plans for integrated testing.

  18. Alternative Water Processor Test Development

    NASA Technical Reports Server (NTRS)

    Pickering, Karen D.; Mitchell, Julie; Vega, Leticia; Adam, Niklas; Flynn, Michael; Wjee (er. Rau); Lunn, Griffin; Jackson, Andrew

    2012-01-01

    The Next Generation Life Support Project is developing an Alternative Water Processor (AWP) as a candidate water recovery system for long duration exploration missions. The AWP consists of biological water processor (BWP) integrated with a forward osmosis secondary treatment system (FOST). The basis of the BWP is a membrane aerated biological reactor (MABR), developed in concert with Texas Tech University. Bacteria located within the MABR metabolize organic material in wastewater, converting approximately 90% of the total organic carbon to carbon dioxide. In addition, bacteria convert a portion of the ammonia-nitrogen present in the wastewater to nitrogen gas, through a combination of nitrogen and denitrification. The effluent from the BWP system is low in organic contaminants, but high in total dissolved solids. The FOST system, integrated downstream of the BWP, removes dissolved solids through a combination of concentration-driven forward osmosis and pressure driven reverse osmosis. The integrated system is expected to produce water with a total organic carbon less than 50 mg/l and dissolved solids that meet potable water requirements for spaceflight. This paper describes the test definition, the design of the BWP and FOST subsystems, and plans for integrated testing.

  19. Phytoremediation -- a practical capping alternative

    SciTech Connect

    Beath, J.M.; Peak, M.J.

    1997-12-31

    Much literature has been devoted recently to the use of various plant species for the uptake of heavy metals and organic contaminants. Other uses for plants as part of the remediation process are growing in perceived effectiveness. Consequently, this paper deals with two other equally important potential uses of plants to address environmental problems that are just now evolving to the field trial stage: the use of plants to remediate organic pollutants; and the use of plants to control the rainfall-driven leaching of contaminants and the subsequent delivery to underlying groundwater. The traditional Resource Conservation and Recovery Act (RCRA) approach to capping landfills will be contrasted with the potential benefits of using plants that can balance incoming rainfall with evapotranspiration, as well as plants which can act on organic constituents in soil or sludge by either uptake or by promoting microbial activity in soil. This paper compares traditional RCRA capping costs to those for a phytoremediation capping alternative, whose benefits include significantly lower implementation cost and continued remediation. This paper discusses important elements of a successful approach to phytoremediation including: species selection, implementation techniques, cost-efficient monitoring, regulatory aspects, project timing, and realistic expectations.

  20. Polyandry and alternative mating tactics

    PubMed Central

    Neff, Bryan D.; Svensson, Erik I.

    2013-01-01

    Many species in the animal kingdom are characterized by alternative mating tactics (AMTs) within a sex. In males, such tactics include mate guarding versus sneaking behaviours, or territorial versus female mimicry. Although AMTs can occur in either sex, they have been most commonly described in males. This sex bias may, in part, reflect the increased opportunity for sexual selection that typically exists in males, which can result in a higher probability that AMTs evolve in that sex. Consequently, females and polyandry can play a pivotal role in governing the reproductive success associated with male AMTs and in the evolutionary dynamics of the tactics. In this review, we discuss polyandry and the evolution of AMTs. First, we define AMTs and review game theoretical and quantitative genetic approaches used to model their evolution. Second, we review several examples of AMTs, highlighting the roles that genes and environment play in phenotype expression and development of the tactics, as well as empirical approaches to differentiating among the mechanisms. Third, ecological and genetic constraints to the evolution of AMTs are discussed. Fourth, we speculate on why female AMTs are less reported on in the literature than male tactics. Fifth, we examine the effects of AMTs on breeding outcomes and female fitness, and as a source, and possibly also a consequence, of sexual conflict. We conclude by suggesting a new model for the evolution of AMTs that incorporates both environmental and genetic effects, and discuss some future avenues of research. PMID:23339236

  1. Alternative Energy for Higher Education

    SciTech Connect

    Michael Cherney, PhD

    2012-02-22

    This project provides educational opportunities creating both a teaching facility and center for public outreach. The facility is the largest solar array in Nebraska. It was designed to allow students to experience a variety of technologies and provide the public with opportunities for exposure to the implementation of an alternative energy installation designed for an urban setting. The project integrates products from 5 panel manufacturers (including monocrystalline, polycrystalline and thin film technologies) mounted on both fixed and tracking structures. The facility uses both micro and high power inverters. The majority of the system was constructed to serve as an outdoor classroom where panels can be monitored, tested, removed and replaced by students. As an educational facility it primarily serves students in the Creighton University and Metropolitan Community College, but it also provides broader educational opportunities. The project includes a real-time dashboard and a historical database of the output of individual inverters and the corresponding meteorological data for researcher and student use. This allows the evaluation of both panel types and the feasibility of installation types in a region of the country subject to significant temperature, wind and precipitation variation.

  2. Economics of alternative energy sources.

    PubMed

    Ryle, M

    1977-05-12

    An important part of the oil and natural gas at present consumed in the UK is used for the heating of buildings, a demand which shows large diurnal, day-to-day and annual fluctuations. The replacement of this energy by nuclear-generated electricity, as at present envisaged, would require the construction of some 250 GW of additional capacity by the end of the century, a progamme which does not seem feasible. By incorporating relatively cheap, short term storage in the form of low-grade heat, the generating capacity required to fulfil peak demand could be reduced by more than 50%. As soon as such storage is provided, however, other sources of energy become viable and attractive alternatives, and the UK is well situated to make use of wind, wave, and tidal power. It seems likely that the value of North Sea oil/gas reserves as feedstock to the chemical industry will rise sufficiently to make an early reduction in their consumption as fuel of great economic importance. PMID:16073407

  3. Alternative to Nitric Acid Passivation

    NASA Technical Reports Server (NTRS)

    Kessel, Kurt R.

    2015-01-01

    The Ground Systems Development and Operations (GSDO) Program at NASA John F. Kennedy Space Center (KSC), Florida, has the primary objective of modernizing and transforming the launch and range complex at KSC to benefit current and future NASA programs along with other emerging users. Described as the launch support and infrastructure modernization program in the NASA Authorization Act of 2010, the GSDO Program will develop and implement shared infrastructure and process improvements to provide more flexible, affordable, and responsive capabilities to a multi-user community. In support of NASA and the GSDO Program, the objective of this project is to qualify citric acid as an environmentally-preferable alternative to nitric acid for passivation of stainless steel alloys. This project is a direct follow-on to United Space Alliance (USA) work at KSC to optimize the parameters for the use of citric acid and verify effectiveness. This project will build off of the USA study to further evaluate citric acids effectiveness and suitability for corrosion protection of a number of stainless steels alloys used by NASA, the Department of Defense (DoD), and the European Space Agency (ESA).

  4. Alternative systematic approaches to training

    SciTech Connect

    Not Available

    1995-01-01

    This handbook is approved for use by all DOE Components and contractors. The handbook was prepared primarily for DOE nuclear facilities, but the information can be effectively used by any other type of facility. DOE nuclear, DOE non-nuclear, commercial nuclear reactor, fuel fabrication, chemical processing, or other types of facilities may also apply the principles of this approach and find it useful and applicable to local needs. The handbook provides DOE and contractor operating organizations with concepts and guidance regarding the use of alternative techniques to implement a systematic approach to training (SAT). The techniques described in this handbook are endorsed by DOE and use of the guidance in this handbook is appropriate for establishment of technical training programs at DOE nuclear facilities. The use of guidance on selection and implementation of appropriate training approaches after consideration of job complexity, the consequences of error based on risk/hazard potential, and available training media should result in effective and efficient training programs. The information presented in this handbook can be used to grade the level of effort and formality used in developing training programs.

  5. Telomere elongation chooses TERRA ALTernatives.

    PubMed

    Arora, Rajika; Azzalin, Claus M

    2015-01-01

    Alternative Lengthening of Telomeres (ALT) mechanisms allow telomerase-negative immortal cells to buffer replicative telomere shortening. ALT is naturally active in a number of human cancers and might be selected upon telomerase inactivation. ALT is thought to operate through homologous recombination (HR) occurring between telomeric repeats from independent chromosome ends. Indeed, suppression of a number of HR factors impairs ALT cell proliferation. Yet, how HR is initiated at ALT telomeres remains elusive. Mounting evidence suggests that the long noncoding telomeric RNA TERRA renders ALT telomeres recombinogenic by forming RNA:DNA hybrids with the telomeric C-rich strand. TERRA and telomeric hybrids act in concert with a number of other factors, including the RNA endoribonuclease RNaseH1 and the single stranded DNA binding protein RPA. The functional interaction network built upon these different players seems indispensable for ALT telomere maintenance, and digging into the molecular details of this previously unappreciated network might open the way to novel avenues for cancer treatments.

  6. Reproductive effects of alternative disinfectants

    SciTech Connect

    Carlton, B.D.; Barlett, P.; Basaran, A.; Colling, K.; Osis, I.; Smith, M.K.

    1986-11-01

    Organohalides formed through the reaction of chlorine and organic compounds in natural and waste waters pose potential health hazards. For this reason, alternative water disinfectants that do not form organohalides are being investigated with great interest. In this laboratory, the authors have examined the reproductive effects of chloramine and chlorine administered by gavage in Long-Evans rats. Animals were treated for a total of 66 to 76 days. Males were treated for 56 days and females for 14 days prior to breeding and throughout the 10-day breeding period. Females were treated throughout gestation and lactation. Following breeding, the males were necropsied and evaluated for sperm parameters and reproductive tract histopathology. Adult females and some pups were necropsied at weaning on postnatal day 21. Other pups were treated postweaning until 28 or 40 days of age. These pups were evaluated for the day of vaginal patency and thyroid hormone levels. No differences were observed between control rats and those rats exposed to up to 5 mg/kg/day chlorine or 10 mg/kg/day chloramine when fertility, viability, litter size, day of eye opening, or day of vaginal patency were evaluated. No alterations in sperm count, sperm direct progressive movement percent motility, or sperm morphology were observed among adult male rats. In addition, male and female reproductive organ weights were comparable to their respective control groups, and no significant histopathologic changes were observed among chlorine- or chloramine-treated male and female rats.

  7. Alternate capacitor dielectric film materials

    SciTech Connect

    Foster, J.C.

    1990-08-01

    New high-temperature, high-energy density, and high-radiation tolerant capacitor applications require the evaluation of alternate dielectric materials. Evaluation work was performed at GE Neutron Devices (GEND) and Sandia National Laboratories (SNL), Albuquerque. US Department of Energy (DOE) requirements for capacitor function and environments are unique, and the representations included in this report do not constitute an endorsement of any material or manufacturer. This report presents data on polymeric dielectric films evaluated to support the design of new high-energy density capacitors. Materials which were evaluated include polycarbonate (two sources), polyphenylene sulfide, polyvinylidene fluoride, polyetherimide (three sources), polyimide (four sources), polyethersulfone, and polyetherether ketone. A polyester was evaluated as the control material since many prior designs utilized this dielectric. The film evaluations were based on dielectric constant and dissipation factor variation as a function of temperature ({minus}55{degree}C to 300{degree}C) as well as dielectric breakdown strength. Additionally, film/foil capacitors in a dry wrap-and-fill configuration were fabricated and tested to determine insulation resistance, breakdown voltage, and radiation hardness. Results are given for all evaluations. 7 refs., 4 figs., 4 tabs.

  8. Alternatives to traditional transportation fuels: An overview

    SciTech Connect

    Not Available

    1994-06-01

    This report presents the first compilation by the Energy Information Administration (EIA) of information on alternatives to gasoline and diesel fuel. The purpose of the report is: (1) to provide background information on alternative transportation fuels and replacement fuels compared with gasoline and diesel fuel, and (2) to furnish preliminary estimates of alternative transportation fuels and alternative fueled vehicles as required by the Energy Policy Act of 1992 (EPACT), Title V, Section 503, ``Replacement Fuel Demand Estimates and Supply Information.`` Specifically, Section 503 requires the EIA to report annually on: (1) the number and type of alternative fueled vehicles in existence the previous year and expected to be in use the following year, (2) the geographic distribution of these vehicles, (3) the amounts and types of replacement fuels consumed, and (4) the greenhouse gas emissions likely to result from replacement fuel use. Alternative fueled vehicles are defined in this report as motorized vehicles licensed for on-road use, which may consume alternative transportation fuels. (Alternative fueled vehicles may use either an alternative transportation fuel or a replacement fuel.) The intended audience for the first section of this report includes the Secretary of Energy, the Congress, Federal and State agencies, the automobile manufacturing industry, the transportation fuel manufacturing and distribution industries, and the general public. The second section is designed primarily for persons desiring a more technical explanation of and background for the issues surrounding alternative transportation fuels.

  9. Alternative Neutron Detection Testing Summary

    SciTech Connect

    Kouzes, Richard T.; Ely, James H.; Erikson, Luke E.; Kernan, Warnick J.; Lintereur, Azaree T.; Siciliano, Edward R.; Stromswold, David C.; Woodring, Mitchell L.

    2010-04-08

    Radiation portal monitors used for interdiction of illicit materials at borders include highly sensitive neutron detection systems. The main reason for having neutron detection capability is to detect fission neutrons from plutonium. Most currently deployed radiation portal monitors (RPMs) use neutron detectors based upon 3He-filled gas proportional counters, which are the most common large area neutron detector. This type of neutron detector is used in the TSA and other RPMs installed in international locations and in the Ludlum and Science Applications International Corporation RPMs deployed primarily for domestic applications. There is a declining supply of 3He in the world and, thus, methods to reduce the use of this gas in RPMs with minimal changes to the current system designs and sensitivity to cargo-borne neutrons are being investigated. Four technologies have been identified as being currently commercially available, potential alternative neutron detectors to replace the use of 3He in RPMs. These technologies are: 1) Boron trifluoride-filled proportional counters, 2) Boron-lined proportional counters, 3) Lithium-loaded glass fibers, and 4) Coated wavelength-shifting plastic fibers. Reported here is a summary of the testing carried out at Pacific Northwest National Laboratory on these technologies to date, as well as measurements on 3He tubes at various pressures. Details on these measurements are available in the referenced reports. Sponsors of these tests include the Department of Energy (DOE), Department of Homeland Security (DHS), and the Department of Defense (DoD), as well as internal Pacific Northwest National Laboratory funds.

  10. Display Sharing: An Alternative Paradigm

    NASA Technical Reports Server (NTRS)

    Brown, Michael A.

    2010-01-01

    The current Johnson Space Center (JSC) Mission Control Center (MCC) Video Transport System (VTS) provides flight controllers and management the ability to meld raw video from various sources with telemetry to improve situational awareness. However, maintaining a separate infrastructure for video delivery and integration of video content with data adds significant complexity and cost to the system. When considering alternative architectures for a VTS, the current system's ability to share specific computer displays in their entirety to other locations, such as large projector systems, flight control rooms, and back supporting rooms throughout the facilities and centers must be incorporated into any new architecture. Internet Protocol (IP)-based systems also support video delivery and integration. IP-based systems generally have an advantage in terms of cost and maintainability. Although IP-based systems are versatile, the task of sharing a computer display from one workstation to another can be time consuming for an end-user and inconvenient to administer at a system level. The objective of this paper is to present a prototype display sharing enterprise solution. Display sharing is a system which delivers image sharing across the LAN while simultaneously managing bandwidth, supporting encryption, enabling recovery and resynchronization following a loss of signal, and, minimizing latency. Additional critical elements will include image scaling support, multi -sharing, ease of initial integration and configuration, integration with desktop window managers, collaboration tools, host and recipient controls. This goal of this paper is to summarize the various elements of an IP-based display sharing system that can be used in today's control center environment.

  11. Planetary Protection Alternate Protocol Certification

    NASA Astrophysics Data System (ADS)

    Baker, Amy; Barengoltz, Jack; Tisdale, David

    The talk presents a standardized approach for new method certification or alterative testing protocol (ATP) certification against the existing U.S. Planetary Protection Standards. In consideration of new method certification there are two phases of activities that are relevant to the certification process. The first is sample acquisition which typically incorporates swab or wipes sampling on relevant hardware, associated facilities and ground support equipment. The sampling methods introduce considerations of field sampling efficiency as it relates to spore distribution on the spacecraft, spacecraft material influences on the ability of the swab or wipe to remove spores from the hardware, the types of swabs and wipes used (polyester, cotton, macrofoam), and human sampling influences. The second portion of a new protocol certification looks specifically at the lab work-up or analysis of the samples provided to the laboratory. Variables in this process include selection of appropriate biomarkers, extraction efficiencies (removal of spores or constituents of interest from the sampling device), and a method's ability to accurately determine the number of spores present in the sample with a statistically valid level of confidence as described by parameters such as precision, accuracy, robustness, specificity and sensitivity. Considerations for alternative testing protocols such as those which utilize bioburden reduction techniques include selection of appropriate biomarkers for testing, test materials and a defined statistical approach that provides sufficient scientific data to support the modification of an existing NASA specification or the generation of a new NASA specification. Synergies between the U.S. and European Space Agency approaches will also be discussed.

  12. Alternative to particle dark matter

    NASA Astrophysics Data System (ADS)

    Khoury, Justin

    2015-01-01

    We propose an alternative to particle dark matter that borrows ingredients of modified Newtonian dynamics (MOND) while adding new key components. The first new feature is a dark matter fluid, in the form of a scalar field with small equation of state and sound speed. This component is critical in reproducing the success of cold dark matter for the expansion history and the growth of linear perturbations, but does not cluster significantly on nonlinear scales. Instead, the missing mass problem on nonlinear scales is addressed by a modification of the gravitational force law. The force law approximates MOND at large and intermediate accelerations, and therefore reproduces the empirical success of MOND at fitting galactic rotation curves. At ultralow accelerations, the force law reverts to an inverse-square law, albeit with a larger Newton's constant. This latter regime is important in galaxy clusters and is consistent with their observed isothermal profiles, provided the characteristic acceleration scale of MOND is mildly varying with scale or mass, such that it is 12 times higher in clusters than in galaxies. We present an explicit relativistic theory in terms of two scalar fields. The first scalar field is governed by a Dirac-Born-Infeld action and behaves as a dark matter fluid on large scales. The second scalar field also has single-derivative interactions and mediates a fifth force that modifies gravity on nonlinear scales. Both scalars are coupled to matter via an effective metric that depends locally on the fields. The form of this effective metric implies the equality of the two scalar gravitational potentials, which ensures that lensing and dynamical mass estimates agree. Further work is needed in order to make both the acceleration scale of MOND and the fraction at which gravity reverts to an inverse-square law explicitly dynamical quantities, varying with scale or mass.

  13. Methods for Characterization of Alternative RNA Splicing.

    PubMed

    Harvey, Samuel E; Cheng, Chonghui

    2016-01-01

    Quantification of alternative splicing to detect the abundance of differentially spliced isoforms of a gene in total RNA can be accomplished via RT-PCR using both quantitative real-time and semi-quantitative PCR methods. These methods require careful PCR primer design to ensure specific detection of particular splice isoforms. We also describe analysis of alternative splicing using a splicing "minigene" in mammalian cell tissue culture to facilitate investigation of the regulation of alternative splicing of a particular exon of interest.

  14. ARI delegation to Japan on Alternative Refrigerants

    SciTech Connect

    Not Available

    1993-02-01

    Researchers from ARI member companies spoke at the International Conference on Alternative Refrigerants in Tokyo and visited several Japanese organizations for the purpose of exchanging information on alternative refrigerants. The specific purpose of the meetings was to review the methods being utilized to screen alternatives to CFCs and HCFCs: materials compatibility screening methods, lubricant testing techniques, as well as flammability studies. A list of papers presented at the conference is included.

  15. Alternative medicine in ancient and medieval history.

    PubMed

    Prioreschi, P

    2000-10-01

    The author, in an attempt to clarify whether the rise of alternative medicine is a phenomenon characteristic of our time or whether it existed in the past as well, has identified at least three alternative medicines, which developed in ancient Rome, ancient India and in the medieval Islamic world. The circumstances leading to the development of alternative medicine in the past and in our time are discussed and compared. PMID:11000060

  16. Cyclic Polymer with Alternating Monomer Sequence.

    PubMed

    Zhu, Wen; Li, Zi; Zhao, Youliang; Zhang, Ke

    2015-11-01

    Cyclic polymers with alternating monomer sequence are synthesized for the first time based on the ring-closure strategy. Well-defined telechelic alternating polymers are synthesized by reversible addition-fragmentation chain transfer polymerization by copolymerizing the electron acceptor monomer of N-benzylmaleimide and donor monomer of styrene with a feed ratio of 1 between them. The corresponding cyclic alternating polymers are then produced by the UV-induced Diels-Alder click reaction to ring-close the linear alternating polymer precursors under highly diluted reaction solution.

  17. Defining alternative rules in water and sanitation.

    PubMed

    Robert, J

    1995-11-01

    While the conventional water and sanitation package remains most prominent, alternatives exist to conventional waterworks and sanitation practices. Some alternate courses of action are considered. Promising an unprecedented availability of piped water, water development projects foster a pump-and-dump mentality even before they are completed. Often simply announcing the impending implementation of such projects encourages the intention among future beneficiaries to waste water resources. Alternatives to the domestic waste of water must be sought and implemented. Disestablishing water development, styles of alternative water technologies, decision-making and decision-makers, water policy scale, regenerating community access to sources, and water pricing are discussed. PMID:12293526

  18. Complementary and Alternative Therapies in ALS

    PubMed Central

    Bedlack, Richard S.; Joyce, Nanette; Carter, Gregory T.; Pagononi, Sabrina; Karam, Chafic

    2015-01-01

    Synopsis Given the severity of their illness and lack of effective disease modifying agents, it is not surprising that most patients with ALS consider trying complementary and alternative therapies. Some of the most commonly considered alternative therapies include special diets, nutritional supplements, cannabis, acupuncture, chelation and energy healing. This chapter reviews these in detail. We also describe 3 models by which physicians may frame discussions about alternative therapies: paternalism, autonomy and shared decision making. Finally, we review a program called ALSUntangled which using shared shared decision making to review alternative therapies for ALS. PMID:26515629

  19. 7 CFR 1220.207 - Alternate members.

    Code of Federal Regulations, 2012 CFR

    2012-01-01

    ... AGREEMENTS AND ORDERS; MISCELLANEOUS COMMODITIES), DEPARTMENT OF AGRICULTURE SOYBEAN PROMOTION, RESEARCH, AND CONSUMER INFORMATION Soybean Promotion and Research Order United Soybean Board § 1220.207 Alternate...

  20. 7 CFR 1220.207 - Alternate members.

    Code of Federal Regulations, 2011 CFR

    2011-01-01

    ... AGREEMENTS AND ORDERS; MISCELLANEOUS COMMODITIES), DEPARTMENT OF AGRICULTURE SOYBEAN PROMOTION, RESEARCH, AND CONSUMER INFORMATION Soybean Promotion and Research Order United Soybean Board § 1220.207 Alternate...

  1. 7 CFR 1220.207 - Alternate members.

    Code of Federal Regulations, 2014 CFR

    2014-01-01

    ... AGREEMENTS AND ORDERS; MISCELLANEOUS COMMODITIES), DEPARTMENT OF AGRICULTURE SOYBEAN PROMOTION, RESEARCH, AND CONSUMER INFORMATION Soybean Promotion and Research Order United Soybean Board § 1220.207 Alternate...

  2. 7 CFR 1220.207 - Alternate members.

    Code of Federal Regulations, 2013 CFR

    2013-01-01

    ... AGREEMENTS AND ORDERS; MISCELLANEOUS COMMODITIES), DEPARTMENT OF AGRICULTURE SOYBEAN PROMOTION, RESEARCH, AND CONSUMER INFORMATION Soybean Promotion and Research Order United Soybean Board § 1220.207 Alternate...

  3. 21 CFR 640.120 - Alternative procedures.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... Alternative procedures. (a) The Director, Center for Biologics Evaluation and Research, may approve an... Regulations regarding blood, blood components, or blood products. Requests for such exceptions or...

  4. The Alternative Low Noise Fan

    NASA Technical Reports Server (NTRS)

    Dittmar, James H.; Elliott, David M.; Jeracki, Robert J.; Moore, Royce D.; Parrott, Tony L.

    2000-01-01

    A 106 bladed fan with a design takeoff tip speed of 1100 ft/sec was hypothesized as reducing perceived noise because of the shift of the blade passing harmonics to frequencies beyond the perceived noise rating range. A 22 in. model of this Alternative Low Noise Fan, ALNF, was tested in the NASA Glenn 9x 15 Wind Tunnel. 'Me fan was tested with a 7 vane long chord stator assembly and a 70 vane conventional stator assembly in both hard and acoustically treated configurations. In addition a partially treated 7 vane configuration was tested wherein the acoustic material between the 7 long chord stators was made inactive. The noise data from the 106 bladed fan with 7 long chord stators in a hard configuration was shown to be around 4 EPNdB quieter than a low tip speed Allison fan at takeoff and around 5 EPNdB quieter at approach. Although the tone noise behaved as hypothesized, the majority of this noise reduction was from reduced broadband noise related to the large number of rotor blades. This 106 bladed ALNF is a research fan designed to push the technology limits and as such is probably not a practical device with present materials technology. However, a low tip speed fan with around 50 blades would be a practical device and calculations indicate that it could be 2 to 3 EPNdB quieter at takeoff and 3 to 4 EPNdB quieter at approach than the Allison fan. 7 vane data compared with 70 vane data indicated that the tone noise was controlled by rotor wake-stator interaction but that the broadband noise is probably controlled by the interaction of the rotor with incoming flows. A possible multiple pure tone noise reduction technique for a fan/acoustic treatment system was identified. The data from the fully treated configuration showed significant noise reductions over a large frequency range thereby providing a real tribute to this bulk absorber treatment design. The tone noise data with the partially treated 7 vane configuration indicated that acoustic material in the

  5. 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.

  6. 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.

  7. 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

  8. 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

  9. 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.

  10. 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.

  11. 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

  12. 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.

  13. 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.

  14. 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

  15. 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.

  16. 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

  17. Complementary and Alternative Therapies for Down Syndrome

    ERIC Educational Resources Information Center

    Roizen, Nancy J.

    2005-01-01

    In their role as committed advocates, parents of children with Down syndrome have always sought alternative therapies, mainly to enhance cognitive function but also to improve their appearance. Nutritional supplements have been the most frequent type of complementary and alternative therapy used. Cell therapy, plastic surgery, hormonal therapy,…

  18. Alternatives for Financing Higher Education Facilities.

    ERIC Educational Resources Information Center

    Leslie, Larry L.; Felix, Frank J.

    1980-01-01

    How state governments should finance public college and university facilities is discussed. A framework for analyzing the capital financing alternatives available to state governments is described. Several alternatives in Arizona--including state appropriations, leasing, and revenue bonding--are considered as an illustration. (Author/MLW)

  19. Pichia pastoris "just in time" alternative respiration.

    PubMed

    Kern, Alexander; Hartner, Franz S; Freigassner, Maria; Spielhofer, Julia; Rumpf, Cornelia; Leitner, Laura; Fröhlich, Kai-Uwe; Glieder, Anton

    2007-04-01

    Alternative oxidases (Aox or Aod) are present in the mitochondria of plants, fungi and many types of yeast. These enzymes transfer electrons from the ubiquinol pool directly to oxygen without contributing to the proton transfer across the mitochondrial membrane. Alternative oxidases are involved in stress responses, programmed cell death and maintenance of the cellular redox balance. The alternative oxidase gene of the methylotrophic yeast Pichia pastoris was isolated and cloned to study its regulation and the effects of deregulation of the alternative respiration by overexpression or disruption of the gene. Both disruption and overexpression had negative effects on the biomass yield; however, the growth rate and substrate uptake rate of the strain overexpressing the alternative oxidase were slightly increased. These effects were even more pronounced when higher glucose concentrations were used. The occurrence of free intracellular radicals and cell death phenomena was investigated using dihydrorhodamine 123 and the TUNEL test. The results suggest a major contribution of the alternative oxidase to P. pastoris cell viability. The negative effects of deregulated alternative respiration clearly indicated the importance of precise regulation of the alternative oxidase in this yeast.

  20. 7 CFR 1216.45 - Alternate members.

    Code of Federal Regulations, 2010 CFR

    2010-01-01

    ... AGREEMENTS AND ORDERS; MISCELLANEOUS COMMODITIES), DEPARTMENT OF AGRICULTURE PEANUT PROMOTION, RESEARCH, AND INFORMATION ORDER Peanut Promotion, Research, and Information Order National Peanut Board § 1216.45 Alternate members. An alternate member of the Board, during the absence of the member for the primary...

  1. 7 CFR 1216.45 - Alternate members.

    Code of Federal Regulations, 2011 CFR

    2011-01-01

    ... AGREEMENTS AND ORDERS; MISCELLANEOUS COMMODITIES), DEPARTMENT OF AGRICULTURE PEANUT PROMOTION, RESEARCH, AND INFORMATION ORDER Peanut Promotion, Research, and Information Order National Peanut Board § 1216.45 Alternate members. An alternate member of the Board, during the absence of the member for the primary...

  2. Alternative Fuels Data Center (Fact Sheet)

    SciTech Connect

    Not Available

    2013-07-01

    Fact sheet describes the Alternative Fuels Data Center, which provides information, data, and tools to help fleets and other transportation decision makers find ways to reduce petroleum consumption through the use of alternative and renewable fuels, advanced vehicles, and other fuel-saving measures.

  3. Alternative Fuels Market and Policy Trends (Presentation)

    SciTech Connect

    Schroeder, A. N.

    2013-09-01

    Market forces and policies are increasing opportunities for alternative fuels. There is no one-size-fits-all, catch-all, silver-bullet fuel. States play a critical role in the alternative fuel market and are taking a leading role.

  4. Responding to Alternative Conceptions in the Classroom.

    ERIC Educational Resources Information Center

    Taber, Keith S.

    2003-01-01

    Explains how students' alternative conceptions about curriculum topics interfere with classroom learning. Describes the Royal Society of Chemistry's Teacher Fellowship Project entitled "Challenging Misconceptions in the Classroom," which develops classroom materials to diagnose and challenge specific alternative conceptions. Uses key ideas of how…

  5. Sodium Bearing Waste Processing Alternatives Analysis

    SciTech Connect

    Murphy, James Anthony; Palmer, Brent J; Perry, Keith Joseph

    2003-12-01

    A multidisciplinary team gathered to develop a BBWI recommendation to DOE-ID on the processing alternatives for the sodium bearing waste in the INTEC Tank Farm. Numerous alternatives were analyzed using a rigorous, systematic approach. The data gathered were evaluated through internal and external peer reviews for consistency and validity. Three alternatives were identified to be top performers: Risk-based Calcination, MACT to WIPP Calcination and Cesium Ion Exchange. A dual-path through early Conceptual design is recommended for MACT to WIPP Calcination and Cesium Ion Exchange since Risk-based Calcination does not require design. If calcination alternatives are not considered based on giving Type of Processing criteria significantly greater weight, the CsIX/TRUEX alternative follows CsIX in ranking. However, since CsIX/TRUEX shares common uncertainties with CsIX, reasonable backups, which follow in ranking, are the TRUEX and UNEX alternatives. Key uncertainties must be evaluated by the decision-makers to choose one final alternative. Those key uncertainties and a path forward for the technology roadmapping of these alternatives is provided.

  6. Context-driven expectations about focus alternatives

    PubMed Central

    Kim, Christina S.; Gunlogson, Christine; Tanenhaus, Michael K.; Runner, Jeffrey T.

    2015-01-01

    What is conveyed by a sentence frequently depends not only on the descriptive content carried by its words, but also on implicit alternatives determined by the context of use. Four visual world eye-tracking experiments examined how alternatives are generated based on aspects of the discourse context and used in interpreting sentences containing the focus operators only and also. Experiment 1 builds on previous reading time studies showing that the interpretations of only sentences are constrained by alternatives explicitly mentioned in the preceding discourse, providing fine-grained time course information about the expectations triggered by only. Experiments 2 and 3 show that, in the absence of explicitly mentioned alternatives, lexical and situation-based categories evoked by the context are possible sources of alternatives. While Experiments 1-3 all demonstrate the discourse dependence of alternatives, only explicit mention triggered expectations about alternatives that were specific to sentences with only. By comparing only with also, Experiment 4 begins to disentangle expectations linked to the meanings of specific operators from those generalizable to the class of focus-sensitive operators. Together, these findings show that the interpretation of sentences with focus operators draws on both dedicated mechanisms for introducing alternatives into the discourse context and general mechanisms associated with discourse processing. PMID:25797456

  7. Adult Education Alternative Diploma. Final Report.

    ERIC Educational Resources Information Center

    Gacka, Richard

    The Adult Education Alternative Diploma project was conducted to develop an alternative program to the General Educational Development (GED) diploma program through which adult students in Pennsylvania could earn actual high school diplomas. During the year-long project, a competency diploma program was developed and implemented in conjunction…

  8. Philadelphia Alternative Diploma Project. 1996-1997.

    ERIC Educational Resources Information Center

    Philadelphia School District, PA.

    A project was conducted in Philadelphia to help adult students obtain a high school diploma as an alternative to or enhancement of a General Educational Development (GED) certificate. During fiscal year 1996-97 the Metropolitan Career Center (MCC), in collaboration with the School District of Philadelphia, designed an Alternative Diploma Program…

  9. Alternatives to traditional transportation fuels 1996

    SciTech Connect

    1997-12-01

    Interest in alternative transportation fuels (ATF`s) has increased in recent years due to the drives for cleaner air and less dependence upon foreign oil. This report, Alternatives to Traditional Transportation Fuels 1996, provides information on ATFs, as well as the vehicles that consume them.

  10. Renewal, resurgence, and alternative reinforcement context.

    PubMed

    Sweeney, Mary M; Shahan, Timothy A

    2015-07-01

    Resurgence, relapse induced by the removal of alternative reinforcement, and renewal, relapse induced by a change in contextual stimuli, are typically studied separately in operant conditioning paradigms. In analogous treatments of operant problem behavior, aspects of both relapse phenomena can operate simultaneously. Therefore, the purpose of this study was to examine a novel method for studying resurgence and renewal in the same experimental preparation. An alternative source of reinforcement was available during extinction for one group of rats (a typical resurgence preparation). Another group experienced an operant renewal preparation in which the extinction context was distinguished via olfactory and visual stimuli. A third group experienced alternative reinforcement delivery in the new context, a novel combination of typical resurgence and renewal preparations. Removal of alternative reinforcement and/or a change in context induced relapse relative to an extinction-only control group. When alternative reinforcement was delivered in a novel context, the alternative response was less persistent relative to when extinction of the alternative response took place in the context in which it was trained. This methodology might be used to illustrate shared (or distinct) mechanisms of resurgence and renewal, and to determine how delivering alternative reinforcement in another context may affect persistence and relapse.

  11. 25 CFR 11.1112 - Dispositional alternatives.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... 25 Indians 1 2014-04-01 2014-04-01 false Dispositional alternatives. 11.1112 Section 11.1112... LAW AND ORDER CODE Minor-in-Need-of-Care Procedure § 11.1112 Dispositional alternatives. (a) If a... court may prescribe; or, if reasonable efforts to have the minor return or remain in his or her own...

  12. 25 CFR 11.1112 - Dispositional alternatives.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... 25 Indians 1 2013-04-01 2013-04-01 false Dispositional alternatives. 11.1112 Section 11.1112... LAW AND ORDER CODE Minor-in-Need-of-Care Procedure § 11.1112 Dispositional alternatives. (a) If a... court may prescribe; or, if reasonable efforts to have the minor return or remain in his or her own...

  13. Demystifying Alternative Education: Considering What Really Works

    ERIC Educational Resources Information Center

    Powell, Diane E.

    2003-01-01

    The effectiveness of alternative programs requires an understanding of both research and practice. Essential components of alternative educational programs must include opportunities to build and foster resilience in our youth. Program elements must be reflective of the quality and standards mandated by the "No Child Left Behind Act of 2001."…

  14. 21 CFR 640.120 - Alternative procedures.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... 21 Food and Drugs 7 2010-04-01 2010-04-01 false Alternative procedures. 640.120 Section 640.120 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) BIOLOGICS ADDITIONAL STANDARDS FOR HUMAN BLOOD AND BLOOD PRODUCTS Alternative Procedures §...

  15. Alternative Asbestos Control Method (AACM), Washington

    EPA Science Inventory

    This presentation describes the status to date of the Alternative Asbestos Control Method research, which is intended as a possible alternative technology for use in the demolition of buildings that contain asbestos and are covered under the regulatory requirements of the Asbesto...

  16. 7 CFR 1205.328 - Alternate members.

    Code of Federal Regulations, 2014 CFR

    2014-01-01

    ... AGREEMENTS AND ORDERS; MISCELLANEOUS COMMODITIES), DEPARTMENT OF AGRICULTURE COTTON RESEARCH AND PROMOTION Cotton Research and Promotion Order Cotton Board § 1205.328 Alternate members. An alternate member of the... member from the same cotton-producing state or region to serve in such member's place and stead of...

  17. 7 CFR 1205.328 - Alternate members.

    Code of Federal Regulations, 2013 CFR

    2013-01-01

    ... AGREEMENTS AND ORDERS; MISCELLANEOUS COMMODITIES), DEPARTMENT OF AGRICULTURE COTTON RESEARCH AND PROMOTION Cotton Research and Promotion Order Cotton Board § 1205.328 Alternate members. An alternate member of the... member from the same cotton-producing state or region to serve in such member's place and stead of...

  18. 7 CFR 1205.328 - Alternate members.

    Code of Federal Regulations, 2011 CFR

    2011-01-01

    ... AGREEMENTS AND ORDERS; MISCELLANEOUS COMMODITIES), DEPARTMENT OF AGRICULTURE COTTON RESEARCH AND PROMOTION Cotton Research and Promotion Order Cotton Board § 1205.328 Alternate members. An alternate member of the... member from the same cotton-producing state or region to serve in such member's place and stead of...

  19. Student Perceptions of the Alternative School

    ERIC Educational Resources Information Center

    Herrington, Tina Sabrina

    2012-01-01

    Some students find it difficult to reach graduation in a regular secondary school setting, but may be successful in an alternative setting. Causes of not graduating could include high absenteeism and behavior problems, which may result in dropping out. This study sought student perceptions of an alternative program in a public school district in…

  20. Alternative Natural Energy Sources in Building Design.

    ERIC Educational Resources Information Center

    Davis, Albert J.; Schubert, Robert P.

    This publication provides a discussion of various energy conserving building systems and design alternatives. The information presented here covers alternative space and water heating systems, and energy conserving building designs incorporating these systems and other energy conserving techniques. Besides water, wind, solar, and bio conversion…

  1. Promoting Excellence: Some Classroom Evaluation Alternatives.

    ERIC Educational Resources Information Center

    Sia, Archibald P.; Sydnor, Darlean

    Following a brief overview of literature on excellence in the classroom, the shortcomings of objective tests as means for evaluating classroom excellence are discussed as well as the need for evaluation alternatives. Descriptions are given of evaluation alternatives, some of which focus upon reading and writing achievement: (1) observation of…

  2. 7 CFR 993.29 - Alternates.

    Code of Federal Regulations, 2010 CFR

    2010-01-01

    ... Regulations of the Department of Agriculture (Continued) AGRICULTURAL MARKETING SERVICE (Marketing Agreements... Order Regulating Handling Prune Marketing Committee § 993.29 Alternates. An alternate for a member of... event of his removal, resignation, disqualification, or death, until a successor for such...

  3. 7 CFR 966.31 - Alternate members.

    Code of Federal Regulations, 2010 CFR

    2010-01-01

    ... 7 Agriculture 8 2010-01-01 2010-01-01 false Alternate members. 966.31 Section 966.31 Agriculture Regulations of the Department of Agriculture (Continued) AGRICULTURAL MARKETING SERVICE (Marketing Agreements... event of the death, removal, resignation, or disqualification of a member, his alternate shall act...

  4. 27 CFR 19.930 - Alternating proprietorship.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... the boundaries of the plant or any part thereof which is to be alternated. (d) Alternation Journal... journal the following information: (1) Name or trade name; (2) Alcohol fuel plant permit number; (3) Date... journal will remain in the possession of the incoming proprietor until the premises are again...

  5. Alternatives to Suspension: A Government Initiative

    ERIC Educational Resources Information Center

    Ryan, Thomas G.; Zoldy, Scott

    2011-01-01

    Student discipline and the ineffectiveness of out-of-school suspension is examined in light of the Ontario (Canada) legislative reform that supported a greater emphasis on progressive discipline alternatives to out-of-school suspension. Alternative discipline herein is explored via the behavior education plan, the school survival group, and…

  6. An Examination of Alternative Instructional Methods

    ERIC Educational Resources Information Center

    Brewer, Peggy D.

    2004-01-01

    Demographic shifts, advances in technology, and changing student perceptions and expectations have influenced the development of alternative means and methods for offering and delivering traditional college courses. This study reports student perceptions of four alternatives at one university where students were asked to rate course offerings…

  7. An Alternative Starting Point for Fraction Instruction

    ERIC Educational Resources Information Center

    Cortina, José Luis; Višnovská, Jana; Zúñiga, Claudia

    2015-01-01

    We analyze the results of a study conducted for the purpose of assessing the viability of an alternative starting point for teaching fractions. The alternative is based on Freudenthal's insights about fraction as comparison. It involves portraying the entities that unit fractions quantify as always being apart from the reference unit, instead of…

  8. 28 CFR 42.734 - Alternative funding.

    Code of Federal Regulations, 2012 CFR

    2012-07-01

    ... 28 Judicial Administration 1 2012-07-01 2012-07-01 false Alternative funding. 42.734 Section 42.734 Judicial Administration DEPARTMENT OF JUSTICE NONDISCRIMINATION; EQUAL EMPLOYMENT OPPORTUNITY...; Implementation of the Age Discrimination Act of 1975 Compliance Procedures § 42.734 Alternative funding....

  9. 28 CFR 42.734 - Alternative funding.

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ... 28 Judicial Administration 1 2010-07-01 2010-07-01 false Alternative funding. 42.734 Section 42.734 Judicial Administration DEPARTMENT OF JUSTICE NONDISCRIMINATION; EQUAL EMPLOYMENT OPPORTUNITY...; Implementation of the Age Discrimination Act of 1975 Compliance Procedures § 42.734 Alternative funding....

  10. 28 CFR 42.734 - Alternative funding.

    Code of Federal Regulations, 2013 CFR

    2013-07-01

    ... 28 Judicial Administration 1 2013-07-01 2013-07-01 false Alternative funding. 42.734 Section 42.734 Judicial Administration DEPARTMENT OF JUSTICE NONDISCRIMINATION; EQUAL EMPLOYMENT OPPORTUNITY...; Implementation of the Age Discrimination Act of 1975 Compliance Procedures § 42.734 Alternative funding....

  11. 28 CFR 42.734 - Alternative funding.

    Code of Federal Regulations, 2011 CFR

    2011-07-01

    ... 28 Judicial Administration 1 2011-07-01 2011-07-01 false Alternative funding. 42.734 Section 42.734 Judicial Administration DEPARTMENT OF JUSTICE NONDISCRIMINATION; EQUAL EMPLOYMENT OPPORTUNITY...; Implementation of the Age Discrimination Act of 1975 Compliance Procedures § 42.734 Alternative funding....

  12. 28 CFR 42.734 - Alternative funding.

    Code of Federal Regulations, 2014 CFR

    2014-07-01

    ... 28 Judicial Administration 1 2014-07-01 2014-07-01 false Alternative funding. 42.734 Section 42.734 Judicial Administration DEPARTMENT OF JUSTICE NONDISCRIMINATION; EQUAL EMPLOYMENT OPPORTUNITY...; Implementation of the Age Discrimination Act of 1975 Compliance Procedures § 42.734 Alternative funding....

  13. Alternative Education Completers: A Phenomenological Study

    ERIC Educational Resources Information Center

    Murray, Becky L.; Holt, Carleton R.

    2014-01-01

    The purpose of this study was to explore the elements of the alternative education experience significant to successful completion of the program. This phenomenological paradigm provided the framework for all aspects of the qualitative study. Students, parents, administrators, and staff members of two alternative programs in the southeast Kansas…

  14. Central Park East: An Alternative Story.

    ERIC Educational Resources Information Center

    Meier, Deborah

    1987-01-01

    Central Park East school was founded in 1974 in Harlem (NY) as an alternative elementary school within the public inner city school system. In 1984 it had expanded into three elementary alternative schools and added a fourth secondary school. It is considered a progressive school following in the tradition of New York city's private schools. (MD)

  15. 7 CFR 1216.45 - Alternate members.

    Code of Federal Regulations, 2014 CFR

    2014-01-01

    ... AGREEMENTS AND ORDERS; MISCELLANEOUS COMMODITIES), DEPARTMENT OF AGRICULTURE PEANUT PROMOTION, RESEARCH, AND INFORMATION ORDER Peanut Promotion, Research, and Information Order National Peanut Board § 1216.45 Alternate members. An alternate member of the Board, during the absence of the member for the primary...

  16. 7 CFR 1216.45 - Alternate members.

    Code of Federal Regulations, 2012 CFR

    2012-01-01

    ... AGREEMENTS AND ORDERS; MISCELLANEOUS COMMODITIES), DEPARTMENT OF AGRICULTURE PEANUT PROMOTION, RESEARCH, AND INFORMATION ORDER Peanut Promotion, Research, and Information Order National Peanut Board § 1216.45 Alternate members. An alternate member of the Board, during the absence of the member for the primary...

  17. 7 CFR 1216.45 - Alternate members.

    Code of Federal Regulations, 2013 CFR

    2013-01-01

    ... AGREEMENTS AND ORDERS; MISCELLANEOUS COMMODITIES), DEPARTMENT OF AGRICULTURE PEANUT PROMOTION, RESEARCH, AND INFORMATION ORDER Peanut Promotion, Research, and Information Order National Peanut Board § 1216.45 Alternate members. An alternate member of the Board, during the absence of the member for the primary...

  18. SKOLE: The Journal of Alternative Education, 1996.

    ERIC Educational Resources Information Center

    Leue, Mary, Ed.

    1996-01-01

    The four issues of "SKOLE" published in 1996 include articles about alternatives to public education, the value of free schools and home-schooling, and children's learning experiences. Feature articles include profiles of alternative schools and educational programs; descriptions of learning experiences and teaching practices; a study of full…

  19. Professional Development in Massachusetts' Public Alternative Schools

    ERIC Educational Resources Information Center

    Farese, Christina Susan

    2013-01-01

    This qualitative study utilized 102 open-ended questionnaires and 15 semi-structured interviews to examine the professional development offered in 27 Massachusetts public alternative schools by answering the major question: What form do professional development programs take in Massachusetts' public, alternative schools? There were many…

  20. Matrix Management: An Organizational Alternative for Libraries.

    ERIC Educational Resources Information Center

    Johnson, Peggy

    1990-01-01

    Describes various organizational structures and models, presents matrix management as an alternative to traditional hierarchical structures, and suggests matrix management as an appropriate organizational alternative for academic libraries. Benefits that are discussed include increased flexibility, a higher level of professional independence, and…