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

  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

    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...

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

    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

  3. 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

  4. 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. PMID:23636690

  5. 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

  6. 2C-Methyl-d-erythritol 4-phosphate enhances and sustains cyclodiphosphate synthase IspF activity

    PubMed Central

    Bitok, J. Kipchirchir; Freel Meyers, Caren

    2012-01-01

    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 IspF by downstream isoprenoid diphosphates. Here, we demonstrate recombinant E. coli IspF is not inhibited by downstream metabolites and 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 2Cmethyl-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

  7. 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

  8. Expression and Molecular Analysis of the Arabidopsis DXR Gene Encoding 1-Deoxy-d-Xylulose 5-Phosphate Reductoisomerase, the First Committed Enzyme of the 2-C-Methyl-d-Erythritol 4-Phosphate Pathway1

    PubMed Central

    Carretero-Paulet, Lorenzo; Ahumada, Iván; Cunillera, Nuria; Rodríguez-Concepción, Manuel; Ferrer, Albert; Boronat, Albert; Campos, Narciso

    2002-01-01

    1-Deoxy-d-xylulose 5-phosphate reductoisomerase (DXR) catalyzes the first committed step of the 2-C-methyl-d-erythritol 4-phosphate pathway for isoprenoid biosynthesis. In Arabidopsis, DXR is encoded by a single-copy gene. We have cloned a full-length cDNA corresponding to this gene. A comparative analysis of all plant DXR sequences known to date predicted an N-terminal transit peptide for plastids, with a conserved cleavage site, and a conserved proline-rich region at the N terminus of the mature protein, which is not present in the prokaryotic DXR homologs. We demonstrate that Arabidopsis DXR is targeted to plastids and localizes into chloroplasts of leaf cells. The presence of the proline-rich region in the mature Arabidopsis DXR was confirmed by detection with a specific antibody. A proof of the enzymatic function of this protein was obtained by complementation of an Escherichia coli mutant defective in DXR activity. The expression pattern of β-glucuronidase, driven by the DXR promoter in Arabidopsis transgenic plants, together with the tissue distribution of DXR transcript and protein, revealed developmental and environmental regulation of the DXR gene. The expression pattern of the DXR gene parallels that of the Arabidopsis 1-deoxy-d-xylulose 5-phosphate synthase gene, but the former is slightly more restricted. These genes are expressed in most organs of the plant including roots, with higher levels in seedlings and inflorescences. The block of the 2-C-methyl-d-erythritol 4-phosphate pathway in Arabidopsis seedlings with fosmidomycin led to a rapid accumulation of DXR protein, whereas the 1-deoxy-d-xylulose 5-phosphate synthase protein level was not altered. Our results are consistent with the participation of the Arabidopsis DXR gene in the control of the 2-C-methyl-d-erythritol 4-phosphate pathway. PMID:12177470

  9. Involvement of 2-C-methyl-D-erythritol-4-phosphate pathway in biosynthesis of aphidicolin-like tetracyclic diterpene of Scoparia dulcis.

    PubMed

    Nkembo, Marguerite Kasidimoko; Lee, Jung-Bum; Nakagiri, Takeshi; Hayashi, Toshimitsu

    2006-05-01

    Specific inhibitors of the MVA pathway (pravastatin) and the MEP pathway (fosmidomycin) were used to interfere with the biosynthetic flux which leads to the production of aphidicolin-like diterpene in leaf organ cultures of Scoparia dulcis. Treatment of leaf organs with fosmidomycin resulted in dose dependent inhibition of chlorophylls, carotenoids, scopadulcic acid B (SDB) and phytol production, and no effect on sterol production was observed. In response to the pravastatin treatment, a significant decrease in sterol and perturbation of SDB production was observed. PMID:16651787

  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. PMID:15384409

  11. [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

  12. 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

  13. 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

  14. Methylerythritol 4-phosphate (MEP) pathway metabolic regulation.

    PubMed

    Banerjee, A; Sharkey, T D

    2014-08-01

    Covering: up to February 2014. The methylerythritol 4-phosphate (MEP) pathway is the recently discovered source of isoprenoid precursors isopentenyl diphosphate (IDP) and dimethylallyl diphosphate (DMADP) in most bacteria, some eukaryotic parasites, and the plastids of plant cells. The precursors lead to the formation of various isoprenoids having diverse roles in different biological processes. Some isoprenoids have important commercial uses. Isoprene, which is made in surprising abundance by some trees, plays a significant role in atmospheric chemistry. The genetic regulation of this pathway has been discussed but information about metabolic regulation is just now becoming available. This review covers metabolic regulation of the MEP pathway starting from the inputs of carbon, ATP, and reducing power. A number of different regulatory mechanisms involving intermediate metabolites and/or enzymes are discussed. Some recent data indicate that methylerythritol cyclodiphosphate (MEcDP), the fifth intermediate of this pathway, is a key metabolite. It has been found to play diverse roles in regulation within the pathway as well as coordinating other biological processes by acting as a stress regulator in bacteria and possibly a retrograde signal from plastids to the nucleus in plants. In this review we focus on the role of the MEP pathway in photosynthetic leaves during isoprene emission and more generally the metabolic regulation of the MEP pathway in both plants and bacteria. PMID:24921065

  15. Probable novel MEP pathway inhibitor and its binding protein, IspG.

    PubMed

    Nakagawa, Kazuya; Takada, Kentaro; Imamura, Nobutaka

    2013-01-01

    A second isoprene unit biosynthetic pathway, via 2-C-methyl-D-erythritol 4-phosphate (MEP), was discovered in the 1990s. We screened and isolated the cyclic dipeptide, maculosin, which is a probable novel MEP pathway inhibitor, from the culture broth of Bacillus subtilis strain KN07. To identify the target enzyme of maculosin, we applied an avidin-biotin complex method using biotinylated maculosin and the lysates of seven Escherichia coli strains, each overexpressing one enzyme of the MEP pathway, and performed quartz crystal microbalance (QCM) experiments using maculosin and each enzyme. The results indicate that IspG, the sixth enzyme on the MEP pathway, was bound to maculosin. PMID:23832336

  16. The plastidial MEP pathway: unified nomenclature and resources.

    PubMed

    Phillips, Michael A; León, Patricia; Boronat, Albert; Rodríguez-Concepción, Manuel

    2008-12-01

    In plants, the plastid-localized 2-C-methyl-d-erythritol 4-phosphate (MEP) pathway provides the precursors for the synthesis of isoprenoid hormones, monoterpenes, carotenoids and the side chain of chlorophylls, tocopherols and prenylquinones. As a result of the fast progress in the elucidation and characterization of the pathway (mainly by genetic approaches in Escherichia coli and Arabidopsis thaliana), different names have been used in the literature to designate the orthologous bacterial and plant genes and the corresponding null and partial loss-of-function mutants. This has led to a confusing variety of naming conventions in this field. Here, we propose a reorganization of the various naming systems with the aim of facilitating the dissemination and sharing of genetic resources and tools central to plant isoprenoid research. PMID:18948055

  17. Engineering the MEP pathway enhanced ajmalicine biosynthesis.

    PubMed

    Chang, Kai; Qiu, Fei; Chen, Min; Zeng, Lingjiang; Liu, Xiaoqiang; Yang, Chunxian; Lan, Xiaozhong; Wang, Qiang; Liao, Zhihua

    2014-01-01

    The 2-C-methyl-D-erythritol-4-phosphate (MEP) pathway genes encoding DXR and MECS from Taxus species and STR from Catharanthus roseus were used to genetically modify the ajmalicine biosynthetic pathway in hairy root cultures of C. roseus. As expected, the STR-overexpressed root cultures showed twofold higher accumulation of ajmalicine than the control. It was important to discover that overexpression of the single DXR or MECS gene from the MEP pathway also remarkably enhanced ajmalicine biosynthesis in transgenic hairy root cultures, and this suggested that engineering the MEP pathway by overexpression of DXR or MECS promoted the metabolic flux into ajmalicine biosynthesis. The transgenic hairy root cultures with co-overexpression of DXR and STR or MECS and STR had higher levels of ajmalicine than those with overexpression of a single gene alone such as DXR, MECS, and STR. It could be concluded that transgenic hairy root cultures harboring both DXR/MECS and STR possessed an increased flux in the terpenoid indole alkaloid biosynthetic pathway that enhanced ajmalicine yield, which was more efficient than cultures harboring only one of the three genes. PMID:24237015

  18. Combination of Entner-Doudoroff pathway with MEP increases isoprene production in engineered Escherichia coli.

    PubMed

    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

  19. 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

  20. 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

  1. 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. PMID:23612965

  2. 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

  3. 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

  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. Structure of the GcpE-HMBPP complex from Thermus thermophilius.

    PubMed

    Rekittke, Ingo; Warkentin, Eberhard; Jomaa, Hassan; Ermler, Ulrich

    2015-03-01

    Isoprenoid biosynthesis in many bacteria, plant chloroplasts and parasitic protozoa but not in humans proceeds via the mevalonate independent 2-C-methyl-D-erythritol-4-phosphate (MEP) pathway. Its penultimate reaction step is catalyzed by (E)-1-hydroxy-2-methyl-but-2-enyl-4-diphosphate (HMBPP) synthase (GcpE/IspG) which transforms 2-C-methyl-D-erythritol-2, 4-cyclo-diphosphate (MEcPP) to HMBPP. In this report we present the structure of GcpE of Thermus thermophiles in complex with its product HMBPP at a resolution of 1.65 Å. The GcpE-HMBPP like the GcpE-MEcPP structure is found in a closed, the ligand-free GcpE structure in an open enzyme state. Imposed by the rigid protein scaffold inside the active site funnel, linear HMBPP and circular MEcPP adopt highly similar conformations. The confined space also determines the conformational freedom of transition state intermediates and the design of anti-infective drugs. The apical Fe of the [4Fe-4S] cluster is coordinated to MEcPP in the GcpE-MEcPP complex and to a hydroxyl/water ligand but not to HMBPP in the GcpE-HMBPP complex. The GcpE-HMBPP structure can be attributed to one step in the currently proposed GcpE reaction cycle. PMID:25660452

  6. 1-Deoxy-D-xylulose 5-phosphate reductoisomerase: an overview.

    PubMed

    Proteau, Philip J

    2004-12-01

    The methylerythritol phosphate pathway to isoprenoids, an alternate biosynthetic route present in many bacteria, algae, plants, and the malarial parasite Plasmodium falciparum, has become an attractive target for the development of new antimalarial and antibacterial compounds. The second enzyme in this pathway, 1-deoxy-D-xylulose 5-phosphate reductoisomerase (DXR; EC 1.1.1.267), has been shown to be the molecular target for fosmidomycin, a promising antimalarial drug. This enzyme converts 1-deoxy-D-xylulose 5-phosphate (DXP) into the branched compound 2-C-methyl-D-erythritol 4-phosphate (MEP). The transformation of DXP into MEP requires an isomerization, followed by a NADPH-dependent reduction. The discovery of DXR, its subsequent characterization, and the identification of inhibitors will be presented. PMID:15530989

  7. Structure of the (E)-4-hydroxy-3-methyl-but-2-enyl-diphosphate reductase from Plasmodium falciparum.

    PubMed

    Rekittke, Ingo; Olkhova, Elena; Wiesner, Jochen; Demmer, Ulrike; Warkentin, Eberhard; Jomaa, Hassan; Ermler, Ulrich

    2013-12-11

    Terpenoid precursor biosynthesis occurs in human and many pathogenic organisms via the mevalonate and 2-C-methyl-d-erythritol-4-phosphate (MEP) pathways, respectively. We determined the X-ray structure of the Fe/S containing (E)-4-hydroxy-3-methyl-but-2-enyl-diphosphate reductase (LytB) of the pathogenic protozoa Plasmodium falciparum which catalyzes the terminal step of the MEP pathway. The cloverleaf fold and the active site of P. falciparum LytB corresponds to those of the Aquifex aeolicus and Escherichia coli enzymes. Its distinct electron donor [2Fe-2S] ferredoxin was modeled to its binding site by docking calculations. The presented structural data provide a platform for a rational search of anti-malarian drugs. PMID:24188825

  8. Mechanism and inhibition of 1-deoxy-D-xylulose-5-phosphate reductoisomerase.

    PubMed

    Murkin, Andrew S; Manning, Kathryn A; Kholodar, Svetlana A

    2014-12-01

    The non-mevalonate or 2-C-methyl-d-erythritol-4-phosphate (MEP) pathway is responsible for generating isoprenoid precursors in plants, protozoa, and bacteria. Because this pathway is absent in humans, its enzymes represent potential targets for the development of herbicides and antibiotics. 1-Deoxy-d-xylulose (DXP) reductoisomerase (DXR) is a particularly attractive target that catalyzes the pathway's first committed step: the sequential isomerization and NADPH-dependent reduction of DXP to MEP. This article provides a comprehensive review of the mechanistic and structural investigations on DXR, including its discovery and validation as a drug target, elucidation of its chemical and kinetic mechanisms, characterization of inhibition by the natural antibiotic fosmidomycin, and identification of structural features that provide the molecular basis for inhibition of and catalysis. PMID:24998420

  9. Pyrethrin biosynthesis and its regulation in Chrysanthemum cinerariaefolium.

    PubMed

    Matsuda, Kazuhiko

    2012-01-01

    Pyrethrins are a natural insecticide biosynthesized by the plant pyrethrum [Chrysanthemum cinerariaefolium (Current species name: Tanacetum cinerariifolium)] of the family Asteraceae. Although pyrethrins have been used to control household pests for the past century, little is known about the mechanism of biosynthesis, contrasting with intensive research on their synthetic analogs, pyrethroids. The author studied pyrethrin biosynthesis in young seedlings of C. cinerariaefolium. The results of experiments using (13)C-labeled glucose as the biosynthesis precursor indicated that the acid and alcohol moieties are biosynthesized via the 2-C-methyl-D: -erythritol 4-phosphate (MEP) and oxylipin pathways, respectively. Further study on the effects of wound-induced signals in leaves showed that biosynthesis is enhanced in response to both volatile and nonvolatile signals. PMID:22006239

  10. 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. PMID:26411373

  11. Metabolic Flux Analysis of Plastidic Isoprenoid Biosynthesis in Poplar Leaves Emitting and Nonemitting Isoprene1[W

    PubMed Central

    Ghirardo, Andrea; Wright, Louwrance Peter; Bi, Zhen; Rosenkranz, Maaria; Pulido, Pablo; Rodríguez-Concepción, Manuel; Niinemets, Ülo; Brüggemann, Nicolas; Gershenzon, Jonathan; Schnitzler, Jörg-Peter

    2014-01-01

    The plastidic 2-C-methyl-d-erythritol-4-phosphate (MEP) pathway is one of the most important pathways in plants and produces a large variety of essential isoprenoids. Its regulation, however, is still not well understood. Using the stable isotope 13C-labeling technique, we analyzed the carbon fluxes through the MEP pathway and into the major plastidic isoprenoid products in isoprene-emitting and transgenic isoprene-nonemitting (NE) gray poplar (Populus × canescens). We assessed the dependence on temperature, light intensity, and atmospheric [CO2]. Isoprene biosynthesis was by far (99%) the main carbon sink of MEP pathway intermediates in mature gray poplar leaves, and its production required severalfold higher carbon fluxes compared with NE leaves with almost zero isoprene emission. To compensate for the much lower demand for carbon, NE leaves drastically reduced the overall carbon flux within the MEP pathway. Feedback inhibition of 1-deoxy-d-xylulose-5-phosphate synthase activity by accumulated plastidic dimethylallyl diphosphate almost completely explained this reduction in carbon flux. Our data demonstrate that short-term biochemical feedback regulation of 1-deoxy-d-xylulose-5-phosphate synthase activity by plastidic dimethylallyl diphosphate is an important regulatory mechanism of the MEP pathway. Despite being relieved from the large carbon demand of isoprene biosynthesis, NE plants redirected only approximately 0.5% of this saved carbon toward essential nonvolatile isoprenoids, i.e. β-carotene and lutein, most probably to compensate for the absence of isoprene and its antioxidant properties. PMID:24590857

  12. Structure of (E)-4-hydroxy-3-methyl-but-2-enyl diphosphate reductase, the terminal enzyme of the non-mevalonate pathway.

    PubMed

    Rekittke, Ingo; Wiesner, Jochen; Röhrich, Rene; Demmer, Ulrike; Warkentin, Eberhard; Xu, Weiya; Troschke, Kathrin; Hintz, Martin; No, Joo Hwan; Duin, Evert C; Oldfield, Eric; Jomaa, Hassan; Ermler, Ulrich

    2008-12-24

    Molecular evolution has evolved two metabolic routes for isoprenoid biosynthesis: the mevalonate and the 2-C-methyl-D-erythritol-4-phosphate (MEP) pathway. The MEP pathway is used by most pathogenic bacteria and some parasitic protozoa (including the malaria parasite, Plasmodium falciparum) as well as by plants, but is not present in animals. The terminal reaction of the MEP pathway is catalyzed by (E)-4-hydroxy-3-methyl-but-2-enyl diphosphate (HMBPP) reductase (LytB), an enzyme that converts HMBPP into isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP). Here, we present the structure of Aquifex aeolicus LytB, at 1.65 A resolution. The protein adopts a cloverleaf or trefoil-like structure with each monomer in the dimer containing three alpha/beta domains surrounding a central [Fe3S4] cluster ligated to Cys13, Cys96, and Cys193. Two highly conserved His (His 42 and His 124) and a totally conserved Glu (Glu126) are located in the same central site and are proposed to be involved in ligand binding and catalysis. Substrate access is proposed to occur from the front-side face of the protein, with the HMBPP diphosphate binding to the two His and the 4OH of HMBPP binding to the fourth iron thought to be present in activated clusters, while Glu126 provides the protons required for IPP/DMAPP formation. PMID:19035630

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

    PubMed Central

    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.

    2011-01-01

    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

  14. 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

  15. 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

  16. 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

  17. Structural studies on Mycobacterium tuberculosis DXR in complex with the antibiotic FR-900098.

    PubMed

    Björkelid, Christofer; Bergfors, Terese; Unge, Torsten; Mowbray, Sherry L; Jones, T Alwyn

    2012-02-01

    A number of pathogens, including the causative agents of tuberculosis and malaria, synthesize the essential isoprenoid precursor isopentenyl diphosphate via the 2-C-methyl-D-erythritol 4-phosphate (MEP) pathway rather than the classical mevalonate pathway that is found in humans. As part of a structure-based drug-discovery program against tuberculosis, DXR, the enzyme that carries out the second step in the MEP pathway, has been investigated. This enzyme is the target for the antibiotic fosmidomycin and its active acetyl derivative FR-900098. The structure of DXR from Mycobacterium tuberculosis in complex with FR-900098, manganese and the NADPH cofactor has been solved and refined. This is a new crystal form that diffracts to a higher resolution than any other DXR complex reported to date. Comparisons with other ternary complexes show that the conformation is that of the enzyme in an active state: the active-site flap is well defined and the cofactor-binding domain has a conformation that brings the NADPH into the active site in a manner suitable for catalysis. The substrate-binding site is highly conserved in a number of pathogens that use this pathway, so any new inhibitor that is designed for the M. tuberculosis enzyme is likely to exhibit broad-spectrum activity. PMID:22281742

  18. 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. PMID:26753746

  19. 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

  20. Functional expression of an orchid fragrance gene in Lactococcus lactis.

    PubMed

    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

  1. Jasmonate-induced biosynthesis of andrographolide in Andrographis paniculata.

    PubMed

    Sharma, Shiv Narayan; Jha, Zenu; Sinha, Rakesh Kumar; Geda, Arvind Kumar

    2015-02-01

    Andrographolide is a prominent secondary metabolite found in Andrographis paniculata that exhibits enormous pharmacological effects. In spite of immense value, the normal biosynthesis of andrographolide results in low amount of the metabolite. To induce the biosynthesis of andrographolide, we attempted elicitor-induced activation of andrographolide biosynthesis in cell cultures of A. paniculata. This was carried out by using methyl jasmonate (MeJA) as an elicitor. Among the various concentrations of MeJA tested at different time periods, 5 µM MeJA yielded 5.25 times more andrographolide content after 24 h of treatment. The accumulation of andrographolide was correlated with the expression level of known regulatory genes (hmgs, hmgr, dxs, dxr, isph and ggps) of mevalonic acid (MVA) and 2-C-methyl-d-erythritol-4-phosphate (MEP) pathways. These results established the involvement of MeJA in andrographolide biosynthesis by inducing the transcription of its biosynthetic pathways genes. The coordination of isph, ggps and hmgs expression highly influenced the andrographolide biosynthesis. PMID:25104168

  2. NMR studies of DOXP reductoisomerase and its inhibitor complex.

    PubMed

    Englert, Nadine E; Richter, Christian; Wiesner, Jochen; Hintz, Martin; Jomaa, Hassan; Schwalbe, Harald

    2011-02-11

    1-Deoxy-D-xylulose 5-phosphate (DOXP) reductoisomerase (EC1.1.1.267) catalyses the second step of the 2-C-methyl-D-erythritol 4-phosphate (MEP) pathway of isoprenoid biosynthesis. The enzyme is used by most bacteria, apicomplexan parasites and the plastids of plants, but not by humans, and therefore represents an attractive target for antibacterial, antiparasitic and herbicidal compounds. Fosmidomycin, an inhibitor of DXR, has been found to be active against bacterial infections and malaria in early clinical studies. Here, we report sample optimisation, partial backbone assignment and secondary-structure prediction of E. coli DXR by heteronuclear NMR analysis for further NMR-aided drug discovery. Perdeuterated (15)N,(13)C-labelled samples were prepared under oxygen exclusion in the presence of Mg(2+), NADPH and the inhibitor FR-900098, a close derivative of fosmidomycin. (1)H and (15)N backbone assignment was achieved for 44 % of the primary structure, and (13)C backbone assignment was achieved for 50 % of the primary structure. Comparison with previously solved crystal structures revealed that the assigned fragments were located mainly in helical regions on the solvent-exposed surface of the enzyme. Torsion angle likelihood obtained from shift and sequence similarity (TALOS) was used for secondary structure prediction, resulting in agreement with eight available crystal structures; deviations could be observed for the catalytic loop region. PMID:21290548

  3. Design of Potential Bisubstrate Inhibitors against Mycobacterium tuberculosis (Mtb) 1-Deoxy-D-Xylulose 5-Phosphate Reductoisomerase (Dxr)-Evidence of a Novel Binding Mode.

    PubMed

    San Jose, Géraldine; Jackson, Emily R; Uh, Eugene; Johny, Chinchu; Haymond, Amanda; Lundberg, Lindsay; Pinkham, Chelsea; Kehn-Hall, Kylene; Boshoff, Helena I; Couch, Robin D; Dowd, Cynthia S

    2013-07-01

    In most bacteria, the nonmevalonate pathway is used to synthesize isoprene units. Dxr, the second step in the pathway, catalyzes the NADPH-dependent reductive isomerization of 1-deoxy-D-xylulose-5-phosphate (DXP) to 2-C-methyl-D-erythritol-4-phosphate (MEP). Dxr is inhibited by natural products fosmidomycin and FR900098, which bind in the DXP binding site. These compounds, while potent inhibitors of Dxr, lack whole cell activity against Mycobacterium tuberculosis (Mtb) due to their polarity. Our goal was to use the Mtb Dxr-fosmidomycin co-crystal structure to design bisubstrate ligands to bind to both the DXP and NADPH sites. Such compounds would be expected to demonstrate improved whole cell activity due to increased lipophilicity. Two series of compounds were designed and synthesized. Compounds from both series inhibited Mtb Dxr. The most potent compound (8) has an IC50 of 17.8 µM. Analysis shows 8 binds to Mtb Dxr via a novel, non-bisubstrate mechanism. Further, the diethyl ester of 8 inhibits Mtb growth making this class of compounds interesting lead molecules in the search for new antitubercular agents. PMID:23914289

  4. MEDICAL EXPENDITURE PANEL SURVEY (MEPS)

    EPA Science Inventory

    The Medical Expenditure Panel Survey, or MEPS as it is commonly called, is the third (and most recent) in a series of national probability surveys conducted by AHRQ on the financing and utilization of medical care in the United States.

  5. Engineering a Platform for Photosynthetic Pigment, Hormone and Cembrane-Related Diterpenoid Production in Nicotiana tabacum.

    PubMed

    Zhang, Hongying; Niu, Dexin; Wang, Jing; Zhang, Songtao; Yang, Yongxia; Jia, Hongfang; Cui, Hong

    2015-11-01

    Plants synthesize a large number of isoprenoids that are of nutritional, medicinal and industrial importance. 1-Deoxy-d-xylulose 5-phosphate reductoisomerase (DXR) catalyzes the first committed step for plastidial isoprenoid biosynthesis. Here, we identified two DXR isogenes, designated NtDXR1 and NtDXR2, from tetraploid common tobacco (Nicotiana tabacum L.). Southern blotting and genotyping analysis revealed that two NtDXR genes existed in the tetraploid tobacco genome; NtDXR1 and NtDXR2 were separately derived from N. tomentosiformis and N. sylvestris. Both NtDXRs were localized in chloroplasts. Expression patterns indicated that NtDXR1 and NtDXR2 had similar expression profiles. NtDXR genes were highly expressed in leaves with or without trichomes; expression was relatively reduced in flowers and stems, weak in leaf trichomes and marginal in roots and seeds. Overexpressing NtDXR1 under control of the 35S promoter resulted in longer primary roots and enhancement of various photosynthetic pigments and hormones in leaves. In contrast, there were no significant changes in cembrane-related diterpenoids synthesized in glandular trichomes. To elucidate further the function of DXR in the biosynthesis of diterpenoids, overexpression vectors for NtDXR1 under the control of a trichome-specific CYP promoter were transferred to tobacco plants. CYP:NtDXR1 tobacco exhibited larger glandular cells and increased cembrane-related diterpenoids in leaf glandular trichomes. Moreover, transcripts of eight MEP (2-C-methyl-d-erythritol 4-phosphate) pathway genes were significantly up-regulated in NtDXR1-overexpressing tobacco plants, indicating that overexpression of NtDXR could boost the expression of downstream genes in the MEP pathway. Our results suggested that overexpression of NtDXR1 could increase the levels of photosynthetic pigments, leaf surface exudates and hormones though the MEP pathway. PMID:26363359

  6. 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. PMID:25926480

  7. 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

  8. 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

  9. 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

  10. [Engineering MEP pathway in Escherichia coli for amorphadiene production and optimizing the bioprocess through glucose feeding control].

    PubMed

    Wang, Jianfeng; Xiong, Zhiqiang; Zhang, Siliang; Wang, Yong

    2014-01-01

    The pathway of 2-methyl-D-erythritol-4-phosphate (MEP) is the exclusive isoprenoid precursor biosynthetic pathway in Escherichia coli, with a higher theoretical yield than mevalonate (MVA) pathway. However, due to lack of information about the regulation of MEP pathway, only engineering MEP pathway in E. coli achieved limited improvement of heterologous isoprenoid production. We used exogenous MEP pathway genes to improve MEP pathway in E. coli and optimized the glucose feeding to release the potential of MEP pathway. The results demonstrate that co-expression of dxs2 from Streptomyces avermitilis and idi from Bacillus subtilis can increase amorphadiene production with 12.2-fold compared with the wild-type strain in shake flask fermentation. Then we established a high-cell density fermentation process for the engineered strain, and found that the phase from 24 to 72 h is important for product biosynthesis. The optimization of glucose feeding rate during 24 to 72 h significantly improved product accumulation, which was improved from 2.5 to 4.85 g/L, within the same process time. Considering the attenuation of strain metabolism after 72 h, this study further modulated the glucose feeding rate during exponential phase to control strain growth and the amorphadiene yield eventually reached to 6.1 g/L. These results provided useful information to develop engineered E. coli for isoprenoid production through MEP pathway engineering. PMID:24818480

  11. 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. PMID:25048025

  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. De Novo Assembly and Transcriptome Analysis of the Rubber Tree (Hevea brasiliensis) and SNP Markers Development for Rubber Biosynthesis Pathways

    PubMed Central

    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. PMID:25048025

  14. 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. PMID:24507902

  15. 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. PMID:23154062

  16. 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. PMID:25736930

  17. Impact of Ammonium Permeases MepA, MepB, and MepC on Nitrogen-Regulated Secondary Metabolism in Fusarium fujikuroi▿

    PubMed Central

    Teichert, Sabine; Rutherford, Julian C.; Wottawa, Marieke; Heitman, Joseph; Tudzynski, Bettina

    2008-01-01

    In Fusarium fujikuroi, the production of gibberellins and bikaverin is repressed by nitrogen sources such as glutamine or ammonium. Sensing and uptake of ammonium by specific permeases play key roles in nitrogen metabolism. Here, we describe the cloning of three ammonium permease genes, mepA, mepB, and mepC, and their participation in ammonium uptake and signal transduction in F. fujikuroi. The expression of all three genes is strictly regulated by the nitrogen regulator AreA. Severe growth defects of ΔmepB mutants on low-ammonium medium and methylamine uptake studies suggest that MepB functions as the main ammonium permease in F. fujikuroi. In ΔmepB mutants, nitrogen-regulated genes such as the gibberellin and bikaverin biosynthetic genes are derepressed in spite of high extracellular ammonium concentrations. mepA mepB and mepC mepB double mutants show a similar phenotype as ΔmepB mutants. All three F. fujikuroi mep genes fully complemented the Saccharomyces cerevisiae mep1 mep2 mep3 triple mutant to restore growth on low-ammonium medium, whereas only MepA and MepC restored pseudohyphal growth in the mep2/mep2 mutant. Overexpression of mepC in the ΔmepB mutants partially suppressed the growth defect but did not prevent derepression of AreA-regulated genes. These studies provide evidence that MepB functions as a regulatory element in a nitrogen sensing system in F. fujikuroi yet does not provide the sensor activity of Mep2 in yeast, indicating differences in the mechanisms by which nitrogen is sensed in S. cerevisiae and F. fujikuroi. PMID:18083831

  18. Biosynthesis of Camptothecin. In Silico and in Vivo Tracer Study from [1-13C]Glucose1

    PubMed Central

    Yamazaki, Yasuyo; Kitajima, Mariko; Arita, Masanori; Takayama, Hiromitsu; Sudo, Hiroshi; Yamazaki, Mami; Aimi, Norio; Saito, Kazuki

    2004-01-01

    Camptothecin derivatives are clinically used antitumor alkaloids that belong to monoterpenoid indole alkaloids. In this study, we investigated the biosynthetic pathway of camptothecin from [1-13C]glucose (Glc) by in silico and in vivo studies. The in silico study measured the incorporation of Glc into alkaloids using the Atomic Reconstruction of Metabolism software and predicted the labeling patterns of successive metabolites from [1-13C]Glc. The in vivo study followed incorporation of [1-13C]Glc into camptothecin with hairy roots of Ophiorrhiza pumila by 13C nuclear magnetic resonance spectroscopy. The 13C-labeling pattern of camptothecin isolated from the hairy roots clearly showed that the monoterpene-secologanin moiety was synthesized via the 2C-methyl-d-erythritol 4-phosphate pathway, not via the mevalonate pathway. This conclusion was supported by differential inhibition of camptothecin accumulation by the pathway-specific inhibitors (fosmidomycin and lovastatin). The quinoline moiety from tryptophan was also labeled as predicted by the Atomic Reconstruction of Metabolism program via the shikimate pathway. These results indicate that camptothecin is formed by the combination of the 2C-methyl-d-erythritol 4-phosphate pathway and the shikimate pathway. This study provides the innovative example for how a computer-aided comprehensive metabolic analysis will refine the experimental design to obtain more precise biological information. PMID:14657405

  19. Mutations within the mepA operator affect binding of the MepR regulatory protein and its induction by MepA substrates in Staphylococcus aureus.

    PubMed

    Schindler, Bryan D; Seo, Susan M; Birukou, Ivan; Brennan, Richard G; Kaatz, Glenn W

    2015-03-01

    The expression of mepA, encoding the Staphylococcus aureus MepA multidrug efflux protein, is repressed by the MarR homologue MepR. Repression occurs through binding of two MepR dimers to an operator with two homologous and closely approximated pseudopalindromic binding sites (site 1 [S1] and site 2 [S2]). MepR binding is impeded in the presence of pentamidine, a MepA substrate. The effects of various mepA operator mutations on MepR binding were determined using electrophoretic mobility shift assays and isothermal titration calorimetry, and an in vivo confirmation of the effects observed was established for a fully palindromic operator mutant. Altering the S1-S2 spacing by 1 to 4 bp severely impaired S2 binding, likely due to a physical collision between adjacent MepR dimers. Extension of the spacing to 9 bp eliminated the S1 binding-mediated DNA allostery required for efficient S2 binding, consistent with positive cooperative binding of MepR dimers. Binding of a single dimer to S1 was maintained when S2 was disrupted, whereas disruption of S1 eliminated any significant binding to S2, also consistent with positive cooperativity. Palindromization of binding sites, especially S2, enhanced MepR affinity for the mepA operator and reduced MepA substrate-mediated MepR induction. As a result, the on-off equilibrium between MepR and its binding sites was shifted toward the on state, resulting in less free MepR being available for interaction with inducing ligand. The selective pressure(s) under which mepA expression is advantageous likely contributed to the accumulation of mutations in the mepA operator, resulting in the current sequence from which MepR is readily induced by MepA substrates. PMID:25583977

  20. 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

  1. Lycopene production in recombinant strains of Escherichia coli is improved by knockout of the central carbon metabolism gene coding for glucose-6-phosphate dehydrogenase.

    PubMed

    Zhou, Yan; Nambou, Komi; Wei, Liujing; Cao, Jingjing; Imanaka, Tadayuki; Hua, Qiang

    2013-12-01

    Genetic manipulation was undertaken in order to understand the mechanism involved in the heterologous synthesis of lycopene in Escherichia coli. Knockout of the central carbon metabolic gene zwf (glucose-6-phosphate dehydrogenase) resulted in the enhancement of lycopene production (above 130 % relative to control). The amplification and overexpression of rate-limiting steps encoded by idi (isopentenyl diphosphate isomerase), dxs (1-deoxyxylulose-5-phosphate synthase) and ispDF (4-diphosphocytidyl-2C-methyl-D-erythritol synthase and 2C-methyl-D-erythritol 2,4-cyclodiphosphate synthase) genes improved lycopene synthesis from 0.89 to 5.39 mg g(-1) DCW. The combination of central metabolic genes knockout with the amplification of MEP pathway genes yielded best amounts of lycopene (6.85-7.55 mg g(-1) DCW). Transcript profiling revealed that idi and dxs were up-regulated in the zwf knock-out strain, providing a plausible explanation for the increase in lycopene yield observed in this strain. An increase in precursor availability might also have contributed to the improved lycopene production. PMID:24062132

  2. Structure of the GcpE (IspG)-MEcPP complex from Thermus thermophilus.

    PubMed

    Rekittke, Ingo; Jomaa, Hassan; Ermler, Ulrich

    2012-09-21

    Isoprenoid precursor biosynthesis occurs through the mevalonate or the methylerythritol phosphate (MEP) pathway, used i.e., by humans and by many human pathogens, respectively. In the MEP pathway, 2-C-methyl-D-erythritol-2,4-cyclo-diphosphate (MEcPP) is converted to (E)-1-hydroxy-2-methyl-but-2-enyl-4-diphosphate (HMBPP) by the iron-sulfur cluster enzyme HMBPP synthase (GcpE). The presented X-ray structure of the GcpE-MEcPP complex from Thermus thermophilus at 1.55Å resolution provides valuable information about the catalytic mechanism and for rational inhibitor design. MEcPP binding inside the TIM-barrel funnel induces a 60° rotation of the [4Fe-4S] cluster containing domain onto the TIM-barrel entrance. The apical iron of the [4Fe-4S] cluster ligates with the C3 oxygen atom of MEcPP. PMID:22967895

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

    PubMed Central

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

    2015-01-01

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

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

    PubMed Central

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

    2016-01-01

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

  5. Phosphatidylinositol 4-phosphate and phosphatidylinositol 3-phosphate regulate phagolysosome biogenesis

    PubMed Central

    Jeschke, Andreas; Zehethofer, Nicole; Lindner, Buko; Krupp, Jessica; Schwudke, Dominik; Haneburger, Ina; Jovic, Marko; Backer, Jonathan M.; Balla, Tamas; Hilbi, Hubert; Haas, Albert

    2015-01-01

    Professional phagocytic cells ingest microbial intruders by engulfing them into phagosomes, which subsequently mature into microbicidal phagolysosomes. Phagosome maturation requires sequential fusion of the phagosome with early endosomes, late endosomes, and lysosomes. Although various phosphoinositides (PIPs) have been detected on phagosomes, it remained unclear which PIPs actually govern phagosome maturation. Here, we analyzed the involvement of PIPs in fusion of phagosomes with various endocytic compartments and identified phosphatidylinositol 4-phosphate [PI(4)P], phosphatidylinositol 3-phosphate [PI(3)P], and the lipid kinases that generate these PIPs, as mediators of phagosome–lysosome fusion. Phagosome–early endosome fusion required PI(3)P, yet did not depend on PI(4)P. Thus, PI(3)P regulates phagosome maturation at early and late stages, whereas PI(4)P is selectively required late in the pathway. PMID:25825728

  6. 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. PMID:18785997

  7. Structural and biochemical characterization of MepR, a multidrug binding transcription regulator of the Staphylococcus aureus multidrug efflux pump MepA

    PubMed Central

    Kumaraswami, Muthiah; Schuman, Jason T.; Seo, Susan M.; Kaatz, Glenn W.; Brennan, Richard G.

    2009-01-01

    MepR is a multidrug binding transcription regulator that represses expression of the Staphylococcus aureus multidrug efflux pump gene, mepA, as well as its own gene. MepR is induced by multiple cationic toxins, which are also substrates of MepA. In order to understand the gene regulatory and drug-binding mechanisms of MepR, we carried out biochemical, in vivo and structural studies. The 2.40 Å resolution structure of drug-free MepR reveals the most open MarR family protein conformation to date, which will require a huge conformational change to bind cognate DNA. DNA-binding data show that MepR uses a dual regulatory binding mode as the repressor binds the mepA operator as a dimer of dimers, but binds the mepR operator as a single dimer. Alignment of the six half sites reveals the consensus MepR binding site, 5'-GTTAGAT-3'. ‘Drug’ binding studies show that MepR binds to ethidium and DAPI with comparable affinities (Kd = 2.6 and 4.5 μM, respectively), but with significantly lower affinity to the larger rhodamine 6G (Kd = 62.6 μM). Mapping clinically relevant or in vitro selected MepR mutants onto the MepR structure suggests that their defective repressor phenotypes are due to structural and allosteric defects. PMID:19129225

  8. Immunochemical characterization of phosphatidylinositol 4-phosphate kinase from rat brain.

    PubMed Central

    van Dongen, C J; Kok, J W; Schrama, L H; Oestreicher, A B; Gispen, W H

    1986-01-01

    Affinity-purified antibodies were used to identify a protein of molecular mass 45 kDa (45 kDa protein) in rat brain cytosol as phosphatidylinositol 4-phosphate (PtdIns4P) kinase. Antibodies were raised in rabbits by immunization with the purified 45 kDa protein. Anti-(45 kDa protein) immunoglobulins were isolated by affinity chromatography of the antiserum on a solid immunosorbent, which was prepared by coupling a soluble rat brain fraction, the DEAE-cellulose pool containing 10-15% 45 kDa protein, to CNBr-activated Sepharose 4B. The purified IgGs were specific for the 45 kDa protein as judged by immunoblot and by immunoprecipitation. The purified anti-(45 kDa protein) IgGs inhibited the enzyme activity of partially purified PtdIns4P kinase, whereas preimmune IgGs were ineffective. Immunoprecipitation of the 45 kDa protein from the partially purified enzyme preparation with the purified IgGs resulted in a concomitant decrease in the amount of 45 kDa protein and in PtdIns4P kinase activity. The amount of 45 kDa protein remaining in the supernatant and the activity of PtdIns4P kinase correlated with a coefficient of r = 0.87. The evidence presented lends further support for the notion that the catalytic activity of PtdIns4P kinase in rat brain cytosol resides in a 45 kDa protein. Images Fig. 1. Fig. 2. PMID:3010943

  9. A chemical rescue screen identifies a Plasmodium falciparum apicoplast inhibitor targeting MEP isoprenoid precursor biosynthesis.

    PubMed

    Wu, Wesley; Herrera, Zachary; Ebert, Danny; Baska, Katie; Cho, Seok H; DeRisi, Joseph L; Yeh, Ellen

    2015-01-01

    The apicoplast is an essential plastid organelle found in Plasmodium parasites which contains several clinically validated antimalarial-drug targets. A chemical rescue screen identified MMV-08138 from the "Malaria Box" library of growth-inhibitory antimalarial compounds as having specific activity against the apicoplast. MMV-08138 inhibition of blood-stage Plasmodium falciparum growth is stereospecific and potent, with the most active diastereomer demonstrating a 50% effective concentration (EC50) of 110 nM. Whole-genome sequencing of 3 drug-resistant parasite populations from two independent selections revealed E688Q and L244I mutations in P. falciparum IspD, an enzyme in the MEP (methyl-d-erythritol-4-phosphate) isoprenoid precursor biosynthesis pathway in the apicoplast. The active diastereomer of MMV-08138 directly inhibited PfIspD activity in vitro with a 50% inhibitory concentration (IC50) of 7.0 nM. MMV-08138 is the first PfIspD inhibitor to be identified and, together with heterologously expressed PfIspD, provides the foundation for further development of this promising antimalarial drug candidate lead. Furthermore, this report validates the use of the apicoplast chemical rescue screen coupled with target elucidation as a discovery tool to identify specific apicoplast-targeting compounds with new mechanisms of action. PMID:25367906

  10. Structural basis for Mep2 ammonium transceptor activation by phosphorylation

    PubMed Central

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

    2016-01-01

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

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

    PubMed

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

    2016-01-01

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

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

    PubMed Central

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

    2014-01-01

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

  13. Structural mechanism of transcription regulation of the Staphylococcus aureus multidrug efflux operon mepRA by the MarR family repressor MepR.

    PubMed

    Birukou, Ivan; Seo, Susan M; Schindler, Bryan D; Kaatz, Glenn W; Brennan, Richard G

    2014-02-01

    The multidrug efflux pump MepA is a major contributor to multidrug resistance in Staphylococcus aureus. MepR, a member of the multiple antibiotic resistance regulator (MarR) family, represses mepA and its own gene. Here, we report the structure of a MepR-mepR operator complex. Structural comparison of DNA-bound MepR with 'induced' apoMepR reveals the large conformational changes needed to allow the DNA-binding winged helix-turn-helix motifs to interact with the consecutive major and minor grooves of the GTTAG signature sequence. Intriguingly, MepR makes no hydrogen bonds to major groove nucleobases. Rather, recognition-helix residues Thr60, Gly61, Pro62 and Thr63 make sequence-specifying van der Waals contacts with the TTAG bases. Removing these contacts dramatically affects MepR-DNA binding activity. The wings insert into the flanking minor grooves, whereby residue Arg87, buttressed by Asp85, interacts with the O2 of T4 and O4' ribosyl oxygens of A23 and T4. Mutating Asp85 and Arg87, both conserved throughout the MarR family, markedly affects MepR repressor activity. The His14':Arg59 and Arg10':His35:Phe108 interaction networks stabilize the DNA-binding conformation of MepR thereby contributing significantly to its high affinity binding. A structure-guided model of the MepR-mepA operator complex suggests that MepR dimers do not interact directly and cooperative binding is likely achieved by DNA-mediated allosteric effects. PMID:24293644

  14. Expression of mep50 in adult and embryos of medaka fish (Oryzias latipes).

    PubMed

    Cheng, Nana; Guo, Maomao; Chang, Pei; Zhang, Xueyan; Zhang, Runshuai; Qi, Chao; Zhong, Xueping; Zhou, Qingchun; Zhao, Haobin

    2016-06-01

    Protein arginine methylation is important for gene regulation and biological processes. Methylosome protein 50 (Mep50) is identified as a partner of protein arginine methyltransferase 5 (Prmt5), a major enzyme capable of symmetric dimethylation, in mammals and Xenopus. The isolation and characterization of medaka mep50 were reported in this paper. Medaka Mep50 is a homolog of human MEP50 with six WD40 domains. Medaka mep50 was ubiquitously expressed in the adult tissues and had maternal origin with continuous and dynamical expression during embryonic development detected by RT-PCR and in situ hybridization. A strong interaction of medaka Mep50 and Prmt5 was shown by yeast two hybridization. The expression pattern of mep50 is similar to that of prmt5 in medaka. The results suggested that medaka Mep50 could be a partner of Prmt5 and might play major roles in a variety of tissues in medaka. PMID:26749004

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

    PubMed

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

    2014-04-15

    The transcript expression of a gene SrUGT85C2 has been documented for direct relation with steviol glycoside content in Stevia plant. Steviol glycoside and gibberellin biosynthetic routes are divergent branches of methyl erythritol-4 phosphate (MEP) pathway. So, SrUGT85C2 might be an influencing gibberellin content. Hence in the present study, transgenic Arabidopsis thaliana overexpressing SrUGT85C2 cDNA from Stevia rebaudiana was developed to check its effect on gibberellin accumulation and related plant growth parameters. The developed transgenics showed a noteworthy decrease of 78-83% in GA3 content. Moreover, the transgenics showed a gibberellin deficient phenotype comprising stunted hypocotyl length, reduced shoot growth and a significant fall in relative water content. Transgenics also showed 17-37 and 64-76% reduction in chlorophyll a and chlorophyll b contents, respectively. Reduction in photosynthetic pigments could be responsible for the noticed significant decrease in plant biomass. Like steviol glycoside and gibberellin biosynthesis, chlorophyll biosynthesis also occurs from the precursors isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP) of MEP pathway in the plastids. The observed downregulated expression of genes encoding MEP pathway enzymes geranyl geranyl diphosphate synthase (GGDPS), copalyl diphosphate synthase (CDPS), kaurenoic acid oxidase (KAO), chlorophyll synthetase and chlorophyll a oxygenase in transgenics overexpressing SrUGT85C2 might be responsible for the reduction in gibberellins as well as chlorophyll. This study has documented for the first time the regulatory role of SrUGT85C2 in the biosynthesis of steviol glycoside, gibberellins and chlorophyll. PMID:24518812

  16. Mep72, a Metzincin Protease That Is Preferentially Secreted by Biofilms of Pseudomonas aeruginosa

    PubMed Central

    Passmore, Ian J.; Nishikawa, Kahoko; Lilley, Kathryn S.; Bowden, Steven D.; Chung, Jade C. S.

    2014-01-01

    In this work, we compared the profile of proteins secreted by planktonic and biofilm cultures of Pseudomonas aeruginosa using two-dimensional difference gel electrophoresis (2D-DiGE). This revealed that a novel metzincin protease, Mep72, was secreted during biofilm growth. Subsequent Western blotting and reverse transcription-PCR (RT-PCR) analyses demonstrated that Mep72 was expressed only during biofilm growth. Mep72 has a tridomain structure comprised of a metzincin protease-like domain and two tandem carbohydrate-binding domains. Unlike the only other metzincin (alkaline protease; AprA) in P. aeruginosa, Mep72 is secreted through the type II pathway and undergoes processing during export. During this processing, the metzincin domain is liberated from the carbohydrate-binding domains. This processing may be self-catalyzed, since purified Mep72 autodegraded in vitro. This autodegradation was retarded in the presence of alginate (an extracellular matrix component of many P. aeruginosa biofilms). The expression of full-length mep72 in Escherichia coli was toxic. However, this toxicity could be alleviated by coexpression of mep72 with the adjacent gene, bamI. Mep72 and BamI were found to form a protein-protein complex in vitro. 2D-DiGE revealed that the electrophoretic mobility of several discrete protein spots was altered in the biofilm secretome of an mep72 mutant, including type III secretion proteins (PopD, PcrV, and ExoS) and a flagellum-associated protein (FliD). Mep72 was found to bind directly to ExoS and PcrV and to affect the processing of these proteins in the biofilm secretome. We conclude that Mep72 is a secreted biofilm-specific regulator that affects the processing of a very specific subset of virulence factors. PMID:25488299

  17. Mep72, a metzincin protease that is preferentially secreted by biofilms of Pseudomonas aeruginosa.

    PubMed

    Passmore, Ian J; Nishikawa, Kahoko; Lilley, Kathryn S; Bowden, Steven D; Chung, Jade C S; Welch, Martin

    2015-02-15

    In this work, we compared the profile of proteins secreted by planktonic and biofilm cultures of Pseudomonas aeruginosa using two-dimensional difference gel electrophoresis (2D-DiGE). This revealed that a novel metzincin protease, Mep72, was secreted during biofilm growth. Subsequent Western blotting and reverse transcription-PCR (RT-PCR) analyses demonstrated that Mep72 was expressed only during biofilm growth. Mep72 has a tridomain structure comprised of a metzincin protease-like domain and two tandem carbohydrate-binding domains. Unlike the only other metzincin (alkaline protease; AprA) in P. aeruginosa, Mep72 is secreted through the type II pathway and undergoes processing during export. During this processing, the metzincin domain is liberated from the carbohydrate-binding domains. This processing may be self-catalyzed, since purified Mep72 autodegraded in vitro. This autodegradation was retarded in the presence of alginate (an extracellular matrix component of many P. aeruginosa biofilms). The expression of full-length mep72 in Escherichia coli was toxic. However, this toxicity could be alleviated by coexpression of mep72 with the adjacent gene, bamI. Mep72 and BamI were found to form a protein-protein complex in vitro. 2D-DiGE revealed that the electrophoretic mobility of several discrete protein spots was altered in the biofilm secretome of an mep72 mutant, including type III secretion proteins (PopD, PcrV, and ExoS) and a flagellum-associated protein (FliD). Mep72 was found to bind directly to ExoS and PcrV and to affect the processing of these proteins in the biofilm secretome. We conclude that Mep72 is a secreted biofilm-specific regulator that affects the processing of a very specific subset of virulence factors. PMID:25488299

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

    ERIC Educational Resources Information Center

    Lee, Beth S.; And Others

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

  19. 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. PMID:25649633

  20. 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

  1. Induction of a Longer Term Component of Isoprene Release in Darkened Aspen Leaves: Origin and Regulation under Different Environmental Conditions1

    PubMed Central

    Rasulov, Bahtijor; Hüve, Katja; Laisk, Agu; Niinemets, Ülo

    2011-01-01

    After darkening, isoprene emission continues for 20 to 30 min following biphasic kinetics. The initial dark release of isoprene (postillumination emission), for 200 to 300 s, occurs mainly at the expense of its immediate substrate, dimethylallyldiphosphate (DMADP), but the origin and controls of the secondary burst of isoprene release (dark-induced emission) between approximately 300 and 1,500 s, are not entirely understood. We used a fast-response gas-exchange system to characterize the controls of dark-induced isoprene emission by light, temperature, and CO2 and oxygen concentrations preceding leaf darkening and the effects of short light pulses and changing gas concentrations during dark-induced isoprene release in hybrid aspen (Populus tremula × Populus tremuloides). The effect of the 2-C-methyl-d-erythritol-4-phosphate pathway inhibitor fosmidomycin was also investigated. The integral of postillumination isoprene release was considered to constitute the DMADP pool size, while the integral of dark-induced emission was defined as the “dark” pool. Overall, the steady-state emission rate in light and the maximum dark-induced emission rate responded similarly to variations in preceding environmental drivers and atmospheric composition, increasing with increasing light, having maxima at approximately 40°C and close to the CO2 compensation point, and were suppressed by lack of oxygen. The DMADP and dark pool sizes were also similar through their environmental dependencies, except for high temperatures, where the dark pool significantly exceeded the DMADP pool. Isoprene release could be enhanced by short lightflecks early during dark-induced isoprene release, but not at later stages. Fosmidomycin strongly suppressed both the isoprene emission rates in light and in the dark, but the dark pool was only moderately affected. These results demonstrate a strong correspondence between the steady-state isoprene emission in light and the dark-induced emission and suggest

  2. Cytidine derivatives as IspF inhibitors of Burkolderia pseudomallei

    PubMed Central

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

    2013-01-01

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

  3. Cytidine derivatives as IspF inhibitors of Burkolderia pseudomallei.

    PubMed

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

    2013-12-15

    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

  4. Identification and validation of a novel lead compound targeting 4-diphosphocytidyl-2-C-methylerythritol synthetase (IspD) of mycobacteria.

    PubMed

    Gao, Peng; Yang, Yanhui; Xiao, Chunling; Liu, Yishuang; Gan, Maoluo; Guan, Yan; Hao, Xueqin; Meng, Jianzhou; Zhou, Shuang; Chen, Xiaojuan; Cui, Jiafei

    2012-11-01

    Tuberculosis is a serious threat to world-wide public health usually caused in humans by Mycobacterium tuberculosis (M. tuberculosis). It exclusively utilizes the methylerythritol phosphate (MEP) pathway for biosynthesis of isopentenyl diphosphate (IPP) and its isomer dimethylallyl diphosphate (DMAPP), the precursors of all isoprenoid compounds. The 4-diphosphocytidyl-2-C-methyl-D-erythritol synthase (IspD; EC 2.7.7.60) is the key enzyme of the MEP pathway. It is also of interest as a new chemotherapeutic target, as the enzyme is absent in mammals and ispD is an essential gene for growth. A high-throughput screening method was therefore developed to identify compounds that inhibit IspD. This process was applied to identify a lead compound, domiphen bromide (DMB), that may effectively inhibit IspD. The inhibitory action of DMB was confirmed by over-expressing or down-regulating IspD in Mycobacterium smegmatis (M. smegmatis), demonstrating that DMB inhibit M. smegmatis growth additionally through an IspD-independent pathway. This also led to higher levels of growth inhibition when combined with IspD knockdown. This novel IspD inhibitor was also reported to exhibit antimycobacterial activity in vitro, an effect that likely occurs as a result of perturbation of cell wall biosynthesis. PMID:22975264

  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. PMID:23816706

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

    PubMed Central

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

    2014-01-01

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

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

    PubMed

    Guggisberg, Ann M; Park, Jooyoung; Edwards, Rachel L; Kelly, Megan L; Hodge, Dana M; Tolia, Niraj H; Odom, Audrey R

    2014-01-01

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

  8. 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.

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

    PubMed Central

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

    2016-01-01

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

  10. Crystal structure of 3,4-dihydroxy-2-butanone 4-phosphate synthase of riboflavin biosynthesis

    SciTech Connect

    Liao, D.-I.; Calabrese, J.C.; Wawrzak, Z.; Viitanen, P.V.; Jordan, D.B.

    2010-03-05

    3,4-Dihydroxy-2-butanone-4-phosphate synthase catalyzes a commitment step in the biosynthesis of riboflavin. On the enzyme, ribulose 5-phosphate is converted to 3,4-dihydroxy-2-butanone 4-phosphate and formate in steps involving enolization, ketonization, dehydration, skeleton rearrangement, and formate elimination. The enzyme is absent in humans and an attractive target for the discovery of antimicrobials for pathogens incapable of acquiring sufficient riboflavin from their hosts. The homodimer of 23 kDa subunits requires Mg{sup 2+} for activity. The first three-dimensional structure of the enzyme was determined at 1.4 {angstrom} resolution using the multiwavelength anomalous diffraction (MAD) method on Escherichia coli protein crystals containing gold. The protein consists of an {alpha} + {beta} fold having a complex linkage of {beta} strands. Intersubunit contacts are mediated by numerous hydrophobic interactions and three hydrogen bond networks. A proposed active site was identified on the basis of amino acid residues that are conserved among the enzyme from 19 species. There are two well-separated active sites per dimer, each of which comprise residues from both subunits. In addition to three arginines and two threonines, which may be used for recognizing the phosphate group of the substrate, the active site consists of three glutamates, two aspartates, two histidines, and a cysteine which may provide the means for general acid and base catalysis and for coordinating the Mg{sup 2+} cofactor within the active site.

  11. Type I phosphatidylinositol 4-phosphate 5-kinase homo- and heterodimerization determines its membrane localization and activity.

    PubMed

    Lacalle, Rosa Ana; de Karam, Juan C; Martínez-Muñoz, Laura; Artetxe, Ibai; Peregil, Rosa M; Sot, Jesús; Rojas, Ana M; Goñi, Félix M; Mellado, Mario; Mañes, Santos

    2015-06-01

    Type I phosphatidylinositol 4-phosphate 5-kinases (PIP5KIs; α, β, and γ) are a family of isoenzymes that produce phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2] using phosphatidylinositol 4-phosphate as substrate. Their structural homology with the class II lipid kinases [type II phosphatidylinositol 5-phosphate 4-kinase (PIP4KII)] suggests that PIP5KI dimerizes, although this has not been formally demonstrated. Neither the hypothetical structural dimerization determinants nor the functional consequences of dimerization have been studied. Here, we used Förster resonance energy transfer, coprecipitation, and ELISA to show that PIP5KIβ forms homo- and heterodimers with PIP5KIγ_i2 in vitro and in live human cells. Dimerization appears to be a general phenomenon for PIP5KI isoenzymes because PIP5KIβ/PIP5KIα heterodimers were also detected by mass spectrometry. Dimerization was independent of actin cytoskeleton remodeling and was also observed using purified proteins. Mutagenesis studies of PIP5KIβ located the dimerization motif at the N terminus, in a region homologous to that implicated in PIP4KII dimerization. PIP5KIβ mutants whose dimerization was impaired showed a severe decrease in PI(4,5)P2 production and plasma membrane delocalization, although their association to lipid monolayers was unaltered. Our results identify dimerization as an integral feature of PIP5K proteins and a central determinant of their enzyme activity. PMID:25713054

  12. 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

  13. 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

  14. 77 FR 23462 - Proposed Information Collection; Comment Request; Manufacturing Extension Partnership (MEP...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2012-04-19

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

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

    NASA Technical Reports Server (NTRS)

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

    1988-01-01

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

  16. 76 FR 43264 - Proposed Information Collection; Comment Request; NIST MEP Client Impact Survey

    Federal Register 2010, 2011, 2012, 2013, 2014

    2011-07-20

    ... National Institute of Standards and Technology Proposed Information Collection; Comment Request; NIST MEP Client Impact Survey AGENCY: National Institute of Standards and Technology (NIST), Commerce. ACTION...@nist.gov . SUPPLEMENTARY INFORMATION: I. Abstract Sponsored by the National Institute of Standards...

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

    PubMed Central

    Jupp, Tim E.; Cox, Peter M.

    2010-01-01

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

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

    PubMed

    Jupp, Tim E; Cox, Peter M

    2010-05-12

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

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

    PubMed

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

    2016-04-01

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

  20. 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

  1. Phosphatidylinositol-4-phosphate 5-Kinase Isoforms Exhibit Acyl Chain Selectivity for Both Substrate and Lipid Activator*

    PubMed Central

    Shulga, Yulia V.; Anderson, Richard A.; Topham, Matthew K.; Epand, Richard M.

    2012-01-01

    Phosphatidylinositol 4,5-bisphosphate is mostly produced in the cell by phosphatidylinositol-4-phosphate 5-kinases (PIP5K) and has a crucial role in numerous signaling events. Here we demonstrate that in vitro all three isoforms of PIP5K, α, β, and γ, discriminate among substrates with different acyl chains for both the substrates phosphatidylinositol 4-phosphate (PtdIns4P) and phosphatidylinositol (PtdIns) although to different extents, with isoform γ being the most selective. Fully saturated dipalmitoyl-PtdIns4P was a poor substrate for all three isoforms, but both the 1-stearoyl-2-arachidonoyl and the 1-stearoyl-2-oleoyl forms of PtdIns4P were good substrates. Vmax was greater for the 1-stearoyl-2-arachidonoyl form compared with the 1-stearoyl-2-oleoyl form, although for PIP5Kβ the difference was small. For the α and γ isoforms, Km was much lower for 1-stearoyl-2-oleoyl PtdIns4P, making this lipid the better substrate of the two under most conditions. Activation of PIP5K by phosphatidic acid is also acyl chain-dependent. Species of phosphatidic acid with two unsaturated acyl chains are much better activators of PIP5K than those containing one saturated and one unsaturated acyl chain. PtdIns is a poor substrate for PIP5K, but it also shows acyl chain selectivity. Curiously, there is no acyl chain discrimination among species of phosphatidic acid in the activation of the phosphorylation of PtdIns. Together, our findings indicate that PIP5K isoforms α, β, and γ act selectively on substrates and activators with different acyl chains. This could be a tightly regulated mechanism of producing physiologically active unsaturated phosphatidylinositol 4,5-bisphosphate species in the cell. PMID:22942276

  2. 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

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

    PubMed

    Kuzuyama, Tomohisa; Seto, Haruo

    2012-01-01

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

  4. 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

  5. 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

  6. 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

  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. PMID:23800667

  8. 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

  9. Overexpression, crystallization and preliminary X-ray crystallographic analysis of erythronate-4-phosphate dehydrogenase from Pseudomonas aeruginosa

    SciTech Connect

    Ha, Jun Yong; Lee, Ji Hyun; Kim, Kyoung Hoon; Kim, Do Jin; Lee, Hyung Ho; Kim, Hye-Kyung; Yoon, Hye-Jin; Suh, Se Won

    2006-02-01

    Erythronate-4-phosphate dehydrogenase from P. aeruginosa was crystallized and X-ray diffraction data were collected to 2.20 Å resolution. The enzyme erythronate-4-phosphate dehydrogenase catalyses the conversion of erythronate-4-phosphate to 3-hydroxy-4-phospho-hydroxy-α-ketobutyrate. It belongs to the d-isomer-specific 2-hydroxyacid dehydrogenase family. It is essential for de novo biosynthesis of vitamin B{sub 6} (pyridoxine). Erythronate-4-phosphate dehydrogenase from Pseudomonas aeruginosa, a homodimeric enzyme consisting of two identical 380-residue subunits, has been overexpressed in Escherichia coli with a C-terminal purification tag and crystallized at 297 K using 0.7 M ammonium dihydrogen phosphate, 0.4 M ammonium tartrate, 0.1 M sodium citrate pH 5.6 and 10 mM cupric chloride. X-ray diffraction data were collected to 2.20 Å from a crystal grown in the presence of NADH. The crystals belong to the orthorhombic space group P2{sub 1}2{sub 1}2{sub 1}, with unit-cell parameters a = 84.77, b = 101.28, c = 142.58 Å. A dimeric molecule is present in the asymmetric unit, giving a crystal volume per protein weight (V{sub M}) of 3.64 Å{sup 3} Da{sup −1} and a solvent content of 66%.

  10. Phosphatidylinositol 4-Phosphate Negatively Regulates Chloroplast Division in Arabidopsis[OPEN

    PubMed Central

    Okazaki, Kumiko; Miyagishima, Shin-ya; Wada, Hajime

    2015-01-01

    Chloroplast division is performed by the constriction of envelope membranes at the division site. Although constriction of a ring-like protein complex has been shown to be involved in chloroplast division, it remains unknown how membrane lipids participate in the process. Here, we show that phosphoinositides with unknown function in envelope membranes are involved in the regulation of chloroplast division in Arabidopsis thaliana. PLASTID DIVISION1 (PDV1) and PDV2 proteins interacted specifically with phosphatidylinositol 4-phosphate (PI4P). Inhibition of phosphatidylinositol 4-kinase (PI4K) decreased the level of PI4P in chloroplasts and accelerated chloroplast division. Knockout of PI4Kβ2 expression or downregulation of PI4Kα1 expression resulted in decreased levels of PI4P in chloroplasts and increased chloroplast numbers. PI4Kα1 is the main contributor to PI4P synthesis in chloroplasts, and the effect of PI4K inhibition was largely abolished in the pdv1 mutant. Overexpression of DYNAMIN-RELATED PROTEIN5B (DRP5B), another component of the chloroplast division machinery, which is recruited to chloroplasts by PDV1 and PDV2, enhanced the effect of PI4K inhibition, whereas overexpression of PDV1 and PDV2 had additive effects. The amount of DRP5B that associated with chloroplasts increased upon PI4K inhibition. These findings suggest that PI4P is a regulator of chloroplast division in a PDV1- and DRP5B-dependent manner. PMID:25736058

  11. 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. PMID:26773155

  12. Spatiotemporal control of phosphatidylinositol 4-phosphate by Sac2 regulates endocytic recycling

    PubMed Central

    Hsu, FoSheng; Hu, Fenghua

    2015-01-01

    It is well established that the spatial- and temporal-restricted generation and turnover of phosphoinositides (PIs) by a cascade of PI-metabolizing enzymes is a key regulatory mechanism in the endocytic pathway. Here, we demonstrate that the Sac1 domain–containing protein Sac2 is a PI 4-phosphatase that specifically hydrolyzes phosphatidylinositol 4-phosphate in vitro. We further show that Sac2 colocalizes with early endosomal markers and is recruited to transferrin (Tfn)-containing vesicles during endocytic recycling. Exogenous expression of the catalytically inactive mutant Sac2C458S resulted in altered cellular distribution of Tfn receptors and delayed Tfn recycling. Furthermore, genomic ablation of Sac2 caused a similar perturbation on Tfn and integrin recycling as well as defects in cell migration. Structural characterization of Sac2 revealed a unique pleckstrin-like homology Sac2 domain conserved in all Sac2 orthologues. Collectively, our findings provide evidence for the tight regulation of PIs by Sac2 in the endocytic recycling pathway. PMID:25869669

  13. 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

  14. 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

  15. 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. PMID:26676327

  16. Eimeria maxima phosphatidylinositol 4-phosphate 5-kinase: locus sequencing, characterization, and cross-phylum comparison.

    PubMed

    Goh, Mei-Yen; Pan, Mei-Zhen; Blake, Damer P; Wan, Kiew-Lian; Song, Beng-Kah

    2011-03-01

    Phosphatidylinositol 4-phosphate 5-kinase (PIP5K) may play an important role in host-cell invasion by the Eimeria species, protozoan parasites which can cause severe intestinal disease in livestock. Here, we report the structural organization of the PIP5K gene in Eimeria maxima (Weybridge strain). Two E. maxima BAC clones carrying the E. maxima PIP5K (EmPIP5K) coding sequences were selected for shotgun sequencing, yielding a 9.1-kb genomic segment. The EmPIP5K coding region was initially identified using in silico gene-prediction approaches and subsequently confirmed by mapping rapid amplification of cDNA ends and RT-PCR-generated cDNA sequence to its genomic segment. The putative EmPIP5K gene was located at position 710-8036 nt on the complimentary strand and comprised of 23 exons. Alignment of the 1147 amino acid sequence with previously annotated PIP5K proteins from other Apicomplexa species detected three conserved motifs encompassing the kinase core domain, which has been shown by previous protein deletion studies to be necessary for PIP5K protein function. Phylogenetic analysis provided further evidence that the putative EmPIP5K protein is orthologous to that of other Apicomplexa. Subsequent comparative gene structure characterization revealed events of intron loss/gain throughout the evolution of the apicomplexan PIP5K gene. Further scrutiny of the genomic structure revealed a possible trend towards "intron gain" between two of the motif regions. Our findings offer preliminary insights into the structural variations that have occurred during the evolution of the PIP5K locus and may aid in understanding the functional role of this gene in the cellular biology of apicomplexan parasites. PMID:20938684

  17. 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

  18. 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

  19. A phase I clinical trial assessing the safety and tolerability of combretastatin A4 phosphate injections.

    PubMed

    Liu, Peng; Qin, Yan; Wu, Lingying; Yang, Sheng; Li, Nan; Wang, Haijun; Xu, Haiyan; Sun, Kelin; Zhang, Shuxiang; Han, Xiaohong; Sun, Yan; Shi, Yuankai

    2014-04-01

    Combretastatin A4 phosphate (CA4P) is a prodrug that selectively destroys tumor blood vessels, and has shown efficacy as a targeted anticancer drug in both animal models and clinical trials. The aims of this single-center, open label, phase I clinical trial were to investigate the safety and tolerability of CA4P administered intravenously to patients aged 18-65 years with advanced solid tumors. Using a dose-escalation protocol, patients were assigned to five groups that received injections with 20 (n=3), 33 (n=3), 50 (n=11), 65 (n=6), or 85 (n=2) mg/m² CA4P. Patients in the 20 and 85 mg/m² groups received a single dose and the other groups received multiple doses. Adverse events (AE), cardiovascular parameters, and biochemical investigations were studied, and the maximum tolerated dose was determined. Of twenty-five patients enrolled, eight were withdrawn/excluded (not because of AE). There were no deaths. A total of 394 AE occurred in the 25 patients, with 89.3% considered related/possibly related to the drug. AE included headache and dizziness (19.8%), tumor-induced pain (14.2%), vascular vagal excitation (10.7%), and vomiting (9.4%). Ninety-five percent of AE were mild (grades 0-II), with only 5% being grade III-IV. Drug administration was associated with biphasic changes in heart rate and blood pressure, and only limited abnormalities in the laboratory investigations performed. The maximum tolerated dose was 65 mg/m². We conclude that CA4P is generally well tolerated, with the vast majority of AE that occurred being of mild severity. Further studies will establish the role of CA4P in cancer therapy. PMID:24500030

  20. 34 CFR 200.89 - MEP allocations; Re-interviewing; Eligibility documentation; and Quality control.

    Code of Federal Regulations, 2012 CFR

    2012-07-01

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

  1. 78 FR 21109 - Manufacturing Extension Partnership (MEP) Center for Nebraska; Availability of Funds

    Federal Register 2010, 2011, 2012, 2013, 2014

    2013-04-09

    ... Federal Register notice of December 17, 2012 (77 FR 74634), are applicable to this notice and the... and the Federal Register notice published on September 14, 2010, at 75 FR 55671. Paperwork Reduction... National Institute of Standards and Technology Manufacturing Extension Partnership (MEP) Center...

  2. Design of the unmanned Multiple Exploratory Probe System (MEPS) for Mars observations

    NASA Technical Reports Server (NTRS)

    1988-01-01

    The unmanned Multiple Exploratory Probe System (MEPS) is designed for Mars observations in preparation for manned missions to the planet early in the 21st century. MEPS will test vehicle systems, provide important data about the Martian surface and atmosphere, and assist the planning of manned missions. This mission will be a precursor to the manned missions. MEPS will consist of six primary systems. A Command Information Center (CIC) will be employed as an onboard mission control, communications link, and observation post. The Space Transportation Main Engine (STME) will be used to provide the thrust for Earth-Mars transit following vehicle construction near the Space Station. A polar lander/Orbital Transfer Vehicle (OTV) will be deployed during transit to achieve a polar orbit about Mars. A secondary propulsion will be used to place MEPS into orbit about Mars; this system and the aerobrake will circularize the orbit. Following orbit circulation, a satellite will be deployed to observe the Martian surface and atmosphere and to study the space environment. Polar and equatorial lander systems will land on Mars with rovers to collect surface and atmospheric samples while on-board laboratories will provide initial sample study. Two solid rocket booster/payload vehicles will launch samples into a low Mars orbit. The OTV will rendezvous with each payload capsule and then transfer the samples to Earth for hands-on observation.

  3. 34 CFR 200.86 - Use of MEP funds in schoolwide projects.

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ... 34 Education 1 2010-07-01 2010-07-01 false Use of MEP funds in schoolwide projects. 200.86 Section 200.86 Education Regulations of the Offices of the Department of Education OFFICE OF ELEMENTARY AND SECONDARY EDUCATION, DEPARTMENT OF EDUCATION TITLE I-IMPROVING THE ACADEMIC ACHIEVEMENT OF THE...

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

    NASA Technical Reports Server (NTRS)

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

    1988-01-01

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

  5. 34 CFR 200.89 - MEP allocations; Re-interviewing; Eligibility documentation; and Quality control.

    Code of Federal Regulations, 2011 CFR

    2011-07-01

    ... documentation; and Quality control. 200.89 Section 200.89 Education Regulations of the Offices of the Department...-interviewing; Eligibility documentation; and Quality control. (a) Allocation of funds under the MEP for fiscal... the identified problems. (2) Prospective re-interviewing. As part of the system of quality...

  6. Kinetic Characterization and Allosteric Inhibition of the Yersinia pestis 1-Deoxy-D-Xylulose 5-Phosphate Reductoisomerase (MEP Synthase)

    PubMed Central

    Haymond, Amanda; Johny, Chinchu; Dowdy, Tyrone; Schweibenz, Brandon; Villarroel, Karen; Young, Richard; Mantooth, Clark J.; Patel, Trishal; Bases, Jessica; Jose, Geraldine San; Jackson, Emily R.; Dowd, Cynthia S.; Couch, Robin D.

    2014-01-01

    The methylerythritol phosphate (MEP) pathway found in many bacteria governs the synthesis of isoprenoids, which are crucial lipid precursors for vital cell components such as ubiquinone. Because mammals synthesize isoprenoids via an alternate pathway, the bacterial MEP pathway is an attractive target for novel antibiotic development, necessitated by emerging antibiotic resistance as well as biodefense concerns. The first committed step in the MEP pathway is the reduction and isomerization of 1-deoxy-D-xylulose-5-phosphate (DXP) to methylerythritol phosphate (MEP), catalyzed by MEP synthase. To facilitate drug development, we cloned, expressed, purified, and characterized MEP synthase from Yersinia pestis. Enzyme assays indicate apparent kinetic constants of KMDXP = 252 µM and KMNADPH = 13 µM, IC50 values for fosmidomycin and FR900098 of 710 nM and 231 nM respectively, and Ki values for fosmidomycin and FR900098 of 251 nM and 101 nM respectively. To ascertain if the Y. pestis MEP synthase was amenable to a high-throughput screening campaign, the Z-factor was determined (0.9) then the purified enzyme was screened against a pilot scale library containing rationally designed fosmidomycin analogs and natural product extracts. Several hit molecules were obtained, most notably a natural product allosteric affector of MEP synthase and a rationally designed bisubstrate derivative of FR900098 (able to associate with both the NADPH and DXP binding sites in MEP synthase). It is particularly noteworthy that allosteric regulation of MEP synthase has not been described previously. Thus, our discovery implicates an alternative site (and new chemical space) for rational drug development. PMID:25171339

  7. Kinetic characterization and allosteric inhibition of the Yersinia pestis 1-deoxy-D-xylulose 5-phosphate reductoisomerase (MEP synthase).

    PubMed

    Haymond, Amanda; Johny, Chinchu; Dowdy, Tyrone; Schweibenz, Brandon; Villarroel, Karen; Young, Richard; Mantooth, Clark J; Patel, Trishal; Bases, Jessica; San Jose, Geraldine; Jackson, Emily R; Dowd, Cynthia S; Couch, Robin D

    2014-01-01

    The methylerythritol phosphate (MEP) pathway found in many bacteria governs the synthesis of isoprenoids, which are crucial lipid precursors for vital cell components such as ubiquinone. Because mammals synthesize isoprenoids via an alternate pathway, the bacterial MEP pathway is an attractive target for novel antibiotic development, necessitated by emerging antibiotic resistance as well as biodefense concerns. The first committed step in the MEP pathway is the reduction and isomerization of 1-deoxy-D-xylulose-5-phosphate (DXP) to methylerythritol phosphate (MEP), catalyzed by MEP synthase. To facilitate drug development, we cloned, expressed, purified, and characterized MEP synthase from Yersinia pestis. Enzyme assays indicate apparent kinetic constants of KMDXP = 252 µM and KMNADPH = 13 µM, IC50 values for fosmidomycin and FR900098 of 710 nM and 231 nM respectively, and Ki values for fosmidomycin and FR900098 of 251 nM and 101 nM respectively. To ascertain if the Y. pestis MEP synthase was amenable to a high-throughput screening campaign, the Z-factor was determined (0.9) then the purified enzyme was screened against a pilot scale library containing rationally designed fosmidomycin analogs and natural product extracts. Several hit molecules were obtained, most notably a natural product allosteric affector of MEP synthase and a rationally designed bisubstrate derivative of FR900098 (able to associate with both the NADPH and DXP binding sites in MEP synthase). It is particularly noteworthy that allosteric regulation of MEP synthase has not been described previously. Thus, our discovery implicates an alternative site (and new chemical space) for rational drug development. PMID:25171339

  8. Magnetic motor evoked potentials (MEP) in diseases of the spinal cord.

    PubMed

    Linden, D; Berlit, P

    1994-11-01

    Transcranial magnetic stimulation (TMS) is a non-invasive diagnostic method particularly suited to investigation of the long motor tracts. The clinical value of this method in many cortical and subcortical diseases has been well established, but comparable studies for most spinal cord diseases have still to be made. Forty patients in whom spinal cord disease was established by clinical examination, cerebrospinal fluid examination, and magnetic resonance imaging (MRI) were studied by means of somatosensory evoked potentials (SEP, median and tibial nerve stimulation) and magnetic motor evoked potentials (MEP, first dorsal interosseus and tibialis anterior muscle recordings after transcranial and spinal stimulation). The underlying pathology was neoplastic (n = 16), inflammatory (n = 15) or ischemic (n = 9). Clinical signs and symptoms ranged from slight sensory disturbances to complete paraplegia and had developed within minutes (ischemia) or over many years (benign neoplastic disease). The overall frequency of pathological SEP was slightly higher than that of MEP (78% vs 68%) which was statistically not significant (p > 0.05). This was also true for the subgroups, except for pure motor disorders, which gave the same yield for both methods. Decreased amplitudes or absence of MEP were more frequent in neoplastic than in inflammatory lesions (75% vs 33%, p < 0.05). In the latter, however, MEP more often occurred with increased latencies (40% vs 31%, p > 0.05, n.s.). Pathological SEP were found in 75% of patients presenting with pure motor abnormalities, while pathological MEP were found in 30% of patients with pure sensory disturbances.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:7887135

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

    PubMed Central

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

    2009-01-01

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

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

    SciTech Connect

    McMahon, James E.

    2004-03-12

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

  11. Phosphatidylserine stimulation of Drs2p·Cdc50p lipid translocase dephosphorylation is controlled by phosphatidylinositol-4-phosphate.

    PubMed

    Jacquot, Aurore; Montigny, Cédric; Hennrich, Hanka; Barry, Raphaëlle; le Maire, Marc; Jaxel, Christine; Holthuis, Joost; Champeil, Philippe; Lenoir, Guillaume

    2012-04-13

    Here, Drs2p, a yeast lipid translocase that belongs to the family of P(4)-type ATPases, was overexpressed in the yeast Saccharomyces cerevisiae together with Cdc50p, its glycosylated partner, as a result of the design of a novel co-expression vector. The resulting high yield allowed us, using crude membranes or detergent-solubilized membranes, to measure the formation from [γ-(32)P]ATP of a (32)P-labeled transient phosphoenzyme at the catalytic site of Drs2p. Formation of this phosphoenzyme could be detected only if Cdc50p was co-expressed with Drs2p but was not dependent on full glycosylation of Cdc50p. It was inhibited by orthovanadate and fluoride compounds. In crude membranes, the phosphoenzyme formed at steady state at 4 °C displayed ADP-insensitive but temperature-sensitive decay. Solubilizing concentrations of dodecyl maltoside left this decay rate almost unaltered, whereas several other detergents accelerated it. Unexpectedly, the dephosphorylation rate for the solubilized Drs2p·Cdc50p complex was inhibited by the addition of phosphatidylserine. Phosphatidylserine exerted its anticipated accelerating effect on the dephosphorylation of Drs2p·Cdc50p complex only in the additional presence of phosphatidylinositol-4-phosphate. These results explain why phosphatidylinositol-4-phosphate tightly controls Drs2p-catalyzed lipid transport and establish the functional relevance of the Drs2p·Cdc50p complex overexpressed here. PMID:22351780

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

    PubMed Central

    Huang, Gan; Mouraux, André

    2015-01-01

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

  13. 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

  14. TIE2-expressing macrophages limit the therapeutic efficacy of the vascular-disrupting agent combretastatin A4 phosphate in mice

    PubMed Central

    Welford, Abigail F.; Biziato, Daniela; Coffelt, Seth B.; Nucera, Silvia; Fisher, Matthew; Pucci, Ferdinando; Di Serio, Clelia; Naldini, Luigi; De Palma, Michele; Tozer, Gillian M.; Lewis, Claire E.

    2011-01-01

    Vascular-disrupting agents (VDAs) such as combretastatin A4 phosphate (CA4P) selectively disrupt blood vessels in tumors and induce tumor necrosis. However, tumors rapidly repopulate after treatment with such compounds. Here, we show that CA4P-induced vessel narrowing, hypoxia, and hemorrhagic necrosis in murine mammary tumors were accompanied by elevated tumor levels of the chemokine CXCL12 and infiltration by proangiogenic TIE2-expressing macrophages (TEMs). Inhibiting TEM recruitment to CA4P-treated tumors either by interfering pharmacologically with the CXCL12/CXCR4 axis or by genetically depleting TEMs in tumor-bearing mice markedly increased the efficacy of CA4P treatment. These data suggest that TEMs limit VDA-induced tumor injury and represent a potential target for improving the clinical efficacy of VDA-based therapies. PMID:21490397

  15. TIE2-expressing macrophages limit the therapeutic efficacy of the vascular-disrupting agent combretastatin A4 phosphate in mice.

    PubMed

    Welford, Abigail F; Biziato, Daniela; Coffelt, Seth B; Nucera, Silvia; Fisher, Matthew; Pucci, Ferdinando; Di Serio, Clelia; Naldini, Luigi; De Palma, Michele; Tozer, Gillian M; Lewis, Claire E

    2011-05-01

    Vascular-disrupting agents (VDAs) such as combretastatin A4 phosphate (CA4P) selectively disrupt blood vessels in tumors and induce tumor necrosis. However, tumors rapidly repopulate after treatment with such compounds. Here, we show that CA4P-induced vessel narrowing, hypoxia, and hemorrhagic necrosis in murine mammary tumors were accompanied by elevated tumor levels of the chemokine CXCL12 and infiltration by proangiogenic TIE2-expressing macrophages (TEMs). Inhibiting TEM recruitment to CA4P-treated tumors either by interfering pharmacologically with the CXCL12/CXCR4 axis or by genetically depleting TEMs in tumor-bearing mice markedly increased the efficacy of CA4P treatment. These data suggest that TEMs limit VDA-induced tumor injury and represent a potential target for improving the clinical efficacy of VDA-based therapies. PMID:21490397

  16. 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

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

    PubMed

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

    2013-09-20

    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. PMID:23994136

  18. 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. PMID:25792735

  19. Structural Basis for Competitive Inhibition of 3,4-Dihydroxy-2-butanone-4-phosphate Synthase from Vibrio cholerae*

    PubMed Central

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

    2015-01-01

    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. PMID:25792735

  20. The mepsMAP server. Mapping epitopes on protein surface: mining annotated proteins.

    PubMed

    Carrabino, D; D'Onorio De Meo, P; Sanna, N; Castrignanò, T; Orsini, M; Floris, M; Tramontano, A

    2007-06-01

    For a growing number of biologists DNA or protein data are typically retrieved and managed on the Web, and not in the laboratory. A large number of bioinformatics datasets from primary and (thousands of) secondary databases are scattered on the Web in various formats. A biologist end-user might need to access and use tens of databases and tools every day. For this reason, the bioinformatics community is developing more and more service-oriented architectures (SOAs): software architecture of loosely coupled software services that can be accessed without knowledge of, or control over, their internal architecture. Data-processing and analysis tasks can be automated by having free access to bioinformatics Web services (WSs) that are the building blocks of the SOAs. In this paper we introduce a new bioinformatics Web server, mepsMAP (mapping epitopes on protein surface: Mining Annotated Proteins), developed to identify the recognition sites between antibodies and their cognate antigens. In some cases, the recognition site is represented by a continuous segment of the antigen sequence, but much more often the epitope is "conformational," i.e., the antibody recognizes the location and type of exposed antigen side chains that are not necessarily contiguous in the antigen's sequence, but brought together by its three-dimensional structure. A facility on the server allows the user to search putative conformational epitopes on protein surface, querying the system for proteins with a given annotation. The mepsMAP server has been implemented as a SOA composed by a database and a set of four WSs. We present here the software architecture of the system with a detailed description of the WS dataflow that has been optimized to provide the best computing performance while maintaining the easiest end-user access to the system via a Web interface. PMID:17695751

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

    PubMed

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

    2016-07-01

    Phthalate plasticizers used in a wide range of common plastic products are released into the environment and may pose a risk of increased incidence of type 2 diabetes. In this work, we studied the effects of monoethyl phthalate (MEP), the metabolite of diethyl phthalate, exposure on 1.1B4 human pancreatic beta cells at low doses (1-1000 nM). We showed that MEP treatment induced proliferation in 1.1B4 cells. Also PCNA protein expression levels were increased related to proliferation induction. It has been noted that phthalates can exert estrogen mediated response by interacting with ER. In our study 24 h MEP treatment decreased ERα protein expression level conversely it increased the same protein expression level after 72 h treatment. Also MEP treatment decreased ERβ expression after 72 h at 1.1B4 cells. Our results further show that insulin content of 1.1B4 cells were increased with low dose MEP treatment. Along with our insulin content results, PDX- 1 expression levels were also increased at 1.1B4 cells with MEP treatment. These findings suggest that MEP acts as an estrogenic compound and PPARγ agonist at lower concentrations. Also it should be noted that PDX-1 may be a critical regulator of 1.1B4 cells treated with MEP. PMID:27133914

  2. 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

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

    ERIC Educational Resources Information Center

    Gilbert, Leslie

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

  4. Osh4p is needed to reduce the level of phosphatidylinositol-4-phosphate on secretory vesicles as they mature

    PubMed Central

    Ling, Yading; Hayano, Scott; Novick, Peter

    2014-01-01

    Phosphatidylinositol-4-phosphate (PI4P) is produced on both the Golgi and the plasma membrane. Despite extensive vesicular traffic between these compartments, genetic analysis suggests that the two pools of PI4P do not efficiently mix with one another. Several lines of evidence indicate that the PI4P produced on the Golgi is normally incorporated into secretory vesicles, but the fate of that pool has been unclear. We show here that in yeast the oxysterol-binding proteins Osh1–Osh7 are collectively needed to maintain the normal distribution of PI4P and that Osh4p is critical in this function. Osh4p associates with secretory vesicles at least in part through its interaction with PI4P and is needed, together with lipid phosphatases, to reduce the level of PI4P as vesicles approach sites of exocytosis. This reduction in PI4P is necessary for a switch in the regulation of the Sec4p exchange protein, Sec2p, from an interaction with the upstream Rab, Ypt31/32, to an interaction with a downstream Sec4p effector, Sec15p. Spatial regulation of PI4P levels thereby plays an important role in vesicle maturation. PMID:25165144

  5. 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

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

    PubMed

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

    2015-06-01

    In this study, we designed biodegradable polymersomes for co-delivery of an antiangiogenic drug combretastatin-A4 phosphate (CA4P) and doxorubicin (DOX) to collapse tumor neovasculature and inhibit cancer cell proliferation with the aim to achieve synergistic antitumor effects. The polymersomes co-encapsulating DOX and CA4P (Ps-DOX-CA4P) were prepared by solvent evaporation method using methoxy poly(ethylene glycol)-b-polylactide (mPEG-PLA) block copolymers as drug carriers. The resulting Ps-DOX-CA4P has vesicles shape with uniform sizes of about 50 nm and controlled co-encapsulation ratios of DOX to CA4P. More importantly, Ps-DOX-CA4P (1:10) showed strong synergistic cytotoxicity (combination index CI = 0.31) against human nasopharyngeal epidermal carcinoma (KB) cells. Furthermore, Ps-DOX-CA4P accumulated remarkably in KB tissues xenografts in nude mice. Consistent with these observations, Ps-DOX-CA4P (1:10) achieved significant antitumor potency because of fast tumor vasculature disruption and sustained tumor cells proliferation inhibition in vivo. The overall findings indicate that co-delivery of an antiangiogenic drug and a chemotherapeutic agent in polymersomes is a potentially promising strategy for cancer therapy. PMID:26353589

  7. Transcriptome Sequencing and Expression Analysis of Terpenoid Biosynthesis Genes in Litsea cubeba

    PubMed Central

    Han, Xiao-Jiao; Wang, Yang-Dong; Chen, Yi-Cun; Lin, Li-Yuan; Wu, Qing-Ke

    2013-01-01

    Background Aromatic essential oils extracted from fresh fruits of Litsea cubeba (Lour.) Pers., have diverse medical and economic values. The dominant components in these essential oils are monoterpenes and sesquiterpenes. Understanding the molecular mechanisms of terpenoid biosynthesis is essential for improving the yield and quality of terpenes. However, the 40 available L. cubeba nucleotide sequences in the public databases are insufficient for studying the molecular mechanisms. Thus, high-throughput transcriptome sequencing of L. cubeba is necessary to generate large quantities of transcript sequences for the purpose of gene discovery, especially terpenoid biosynthesis related genes. Results Using Illumina paired-end sequencing, approximately 23.5 million high-quality reads were generated. De novo assembly yielded 68,648 unigenes with an average length of 834 bp. A total of 38,439 (56%) unigenes were annotated for their functions, and 35,732 and 25,806 unigenes could be aligned to the GO and COG database, respectively. By searching against the Kyoto Encyclopedia of Genes and Genomes Pathway database (KEGG), 16,130 unigenes were assigned to 297 KEGG pathways, and 61 unigenes, which contained the mevalonate and 2-C-methyl-D-erythritol 4-phosphate pathways, could be related to terpenoid backbone biosynthesis. Of the 12,963 unigenes, 285 were annotated to the terpenoid pathways using the PlantCyc database. Additionally, 14 terpene synthase genes were identified from the transcriptome. The expression patterns of the 16 genes related to terpenoid biosynthesis were analyzed by RT-qPCR to explore their putative functions. Conclusion RNA sequencing was effective in identifying a large quantity of sequence information. To our knowledge, this study is the first exploration of the L. cubeba transcriptome, and the substantial amount of transcripts obtained will accelerate the understanding of the molecular mechanisms of essential oils biosynthesis. The results may help

  8. [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

  9. 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

  10. Investigation on traditional medicines of Guarany Indio and studies on diterpenes from Scoparia dulcis.

    PubMed

    Hayashi, Toshimitsu

    2011-01-01

      In interviews on the traditional herbal medicines of Tupi-Guarany Indians at the herbal market of Asuncion and questionnaire from their users, it was clarified that various useful medicinal plants are available in Paraguay and most of them are generally used without drying. In the search for bioactive substances from those plants, a β-glucuronidase-inhibitory diterpene called scoparic acid A (SA) was isolated from Scoparia dulcis L. together with scoparic acid B, scoparic acid C, and the aphidicolin-like tetracyclic diterpenes scopadulcic acid A (SDA) and scopadulcic acid B (SDB). HPLC analysis of diterpenes in the individual plants of Paraguayan and Asian S. dulcis revealed the presence of three chemotypes based on major component, i.e., SA type, SDB type, and SDX type containing mainly scopadiol and scopadulciol (SDC). SA and SDB were elucidated to be mainly biosynthesized in the leaves via 2-C-methyl-D-erythritol- 4-phosphate pathway, and a leaf organ culture system containing methyl jasmonate 10 µM was found to enhance the production of diterpenes by activation of Ca-signal transduction systems such as calmodulin and protein kinase C. On the other hand, SDB and SDC were found to show multifaceted pharmacological effects such as inhibitory effects on gastric acid excretion, bone resorption, replication of herpes simplex virus type 1 (HSV-1), etc. In addition, SDC was suggested to be applicable to cancer gene therapy using ganciclovir or acyclovir and the HSV-1 thymidine kinase gene called the suicide gene. PMID:21881299

  11. 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

  12. 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. PMID:24943221

  13. Mutagenesis and Modeling To Predict Structural and Functional Characteristics of the Staphylococcus aureus MepA Multidrug Efflux Pump

    PubMed Central

    Schindler, Bryan D.; Patel, Diixa; Seo, Susan M.

    2013-01-01

    MepA is a multidrug and toxin extrusion (MATE) family protein and the only MATE protein encoded within the Staphylococcus aureus genome. Structural data for MATE proteins are limited to a single high-resolution example, NorM of Vibrio cholerae. Substitution mutations were created in MepA using gradient plates containing both a substrate and reserpine as an efflux pump inhibitor. Site-directed mutagenesis of plasmid-based mepA was used to reproduce these mutations, as well as unique or low-frequency mutations identified in mepA-overexpressing clinical strains, and to mutagenize conserved acidic residues. The effect of these changes on protein function was quantitated in a norA-disrupted host strain by susceptibility testing with and without inhibitors and by determining the proficiency of ethidium efflux. Up-function substitutions clustered in the carboxy half of MepA, near the cytoplasmic face of the protein. Repeated application of the same gradient plate conditions frequently reproduced identical substitution mutations, suggesting that individual residues are required for interaction with specific substrates. Acidic residues critical to protein function were identified in helices 4 and 5. In silico modeling revealed an outward-facing molecule, with helices 1, 2, 4, 7, 8, and 10 having contact with a central cavity that may represent a substrate translocation pathway. Functionally important residues within this cavity included S81, A161, M291, and A302. These data provide a critical starting point for understanding how MATE multidrug efflux proteins function and will be useful in refining crystallographic data when they are available. PMID:23175649

  14. Nanoscale analysis reveals agonist-sensitive and heterogeneous pools of phosphatidylinositol 4-phosphate in the plasma membrane.

    PubMed

    Yoshida, Akane; Shigekuni, Mikiko; Tanabe, Kenji; Fujita, Akikazu

    2016-06-01

    Phosphatidylinositol 4-phosphate [PtdIns(4)P] is the immediate precursor of phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P2], which is localized to the cytoplasmic leaflet of the plasma membrane and has been reported to possess multiple cell biological functions. Direct evidence showing the distribution of PtdIns(4)P pools at a nanoscale when the plasma membrane PtdIns(4,5)P2 is hydrolyzed by agonist stimulation is lacking. To analyze the distribution of PtdIns(4)P at a nanoscale, we employed an electron microscopy technique that specifically labels PtdIns(4)P on the freeze-fracture replica of the plasma membrane. This method minimizes the possibility of artificial perturbation, because molecules in the membrane are physically immobilized in situ. Using this technique, we observed no PtdIns(4)P in the caveolae of normal cultured human fibroblasts, although PtdIns(4,5)P2 has been shown to be highly concentrated in them in our previous report. When cells were stimulated with angiotensin II, the level of PtdIns(4)P in the undifferentiated membrane transiently decreased to 64.3% at 10 s, began to increase at 30 s and largely increased to 341.9% at 40 s, and then returned to the initial level at 130 s after the stimulation. Interestingly, PtdIns(4)P localized at the caveolae at 70 and 130 s after the stimulation. These results suggest that the level of the PtdIns(4)P pool in the plasma membrane is sensitive and the distribution of PtdIns(4)P dramatically changes by agonist stimulation, and there are active sites of production or replenishment of PtdIns(4)P at undifferentiated membrane and caveolar areas. PMID:26972044

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

    PubMed

    Liao, Der-Ing; Zheng, Ya-Jun; Viitanen, Paul V; Jordan, Douglas B

    2002-02-12

    X-ray crystal structures of L-3,4-dihydroxy-2-butanone-4-phosphate synthase from Magnaporthe grisea are reported for the E-SO(4)(2-), E-SO(4)(2-)-Mg(2+), E-SO(4)(2)(-)-Mn(2+), E-SO(4)(2)(-)-Mn(2+)-glycerol, and E-SO(4)(2)(-)-Zn(2+) complexes with resolutions that extend to 1.55, 0.98, 1.60, 1.16, and 1.00 A, 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(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(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. PMID:11827524

  16. 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.

  17. 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.

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

    PubMed Central

    He, Ju; Scott, Jordan L.; Heroux, Annie; Roy, Siddhartha; Lenoir, Marc; Overduin, Michael; Stahelin, Robert V.; Kutateladze, Tatiana G.

    2011-01-01

    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 Å 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 β-barrel capped by an α-helix at one edge, whereas the opposite edge is flanked by three loops and the β4 and β7 strands that form a lipid-binding pocket within the β-barrel. The ARF1-binding site is located on the outer side of the β-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. PMID:21454700

  19. Molecular basis of phosphatidylinositol 4-phosphate and ARF1 GTPase recognition by the FAPP1 pleckstrin homology (PH) domain.

    PubMed

    He, Ju; Scott, Jordan L; Heroux, Annie; Roy, Siddhartha; Lenoir, Marc; Overduin, Michael; Stahelin, Robert V; Kutateladze, Tatiana 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 Å 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 β-barrel capped by an α-helix at one edge, whereas the opposite edge is flanked by three loops and the β4 and β7 strands that form a lipid-binding pocket within the β-barrel. The ARF1-binding site is located on the outer side of the β-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. PMID:21454700

  20. Oxysterol-binding Protein Activation at Endoplasmic Reticulum-Golgi Contact Sites Reorganizes Phosphatidylinositol 4-Phosphate Pools.

    PubMed

    Goto, Asako; Charman, Mark; Ridgway, Neale D

    2016-01-15

    Oxysterol-binding protein (OSBP) exchanges cholesterol and phosphatidylinositol 4-phosphate (PI-4P) at contact sites between the endoplasmic reticulum (ER) and the trans-Golgi/trans-Golgi network. 25-Hydroxycholesterol (25OH) competitively inhibits this exchange reaction in vitro and causes the constitutive localization of OSBP at the ER/Golgi interface and PI-4P-dependent recruitment of ceramide transfer protein (CERT) for sphingomyelin synthesis. We used PI-4P probes and mass analysis to determine how OSBP controls the availability of PI-4P for this metabolic pathway. Treatment of fibroblasts or Chinese hamster ovary (CHO) cells with 25OH caused a 50-70% reduction in Golgi-associated immunoreactive PI-4P that correlated with Golgi localization of OSBP. In contrast, 25OH caused an OSBP-dependent enrichment in Golgi PI-4P that was detected with a pleckstrin homology domain probe. The cellular mass of phosphatidylinositol monophosphates and Golgi PI-4P measured with an unbiased PI-4P probe (P4M) was unaffected by 25OH and OSBP silencing, indicating that OSBP shifts the distribution of PI-4P upon localization to ER-Golgi contact sites. The PI-4P and sterol binding activities of OSBP were both required for 25OH activation of sphingomyelin synthesis, suggesting that 25OH must be exchanged for PI-4P to be concentrated at contact sites. We propose a model wherein 25OH activation of OSBP promotes the binding and retention of PI-4P at ER-Golgi contact sites. This pool of PI-4P specifically recruits pleckstrin homology domain-containing proteins involved in lipid transfer and metabolism, such as CERT. PMID:26601944

  1. Tumor Antivascular Effects of Radiotherapy Combined with Combretastatin A4 Phosphate in Human Non-Small-Cell Lung Cancer

    SciTech Connect

    Ng, Q.-S.; Goh, Vicky; Carnell, Dawn; Meer, Khalda; Padhani, Anwar R.; Saunders, Michele I.; Hoskin, Peter J. . E-mail: peterhoskin@nhs.net

    2007-04-01

    Purpose: The tumor vascular effects of radiotherapy and subsequent administration of the vascular disrupting agent combretastatin A4 phosphate (CA4P) were studied in patients with advanced non-small-cell lung cancer using volumetric dynamic contrast-enhanced computed tomography (CT). Patients and Methods: Following ethical committee approval and informed consent, 8 patients receiving palliative radiotherapy (27 Gy in six fractions, twice weekly) also received CA4P (50 mg/m{sup 2}) after the second fraction of radiotherapy. Changes in dynamic CT parameters of tumor blood volume (BV) and permeability surface area product (PS) were measured for the whole tumor volume, tumor rim, and center after radiotherapy alone and after radiotherapy in combination with CA4P. Results: After the second fraction of radiotherapy, 6 of the 8 patients showed increases in tumor PS (23.6%, p = 0.011). Four hours after CA4P, a reduction in tumor BV (22.9%, p < 0.001) was demonstrated in the same 6 patients. Increase in PS after radiotherapy correlated with reduction in BV after CA4P (r = 0.77, p = 0.026). At 72 h after CA4P, there was a sustained reduction in tumor BV of 29.4% (p < 0.001). Both increase in PS after radiotherapy and reduction in BV after CA4P were greater at the rim of the tumor. The BV reduction at the rim was sustained to 72 h (51.4%, p 0.014). Conclusion: Radiotherapy enhances the tumor antivascular activity of CA4P in human non-small-cell lung cancer, resulting in sustained tumor vascular shutdown.

  2. Synthesis of unsaturated phosphatidylinositol 4-phosphates and the effects of substrate unsaturation on SopB phosphatase activity.

    PubMed

    Furse, Samuel; Mak, LokHang; Tate, Edward W; Templer, Richard H; Ces, Oscar; Woscholski, Rüdiger; Gaffney, Piers R J

    2015-02-21

    In this paper evidence is presented that the fatty acid component of an inositide substrate affects the kinetic parameters of the lipid phosphatase Salmonella Outer Protein B (SopB). A succinct route was used to prepare the naturally occurring enantiomer of phosphatidylinositol 4-phosphate (PI-4-P) with saturated, as well as singly, triply and quadruply unsaturated, fatty acid esters, in four stages: (1) The enantiomers of 2,3:5,6-O-dicyclohexylidene-myo-inositol were resolved by crystallisation of their di(acetylmandelate) diastereoisomers. (2) The resulting diol was phosphorylated regio-selectively exclusively on the 1-O using the new reagent tri(2-cyanoethyl)phosphite. (3) With the 4-OH still unprotected, the glyceride was coupled using phosphate tri-ester methodology. (4) A final phosphorylation of the 4-O, followed by global deprotection under basic then acidic conditions, provided PI-4-P bearing a range of sn-1-stearoyl, sn-2-stearoyl, -oleoyl, -γ-linolenoyl and arachidonoyl, glycerides. Enzymological studies showed that the introduction of cis-unsaturated bonds has a measurable influence on the activity (relative Vmax) of SopB. Mono-unsaturated PI-4-P exhibited a five-fold higher activity, with a two-fold higher KM, over the saturated substrate, when presented in DOPC vesicles. Poly-unsaturated PI-4-P showed little further change with respect to the singly unsaturated species. This result, coupled with our previous report that saturated PI-4-P has much higher stored curvature elastic stress than PI, supports the hypothesis that the activity of inositide phosphatase SopB has a physical role in vivo. PMID:25515724

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

    PubMed Central

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

    2016-01-01

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

  4. skittles, a Drosophila phosphatidylinositol 4-phosphate 5-kinase, is required for cell viability, germline development and bristle morphology, but not for neurotransmitter release.

    PubMed Central

    Hassan, B A; Prokopenko, S N; Breuer, S; Zhang, B; Paululat, A; Bellen, H J

    1998-01-01

    The phosphatidylinositol pathway is implicated in the regulation of numerous cellular functions and responses to extracellular signals. An important branching point in the pathway is the phosphorylation of phosphatidylinositol 4-phosphate by the phosphatidylinositol 4-phosphate 5-kinase (PIP5K) to generate the second messenger phosphatidylinositol 4,5-bis-phosphate (PIP2). PIP5K and PIP2 have been implicated in signal transduction, cytoskeletal regulation, DNA synthesis, and vesicular trafficking. We have cloned and generated mutations in a Drosophila PIP5K type I (skittles). Our analysis indicates that skittles is required for cell viability, germline development, and the proper structural development of sensory bristles. Surprisingly, we found no evidence for PIP5KI involvement in neural secretion. PMID:9832529

  5. Eps15 Homology Domain 1-associated Tubules Contain Phosphatidylinositol-4-Phosphate and Phosphatidylinositol-(4,5)-Bisphosphate and Are Required for Efficient Recycling

    PubMed Central

    Jović, Marko; Kieken, Fabien; Naslavsky, Naava

    2009-01-01

    The C-terminal Eps15 homology domain (EHD) 1/receptor-mediated endocytosis-1 protein regulates recycling of proteins and lipids from the recycling compartment to the plasma membrane. Recent studies have provided insight into the mode by which EHD1-associated tubular membranes are generated and the mechanisms by which EHD1 functions. Despite these advances, the physiological function of these striking EHD1-associated tubular membranes remains unknown. Nuclear magnetic resonance spectroscopy demonstrated that the Eps15 homology (EH) domain of EHD1 binds to phosphoinositides, including phosphatidylinositol-4-phosphate. Herein, we identify phosphatidylinositol-4-phosphate as an essential component of EHD1-associated tubules in vivo. Indeed, an EHD1 EH domain mutant (K483E) that associates exclusively with punctate membranes displayed decreased binding to phosphatidylinositol-4-phosphate and other phosphoinositides. Moreover, we provide evidence that although the tubular membranes to which EHD1 associates may be stabilized and/or enhanced by EHD1 expression, these membranes are, at least in part, pre-existing structures. Finally, to underscore the function of EHD1-containing tubules in vivo, we used a small interfering RNA (siRNA)/rescue assay. On transfection, wild-type, tubule-associated, siRNA-resistant EHD1 rescued transferrin and β1 integrin recycling defects observed in EHD1-depleted cells, whereas expression of the EHD1 K483E mutant did not. We propose that phosphatidylinositol-4-phosphate is an essential component of EHD1-associated tubules that also contain phosphatidylinositol-(4,5)-bisphosphate and that these structures are required for efficient recycling to the plasma membrane. PMID:19369419

  6. Identifying Episodes of Back Pain Using Medical Expenditures Panel Survey (MEPS) Data: Patient Experience, Use of Services, and Chronicity

    PubMed Central

    Monica, Smith

    2010-01-01

    Objective To explore the correspondence between “episodes of pain” and “episodes of care” for individuals with back pain. Data Source Secondary analysis of Medical Expenditures Panel Survey (MEPS) 2-year longitudinal data. Study Design Individual use and utilization of back pain services were examined across ambulatory settings and providers, and linked to MEPS medical condition data to identify individuals with back pain who do not use, or who delay or discontinue utilization of health services for back pain. Data Collection/Extraction Methods Episodes-of-care and episodes-of-pain were approximated through round-by-round temporal mapping of MEPS back pain utilization events data and medical conditions data. Principal Findings Of 10,193 individuals with back pain, approximately one fifth did not actively seek care for their back pain. Utilization of services for back pain (episodes-of-care) does not always correspond with an individual’s full experience of back pain (episodes-of-pain). Upwards of 20% of MEPS respondents who use services for some back pain episodes, reported additional episodes for which they do not use services. Conclusions These findings suggest that other longitudinal studies based only on data that reflect service use, e.g., claims data, may incorrectly infer the nature of back pain and back pain episodes. Many individuals report ongoing back pain that continues beyond their episodes-of-care, and many individuals with persistent back pain may use prescription drugs, medical services, and other health services only intermittently. PMID:21036278

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

    PubMed

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

    2016-10-01

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

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

    PubMed Central

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

    2015-01-01

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

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

    PubMed

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

    2014-11-01

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

  10. A split-ubiquitin two-hybrid screen for proteins physically interacting with the yeast amino acid transceptor Gap1 and ammonium transceptor Mep2.

    PubMed

    Van Zeebroeck, Griet; Kimpe, Marlies; Vandormael, Patrick; Thevelein, Johan M

    2011-01-01

    Several nutrient permeases have been identified in yeast, which combine a transport and receptor function, and are called transceptors. The Gap1 general amino acid permease and the Mep2 ammonium permease mediate rapid activation by amino acids and by ammonium, respectively, of the protein kinase A (PKA) pathway in nitrogen-starved cells. Their mode of action is not well understood. Both proteins are subject to complex controls governing their intracellular trafficking. Using a split-ubiquitin yeast two-hybrid screen with Gap1 or Mep2 as bait, we identified proteins putatively interacting with Gap1 and/or Mep2. They are involved in glycosylation, the secretory pathway, sphingolipid biosynthesis, cell wall biosynthesis and other processes. For several candidate interactors, determination of transport and signaling capacity, as well as localization of Gap1 or Mep2 in the corresponding deletion strains, confirmed a functional interaction with Gap1 and/or Mep2. Also common interacting proteins were identified. Transport and signaling were differentially affected in specific deletion strains, clearly separating the two functions of the transceptors and confirming that signaling does not require transport. We identified two new proteins, Bsc6 and Yir014w, that affect trafficking or downregulation of Gap1. Deletion of EGD2, YNL024c or SPC2 inactivates Gap1 transport and signaling, while its plasma membrane level appears normal.. Vma4 is required for Mep2 expression, while Gup1 appears to be required for proper distribution of Mep2 over the plasma membrane. Some of the interactions were confirmed by GST pull-down assay, using the C-terminal tail of Gap1 or Mep2 expressed in E.coli. Our results reveal the effectiveness of split-ubiquitin two-hybrid screening for identification of proteins functionally interacting with membrane proteins. They provide several candidate proteins involved in the transport and signaling function or in the complex trafficking control of the Gap1

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

    SciTech Connect

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

    2008-03-01

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

  12. Conformational analysis, X-ray crystallographic, FT-IR, FT-Raman, DFT, MEP and molecular docking studies on 1-(1-(3-methoxyphenyl) ethylidene) thiosemicarbazide

    NASA Astrophysics Data System (ADS)

    Saravanan, R. R.; Seshadri, S.; Gunasekaran, S.; Mendoza-Meroño, R.; Garcia-Granda, S.

    2015-03-01

    Conformational analysis, X-ray crystallographic, FT-IR, FT-Raman, DFT, MEP and molecular docking studies on 1-(1-(3-methoxyphenyl) ethylidene) thiosemicarbazide (MPET) are investigated. From conformational analysis the examination of the positions of a molecule taken and the energy changes is observed. The docking studies of the ligand MPET with target protein showed that this is a good molecule which docks well with target related to HMG-CoA. Hence MPET can be considered for developing into a potent anti-cholesterol drug. MEP assists in optimization of electrostatic interactions between the protein and the ligand. The MEP surface displays the molecular shape, size and electrostatic potential values. The optimized geometry of the compound was calculated from the DFT-B3LYP gradient calculations employing 6-31G (d, p) basis set and calculated vibrational frequencies are evaluated via comparison with experimental values.

  13. Conformational analysis, X-ray crystallographic, FT-IR, FT-Raman, DFT, MEP and molecular docking studies on 1-(1-(3-methoxyphenyl) ethylidene) thiosemicarbazide.

    PubMed

    Saravanan, R R; Seshadri, S; Gunasekaran, S; Mendoza-Meroño, R; Garcia-Granda, S

    2015-03-15

    Conformational analysis, X-ray crystallographic, FT-IR, FT-Raman, DFT, MEP and molecular docking studies on 1-(1-(3-methoxyphenyl) ethylidene) thiosemicarbazide (MPET) are investigated. From conformational analysis the examination of the positions of a molecule taken and the energy changes is observed. The docking studies of the ligand MPET with target protein showed that this is a good molecule which docks well with target related to HMG-CoA. Hence MPET can be considered for developing into a potent anti-cholesterol drug. MEP assists in optimization of electrostatic interactions between the protein and the ligand. The MEP surface displays the molecular shape, size and electrostatic potential values. The optimized geometry of the compound was calculated from the DFT-B3LYP gradient calculations employing 6-31G (d, p) basis set and calculated vibrational frequencies are evaluated via comparison with experimental values. PMID:25574651

  14. 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

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

    PubMed Central

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

    2015-01-01

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

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

    PubMed

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

    2015-01-01

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

  17. 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

  18. Mechanistic insights into 1-deoxy-D-xylulose 5-phosphate reductoisomerase, a key enzyme of the MEP terpenoid biosynthetic pathway.

    PubMed

    Li, Heng; Tian, Jie; Sun, Wei; Qin, Wei; Gao, Wen-Yun

    2013-11-01

    The binding mode of 1-deoxy-D-xylulose 5-phosphate (DXP) to 1-deoxy-D-xylulose 5-phosphate reductoisomerase (DXR) (EC 1.1.1.267) from Escherichia coli was investigated via (18) O isotope exchange experiments and determination of the kinetic parameters of the reaction. The results support a C3-C4 substrate binding mode in which DXP chelates a DXR-bound divalent cation via its hydroxyl groups at C3 and C4. Based on this binding mode and the early results, a catalytic cycle for the conversion of DXP to 2-methyl-D-erythritol 4-phosphate mediated by DXR including a pseudo-single molecule transition state of the retro-aldol intermediates is proposed. Taking into account the binding mode of DXP and the catalytic cycle of DXR, the mechanistic insights of DXR are disclosed and the current discrepancies concerning the catalysis of this enzyme are interpreted within the accepted retro-aldol/aldol sequence. PMID:24010408

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

    PubMed

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

    2016-01-01

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

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

    PubMed Central

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

    2016-01-01

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

  1. 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.

  2. Phosphatidylinositol4-phosphate 5-kinase prevents the decrease in the HERG potassium current induced by Gq protein-coupled receptor stimulation.

    PubMed

    Kubo, Taeko; Ding, Wei-Guang; Toyoda, Futoshi; Fujii, Yusuke; Omatsu-Kanbe, Mariko; Matsuura, Hiroshi

    2015-01-01

    The human ether-a-go-go-related gene (HERG) potassium current (IHERG) has been shown to decrease in amplitude following stimulation with Gq protein-coupled receptors (GqRs), such as α1-adrenergic and M1-muscarinic receptors (α1R and M1R, respectively), at least partly via the reduction of membrane phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2). The present study was designed to investigate the modulation of HERG channels by PI(4,5)P2 and phosphatidylinositol4-phosphate 5-kinase (PI(4)P5-K), a synthetic enzyme of PI(4,5)P2. Whole-cell patch-clamp recordings were used to examine the activity of HERG channels expressed heterologously in Chinese Hamster Ovary cells. The stimulation of α1R with phenylephrine or M1R with acetylcholine decreased the amplitude of IHERG accompanied by a significant acceleration of deactivation kinetics and the effects on IHERG were significantly attenuated in cells expressing PI(4)P5-K. The density of IHERG in cells expressing GqRs alone was significantly increased by the coexpression of PI(4)P5-K without significant differences in the voltage dependence of activation and deactivation kinetics. The kinase-deficient substitution mutant, PI(4)P5-K-K138A did not have these counteracting effects on the change in IHERG by M1R stimulation. These results suggest that the current density of IHERG is closely dependent on the membrane PI(4,5)P2 level, which is regulated by PI(4)P5-K and GqRs and that replenishing PI(4,5)P2 by PI(4)P5-K recovers IHERG. PMID:25704028

  3. 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

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

    NASA Astrophysics Data System (ADS)

    Yurova, Alla

    2016-04-01

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

  5. Biosynthesis of vitamin B6: direct identification of the product of the PdxA-catalyzed oxidation of 4-hydroxy-l-threonine-4-phosphate using electrospray ionization mass spectrometry.

    PubMed

    Banks, Jerel; Cane, David E

    2004-04-01

    PdxA (E.C. 1.1.1.262) catalyzes a key step in the biosynthesis of vitamin B(6): the nicotinamide-dependent oxidation of 4-hydroxy-l-threonine-4-phosphate (HTP) to a product tentatively identified as 3-amino-1-hydroxyacetone 1-phosphate (AHAP). To date, the evidence for the formation of AHAP, while self-consistent, has been largely circumstantial, and does not exclude the possibility that the actual product of the enzyme-catalyzed oxidation of HTP might be 2-amino-3-oxo-4-hydroxybutyric acid 4-phosphate which could undergo rapid, non-enzyme-catalyzed decarboxylation once released from the protein. Use of negative ion electrospray ionization mass spectrometry (ESI-MS) and tandem mass spectrometric analysis (MS-MS) confirms that AHAP is the product of the PdxA-catalyzed reaction. PMID:15026039

  6. Rapid breakdown of phosphatidylinositol 4-phosphate and phosphatidylinositol 4,5-bisphosphate in rat hepatocytes stimulated by vasopressin and other Ca2+-mobilizing hormones.

    PubMed Central

    Creba, J A; Downes, C P; Hawkins, P T; Brewster, G; Michell, R H; Kirk, C J

    1983-01-01

    Rat hepatocytes rapidly incorporate [32P]Pi into phosphatidylinositol 4-phosphate (PtdIns4P) and phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P2]; their monoester phosphate groups approach isotopic equilibrium with the cellular precursor pools within 1 h. Upon stimulation of these prelabelled cells with Ca2+-mobilizing stimuli (V1-vasopressin, angiotensin, alpha 1-adrenergic, ATP) there is a rapid fall in the labelling of PtdIns4P and PtdIns(4,5)P2. Pharmacological studies suggest that each of the four stimuli acts at a different population of receptors. Insulin, glucagon and prolactin do not provoke disappearance of labelled PtdIns4P and PtdIns(4,5)P2. The labelling of PtdIns4P and PtdIns(4,5)P2 in cells stimulated with vasopressin or angiotensin initially declines at a rate of 0.5-1.0% per s, reaches a minimum after 1-2 min and then returns towards the initial value. The dose-response curves for the vasopressin- and angiotensin-stimulated responses lie close to the respective receptor occupation curves, rather than at the lower hormone concentrations needed to evoke activation of glycogen phosphorylase. Disappearance of labelled PtdIns4P and PtdIns(4,5)P2 is not observed when cells are incubated with the ionophore A23187. The hormone-stimulated polyphosphoinositide disappearance is reduced, but not abolished, in Ca2+-depleted cells. These hormonal effects are not modified by 8-bromo cyclic GMP, cycloheximide or delta-hexachlorocyclohexane. The absolute rate of polyphosphoinositide breakdown in stimulated cells is similar to the rate previously reported for the disappearance of phosphatidylinositol [Kirk, Michell & Hems (1981) Biochem. J. 194, 155-165]. It seems likely that these changes in polyphosphoinositide labelling are caused by hormonal activation of the breakdown of PtdIns(4,5)P2 (and may be also PtdIns4P) by the action of a polyphosphoinositide phosphodiesterase. We therefore suggest that the initial response to hormones is breakdown of PtdIns(4,5)P2

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

    PubMed Central

    Bach, Thomas J

    2013-01-01

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

  8. Microextraction by packed sorbent (MEPS)-UHPLC-UV: A simple and efficient method for the determination of five benzodiazepines in an alcoholic beverage.

    PubMed

    Magrini, Laura; Cappiello, Achille; Famiglini, Giorgio; Palma, Pierangela

    2016-06-01

    This article describes a nano-scale method for the determination and quantification of five benzodiazepines (BDZ) in an alcoholic grappa drink (chlordiazepoxide; lorazepam; diazepam; oxazepam; medazepam). BDZ are typically used in drug-facilitated crimes (DFC) for their accessibility and synergistic effects with alcohol. Specimens collected on the crime scene must be rapidly analyzed to prove the crime, though, in most cases, a very small amount is available. Off-line MEPS extraction of diluted grappa samples proved to be an efficient and reliable method for the recovery of the selected compounds. Requiring a very small amount of extraction solvents, MEPS is an environment-friendly technique. LC separation with UV detection was used as the analytical technique because it is simple, robust, relatively economic and easy-to-find in most laboratories. The method was validated in terms of precision, accuracy and recovery. Limits of detection and quantitation were in the range of 0.5-2ng/μL. Linearity (R(2)) spanned from 0.9994 and 1.0000. Intra- and inter-day repeatabilities were lower than 12% at any concentration. Recovery percentages of an equivalent-to-real sample at three different concentrations were between 70.7 and 74.1%. PMID:27003119

  9. Do Organometallic CH4-Me(+p) Adducts and X4H(+) (X = P, As) Clusters Undergo Two-Electron Three-Center Interactions? Some Aspects of Discussion.

    PubMed

    Lobayan, Rosana M; Bochicchio, Roberto C

    2015-07-01

    Most of the systems possessing true two-electron three-center interactions are electron deficient compounds like boron hydrids, closo-boranes, and some organic ions such as butonium cations. In this work, we perform a detailed study of the electron distribution for two different types of systems to which likewise interactions has been adjudicated: organometallic CH4-Me(+p) (p = 1, 2) adducts with Me, alkaline and earth alkaline metallic ions of Li, Na, K, Be, Mg, Ca in their stable gaseous phase and X4H(+) (X = P, As) simple clusters. For this purpose, topological analysis of the electron density decomposed into its effectively paired and unpaired contributions has been carried out looking for complex interactions. PMID:26061421

  10. Microextraction by Packed Sorbent (MEPS) and Solid-Phase Microextraction (SPME) as Sample Preparation Procedures for the Metabolomic Profiling of Urine

    PubMed Central

    Silva, Catarina; Cavaco, Carina; Perestrelo, Rosa; Pereira, Jorge; Câmara, José S.

    2014-01-01

    For a long time, sample preparation was unrecognized as a critical issue in the analytical methodology, thus limiting the performance that could be achieved. However, the improvement of microextraction techniques, particularly microextraction by packed sorbent (MEPS) and solid-phase microextraction (SPME), completely modified this scenario by introducing unprecedented control over this process. Urine is a biological fluid that is very interesting for metabolomics studies, allowing human health and disease characterization in a minimally invasive form. In this manuscript, we will critically review the most relevant and promising works in this field, highlighting how the metabolomic profiling of urine can be an extremely valuable tool for the early diagnosis of highly prevalent diseases, such as cardiovascular, oncologic and neurodegenerative ones. PMID:24958388

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

    PubMed

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

    2012-06-01

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

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

    PubMed

    Tanak, Hasan; Toy, Mehmet

    2013-11-01

    The molecular geometry and vibrational frequencies of bis[2-hydroxy-кO-N-(2-pyridyl)-1-naphthaldiminato-кN]zinc(II) in the ground state have been calculated by using the Hartree-Fock (HF) and density functional method (B3LYP) with 6-311G(d,p) basis set. The results of the optimized molecular structure are presented and compared with the experimental X-ray diffraction. The energetic and atomic charge behavior of the title compound in solvent media has been examined by applying the Onsager and the polarizable continuum model. To investigate second order nonlinear optical properties of the title compound, the electric dipole (μ), linear polarizability (α) and first-order hyperpolarizability (β) were computed using the density functional B3LYP and CAM-B3LYP methods with the 6-31+G(d) basis set. According to our calculations, the title compound exhibits nonzero (β) value revealing second order NLO behavior. In addition, DFT calculations of the title compound, molecular electrostatic potential (MEP), frontier molecular orbitals, and thermodynamic properties were performed at B3LYP/6-311G(d,p) level of theory. PMID:23832222

  13. Development and validation of a MEPS-UHPLC-PDA method for determination of ulifloxacin in human plasma and urine of patients with peripheral arterial disease.

    PubMed

    Ferrone, Vincenzo; Carlucci, Maura; Palumbo, Paola; Carlucci, Giuseppe

    2016-09-01

    A novel sensitive analytical method based on the use of a semi-automatic microextraction by packed sorbents (MEPS) techniques combined with ultra high-performance liquid chromatography (UHPLC) with PDA detection has been developed and validate for the analysis of ulifloxacin, the active metabolite of prulifloxacin using danofloxacin as internal standard in human plasma and urine. Different experimental parameters were optimized and validated according to international guidelines. Complete separation of the analytes was achieved with a Waters BEH C18 (50×2.1mm I.D., 1.7μm particle size) analytical column, a mixture of 10mM ammonium acetate (pH 3.0) (A) with and acetonitrile (B) both containing 1% triethylamine were used as mobile phase, at a flow rate of 0.6mL/min in gradient elution, and detection wavelength of 272nm. This method is linear in concentration range of 0.02-10.0μg/mL for plasma and urine, respectively. The limit of quantitation was 20ng/mL for the two fluids. The recoveries of the method were 95% for ulifloxacin in human plasma and urine and 95.5% for the internal standard. Intra- and inter- assay precision and accuracy for ulifloxacin were lower than 10% at all tested concentrations. The proposed method was successfully applied to measure plasma and urine concentrations of ulifloxacin in patients suffering from Peripheral Arterial Disease and for pharmacokinetics study. PMID:27318081

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

    PubMed

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

    2016-09-01

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

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

    NASA Astrophysics Data System (ADS)

    Tanak, Hasan; Toy, Mehmet

    2013-11-01

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

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

    NASA Astrophysics Data System (ADS)

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

    2016-09-01

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

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

    PubMed

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

    2016-05-01

    The transcripts of three putative ammonia (NH3/NH4 (+)) transporters, Rhesus-like glycoproteins AeRh50-1, AeRh50-2 and Amt/Mep-like AeAmt1 were detected in the anal papillae of larval Aedes aegypti Quantitative PCR studies revealed 12-fold higher transcript levels of AeAmt1 in anal papillae relative to AeRh50-1, and levels of AeRh50-2 were even lower. Immunoblotting revealed AeAmt1 in anal papillae as a pre-protein with putative monomeric and trimeric forms. AeAmt1 was immunolocalized to the basal side of the anal papillae epithelium where it co-localized with Na(+)/K(+)-ATPase. Ammonium concentration gradients were measured adjacent to anal papillae using the scanning ion-selective electrode technique (SIET) and used to calculate ammonia efflux by the anal papillae. dsRNA-mediated reductions in AeAmt1 decreased ammonia efflux at larval anal papillae and significantly increased ammonia levels in hemolymph, indicating a principal role for AeAmt1 in ammonia excretion. Pharmacological characterization of ammonia transport mechanisms in the anal papillae suggests that, in addition to AeAmt1, the ionomotive pumps V-type H(+)-ATPase and Na(+)/K(+)-ATPase as well as NHE3 are involved in ammonia excretion at the anal papillae. PMID:26944496

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

    PubMed

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

    2013-11-15

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

  19. Vegetable Crop Pests. MEP 311.

    ERIC Educational Resources Information Center

    Kantzes, James G.; And Others

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

  20. Fruit Crop Pests. MEP 312.

    ERIC Educational Resources Information Center

    Weaver, Leslie O.; And Others

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

  1. 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

  2. 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. PMID:25148954

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

    NASA Astrophysics Data System (ADS)

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

    2015-02-01

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

  4. Molecular structure, FT-IR, vibrational assignments, HOMO-LUMO, MEP, NBO analysis and molecular docking study of ethyl-6-(4-chlorophenyl)-4-(4-fluorophenyl)-2-oxocyclohex-3-ene-1-carboxylate

    NASA Astrophysics Data System (ADS)

    Sheena Mary, Y.; Yohannan Panicker, C.; Sapnakumari, M.; Narayana, B.; Sarojini, B. K.; Al-Saadi, Abdulaziz A.; Van Alsenoy, Christian; War, Javeed Ahmad

    2015-03-01

    The optimized molecular structure, vibrational frequencies, corresponding vibrational assignments of ethyl-6-(4-chlorophenyl)-4-(4-fluoro-phenyl)-2-oxocyclohex-3-ene-1-carboxylate have been investigated experimentally and theoretically using Gaussian09 software. The title compound was optimized using the HF and DFT levels of theory. The geometrical parameters are in agreement with the XRD data. The stability of the molecule has been analyzed by NBO analysis. The HOMO and LUMO analysis is used to determine the charge transfer within the molecule. Molecular electrostatic potential was performed by the DFT method. As can be seen from the MEP map of the title compound, regions having the negative potential are over the electro negative atoms, the region having the positive potential are over the phenyl rings and the remaining species are surrounded by zero potential. First hyperpolarizability is calculated in order to find its role in non linear optics. The title compound binds at the active sites of both CypD and β-secretase and the molecular docking results draw the conclusion that the compound might exhibit β-secretase inhibitory activity which could be utilized for development of new anti-alzheimeric drugs with mild CypD inhibitory activity.

  5. Molecular structure, FT-IR, vibrational assignments, HOMO-LUMO, MEP, NBO analysis and molecular docking study of ethyl-6-(4-chlorophenyl)-4-(4-fluorophenyl)-2-oxocyclohex-3-ene-1-carboxylate.

    PubMed

    Sheena Mary, Y; Yohannan Panicker, C; Sapnakumari, M; Narayana, B; Sarojini, B K; Al-Saadi, Abdulaziz A; Van Alsenoy, Christian; War, Javeed Ahmad

    2015-03-01

    The optimized molecular structure, vibrational frequencies, corresponding vibrational assignments of ethyl-6-(4-chlorophenyl)-4-(4-fluoro-phenyl)-2-oxocyclohex-3-ene-1-carboxylate have been investigated experimentally and theoretically using Gaussian09 software. The title compound was optimized using the HF and DFT levels of theory. The geometrical parameters are in agreement with the XRD data. The stability of the molecule has been analyzed by NBO analysis. The HOMO and LUMO analysis is used to determine the charge transfer within the molecule. Molecular electrostatic potential was performed by the DFT method. As can be seen from the MEP map of the title compound, regions having the negative potential are over the electro negative atoms, the region having the positive potential are over the phenyl rings and the remaining species are surrounded by zero potential. First hyperpolarizability is calculated in order to find its role in non linear optics. The title compound binds at the active sites of both CypD and β-secretase and the molecular docking results draw the conclusion that the compound might exhibit β-secretase inhibitory activity which could be utilized for development of new anti-alzheimeric drugs with mild CypD inhibitory activity. PMID:25463053

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

    PubMed Central

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

    1993-01-01

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

  7. Field and Forage Crop Pests. MEP 310.

    ERIC Educational Resources Information Center

    Morgan, Omar, D.; And Others

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

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

    ERIC Educational Resources Information Center

    Morgan, Omar D.; And Others

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

  9. Household and Structural Pests. MEP 307.

    ERIC Educational Resources Information Center

    Wood, F. E.

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

  10. Effects of Feeding Spodoptera littoralis on Lima Bean Leaves: IV. Diurnal and Nocturnal Damage Differentially Initiate Plant Volatile Emission1[W][OA

    PubMed Central

    Arimura, Gen-ichiro; Köpke, Sabrina; Kunert, Maritta; Volpe, Veronica; David, Anja; Brand, Peter; Dabrowska, Paulina; Maffei, Massimo E.; Boland, Wilhelm

    2008-01-01

    Continuous mechanical damage initiates the rhythmic emission of volatiles in lima bean (Phaseolus lunatus) leaves; the emission resembles that induced by herbivore damage. The effect of diurnal versus nocturnal damage on the initiation of plant defense responses was investigated using MecWorm, a robotic device designed to reproduce tissue damage caused by herbivore attack. Lima bean leaves that were damaged by MecWorm during the photophase emitted maximal levels of β-ocimene and (Z)-3-hexenyl acetate in the late photophase. Leaves damaged during the dark phase responded with the nocturnal emission of (Z)-3-hexenyl acetate, but with only low amounts of β-ocimene; this emission was followed by an emission burst directly after the onset of light. In the presence of 13CO2, this light-dependent synthesis of β-ocimene resulted in incorporation of 75% to 85% of 13C, demonstrating that biosynthesis of β-ocimene is almost exclusively fueled by the photosynthetic fixation of CO2 along the plastidial 2-C-methyl-d-erythritol 4-P pathway. Jasmonic acid (JA) accumulated locally in direct response to the damage and led to immediate up-regulation of the P. lunatus β-ocimene synthase gene (PlOS) independent of the phase, that is, light or dark. Nocturnal damage caused significantly higher concentrations of JA (approximately 2–3 times) along with enhanced expression levels of PlOS. Transgenic Arabidopsis thaliana transformed with PlOS promoter∷β-glucuronidase fusion constructs confirmed expression of the enzyme at the wounded sites. In summary, damage-dependent JA levels directly control the expression level of PlOS, regardless of light or dark conditions, and photosynthesis is the major source for the early precursors of the 2-C-methyl-d-erythritol 4-P pathway. PMID:18165324

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

    NASA Astrophysics Data System (ADS)

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

    2015-03-01

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

  12. Decree No. 18274-S-MEP, 27 June 1988.

    PubMed

    1988-01-01

    This Decree creates in Costa Rica an Interinstitutional Commission of Prevention and Integral Attention with Respect to Abandoned Children and Adolescents with Physical and Mental Limitations, composed of the representatives of various government and nongovernment agencies. The functions of the Commission are 1) to create and put into operation a program of prevention and integral attention with respect to such children; 2) to give advice on and supervise the execution of the program; and 3) to promote the establishment of participation agreements between public and private, national and international bodies with respect to the issue. PMID:12289154

  13. Momentous discoveries on Mars: science outside MEP pathways

    NASA Technical Reports Server (NTRS)

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

    2003-01-01

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

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

    ERIC Educational Resources Information Center

    Council for Educational Technology, London (England).

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

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

    PubMed Central

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

    2016-01-01

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

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

    PubMed

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

    2016-01-01

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

  17. 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. PMID:25184438

  18. 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

  19. 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.

  20. 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.

  1. 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. PMID:25868383

  2. 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

  3. 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

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

    PubMed

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

    2015-12-01

    The biosynthetic pathway of terpene trilactones of Ginkgo biloba is unclear. In this present study, suspension cultured cells of G. biloba were used to explore the regulation of the mevalonic acid (MVA) and methylerythritol 4-phosphate (MEP) pathways in response to specific enzyme inhibitors (lovastatin and clomazone). The results showed that the biosynthesis of bilobalide was more highly correlated with the MVA pathway, and the biosynthesis of ginkgolides was more highly correlated with the MEP pathway. Meanwhile, according to the results, it could be speculated that bilobalide might be a product of ginkgolide metabolism. PMID:26882658

  5. Organometallic mechanism of action and inhibition of the 4Fe-4S isoprenoid biosynthesis protein GcpE (IspG)

    PubMed Central

    Wang, Weixue; Li, Jikun; Wang, Ke; Huang, Cancan; Zhang, Yong; Oldfield, Eric

    2010-01-01

    We report the results of a series of chemical, EPR, ENDOR, and HYSCORE spectroscopic investigations of the mechanism of action (and inhibition) of GcpE, E-1-hydroxy-2-methyl-but-2-enyl-4-diphosphate (HMBPP) synthase, also known as IspG, an Fe4S4 cluster-containing protein. We find that the epoxide of HMBPP when reduced by GcpE generates the same transient EPR species as observed on addition of the substrate, 2-C-methyl-D-erythritol-2, 4-cyclo-diphosphate. ENDOR and HYSCORE spectra of these transient species (using 2H, 13C and 17O labeled samples) indicate formation of an Fe-C-H containing organometallic intermediate, most likely a ferraoxetane. This is then rapidly reduced to a ferracyclopropane in which the HMBPP product forms an η2-alkenyl π- (or π/σ) complex with the 4th Fe in the Fe4S4 cluster, and a similar “metallacycle” also forms between isopentenyl diphosphate (IPP) and GcpE. Based on this metallacycle concept, we show that an alkyne (propargyl) diphosphate is a good (Ki ∼ 300 nM) GcpE inhibitor, and supported again by EPR and ENDOR results (a 13C hyperfine coupling of ∼7 MHz), as well as literature precedent, we propose that the alkyne forms another π/σ metallacycle, an η2-alkynyl, or ferracyclopropene. Overall, the results are of broad general interest because they provide new mechanistic insights into GcpE catalysis and inhibition, with organometallic bond formation playing, in both cases, a key role. PMID:20534554

  6. Interfacial hydrolysis of phosphatidylinositol 4-phosphate and phosphatidylinositol 4,5-bisphosphate by turkey erythrocyte phospholipase C.

    PubMed Central

    James, S R; Demel, R A; Downes, C P

    1994-01-01

    The activity of a beta-isoform of phospholipase C (PLC) partially purified from turkey erythrocyte cytosol was assayed using phospholipid monolayers formed at an air-water interface. PLC was rapidly purified at least 8000-fold by a sequence of ion-exchange, hydrophobic and heparin chromatographies. 33P-labelled substrates were prepared using partially purified PtdIns kinase and PtdIns4P 5-kinases, respectively, and purified by h.p.l.c. using an amino-cyano analytical column. Using such 33P-labelled phosphoinositides of high specific radioactivity, PLC activity was monitored directly by measuring the loss of radioactivity from monolayers as a result of the release of inositol phosphates and their subsequent dissolution and quenching in the subphase. Under these conditions, PtdIns4P hydrolysis obeyed approximately first-order kinetics whereas PtdIns(4,5)P2 hydrolysis was zero-order at least until 80% of the substrate had been degraded. PLC activity was markedly affected by the surface pressure of the monolayer, with reduced activity at extremes of initial pressure and with the most permissive pressures in the middle of the range investigated. The optimum surface pressure for hydrolysis of PtdIns4P was approx. 25 mN/m, but for PtdIns(4,5)P2 the maximum activity occurred at the markedly higher surface pressure of 30 mN/m. These data are discussed in terms of the substrate specificity and likely regulation of PLC beta isoforms engaged in degrading their substrate in biological membranes. PMID:8135761

  7. Vacuum Ultraviolet and Ultraviolet Spectroscopy of Tb3+- and Eu3+-Doped Na(Sr,Ba)PO4 Phosphate

    NASA Astrophysics Data System (ADS)

    Huang, Yanlin; Cao, Yonggang; Jiang, Chuanfang; Shi, Liang; Tao, Ye; Seo, Hyo Jin

    2008-08-01

    The Eu3+- and Tb3+-doped NaSr0.65Ba0.35(PO4) phosphors were prepared by high-temperature solid-state reaction. X-ray powder diffraction (XRD) analyses confirm the pure crystalline phase of Olgite mineral. Scanning electron microscopy (SEM) measurements indicate that these phosphors have good surface-crystalline with a narrow size distribution. Vacuum ultraviolet (VUV) synchrotron radiation and ultraviolet (UV) spectroscopy were applied to study the luminescence properties of these phosphors. The bands near 147 nm in the VUV excitation spectra of those doped samples are attributed to the PO43- absorption in the host. The excitation and emission spectra indicate that the phosphors can be effectively excited for NaSr0.65Ba0.35(PO4):Eu3+ and NaSr0.65Ba0.35(PO4):Tb3+ by 147 nm and exhibit a red and green light, respectively. The Commission International de I'Eclairage (CIE) chromaticity coordinates for Eu3+- and Tb3+-activated phosphors are calculated. Considering the high luminescence intensity, excellent color purity and chemical stability, NaSr0.65Ba0.35(PO4):RE (RE = Eu3+, Tb3+) are attractive red- and green-emitting plasma display panel phosphors.

  8. 78 FR 34346 - Proposed Information Collection; Comment Request; NIST MEP Advanced Manufacturing Jobs and...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2013-06-07

    ... Advanced Manufacturing Jobs and Innovation Accelerator Challenge (AMJIAC) Client Impact Survey AGENCY... information collection. The purpose of the Advanced Manufacturing Jobs and Innovation Accelerator Challenge... to support job creation, encourage economic development, and enhance the competitiveness of...

  9. 77 FR 37653 - Manufacturing Extension Partnership (MEP) Centers for Arizona, Maryland and Rhode Island...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2012-06-22

    ..., which are contained in the Federal Register notice of February 11, 2008 (73 FR 7696), are applicable to this competition and are available at http://www.gpo.gov/fdsys/pkg/FR-2008-02-11/pdf/E8-2482.pdf... the Federal Register notice published on September 14, 2010, at 75 FR 55671. Paperwork Reduction...

  10. 34 CFR 200.84 - Responsibilities of SEAs for evaluating the effectiveness of the MEP.

    Code of Federal Regulations, 2011 CFR

    2011-07-01

    ... ACADEMIC ACHIEVEMENT OF THE DISADVANTAGED Migrant Education Program § 200.84 Responsibilities of SEAs for...'s performance targets in § 200.83(a)(1), particularly for those students who have priority...

  11. 75 FR 6355 - Manufacturing Extension Partnership (MEP) Availability of Funds for Three Regions Including the...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2010-02-09

    ... methodology for delivering advanced manufacturing technology to small- and medium-sized manufacturers and... the Federal Register Notice of February 11, 2008 (73 FR 7696), are applicable to this notice. Please refer to http://www.gpoaccess.gov/fr/ . Dun and Bradstreet Data Universal Numbering System: On the...

  12. 34 CFR 200.84 - Responsibilities of SEAs for evaluating the effectiveness of the MEP.

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ... ACADEMIC ACHIEVEMENT OF THE DISADVANTAGED Migrant Education Program § 200.84 Responsibilities of SEAs for...'s performance targets in § 200.83(a)(1), particularly for those students who have priority...

  13. 75 FR 33766 - Manufacturing Extension Partnership (MEP) Availability of Funds for Projects To Develop Client...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2010-06-15

    ... Cooperative Agreements, which are contained in the Federal Register Notice of February 11, 2008 (73 FR 7696), are applicable to this notice. Please refer to http://www.gpoaccess.gov/fr/ . Dun and Bradstreet Data... productivity, technological performance, and strengthen the global competitiveness of small- and medium-sized...

  14. 75 FR 33769 - Manufacturing Extension Partnership (MEP) Availability of Funds for Projects To Develop and...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2010-06-15

    ... Acceleration, Workforce and Continuous Improvement). Funded projects will improve the competitiveness of... focus are: Supply Chain, Sustainability, Technology Acceleration, Workforce and Continuous Improvement... Growth Areas (Supply Chain, Sustainability, Technology Acceleration, Workforce and Continuous...

  15. 77 FR 12041 - Applications for New Awards; Migrant Education Program (MEP) Consortium Incentive Grants Program

    Federal Register 2010, 2011, 2012, 2013, 2014

    2012-02-28

    ... notice of final requirements for this program, published in the Federal Register on March 3, 2004 (69 FR... FR 13217). Absolute Priorities: For FY 2012, these priorities are absolute priorities. Under 34 CFR... notice of final requirements published in the Federal Register on March 3, 2004 (69 FR 10110); and...

  16. 34 CFR 200.89 - MEP allocations; Re-interviewing; Eligibility documentation; and Quality control.

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ... the identified problems. (2) Prospective re-interviewing. As part of the system of quality controls... information on which the determination was based were true and correct; (v) Stop serving any children found... children identified in the State. (d) Responsibilities of an SEA to establish and implement a system...

  17. 34 CFR 200.89 - MEP allocations; Re-interviewing; Eligibility documentation; and Quality control.

    Code of Federal Regulations, 2014 CFR

    2014-07-01

    ... the identified problems. (2) Prospective re-interviewing. As part of the system of quality controls... information on which the determination was based were true and correct; (v) Stop serving any children found... children identified in the State. (d) Responsibilities of an SEA to establish and implement a system...

  18. 34 CFR 200.89 - MEP allocations; Re-interviewing; Eligibility documentation; and Quality control.

    Code of Federal Regulations, 2013 CFR

    2013-07-01

    ... the identified problems. (2) Prospective re-interviewing. As part of the system of quality controls... information on which the determination was based were true and correct; (v) Stop serving any children found... children identified in the State. (d) Responsibilities of an SEA to establish and implement a system...

  19. 77 FR 12563 - Manufacturing Extension Partnership (MEP) Centers for South Dakota and Kentucky; Availability of...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2012-03-01

    ... Program and the following applicable Federal cost principles: (1) Institutions of Higher Education: 2 CFR... INFORMATION CONTACT: Administrative, budget, cost-sharing, and eligibility questions and other programmatic... cost effective, local, leveraged resources for those services through the coordinated efforts of...

  20. Deoxyxylulose 5-phosphate reductoisomerase is not a rate-determining enzyme for essential oil production in spike lavender.

    PubMed

    Mendoza-Poudereux, Isabel; Muñoz-Bertomeu, Jesús; Arrillaga, Isabel; Segura, Juan

    2014-11-01

    Spike lavender (Lavandula latifolia) is an economically important aromatic plant producing essential oils, whose components (mostly monoterpenes) are mainly synthesized through the plastidial methylerythritol 4-phosphate (MEP) pathway. 1-Deoxy-D-xylulose-5-phosphate (DXP) synthase (DXS), that catalyzes the first step of the MEP pathway, plays a crucial role in monoterpene precursors biosynthesis in spike lavender. To date, however, it is not known whether the DXP reductoisomerase (DXR), that catalyzes the conversion of DXP into MEP, is also a rate-limiting enzyme for the biosynthesis of monoterpenes in spike lavender. To investigate it, we generated transgenic spike lavender plants constitutively expressing the Arabidopsis thaliana DXR gene. Although two out of the seven transgenic T0 plants analyzed accumulated more essential oils than the controls, this is hardly imputable to the DXR transgene effect since a clear correlation between transcript accumulation and monoterpene production could not be established. Furthermore, these increased essential oil phenotypes were not maintained in their respective T1 progenies. Similar results were obtained when total chlorophyll and carotenoid content in both T0 transgenic plants and their progenies were analyzed. Our results then demonstrate that DXR enzyme does not play a crucial role in the synthesis of plastidial monoterpene precursors, suggesting that the control flux of the MEP pathway in spike lavender is primarily exerted by the DXS enzyme. PMID:25151124

  1. A simple and visible colorimetric method through Zr(4+)-phosphate coordination for the assay of protein tyrosine phosphatase 1B and screening of its inhibitors.

    PubMed

    Zhang, Juan; Lv, Jun; Wang, Xiaonan; Li, Defeng; Wang, Zhaoxia; Li, Genxi

    2015-08-21

    Inhibitors of protein tyrosine phosphatase 1B (PTP1B) are promising agents for the treatment of type 2 diabetes and obesity, so a colorimetric method has been developed in this work for PTP1B assay and screening of its inhibitors. The method is based on the chelation effect of zirconium (Zr(4+)) ions on the phosphate group, which may induce aggregation of 4-aminophenylphosphate-functionalized gold nanoparticles (APP/AuNPs) and the corresponding color change of the testing solution. Owing to the dephosphorylation of PTP1B, the aggregation of AuNPs will be influenced by PTP1B since there is no coordination reactivity between Zr(4+) ions and 4-aminophenol, the hydrolyzed product of APP catalyzed by the enzyme. Therefore, a simple colorimetric method for the assay of PTP1B activity can be developed. Under the optimized experimental conditions, the ratios of absorbance at a wavelength of 650 nm to that at 522 nm vary linearly with the PTP1B activity in the range from 0.005 to 0.18 U mL(-1) with the lowest detection limit of 0.0017 U mL(-1). Moreover, using this proposed method, the inhibition effect of 6-chloro-3-formyl-7-methylchromone, betulinic acid, ursolic acid, and sodium orthovanadate on PTP1B activity can be tested with IC50 values of 10, 13, 9, and 1.1 μM, respectively. Therefore, this new method has great potential not only for the detection of PTP1B activity but also for the screening of the inhibitors. PMID:26153652

  2. Mechanisms associated with tumor vascular shut-down induced by combretastatin A-4 phosphate: intravital microscopy and measurement of vascular permeability.

    PubMed

    Tozer, G M; Prise, V E; Wilson, J; Cemazar, M; Shan, S; Dewhirst, M W; Barber, P R; Vojnovic, B; Chaplin, D J

    2001-09-01

    The tumor vascular effects of the tubulin destabilizing agent disodium combretastatinA-4 3-O-phosphate (CA-4-P) were investigated in the rat P22 tumor growing in a dorsal skin flap window chamber implanted into BD9 rats. CA-4-P is in clinical trial as a tumor vascular targeting agent. In animal tumors, it can cause the shut-down of blood flow, leading to extensive tumor cell necrosis. However, the mechanisms leading to vascular shut-down are still unknown. Tumor vascular effects were visualized and monitored on-line before and after the administration of two doses of CA-4-P (30 and 100 mg/kg) using intravital microscopy. The combined effect of CA-4-P and systemic nitric oxide synthase (NOS) inhibition using N(omega)-nitro-L-arginine (L-NNA) was also assessed, because this combination has been shown previously to have a potentiating effect. The early effect of CA-4-P on tumor vascular permeability to albumin was determined to assess whether this could be involved in the mechanism of action of the drug. Tumor blood flow reduction was extremely rapid after CA-4-P treatment, with red cell velocity decreasing throughout the observation period and dropping to <5% of the starting value by 1 h. NOS inhibition alone caused a 50% decrease in red cell velocity, and the combined treatment of CA-4-P and NOS inhibition was approximately additive. The mechanism of blood flow reduction was very different for NOS inhibition and CA-4-P. That of NOS inhibition could be explained by a decrease in vessel diameter, which was most profound on the arteriolar side of the tumor circulation. In contrast, the effects of CA-4-P resembled an acute inflammatory reaction resulting in a visible loss of a large proportion of the smallest blood vessels. There was some return of visible vasculature at 1 h after treatment, but the blood in these vessels was static or nearly so, and many of the vessels were distended. The hematocrit within larger draining tumor venules tended to increase at early times after CA-4-P, suggesting fluid loss from the blood. The stacking of red cells to form rouleaux was also a common feature, coincident with slowing of blood flow; and these two factors would lead to an increase in viscous resistance to blood flow. Tumor vascular permeability to albumin was increased to approximately 160% of control values at 1 and 10 min after treatment. This could lead to an early decrease in tumor blood flow via an imbalance between intravascular and tissue pressures and/or an increase in blood viscosity as a result of increased hematocrit. These results suggest a mechanism of action of CA-4-P in vivo. Combination of CA-4-P with a NOS inhibitor has an additive effect, which it may be possible to exploit therapeutically. PMID:11522635

  3. Phosphatidylinositol 4-phosphate 5-kinases 1 and 2 are involved in the regulation of vacuole morphology during Arabidopsis thaliana pollen development.

    PubMed

    Ugalde, José-Manuel; Rodriguez-Furlán, Cecilia; Rycke, Riet De; Norambuena, Lorena; Friml, Jiří; León, Gabriel; Tejos, Ricardo

    2016-09-01

    The pollen grains arise after meiosis of pollen mother cells within the anthers. A series of complex structural changes follows, generating mature pollen grains capable of performing the double fertilization of the female megasporophyte. Several signaling molecules, including hormones and lipids, have been involved in the regulation and appropriate control of pollen development. Phosphatidylinositol 4-phophate 5-kinases (PIP5K), which catalyze the biosynthesis of the phosphoinositide PtdIns(4,5)P2, are important for tip polar growth of root hairs and pollen tubes, embryo development, vegetative plant growth, and responses to the environment. Here, we report a role of PIP5Ks during microgametogenesis. PIP5K1 and PIP5K2 are expressed during early stages of pollen development and their transcriptional activity respond to auxin in pollen grains. Early male gametophytic lethality to certain grade was observed in both pip5k1(-/-) and pip5k2(-/-) single mutants. The number of pip5k mutant alleles is directly related to the frequency of aborted pollen grains suggesting the two genes are involved in the same function. Indeed PIP5K1 and PIP5K2 are functionally redundant since homozygous double mutants did not render viable pollen grains. The loss of function of PIP5K1 and PIP5K2results in defects in vacuole morphology in pollen at the later stages and epidermal root cells. Our results show that PIP5K1, PIP5K2 and phosphoinositide signaling are important cues for early developmental stages and vacuole formation during microgametogenesis. PMID:27457979

  4. Enhanced production of coenzyme Q10 by self-regulating the engineered MEP pathway in Rhodobacter sphaeroides.

    PubMed

    Lu, Wenqiang; Ye, Lidan; Xu, Haoming; Xie, Wenping; Gu, Jiali; Yu, Hongwei

    2014-04-01

    Fine-tuning the expression level of an engineered pathway is crucial for the metabolic engineering of a host toward a desired phenotype. However, most engineered hosts suffer from nonfunctional protein expression, metabolic imbalance, cellular burden or toxicity from intermediates when an engineered pathway is first introduced, which can decrease production of the desired product. To circumvent these obstacles, we developed a self-regulation system utilizing the trc/tac promoter, LacI(q) protein and ribosomal binding sites (RBS). With the purpose of improving coenzyme Q10 (CoQ10 ) production by increasing the decaprenyl diphosphate supplement, enzymes DXS, DXR, IDI, and IspD were constitutively overexpressed under the control of the trc promoter in Rhodobacter sphaeroides. Then, a self-regulation system combining a set of RBSs for adjusting the expression of the LacI(q) protein was applied to tune the expression of the four genes, resulting in improved CoQ10 production. Finally, another copy of the tac promoter with the UbiG gene (involved in the ubiquinone pathway of CoQ10 biosynthesis) was introduced into the engineered pathway. By optimizing the expression level of both the upstream and downstream pathway, CoQ10 production in the mutants was improved up to 93.34 mg/L (7.16 mg/g DCW), about twofold of the wild-type (48.25 mg/L, 3.24 mg/g DCW). PMID:24122603

  5. Differential Subplastidial Localization and Turnover of Enzymes Involved in Isoprenoid Biosynthesis in Chloroplasts

    PubMed Central

    Perello, Catalina; Llamas, Ernesto; Burlat, Vincent; Ortiz-Alcaide, Miriam; Phillips, Michael A.; Pulido, Pablo; Rodriguez-Concepcion, Manuel

    2016-01-01

    Plastidial isoprenoids are a diverse group of metabolites with roles in photosynthesis, growth regulation, and interaction with the environment. The methylerythritol 4-phosphate (MEP) pathway produces the metabolic precursors of all types of plastidial isoprenoids. Proteomics studies in Arabidopsis thaliana have shown that all the enzymes of the MEP pathway are localized in the plastid stroma. However, immunoblot analysis of chloroplast subfractions showed that the first two enzymes of the pathway, deoxyxylulose 5-phosphate synthase (DXS) and reductoisomerase (DXR), can also be found in non-stromal fractions. Both transient and stable expression of GFP-tagged DXS and DXR proteins confirmed the presence of the fusion proteins in distinct subplastidial compartments. In particular, DXR-GFP was found to accumulate in relatively large vesicles that could eventually be released from chloroplasts, presumably to be degraded by an autophagy-independent process. Together, we propose that protein-specific mechanisms control the localization and turnover of the first two enzymes of the MEP pathway in Arabidopsis chloroplasts. PMID:26919668

  6. Differential Subplastidial Localization and Turnover of Enzymes Involved in Isoprenoid Biosynthesis in Chloroplasts.

    PubMed

    Perello, Catalina; Llamas, Ernesto; Burlat, Vincent; Ortiz-Alcaide, Miriam; Phillips, Michael A; Pulido, Pablo; Rodriguez-Concepcion, Manuel

    2016-01-01

    Plastidial isoprenoids are a diverse group of metabolites with roles in photosynthesis, growth regulation, and interaction with the environment. The methylerythritol 4-phosphate (MEP) pathway produces the metabolic precursors of all types of plastidial isoprenoids. Proteomics studies in Arabidopsis thaliana have shown that all the enzymes of the MEP pathway are localized in the plastid stroma. However, immunoblot analysis of chloroplast subfractions showed that the first two enzymes of the pathway, deoxyxylulose 5-phosphate synthase (DXS) and reductoisomerase (DXR), can also be found in non-stromal fractions. Both transient and stable expression of GFP-tagged DXS and DXR proteins confirmed the presence of the fusion proteins in distinct subplastidial compartments. In particular, DXR-GFP was found to accumulate in relatively large vesicles that could eventually be released from chloroplasts, presumably to be degraded by an autophagy-independent process. Together, we propose that protein-specific mechanisms control the localization and turnover of the first two enzymes of the MEP pathway in Arabidopsis chloroplasts. PMID:26919668

  7. Transcript Quantification of Genes Involved in Steviol Glycoside Biosynthesis in Stevia rebaudiana Bertoni by Real-Time Polymerase Chain Reaction (RT-PCR).

    PubMed

    Modi, Arpan; Kumar, Nitish; Narayanan, Subhash

    2016-01-01

    Stevia (Stevia rebaudiana Bertoni) is a medicinal plant having sweet, diterpenoid glycosides known as steviol glycosides which are 200-300 times sweeter than sucrose (0.4 % solution). They are synthesized mainly in the leaves via plastid localized 2-C-methyl-D-erythrose-4-phosphate pathway (MEP pathway). Fifteen genes are involved in the formation of these glycosides. In the present protocol, a method for the quantification of transcripts of these genes is shown. The work involves RNA extraction and cDNA preparation, and therefore, procedures for the confirmation of DNA-free cDNA preparation have also been illustrated. Moreover, details of plant treatments are not mentioned as this protocol may apply to relative gene expression profile in any medicinal plant with any treatment. The treatments are numbered as T0 (Control), T1, T2, T3, and T4. PMID:27108325

  8. Construction and Evaluation of Desulfovibrio vulgaris Whole-Genome Oligonucleotide Microarrays

    SciTech Connect

    Z. He; Q. He; L. Wu; M.E. Clark; J.D. Wall; Jizhong Zhou; Matthew W. Fields

    2004-03-17

    Desulfovibrio vulgaris Hildenborough has been the focus of biochemical and physiological studies in the laboratory, and the metabolic versatility of this organism has been largely recognized, particularly the reduction of sulfate, fumarate, iron, uranium and chromium. In addition, a Desulfovibrio sp. has been shown to utilize uranium as the sole electron acceptor. D. vulgaris is a d-Proteobacterium with a genome size of 3.6 Mb and 3584 ORFs. The whole-genome microarrays of D. vulgaris have been constructed using 70mer oligonucleotides. All ORFs in the genome were represented with 3471 (97.1%) unique probes and 103 (2.9%) non-specific probes that may have cross-hybridization with other ORFs. In preparation for use of the experimental microarrays, artificial probes and targets were designed to assess specificity and sensitivity and identify optimal hybridization conditions for oligonucleotide microarrays. The results indicated that for 50mer and 70mer oligonucleotide arrays, hybridization at 45 C to 50 C, washing at 37 C and a wash time of 2.5 to 5 minutes obtained specific and strong hybridization signals. In order to evaluate the performance of the experimental microarrays, growth conditions were selected that were expected to give significant hybridization differences for different sets of genes. The initial evaluations were performed using D. vulgaris cells grown at logarithmic and stationary phases. Transcriptional analysis of D. vulgaris cells sampled during logarithmic phase growth indicated that 25% of annotated ORFs were up-regulated and 3% of annotated ORFs were downregulated compared to stationary phase cells. The up-regulated genes included ORFs predicted to be involved with acyl chain biosynthesis, amino acid ABC transporter, translational initiation factors, and ribosomal proteins. In the stationary phase growth cells, the two most up-regulated ORFs (70-fold) were annotated as a carboxynorspermidine decarboxylase and a 2C-methyl-D-erythritol-2

  9. A combination of whey protein and potassium bicarbonate supplements during head-down-tilt bed rest: Presentation of a multidisciplinary randomized controlled trial (MEP study)

    NASA Astrophysics Data System (ADS)

    Buehlmeier, Judith; Mulder, Edwin; Noppe, Alexandra; Frings-Meuthen, Petra; Angerer, Oliver; Rudwill, Floriane; Biolo, Gianni; Smith, Scott M.; Blanc, Stéphane; Heer, Martina

    2014-02-01

    Inactivity, as it appears during space flight and in bed rest, induces reduction of lean body and bone mass, glucose intolerance, and weakening of the cardiovascular system. Increased protein intake, whey protein in particular, has been proposed to counteract some of these effects, but has also been associated with negative effects on bone, likely caused by a correspondingly high ratio of acid to alkali precursors in the diet.

  10. Molecular structure, vibrational spectra, MEP, HOMO-LUMO and NBO analysis of Hf(SeO3)(SeO4)(H2O)4

    NASA Astrophysics Data System (ADS)

    Yankova, Rumyana; Genieva, Svetlana; Halachev, Nenko; Dimitrova, Ginka

    2016-02-01

    Hf(SeO3)(SeO4)(H2O)4 was obtained with the hydrothermal synthesis. The geometry optimization of this molecule was done by Density Functional Theory (DFT/B3LYP) method with 6-31G(d) basis set and LANL2DZ for Hf. The experimental infrared spectrum was compared with calculated and complete vibrational assignment was provided. The bond orders and the electronic properties of the molecule were calculated. The natural bond orbital analysis (NBO) was performed in order to study the intramolecular bonding interactions among bonds and delocalization of unpaired electrons. The calculated highest occupied molecular orbital (HOMO) and lowest unoccupied molecular orbital (LUMO) with frontier orbital gap were presented. The electrostatic potential was calculated in order to investigate the reaction properties of the molecule. The thermodynamic properties of the studied compound at different temperatures were calculated.

  11. Synthetic Routes to Methylerythritol Phosphate Pathway Intermediates and Downstream Isoprenoids

    PubMed Central

    Jarchow-Choy, Sarah K; Koppisch, Andrew T; Fox, David T

    2014-01-01

    Isoprenoids constitute the largest class of natural products with greater than 55,000 identified members. They play essential roles in maintaining proper cellular function leading to maintenance of human health, plant defense mechanisms against predators, and are often exploited for their beneficial properties in the pharmaceutical and nutraceutical industries. Most impressively, all known isoprenoids are derived from one of two C5-precursors, isopentenyl diphosphate (IPP) or dimethylallyl diphosphate (DMAPP). In order to study the enzyme transformations leading to the extensive structural diversity found within this class of compounds there must be access to the substrates. Sometimes, intermediates within a biological pathway can be isolated and used directly to study enzyme/pathway function. However, the primary route to most of the isoprenoid intermediates is through chemical catalysis. As such, this review provides the first exhaustive examination of synthetic routes to isoprenoid and isoprenoid precursors with particular emphasis on the syntheses of intermediates found as part of the 2C-methylerythritol 4-phosphate (MEP) pathway. In addition, representative syntheses are presented for the monoterpenes (C10), sesquiterpenes (C15), diterpenes (C20), triterpenes (C30) and tetraterpenes (C40). Finally, in some instances, the synthetic routes to substrate analogs found both within the MEP pathway and downstream isoprenoids are examined. PMID:25009443

  12. Environmental and Genetic Factors Associated with Solanesol Accumulation in Potato Leaves

    PubMed Central

    Campbell, Raymond; Freitag, Sabine; Bryan, Glenn J.; Stewart, Derek; Taylor, Mark A.

    2016-01-01

    Solanesol is a high value 45-carbon, unsaturated, all-trans-nonaprenol isoprenoid. Recently solanesol has received particular attention because of its utility, both in its own right and as a precursor in the production of numerous compounds used in the treatment of disease states. Solanesol is found mainly in solanaceous crops such as potato, tomato, tobacco and pepper where it accumulates in the foliage. There is considerable potential to explore the extraction of solanesol from these sources as a valuable co-product. In this study we have characterized the genetic variation in leaf solanesol content in a biparental, segregating diploid potato population. We demonstrate that potato leaf solanesol content is genetically controlled and identify several quantitative trait loci associated with leaf solanesol content. Transient over-expression of genes from the methylerythritol 4-phosphate (MEP) and mevalonic acid (MVA) pathways, either singly or in combination, resulted in enhanced accumulation of solanesol in leaves of Nicotiana benthamiana, providing insights for genetically engineering the pathway. We also demonstrate that in potato, leaf solanesol content is enhanced by up to six-fold on exposure to moderately elevated temperature and show corresponding changes in expression patterns of MEP and MVA genes. Our combined approaches offer new insights into solanesol accumulation and strategies for developing a bio-refinery approach to potato production. PMID:27610114

  13. Carbon partitioning to the terpenoid biosynthetic pathway enables heterologous β-phellandrene production in Escherichia coli cultures.

    PubMed

    Formighieri, Cinzia; Melis, Anastasios

    2014-12-01

    Escherichia coli was used as a microbial system for the heterologous synthesis of β-phellandrene, a monoterpene of plant origin with several potential commercial applications. Expression of Lavandula angustifolia β-phellandrene synthase (PHLS), alone or in combination with Picea abies geranyl-diphosphate synthase in E. coli, resulted in no β-phellandrene accumulation, in sharp contrast to observations with PHLS-transformed cyanobacteria. Lack of β-phellandrene biosynthesis in E. coli was attributed to the limited endogenous carbon partitioning through the native 2-C-methylerythritol-4-phosphate (MEP) pathway. Heterologous co-expression of the mevalonic acid pathway, enhancing cellular carbon partitioning and flux toward the universal isoprenoid precursors, isopentenyl-diphosphate and dimethylallyl-diphosphate, was required to confer β-phellandrene production. Differences in endogenous carbon flux toward the synthesis of isoprenoids between photosynthetic (Synechocystis) and non-photosynthetic bacteria (E. coli) are discussed in terms of differences in the regulation of carbon partitioning through the MEP biosynthetic pathway in the two systems. PMID:25116411

  14. Environmental and Genetic Factors Associated with Solanesol Accumulation in Potato Leaves.

    PubMed

    Campbell, Raymond; Freitag, Sabine; Bryan, Glenn J; Stewart, Derek; Taylor, Mark A

    2016-01-01

    Solanesol is a high value 45-carbon, unsaturated, all-trans-nonaprenol isoprenoid. Recently solanesol has received particular attention because of its utility, both in its own right and as a precursor in the production of numerous compounds used in the treatment of disease states. Solanesol is found mainly in solanaceous crops such as potato, tomato, tobacco and pepper where it accumulates in the foliage. There is considerable potential to explore the extraction of solanesol from these sources as a valuable co-product. In this study we have characterized the genetic variation in leaf solanesol content in a biparental, segregating diploid potato population. We demonstrate that potato leaf solanesol content is genetically controlled and identify several quantitative trait loci associated with leaf solanesol content. Transient over-expression of genes from the methylerythritol 4-phosphate (MEP) and mevalonic acid (MVA) pathways, either singly or in combination, resulted in enhanced accumulation of solanesol in leaves of Nicotiana benthamiana, providing insights for genetically engineering the pathway. We also demonstrate that in potato, leaf solanesol content is enhanced by up to six-fold on exposure to moderately elevated temperature and show corresponding changes in expression patterns of MEP and MVA genes. Our combined approaches offer new insights into solanesol accumulation and strategies for developing a bio-refinery approach to potato production. PMID:27610114

  15. Predictability of Motor Outcome According to the Time of Motor Evoked Potentials From the Onset of Stroke in Patients With Putaminal Hemorrhage

    PubMed Central

    Kwon, Yong Min; Jang, Sung Ho

    2015-01-01

    Objective To determine the predictability of motor evoked potentials (MEP) in patients with putaminal hemorrhage (PH) according to the time of MEP from the onset of stroke. Methods Sixty consecutive patients with PH from January 2006 to November 2013 were retrospectively reviewed. Motor function of affected extremities was measured at onset time and at six months after the onset. Patients were classified into two groups according to the time of MEP from the onset of stroke: early MEP group (within 15 days from onset) and late MEP group (16-30 days from onset). Patients were also classified into two groups according to the presence of MEP on the affected abductor pollicis brevis (APB): MEP (+) group-patients (showing MEP in the affected APB) and MEP (-) group-patients (no MEP in the affected APB). Motor outcome was compared between the two early and late MEP groups or between the presence and absence of MEP in the affected APB groups. Results For patients with MEP (+), a larger portion in the late MEP group showed good prognosis compared to the early MEP group (late MEP, 94.4%; early MEP, 80%). In contrast, in patients with MEP (-), a larger portion of patients in the late MEP group showed bad prognosis compared to the early MEP group (late MEP, 80%; early MEP, 71.4%). No significant improvement of MI between MEP (+) and MEP (-) was observed when MEP was performed early or late. Conclusion Our results revealed that the predictability of motor outcome might be better if MEP is performed late compared to that when MEP is performed early in patients with PH. PMID:26361591

  16. Evolutionary diversification and characterization of the eubacterial gene family encoding DXR type II, an alternative isoprenoid biosynthetic enzyme

    PubMed Central

    2013-01-01

    Background Isoprenoids constitute a vast family of natural compounds performing diverse and essential functions in all domains of life. In most eubacteria, isoprenoids are synthesized through the methylerythritol 4-phosphate (MEP) pathway. The production of MEP is usually catalyzed by deoxyxylulose 5-phosphate reductoisomerase (DXR-I) but a few organisms use an alternative DXR-like enzyme (DXR-II). Results Searches through 1498 bacterial complete proteomes detected 130 sequences with similarity to DXR-II. Phylogenetic analysis identified three well-resolved clades: the DXR-II family (clustering 53 sequences including eleven experimentally verified as functional enzymes able to produce MEP), and two previously uncharacterized NAD(P)-dependent oxidoreductase families (designated DLO1 and DLO2 for DXR-II-like oxidoreductases 1 and 2). Our analyses identified amino acid changes critical for the acquisition of DXR-II biochemical function through type-I functional divergence, two of them mapping onto key residues for DXR-II activity. DXR-II showed a markedly discontinuous distribution, which was verified at several levels: taxonomic (being predominantly found in Alphaproteobacteria and Firmicutes), metabolic (being mostly found in bacteria with complete functional MEP pathways with or without DXR-I), and phenotypic (as no biological/phenotypic property was found to be preferentially distributed among DXR-II-containing strains, apart from pathogenicity in animals). By performing a thorough comparative sequence analysis of GC content, 3:1 dinucleotide frequencies, codon usage and codon adaptation indexes (CAI) between DXR-II sequences and their corresponding genomes, we examined the role of horizontal gene transfer (HGT), as opposed to an scenario of massive gene loss, in the evolutionary origin and diversification of the DXR-II subfamily in bacteria. Conclusions Our analyses support a single origin of the DXR-II family through functional divergence, in which constitutes

  17. Engineering of Recombinant Poplar Deoxy-D-Xylulose-5-Phosphate Synthase (PtDXS) by Site-Directed Mutagenesis Improves Its Activity

    PubMed Central

    Banerjee, Aparajita; Preiser, Alyssa L.

    2016-01-01

    Deoxyxylulose 5-phosphate synthase (DXS), a thiamine diphosphate (ThDP) dependent enzyme, plays a regulatory role in the methylerythritol 4-phosphate (MEP) pathway. Isopentenyl diphosphate (IDP) and dimethylallyl diphosphate (DMADP), the end products of this pathway, inhibit DXS by competing with ThDP. Feedback inhibition of DXS by IDP and DMADP constitutes a significant metabolic regulation of this pathway. The aim of this work was to experimentally test the effect of key residues of recombinant poplar DXS (PtDXS) in binding both ThDP and IDP. This work also described the engineering of PtDXS to improve the enzymatic activity by reducing its inhibition by IDP and DMADP. We have designed and tested modifications of PtDXS in an attempt to reduce inhibition by IDP. This could possibly be valuable by removing a feedback that limits the usefulness of the MEP pathway in biotechnological applications. Both ThDP and IDP use similar interactions for binding at the active site of the enzyme, however, ThDP being a larger molecule has more anchoring sites at the active site of the enzyme as compared to the inhibitors. A predicted enzyme structure was examined to find ligand-enzyme interactions, which are relatively more important for inhibitor-enzyme binding than ThDP-enzyme binding, followed by their modifications so that the binding of the inhibitors can be selectively affected compared to ThDP. Two alanine residues important for binding ThDP and the inhibitors were mutated to glycine. In two of the cases, both the IDP inhibition and the overall activity were increased. In another case, both the IDP inhibition and the overall activity were reduced. This provides proof of concept that it is possible to reduce the feedback from IDP on DXS activity. PMID:27548482

  18. Pheophytinase Knockdown Impacts Carbon Metabolism and Nutraceutical Content Under Normal Growth Conditions in Tomato.

    PubMed

    Lira, Bruno Silvestre; Rosado, Daniele; Almeida, Juliana; de Souza, Amanda Pereira; Buckeridge, Marcos Silveira; Purgatto, Eduardo; Guyer, Luzia; Hörtensteiner, Stefan; Freschi, Luciano; Rossi, Magdalena

    2016-03-01

    Although chlorophyll (Chl) degradation is an essential biochemical pathway for plant physiology, our knowledge regarding this process still has unfilled gaps. Pheophytinase (PPH) was shown to be essential for Chl breakdown in dark-induced senescent leaves. However, the catalyzing enzymes involved in pigment turnover and fruit ripening-associated degreening are still controversial. Chl metabolism is closely linked to the biosynthesis of other isoprenoid-derived compounds, such as carotenoids and tocopherols, which are also components of the photosynthetic machinery. Chls, carotenoids and tocopherols share a common precursor, geranylgeranyl diphosphate, produced by the plastidial methylerythritol 4-phosphate (MEP) pathway. Additionally, the Chl degradation-derived phytol can be incorporated into tocopherol biosynthesis. In this context, tomato turns out to be an interesting model to address isoprenoid-metabolic cross-talk since fruit ripening combines degreening and an intensely active MEP leading to carotenoid accumulation. Here, we investigate the impact of PPH deficiency beyond senescence by the comprehensive phenotyping of SlPPH-knockdown tomato plants. In leaves, photosynthetic parameters indicate altered energy usage of excited Chl. As a mitigatory effect, photosynthesis-associated carotenoids increased while tocopherol content remained constant. Additionally, starch and soluble sugar profiles revealed a distinct pattern of carbon allocation in leaves that suggests enhanced sucrose exportation. The higher levels of carbohydrates in sink organs down-regulated carotenoid biosynthesis. Additionally, the reduction in Chl-derived phytol recycling resulted in decreased tocopherol content in transgenic ripe fruits. Summing up, tocopherol and carotenoid metabolism, together with the antioxidant capacity of the hydrophilic and hydrophobic fractions, were differentially affected in leaves and fruits of the transgenic plants. Thus, in tomato, PPH plays a role beyond

  19. Isopentenyl diphosphate (IPP)-bypass mevalonate pathways for isopentenol production.

    PubMed

    Kang, Aram; George, Kevin W; Wang, George; Baidoo, Edward; Keasling, Jay D; Lee, Taek Soon

    2016-03-01

    Branched C5 alcohols are promising biofuels with favorable combustion properties. A mevalonate (MVA)-based isoprenoid biosynthetic pathway for C5 alcohols was constructed in Escherichia coli using genes from several organisms, and the pathway was optimized to achieve over 50% theoretical yield. Although the MVA pathway is energetically less efficient than the native methylerythritol 4-phosphate (MEP) pathway, implementing the MVA pathway in bacterial hosts such as E. coli is advantageous due to its lack of endogenous regulation. The MVA and MEP pathways intersect at isopentenyl diphosphate (IPP), the direct precursor to isoprenoid-derived C5 alcohols and initial precursor to longer chain terpenes, which makes independent regulation of the pathways difficult. In pursuit of the complete "decoupling" of the MVA pathway from native cellular regulation, we designed novel IPP-bypass MVA pathways for C5 alcohol production by utilizing promiscuous activities of two enzymes, phosphomevalonate decarboxylase (PMD) and an E. coli-endogenous phosphatase (AphA). These bypass pathways have reduced energetic requirements, are further decoupled from intrinsic regulation, and are free from IPP-related toxicity. In addition to these benefits, we demonstrate that reduced aeration rate has less impact on the bypass pathway than the original MVA pathway. Finally, we showed that performance of the bypass pathway was primarily determined by the activity of PMD. We designed PMD mutants with improved activity and demonstrated titer increases in the mutant strains. These modified pathways would be a good platform for industrial production of isopentenol and related chemicals such as isoprene. PMID:26708516

  20. Novel insights into structure–function mechanism and tissue-specific expression profiling of full-length dxr gene from Cymbopogon winterianus

    PubMed Central

    Devi, Kamalakshi; Dehury, Budheswar; Phukon, Munmi; Modi, Mahendra Kumar; Sen, Priyabrata

    2015-01-01

    The 1-deoxy-d-xylulose-5-phosphate reductoisomerase (DXR; EC1.1.1.267), an NADPH-dependent reductase, plays a pivotal role in the methylerythritol 4-phosphate pathway (MEP), in the conversion of 1-deoxy-d-xylulose-5-phosphate (DXP) into MEP. The sheath and leaf of citronella (Cymbopogon winterianus) accumulates large amount of terpenes and sesquiterpenes with proven medicinal value and economic uses. Thus, sequencing of full length dxr gene and its characterization seems to be a valuable resource in metabolic engineering to alter the flux of isoprenoid active ingredients in plants. In this study, full length DXR from citronella was characterized through in silico and tissue-specific expression studies to explain its structure–function mechanism, mode of cofactor recognition and differential expression. The modelled DXR has a three-domain architecture and its active site comprised of a cofactor (NADPH) binding pocket and the substrate-binding pocket. Molecular dynamics simulation studies indicated that DXR model retained most of its secondary structure during 10 ns simulation in aqueous solution. The modelled DXR superimposes well with its closest structural homolog but subtle variations in the charge distribution over the cofactor recognition site were noticed. Molecular docking study revealed critical residues aiding tight anchoring NADPH within the active pocket of DXR. Tissue-specific differential expression analysis using semi-quantitative RT-PCR and qRT-PCR in various tissues of citronella plant revealed distinct differential expression of DXR. To our knowledge, this is the first ever report on DXR from the important medicinal plant citronella and further characterization of this gene will open up better avenues for metabolic engineering of secondary metabolite pathway genes from medicinal plants in the near future. PMID:25941629

  1. An in silico structural insights into Plasmodium LytB protein and its inhibition.

    PubMed

    Bhuyan, Rajabrata; Nandy, Suman Kumar; Seal, Alpana

    2015-01-01

    In most of the pathogenic organisms including Plasmodium falciparum, isoprenoids are synthesized via MEP (MethylErythritol 4-Phosphate) pathway. LytB is the last enzyme of this pathway which catalyzes the conversion of (E)-4-hydroxy-3-methylbut-2-en-1-yl diphosphate (HMBPP) into the two isoprenoid precursors: isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP). Since the MEP pathway is not used by humans, it represents an attractive target for the development of new anti-malarial compounds or inhibitors. Here a systematic in silico study has been conducted to get an insight into the structure of Plasmodium lytB as well as its affinities towards different inhibitors. We used comparative modeling technique to predict the three-dimensional (3D) structure of Plasmodium LytB taking Escherichia coli LytB protein (PDB ID: 3KE8) as template and the model was subsequently refined through molecular dynamics (MD) simulation. A large ligand data-set containing diphospate group was subjected for virtual screening against the target using GOLD 5.2 program. Considering the mode of binding and affinities, 17 leads were selected on basis of binding energies in comparison to its substrate HMBPP (Gold.Chemscore.DG: -20.9734 kcal/mol). Among them, five were discarded because of their inhibitory activity towards other human enzymes. The rest 12 potential leads carry all the properties of any "drug like" molecule and the knowledge of Plasmodium LytB-inhibitory mechanism which can provide valuable support for the anti-malarial-inhibitor design in future. PMID:25011618

  2. Vibrational (FT-IR, FT-Raman) and UV-Visible spectroscopic studies, HOMO-LUMO, NBO, NLO and MEP analysis of Benzyl (imino (1H-pyrazol-1-yl) methyl) carbamate using DFT calculaions

    NASA Astrophysics Data System (ADS)

    Shankar Rao, Y. B.; Prasad, M. V. S.; Udaya Sri, N.; Veeraiah, V.

    2016-03-01

    This paper contains a combined experimental and theoretical study of vibrational and electronic properties of Benzyl(imino(1H-pyrazol-1-yl)methyl)carbamate (BPMC) molecule. The FT-IR and FT-Raman spectra of the title molecule in solid phase were recorded in the region 4000-400 cm-1 and 4000-50 cm-1, respectively. The UV absorption spectrum of the studied compound dissolved in ethanol was recorded in the range of 180-400 nm. The molecular geometries calculated using density functional theory (DFT) was compared with available experimental data. The vibrational spectra calculated at the B3LYP/6-31G(d,p) level were compared with the experimental spectra and assignment to each vibrational frequency was assigned on the basis of potential energy distribution (PED). The calculated electronic and nonlinear optical properties of the title molecule were reported. Furthermore, the thermodynamic properties of the molecule were discussed.

  3. FT-IR, NBO, HOMO-LUMO, MEP analysis and molecular docking study of Methyl N-({[2-(2-methoxyacetamido)-4-(phenylsulfanyl)phenyl]amino}[(methoxycarbonyl) imino]methyl)carbamate

    NASA Astrophysics Data System (ADS)

    Panicker, C. Yohannan; Varghese, Hema Tresa; Narayana, B.; Divya, K.; Sarojini, B. K.; War, Javeed Ahmad; Van Alsenoy, C.; Fun, H. K.

    2015-09-01

    The optimized molecular structure, vibrational frequencies, corresponding vibrational assignments of Methyl N-({[2-(2-methoxyacetamido)-4-(phenylsulfanyl) phenyl]amino} [(methoxycarbonyl)imino]methyl)carbamate have been investigated using HF and DFT levels of calculations. The geometrical parameters are in agreement with XRD data. The stability of the molecule arising from hyper-conjugative interaction and charge delocalization has been analyzed using NBO analysis. The HOMO and LUMO analysis is used to determine the charge transfer within the molecule. Molecular electrostatic potential study was also performed. The first and second hyperpolarizability was calculated in order to find its role in nonlinear optics. Molecular docking studies are also reported. Prediction of Activity Spectra analysis of the title compound predicts anthelmintic and antiparasitic activity as the most probable activity with Pa (probability to be active) value of 0.808 and 0.797, respectively. Molecular docking studies show that both the phenyl groups and the carbonyl oxygens of the molecule are crucial for bonding and these results draw us to the conclusion that the compound might exhibit pteridine reductase inhibitory activity.

  4. Vibrational assignments, spectroscopic investigation (FT-IR and FT-Raman), NBO, MEP, HOMO‒LUMO analysis and intermolecular hydrogen bonding interactions of 7-fluoroisatin, 7-bromoisatin and 1-methylisatin ‒ A comparative study

    NASA Astrophysics Data System (ADS)

    Polat, Turgay; Bulut, Fatih; Arıcan, Ilknur; Kandemirli, Fatma; Yildirim, Gürcan

    2015-12-01

    In this comprehensive study, theoretical and experimental studies were carried out on 7-fluoroisatin, 7-bromoisatin and 1-methylisatin using FT-Raman and FT-IR spectra. The optimized geometrical parameters and theoretical vibrational frequencies were calculated by means of density functional theory (DFT/B3LYP) with 6-311++G(d,p) basis set based on scaled quantum mechanical (SQM) method for the first time. The relative abundances of the possible tautomers or conformers found were calculated with respect to the Boltzmann distribution. Moreover, the harmonic vibrational frequencies including IR and Raman intensities, thermodynamic and electronic parameters were computed in detail. The effects of substituents -F, ‒Br and -CH3 on the crucial characteristics pertaining to the title compound of isatin were investigated, and the obtained data were compared with each other. Natural bond orbital (NBO) analysis was applied to study the stability arising from charge delocalization along with the compound. The chemical reactivity parameters (chemical hardness and softness, electronegativity, chemical potential and electrophilicity index) were discussed clearly. The HOMO and LUMO energies determined showed that the serious charge transfer occurs in the title molecules studied. Furthermore, the size, shape, charge density distributions and chemical reactivity sites belonging to the molecules were obtained by mapping electron density isosurface with electrostatic potential surfaces (ESP). Additionally, the hydrogen-bonded complexes were simulated to describe the roles of intermolecular hydrogen bonding on the molecular structures and vibrational frequencies.

  5. Crystallographic, experimental (FT-IR and FT-RS) and theoretical (DFT) investigation, UV-Vis, MEP, HOMO-LUMO and NBO/NLMO of (E)-1-[1-(4-Chlorophenyl)ethylidene]thiosemicarbazide

    NASA Astrophysics Data System (ADS)

    Saravanan, R. R.; Seshadri, S.; Gunasekaran, S.; Mendoza-Meroño, R.; Garcia-Granda, S.

    2014-03-01

    Crystallographic, experimental (FT-IR and FT-RS) and theoretical density function theory (DFT) and UV-Vis spectra of (E)-1-[1-(4-Chlorophenyl)ethylidene]thiosemicarbazide) (ECET) are investigated. The optimized geometry of the compound was calculated from the DFT-B3LYP gradient calculations employing 6-31G (d,p) basis set and calculated vibrational frequencies are evaluated via comparison with experimental values. Molecular stability has been analyzed using Natural Bond Orbital (NBO) and Natural Localized Molecular Orbital (NLMO) analysis and the limits of the molecular electrostatic potential observed. The calculated HOMO and LUMO energies show the charge transfer occurs within the molecule.

  6. Synthesis, growth, structural and HOMO and LUMO, MEP analysis of a new stilbazolium derivative crystal: A enhanced third-order NLO properties with a high laser-induced damage threshold for NLO applications

    NASA Astrophysics Data System (ADS)

    Senthil, K.; Kalainathan, S.; Hamada, F.; Yamada, M.; Aravindan, P. G.

    2015-08-01

    A new organic third-order nonlinear optical crystal from stilbazolium family 2-[2-(4-methoxy-phenyl) vinyl]-1-methyl-pyridinium tetrafluoroborate (4MSTB) has been synthesized and grown by slow evaporation method for the first time. The grown crystal structure was confirmed by single crystal X-ray diffraction analysis, and it is revealed that the grown crystal crystallized in a triclinic crystal system with centrosymmetric space group P 1 bar . The HOMO and LUMO energies were calculated for the grown crystal explains charge transfer takes place within the molecule and confirms the suitability of the title crystal for NLO applications. The presence of various vibration modes of expected functional groups was identified by FT-IR analysis. The transmittance ability of the grown crystal was also analyzed by using UV-Vis-NIR spectral studies and shows that the crystal has no absorption of light in the entire Vis-NIR region. The thermal stability of the title crystal has been investigated by TGA/DTA studies and revealed that the material was thermally stable up to the melting point, 193 °C. The hardness number, Meyer index, yield strength, and elastic stiffness constant has been estimated for the grown 4MSTB crystal using Vickers microhardness tester. Photoluminescence excitation studies showed green emission radiation occurred at 517 nm. The dielectric properties of the grown crystal have been analyzed as a function of temperature over a wide range of frequency (50 Hz-5 MHz) by using LCR meter. The result of ac electrical conductivity of 4MSTB was found to be 5.25 × 10-5 (Ω m)-1. The laser damage threshold (LDT) energy for the grown crystal has been measured by using a Q-switched Nd:YAG laser as a source in single-shot mode (1064 nm, 10 Hz, 420 mJ). The result of LDT indicates that grown title crystal has excellent resistance to laser radiation than those of known some inorganic NLO materials. The chemical etching studies were carried out to assess the perfection of the grown crystals. Its third-order nonlinear optical properties were investigated by Z-scan technique and proved that the 4MSTB crystal possesses two-photon absorptions (TPA) and the self-defocusing effect. The second-order molecular hyperpolarizability (γ) value at 632.8 nm is calculated to be 3.555 × 10-34 esu. The photoconductivity study of 4MSTB reveals that the negative photoconducting nature. The obtained all the results in the present work are making a 4MSTB promising candidate for the possible applications in optical switches, optical power limiter, and non-linear optical applications.

  7. De Novo Transcriptome and Expression Profile Analysis to Reveal Genes and Pathways Potentially Involved in Cantharidin Biosynthesis in the Blister Beetle Mylabris cichorii.

    PubMed

    Huang, Yi; Wang, Zhongkang; Zha, Shenfang; Wang, Yu; Jiang, Wei; Liao, Yufeng; Song, Zhangyong; Qi, Zhaoran; Yin, Youping

    2016-01-01

    The dried body of Mylabris cichorii is well-known Chinese traditional medicine. The sesquiterpenoid cantharidin, which is secreted mostly by adult male beetles, has recently been used as an anti-cancer drug. However, little is known about the mechanisms of cantharidin biosynthesis. Furthermore, there is currently no genomic or transcriptomic information for M. cichorii. In this study, we performed de novo assembly transcriptome of M. cichorii using the Illumina Hiseq2000. A single run produced 9.19 Gb of clean nucleotides comprising 29,247 sequences, including 23,739 annotated sequences (about 81%). We also constructed two expression profile libraries (20-25 day-old adult males and 20-25 day-old adult females) and discovered 2,465 significantly differentially-expressed genes. Putative genes and pathways involved in the biosynthesis of cantharidin were then characterized. We also found that cantharidin biosynthesis in M. cichorii might only occur via the mevalonate (MVA) pathway, not via the methylerythritol 4-phosphate/deoxyxylulose 5-phosphate (MEP/DOXP) pathway or a mixture of these. Besides, we considered that cantharidin biosynthesis might be related to the juvenile hormone (JH) biosynthesis or degradation. The results of transcriptome and expression profiling analysis provide a comprehensive sequence resource for M. cichorii that could facilitate the in-depth study of candidate genes and pathways involved in cantharidin biosynthesis, and may thus help to improve our understanding of the mechanisms of cantharidin biosynthesis in blister beetles. PMID:26752526

  8. De Novo Transcriptome and Expression Profile Analysis to Reveal Genes and Pathways Potentially Involved in Cantharidin Biosynthesis in the Blister Beetle Mylabris cichorii

    PubMed Central

    Huang, Yi; Wang, Zhongkang; Zha, Shenfang; Wang, Yu; Jiang, Wei; Liao, Yufeng; Song, Zhangyong; Qi, Zhaoran; Yin, Youping

    2016-01-01

    The dried body of Mylabris cichorii is well-known Chinese traditional medicine. The sesquiterpenoid cantharidin, which is secreted mostly by adult male beetles, has recently been used as an anti-cancer drug. However, little is known about the mechanisms of cantharidin biosynthesis. Furthermore, there is currently no genomic or transcriptomic information for M. cichorii. In this study, we performed de novo assembly transcriptome of M. cichorii using the Illumina Hiseq2000. A single run produced 9.19 Gb of clean nucleotides comprising 29,247 sequences, including 23,739 annotated sequences (about 81%). We also constructed two expression profile libraries (20–25 day-old adult males and 20–25 day-old adult females) and discovered 2,465 significantly differentially-expressed genes. Putative genes and pathways involved in the biosynthesis of cantharidin were then characterized. We also found that cantharidin biosynthesis in M. cichorii might only occur via the mevalonate (MVA) pathway, not via the methylerythritol 4-phosphate/deoxyxylulose 5-phosphate (MEP/DOXP) pathway or a mixture of these. Besides, we considered that cantharidin biosynthesis might be related to the juvenile hormone (JH) biosynthesis or degradation. The results of transcriptome and expression profiling analysis provide a comprehensive sequence resource for M. cichorii that could facilitate the in-depth study of candidate genes and pathways involved in cantharidin biosynthesis, and may thus help to improve our understanding of the mechanisms of cantharidin biosynthesis in blister beetles. PMID:26752526

  9. Synthesis and bioactivity of β-substituted fosmidomycin analogues targeting 1-deoxy-D-xylulose-5-phosphate reductoisomerase.

    PubMed

    Chofor, René; Sooriyaarachchi, Sanjeewani; Risseeuw, Martijn D P; Bergfors, Terese; Pouyez, Jenny; Johny, Chinchu; Haymond, Amanda; Everaert, Annelien; Dowd, Cynthia S; Maes, Louis; Coenye, Tom; Alex, Alexander; Couch, Robin D; Jones, T Alwyn; Wouters, Johan; Mowbray, Sherry L; Van Calenbergh, Serge

    2015-04-01

    Blocking the 2-C-methyl-d-erythrithol-4-phosphate (MEP) pathway for isoprenoid biosynthesis offers interesting prospects for inhibiting Plasmodium or Mycobacterium spp. growth. Fosmidomycin (1) and its homologue FR900098 (2) potently inhibit 1-deoxy-d-xylulose-5-phosphate reductoisomerase (Dxr), a key enzyme in this pathway. Here we introduced aryl or aralkyl substituents at the β-position of the hydroxamate analogue of 2. While direct addition of a β-aryl moiety resulted in poor inhibition, longer linkers between the carbon backbone and the phenyl ring were generally associated with better binding to the enzymes. X-ray structures of the parasite Dxr-inhibitor complexes show that the "longer" compounds generate a substantially different flap structure, in which a key tryptophan residue is displaced, and the aromatic group of the ligand lies between the tryptophan and the hydroxamate's methyl group. Although the most promising new Dxr inhibitors lack activity against Escherichia coli and Mycobacterium smegmatis, they proved to be highly potent inhibitors of Plasmodium falciparum in vitro growth. PMID:25781377

  10. Identification and elimination of metabolic bottlenecks in the quinone modification pathway for enhanced coenzyme Q10 production in Rhodobacter sphaeroides.

    PubMed

    Lu, Wenqiang; Ye, Lidan; Lv, Xiaomei; Xie, Wenping; Gu, Jiali; Chen, Zhaofeng; Zhu, Yongqiang; Li, Aipeng; Yu, Hongwei

    2015-05-01

    In this report, UbiE and UbiH in the quinone modification pathway (QMP) were identified in addition to UbiG as bottleneck enzymes in the CoQ10 biosynthesis by Rhodobacter sphaeroides. The CoQ10 content was enhanced after co-overexpression of UbiE and UbiG, however, accompanied by the accumulation of the intermediate 10P-MMBQ. UbiH was then co-overexpressed to pull the metabolic flux towards downstream, resulting in an elevated CoQ10 productivity and decreased biomass. On the other hand, the expression levels of UbiE and UbiG were tuned to eliminate the intermediate accumulation, however at the sacrifice of productivity. To alleviate the detrimental effect on either productivity or cell growth, we tried to fuse UbiG with UbiE and localize them onto the membrane to elevate intermediate conversion. By fusing UbiE and UbiG to pufX, CoQ10 was accumulated to 108.51±2.76mg/L with a biomass of 12.2±0.9g/L. At last, we combined the optimized QMP and the previously engineered 2-methyl-d-erythritol-4-phosphate pathway (MEP) to further boost CoQ10 biosynthesis, resulting in a strain with 138±2.64mg/L CoQ10 production. PMID:25817210

  11. Transcriptome analysis of bitter acid biosynthesis and precursor pathways in hop (Humulus lupulus)

    PubMed Central

    2013-01-01

    Background Bitter acids (e.g. humulone) are prenylated polyketides synthesized in lupulin glands of the hop plant (Humulus lupulus) which are important contributors to the bitter flavour and stability of beer. Bitter acids are formed from acyl-CoA precursors derived from branched-chain amino acid (BCAA) degradation and C5 prenyl diphosphates from the methyl-D-erythritol 4-phosphate (MEP) pathway. We used RNA sequencing (RNA-seq) to obtain the transcriptomes of isolated lupulin glands, cones with glands removed and leaves from high α-acid hop cultivars, and analyzed these datasets for genes involved in bitter acid biosynthesis including the supply of major precursors. We also measured the levels of BCAAs, acyl-CoA intermediates, and bitter acids in glands, cones and leaves. Results Transcripts encoding all the enzymes of BCAA metabolism were significantly more abundant in lupulin glands, indicating that BCAA biosynthesis and subsequent degradation occurs in these specialized cells. Branched-chain acyl-CoAs and bitter acids were present at higher levels in glands compared with leaves and cones. RNA-seq analysis showed the gland-specific expression of the MEP pathway, enzymes of sucrose degradation and several transcription factors that may regulate bitter acid biosynthesis in glands. Two branched-chain aminotransferase (BCAT) enzymes, HlBCAT1 and HlBCAT2, were abundant, with gene expression quantification by RNA-seq and qRT-PCR indicating that HlBCAT1 was specific to glands while HlBCAT2 was present in glands, cones and leaves. Recombinant HlBCAT1 and HlBCAT2 catalyzed forward (biosynthetic) and reverse (catabolic) reactions with similar kinetic parameters. HlBCAT1 is targeted to mitochondria where it likely plays a role in BCAA catabolism. HlBCAT2 is a plastidial enzyme likely involved in BCAA biosynthesis. Phylogenetic analysis of the hop BCATs and those from other plants showed that they group into distinct biosynthetic (plastidial) and catabolic (mitochondrial

  12. Facilitation of magnetic motor evoked potentials during the mixed nerve silent period.

    PubMed

    Young, M S; Triggs, W J; Gerstle, G

    1995-11-01

    We studied motor neuron excitability during the mixed nerve silent period (MNSP) in a hand muscle using magnetic motor evoked potentials (MEPs) and F-waves. MEPs elicited between the V1 and V2 potentials of the MNSP were much larger than control MEPs elicited at rest, and were even comparable in size to control MEPs elicited during voluntary contraction. This facilitation of MEPs occurred without shortening of MEP latency, suggesting a supraspinal mechanism. MEPs were facilitated during the MNSP when elicited with a figure-8-shaped coil in a posterior-anterior orientation, but not when MEPs of the same size were elicited with the coil held in a lateral-medial orientation. F-waves elicited during the MNSP were variable between subjects, and not consistently different from control F-waves elicited at rest. Our findings may reflect increased cortical motor excitability during the MNSP, possibly related to activation of muscle afferents by mixed nerve stimulation. PMID:7565926

  13. Use of a cloned multidrug resistance gene for coamplification and overproduction of major excreted protein, a transformation-regulated secreted acid protease

    SciTech Connect

    Kane, S.E.; Troen, B.R.; Gal, S.; Ueda, K.; Pastan, I.; Gottesman, M.M.

    1988-08-01

    Malignantly transformed mouse fibroblasts synthesize and secrete large amounts of major excreted protein (MEP), a 39,000-dalton precursor to an acid protease (cathepsin L). To evaluate the possible role of this protease in the transformed phenotype, the authors transfected cloned genes for mouse or human MEP into mouse MIH 3T3 cells with an expression vector for the dominant, selectable human multidrug resistance (MDR1) gene. The cotransfected MEP sequences were efficiently coamplified and transcribed during stepwise selection for multidrug resistance in colchicine. The transfected NIH 3T3 cell lines containing amplified MEP sequences synthesized as much MEP as did Kirsten sarcoma virus-transformed NIH 3T3 cells. The MEP synthesized by cells transfected with the cloned mouse and human MEP genes were also secreted. Elevated synthesis and secretion of MEP by NIH 3T3 cells did not change the nontransformed phenotype of these cells.

  14. 16 CFR 1203.4 - Definitions.

    Code of Federal Regulations, 2010 CFR

    2010-01-01

    ... a polyurethane rubber, used as a consistent impact medium for the systems check procedure. The MEP.... The MEP shall be affixed to the top surface of a flat 6.35 mm (1/4 in) thick aluminum plate....

  15. 16 CFR 1203.4 - Definitions.

    Code of Federal Regulations, 2012 CFR

    2012-01-01

    ... a polyurethane rubber, used as a consistent impact medium for the systems check procedure. The MEP.... The MEP shall be affixed to the top surface of a flat 6.35 mm (1/4 in) thick aluminum plate....

  16. Migrant Children: Education and HHS Need To Improve the Exchange of Participant Information. Report to Congressional Requesters.

    ERIC Educational Resources Information Center

    Fagnoni, Cynthia M.

    The Migrant Education Program (MEP) and the Migrant Head Start (MHS) program have served millions of migrant children, but little is known about program outcomes. The Government Accounting Office examined: (1) MEP and MHS goals, operations, clients, and services; (2) coordination of MEP and MHS services within and between programs by the…

  17. Identification and Recruitment: Trends and Issues.

    ERIC Educational Resources Information Center

    Duron, Susan

    This chapter examines trends and issues related to the identification and recruitment of migrant children and youth for the Migrant Education Program (MEP). The MEP is federally funded and implemented by the states. Requirements and definitions of student eligibility and the functions of state MEP programs are spelled out in the Elementary and…

  18. Bacterial population succession and adaptation affected by insecticide application and soil spraying history

    PubMed Central

    Itoh, Hideomi; Navarro, Ronald; Takeshita, Kazutaka; Tago, Kanako; Hayatsu, Masahito; Hori, Tomoyuki; Kikuchi, Yoshitomo

    2014-01-01

    Although microbial communities have varying degrees of exposure to environmental stresses such as chemical pollution, little is known on how these communities respond to environmental disturbances and how past disturbance history affects these community-level responses. To comprehensively understand the effect of organophosphorus insecticide application on microbiota in soils with or without insecticide-spraying history, we investigated the microbial succession in response to the addition of fenitrothion [O,O-dimethyl O-(3-methyl-p-nitrophenyl) phosphorothioate, abbreviated as MEP] by culture-dependent experiments and deep sequencing of 16S rRNA genes. Despite similar microbial composition at the initial stage, microbial response to MEP application was remarkably different between soils with and without MEP-spraying history. MEP-degrading microbes more rapidly increased in the soils with MEP-spraying history, suggesting that MEP-degrading bacteria might already exist at a certain level and could quickly respond to MEP re-treatment in the soil. Culture-dependent and -independent evaluations revealed that MEP-degrading Burkholderia bacteria are predominant in soils after MEP application, limited members of which might play a pivotal role in MEP-degradation in soils. Notably, deep sequencing also revealed that some methylotrophs dramatically increased after MEP application, strongly suggesting that these bacteria play a role in the consumption and removal of methanol, a harmful derivative from MEP-degradation, for better growth of MEP-degrading bacteria. This comprehensive study demonstrated the succession and adaptation processes of microbial communities under MEP application, which were critically affected by past experience of insecticide-spraying. PMID:25221549

  19. [Prognostic evaluation of patients with severe head injury by motor evoked potentials induced by transcranial magnetic stimulation--combined analysis with brainstem auditory evoked potentials].

    PubMed

    Kaneko, M

    1995-05-01

    Prognostic evaluation of severe head injury was performed on the basis of transcranial magnetic motor evoked potentials (MEPs) and brainstem auditory evoked potentials (BAEPs). The subjects were 43 severe head injury patients with Glasgow Coma Scale Scores (GCS) of 9 or less. MEPs were recorded within 3 days after the injury. Patient outcome at 1, 6 and 12 months after the injury was correlated with the MEPs and BAEPs. Differences between MEP and BAEP findings in focal lesions and diffuse lesions also were analyzed. MEP wave latencies and inter-peak latencies between BAEP waves IV and V and between waves I and V were evaluated. There was a closer relationship between MEP latency and GOS, especially between the good recovery group and other outcome groups. At 1 month after the injury, there was a closer correlation between MEP latency and BAEP latency in those who died than in those who survived, and this tendency was more evident with regard to focal lesions. However, there was no significant correlation between MEP and patient outcome when the lesions were diffuse. There was no correlation between BAEP latencies and patient outcome, but there was a good, close correlation between prolonged MEP latency and unfavorable outcome at 1, 6, 12 months after injury. In conclusion, the combined use of BAEPs and MEPs induced by transcranial magnetic stimulation is useful in prognostic evaluation of acute head injury patients, especially when the brain lesions are focal. PMID:7786626

  20. Comparison of false-negative/positive results of intraoperative evoked potential monitoring between no and partial neuromuscular blockade in patients receiving propofol/remifentanil-based anesthesia during cerebral aneurysm clipping surgery: A retrospective analysis of 685 patients.

    PubMed

    Kim, Sung-Hoon; Jin, Seok-Joon; Karm, Myong-Hwan; Moon, Young-Jin; Jeong, Hye-Won; Kim, Jae-Won; Ha, Seung-Il; Kim, Joung-Uk

    2016-08-01

    Although the elicited responses of motor evoked potential (MEP) monitoring are very sensitive to suppression by anesthetic agents and muscle relaxants, the use of neuromuscular blockade (NMB) during MEP monitoring is still controversial because of serious safety concerns and diagnostic accuracy. Here, we evaluated the incidence of unacceptable movement and compared false-negative MEP results between no and partial NMB during cerebral aneurysm clipping surgery. We reviewed patient medical records for demographic data, anesthesia regimen, neurophysiology event logs, MEP results, and clinical outcomes. Patients were divided into 2 groups according to the intraoperative use of NMB: no NMB group (n = 276) and partial NMB group (n = 409). We compared the diagnostic accuracy of MEP results to predict postoperative outcomes between both groups. Additionally, we evaluated unwanted patient movement during MEP monitoring in both groups. Of the 685 patients, 622 (90.8%) manifested no intraoperative changes in MEP and no postoperative motor deficits. Twenty patients showed postoperative neurologic deficits despite preserved intraoperative MEP. False-positive MEP results were 3.6% in the no NMB group and 3.9% in the partial NMB group (P = 1.00). False-negative MEP results were 1.1% in the no NMB group and 4.2% in the partial NMB group (P = 0.02). No spontaneous movement or spontaneous respiration was observed in either group. Propofol/remifentanil-based anesthesia without NMB decreases the stimulation intensity of MEPs, which may reduce the false-negative ratio of MEP monitoring during cerebral aneurysm surgery. Our anesthetic protocol enabled reliable intraoperative MEP recording and patient immobilization during cerebral aneurysm clipping surgery. PMID:27559984

  1. Molecular characterization of a family of metalloendopeptidases from the intestinal brush border of Haemonchus contortus.

    PubMed

    Newlands, G F J; Skuce, P J; Nisbet, A J; Redmond, D L; Smith, S K; Pettit, D; Smith, W D

    2006-09-01

    Substantial protection against the economically important parasitic nematode Haemonchus contortus has been achieved by immunizing sheep with a glycoprotein fraction isolated from the intestinal membranes of the worm (H-gal-GP). Previous studies showed that one of the major components of H-gal-GP is a family of at least 4 zinc metalloendopeptidases, designated MEPs 1-4. This paper describes aspects of the molecular architecture of this protease family, including the proteomic analysis of the MEP fraction of the H-gal-GP complex. These enzymes belong to the M13 zinc metalloendopeptidase family (EC 3.4.24.11), also known as neutral endopeptidases or neprilysins. The sequences of MEPs 1 and 3 suggested a typical Type II integral membrane protein structure, whilst MEPs 2 and 4 had putative cleavable signal peptides, typical of secreted proteins. Proteomic analysis of H-gal-GP indicated that the extracellular domain of all 4 MEPs had been cleaved close to the transmembrane region/signal peptide with additional cleavage sites mid-way along the polypeptide. MEP3 was present as a homo-dimer in H-gal-GP, whereas MEP1 or MEP2 formed hetero-dimers with MEP4. It was found that expression of MEP3 was confined to developing 4th-stage larvae and to adult worms, the stages of Haemonchus which feed on blood. MEP-like activity was detected in the H-gal-GP complex over a broad pH range (5-9). Since all 4 MEPs must share a similar microenvironment in the complex, this suggests that each might have a different substrate specificity. PMID:16740178

  2. Secreted Metalloprotease Gene Family of Microsporum canis

    PubMed Central

    Brouta, Frédéric; Descamps, Frédéric; Monod, Michel; Vermout, Sandy; Losson, Bertrand; Mignon, Bernard

    2002-01-01

    Keratinolytic proteases secreted by dermatophytes are likely to be virulence-related factors. Microsporum canis, the main agent of dermatophytosis in dogs and cats, causes a zoonosis that is frequently reported. Using Aspergillus fumigatus metalloprotease genomic sequence (MEP) as a probe, three genes (MEP1, MEP2, and MEP3) were isolated from an M. canis genomic library. They presented a quite-high percentage of identity with both A. fumigatus MEP and Aspergillus oryzae neutral protease I genes. At the amino acid level, they all contained an HEXXH consensus sequence, confirming that these M. canis genes (MEP genes) encode a zinc-containing metalloprotease gene family. Furthermore, MEP3 was found to be the gene encoding a previously isolated M. canis 43.5-kDa keratinolytic metalloprotease, and was successfully expressed as an active recombinant enzyme in Pichia pastoris. Reverse transcriptase nested PCR performed on total RNA extracted from the hair of M. canis-infected guinea pigs showed that at least MEP2 and MEP3 are produced during the infection process. This is the first report describing the isolation of a gene family encoding potential virulence-related factors in dermatophytes. PMID:12228297

  3. 78 FR 52132 - Submission for OMB Review; Comment Request

    Federal Register 2010, 2011, 2012, 2013, 2014

    2013-08-22

    ... Technology (NIST) Title: NIST MEP Advanced Manufacturing Jobs and Innovation Accelerator Challenge (AMJIAC... advanced manufacturing. The objectives of the challenge are to support job creation, encourage...

  4. Consistency of evoked responses to dual-stimulator, single-pulse transcranial magnetic stimulation in the lower limb of people with multiple sclerosis.

    PubMed

    Meaney, A; Collett, J; Dawes, H; Howells, K; Izadi, H

    2015-09-01

    The purpose of this study was to explore the within session and test-retest consistency of motor evoked potentials (MEP) elicited by transcranial magnetic stimulation (TMS) from the resting tibialis anterior (TA) muscle of 10 patients (two men, eight women) with clinically definite multiple sclerosis (MS). Dual stimulators were configured to produce a single pulse (DS/SP) through a hand-held coil. MEP were recorded in five blocks of five trials with a repeat test occurring 7-14 days later. Analysis of a trial sequence revealed the area of the first MEP trial of each block to be significantly different to subsequent trials (trials 2-5; p<0.05). We therefore discarded T1 from further analysis. Thereafter, repeated measures of analysis of variance of MEP characteristics and blocks of MEP (average of four trials) revealed no significant differences (p>0.05). The results of the repeat session revealed no significant differences in motor thresholds, MEP latency, MEP amplitude or MEP area between sessions (p>0.05). Test-retest intra-class coefficients of correlation and their 95% confidence intervals indicated high reliability (>0.80). Our results show that consistent, repeatable TMS measures can be obtained from the resting TA of MS patients using the DS/SP method. PMID:26154149

  5. Title I Migrant Education Program, National Trends Report: 1998-2001. Doc # 2003-16

    ERIC Educational Resources Information Center

    US Department of Education, 2004

    2004-01-01

    This report summarizes Title I, Part C, Consolidated Performance Report information for the Migrant Education Program (MEP) submitted by the states for the 2000-01 school year. Also presented are national trends on participation in the MEP for the three years spanning 1998-99 through 2000-01. In addition to providing a national description of the…

  6. Improving the Academic Achievement of the Disadvantaged; Migrant Education Program. Final Rule. Federal Register, Department of Education, 34 CFR Part 200

    ERIC Educational Resources Information Center

    National Archives and Records Administration, 2008

    2008-01-01

    The Secretary amends the regulations governing the Migrant Education Program (MEP) administered under Part C of Title I of the Elementary and Secondary Education Act of 1965, as amended (ESEA). These final regulations adjust the base amounts of the MEP Basic State Formula grant allocations for fiscal year (FY) 2006 and subsequent years (as well…

  7. State Auditor's Review of the Vermont Migrant Education Program, July 1, 1997 to December 31, 1999.

    ERIC Educational Resources Information Center

    Vermont State Office of the State Auditor, Montpelier.

    Since 1992, the Vermont Department of Education has granted funds to the University of Vermont (UVM) to operate the Migrant Education Program (MEP). This review by the State Auditor found that prior to fiscal year 1999, the Department did not make a concerted effort to oversee the MEP and, as a result, several critical programs components were not…

  8. Improving the Retention and Graduation of Minorities in Engineering. Handbook.

    ERIC Educational Resources Information Center

    Landis, Raymond B., Ed.

    Procedures for operating undergraduate minority engineering programs (MEPs) are provided in this handbook. The findings and suggestions presented are based on the experiences of MEP directors who have managed support programs, which focus on improving the retention of minority students within engineering. The titles and authors of the handbook's…

  9. Intraoperative monitoring of spinal cord function using motor evoked potentials via transcutaneous epidural electrode during anterior cervical spinal surgery.

    PubMed

    Gokaslan, Z L; Samudrala, S; Deletis, V; Wildrick, D M; Cooper, P R

    1997-08-01

    Because false-positive results are not infrequent when monitoring somatosensory evoked potentials during surgery, monitoring of motor evoked potentials (MEPs) has been proposed and successfully used during the removal of spinal cord tumors. However, this often requires direct visual placement of an epidural electrode after a laminectomy. We evaluated the use of MEPs, recorded via a transcutaneously placed epidural electrode, to monitor motor pathway functional integrity during surgery on the anterior cervical spine. Sixteen patients underwent anterior cervical vertebral decompression and fusion for cervical myelopathy and/or radiculopathy. Before surgery, an epidural monitoring electrode was placed transcutaneously at the midthoracic level and was used to record MEPs after transcranial cortical electrical stimulation. Electrode placement was successful in all patients but one, and satisfactory baseline spinal MEPs were obtained except for one patient who had cerebral palsy with significant motor dysfunction. Patients showed no significant changes in spinal MEPs during surgery, and all had baseline or better motor function postoperatively. None had complications from epidural electrode placement or electrical stimulation. We conclude that motor pathways can be monitored safely during anterior cervical spinal surgery using spinal MEPs recorded via a transcutaneously placed epidural electrode, that MEP preservation during surgery correlates with good postoperative motor function, and that cerebral palsy patients may possess too few functional motor fibers to allow MEP recording. PMID:9278914

  10. Monitoring of Motor and Somatosensory Evoked Potentials During Spine Surgery: Intraoperative Changes and Postoperative Outcomes

    PubMed Central

    2016-01-01

    Objective To evaluate whether the combination of muscle motor evoked potentials (mMEPs) and somatosensory evoked potentials (SEPs) measured during spinal surgery can predict immediate and permanent postoperative motor deficits. Methods mMEP and SEP was monitored in patients undergoing spinal surgery between November 2012 and July 2014. mMEPs were elicited by a train of transcranial electrical stimulation over the motor cortex and recorded from the upper/lower limbs. SEPs were recorded by stimulating the tibial and median nerves. Results Combined mMEP/SEP recording was successfully achieved in 190 operations. In 117 of these, mMEPs and SEPs were stable and 73 showed significant changes. In 20 cases, motor deficits in the first 48 postoperative hours were observed and 6 patients manifested permanent neurological deficits. The two potentials were monitored in a number of spinal surgeries. For surgery on spinal deformities, the sensitivity and specificity of combined mMEP/SEP monitoring were 100% and 92.4%, respectively. In the case of spinal cord tumor surgeries, sensitivity was only 50% but SEP changes were observed preceding permanent motor deficits in some cases. Conclusion Intraoperative monitoring is a useful tool in spinal surgery. For spinal deformity surgery, combined mMEP/SEP monitoring showed high sensitivity and specificity; in spinal tumor surgery, only SEP changes predicted permanent motor deficits. Therefore, mMEP, SEP, and joint monitoring may all be appropriate and beneficial for the intraoperative monitoring of spinal surgery. PMID:27446784

  11. Microelectronics in Education--A Changing Future.

    ERIC Educational Resources Information Center

    Humphries, Chris

    1985-01-01

    Describes major program activities of Microelectronics Education Programme (MEP) for England, Wales, and Northern Ireland, which is designed to help schools prepare children for society with microelectronics. The core elements and potential consequences of the Microelectronics Support Unit, a follow-up to MEP which will conclude activities in…

  12. 75 FR 19941 - Manufacturing Extension Partnership Advisory Board

    Federal Register 2010, 2011, 2012, 2013, 2014

    2010-04-16

    .... ADDRESSES: The meeting will be held at Orlando World Center Marriott Resort & Convention Center, 8701 World Center Drive, Orlando, Florida 32821. Anyone wishing to attend this meeting should submit name, e-mail... MEP's National Conference in Orlando, FL. The MEP Advisory Board is composed of 10 members,...

  13. 76 FR 22674 - Manufacturing Extension Partnership Advisory Board

    Federal Register 2010, 2011, 2012, 2013, 2014

    2011-04-22

    .... ADDRESSES: The meeting will be held at the Orlando World Center Marriott Resort and Convention Center, 8701 World Center Drive, Orlando, Florida 32821. Anyone wishing to attend this meeting should submit their... conjunction with MEP's Manufacturing Innovations 2011 Conference in Orlando, Florida. The MEP Advisory...

  14. National Coordination Forum. A Collaborative Effort between Migrant Education and Migrant Head Start. Proceedings Report (Washington, D.C., February 6-7, 1993).

    ERIC Educational Resources Information Center

    State Univ. of New York, Oneonta. Coll. at Oneonta. Eastern Stream Center on Resources and Training.

    The National Coordination Forum was held to coordinate efforts of Migrant Education Preschool (MEP) and Migrant Head Start (MHS) to better serve the migrant preschool child. The introduction highlights the major differences between MEP and MHS (in authorizing legislation, eligibility, and program organization and administration) and the problems…

  15. Effect of Training Frequency on Maximum Expiratory Pressure

    ERIC Educational Resources Information Center

    Anand, Supraja; El-Bashiti, Nour; Sapienza, Christine

    2012-01-01

    Purpose: To determine the effects of expiratory muscle strength training (EMST) frequency on maximum expiratory pressure (MEP). Method: We assigned 12 healthy participants to 2 groups of training frequency (3 days per week and 5 days per week). They completed a 4-week training program on an EMST trainer (Aspire Products, LLC). MEP was the primary…

  16. Botulinum Toxin Type A Injections in the Psoas Muscle of Children with Cerebral Palsy: Muscle Atrophy after Motor End Plate-Targeted Injections

    ERIC Educational Resources Information Center

    Van Campenhout, Anja; Verhaegen, Ann; Pans, Steven; Molenaers, Guy

    2013-01-01

    MEP targeting during BoNT-A injections has been demonstrated to improve outcome. Two injection techniques of the psoas muscle--proximal MEP targeting versus a widely used more distal injection technique--are compared using muscle volume assessment by digital MRI segmentation as outcome measure. Method: 7 spastic diplegic children received…

  17. Inter-pulse Interval Affects the Size of Single-pulse TMS-induced Motor Evoked Potentials: A Reliability Study

    PubMed Central

    Vaseghi, Bita; Zoghi, Maryam; Jaberzadeh, Shapour

    2015-01-01

    Introduction: Measuring the size of motor evoked potentials (MEPs) induced by transcranial magnetic stimulation (TMS) is an investigational technique to show the level of corticospinal excitability; however, some of the fundamental methodological aspects of TMS (such as the effects of inter-pulse intervals (IPI) on MEP size) are not fully understood, this issue raises concerns about the reliability of MEPs, especially in pre-test post-test studies. Methods: MEP size at short and long IPIs was assessed during two separate sessions. Inter- and intra-session reliability of MEP size also was assessed at both short and long IPIs. Results: The results indicated that long IPIs induced larger MEPs (P< 0.05) across all time points. The intra-class correlation coefficient (ICC) indicated high intra- and inter-session reliability for short (0.87 to 0.96) and long (0.80 to 0.97) IPIs respectively. The amplitude of MEPs also had high intersession reliability for short (ICC=0.87) and long (ICC = 0.80) IPIs. Discussion: This study provides evidence that the length of IPIs determines the size of MEPs. As a result, it is recommended to add the length of IPI to the international checklist of considerations for TMS application.

  18. Using Transcranial Magnetic Stimulation to Evaluate the Motor Pathways After an Intraoperative Spinal Cord Injury and to Predict the Recovery of Intraoperative Transcranial Electrical Motor Evoked Potentials: A Case Report.

    PubMed

    Grover, Helen J; Thornton, Rachel; Lutchman, Lennel N; Blake, Julian C

    2016-06-01

    The authors report a case of unilateral loss of intraoperative transcranial electrical motor evoked potentials (TES MEP) associated with a spinal cord injury during scoliosis correction and the subsequent use of extraoperative transcranial magnetic stimulation to monitor the recovery of spinal cord function. The authors demonstrate the absence of TES MEPs and absent transcranial magnetic stimulation responses in the immediate postoperative period, and document the partial recovery of transcranial magnetic stimulation responses, which corresponded to partial recovery of TES MEPs. Intraoperative TES MEPs were enhanced using spatial facilitation technique, which enabled the patient to undergo further surgery to stabilize the spine and correct her scoliosis. This case report supports evidence of the use of extraoperative transcranial magnetic stimulation to predict the presence of intraoperative TES responses and demonstrates the usefulness of spatial facilitation to monitor TES MEPs in a patient with a preexisting spinal cord injury. PMID:26061481

  19. Basic study on the influence of inhibition induced by the magnetic stimulation on the peripheral nerve

    NASA Astrophysics Data System (ADS)

    Sato, Aya; Torii, Tetsuya; Iwahashi, Masakuni; Iramina, Keiji

    2015-05-01

    The purpose of this study is to analyze the inhibition mechanism of magnetic stimulation on motor function. A magnetic stimulator with a flat figure-eight coil was used to stimulate the peripheral nerve of the antebrachium. The intensity of magnetic stimulation was 0.8 T, and the stimulation frequency was 1 Hz. The amplitudes of the motor-evoked potentials (MEPs) at the abductor pollicis brevis muscle and first dorsal interosseous muscle were used to evaluate the effects of magnetic stimulation. The effects of magnetic stimulation were evaluated by analyzing the MEP amplitude before and after magnetic stimulation to the primary motor cortex. The results showed that MEP amplitude after magnetic stimulation compared with before magnetic stimulation decreased. Because there were individual differences in MEP amplitude induced by magnetic stimulation, the MEP amplitude after stimulation was normalized by the amplitude of each participant before stimulation. The MEP amplitude after stimulation decreased by approximately 58% (p < 0.01) on average compared with before stimulation. Previous studies suggested that magnetic stimulation to the primary motor cortex induced an increase or a decrease in MEP amplitude. Furthermore, previous studies have shown that the alteration in MEP amplitude was induced by cortical excitability based on magnetic stimulation. The results of this study showed that MEP amplitude decreased following magnetic stimulation to the peripheral nerve. We suggest that the decrease in MEP amplitude found in this study was obtained via the feedback from a peripheral nerve through an afferent nerve to the brain. This study suggests that peripheral excitement by magnetic stimulation of the peripheral nerve may control the central nervous system via afferent feedback.

  20. How Do I Feel About Feelings? Emotion Socialization in Families of Depressed and Healthy Adolescents

    PubMed Central

    Hunter, Erin C.; Katz, Lynn Fainsilber; Shortt, Joann Wu; Davis, Betsy; Leve, Craig; Allen, Nicholas B.

    2010-01-01

    Emotional and cognitive changes that occur during adolescence set the stage for the development of adaptive or maladaptive beliefs about emotions. Although research suggests that parents’ behaviors and beliefs about emotions relate to children’s emotional abilities, few studies have looked at parental socialization of children’s emotions, particularly in families with depressed adolescents. The present study examined associations between parent and adolescent meta-emotion philosophies (MEP), defined as thoughts, reactions, and feelings about their own emotions. Additionally, adolescent depressive status was tested as a moderator of relationships between parents’ and adolescents’ MEP. One hundred and 52 adolescents, aged 14–18 (65.8% female), and their parents (148 mothers, 106 fathers) participated in a study on emotion socialization in families of depressed and healthy adolescents. Depressed adolescents (n = 75) and matched healthy adolescents (n = 77) were recruited based on research criteria for mental health status. The sample was largely Caucasian (82%) and of middle socioeconomic class status. Results indicated that mothers’ and fathers’ MEP about their children’s emotions were associated with adolescents’ MEP, although parents’ MEP about their own emotions was unrelated to adolescents’ MEP. Fathers’ MEP about children’s emotions made unique contributions to adolescents’ MEP across both adolescent groups. Adolescents’ depressive status moderated the relationship between mothers’ and adolescents’ MEP such that mothers’ MEP was particularly relevant for depressed adolescents. The continued influence of parents in the emotional lives of adolescents is discussed as well as differences in emotion socialization in families with depressed and healthy adolescents. PMID:20473712

  1. Usefulness of Transcranial Magnetic Stimulation to Assess Motor Function in Patients With Parkinsonism

    PubMed Central

    Park, Jaechan; Cho, Jin Whan; Youn, Jinyoung; Kim, Yun Kwan; Kim, Sun Woong; Kim, Yun-Hee

    2016-01-01

    Objective To investigate the clinical significance of upper and lower extremity transcranial magnetic stimulation (TMS)-induced motor evoked potentials (MEPs) in patients with parkinsonism. Methods Twenty patients (14 men, 6 women; mean age 70.5±9.1 years) suffering from parkinsonism were included in this study. All participants underwent single-pulse TMS session to assess the corticospinal excitability of the upper and lower extremity motor cortex. The resting motor threshold (RMT) was defined as the lowest stimulus intensity able to evoke MEPs of an at least 50 µV peak-to-peak amplitude in 5 of 10 consecutive trials. Five sweeps of MEPs at 120% of the RMT were performed, and the mean amplitude and latency of the MEPs were calculated. Patients were also assessed using the Unified Parkinson's Disease Rating Scale part III (UPDRS-III) and the 5-meter Timed Up and Go (5m-TUG) test. Results There was a significant positive correlation between the RMTs of MEPs in the upper and lower extremities (r=0.612, p=0.004) and between the amplitude of MEPs in the upper and lower extremities (r=0.579, p=0.007). The RMT of upper extremity MEPs showed a significant negative relationship with the UPDRS-III score (r=–0.516, p=0.020). In addition, RMTs of lower extremity MEPs exhibited a negative relationship with the UPDRS-III score, but the association was not statistically significant (r=–406, p=0.075). Conclusion These results indicated that the RMT of MEPs reflect the severity of motor dysfunction in patients with parkinsonism. MEP is a potential quantitative, electrodiagnostic method to assess motor function in patients with parkinsonism. PMID:26949673

  2. Transcranial Motor Evoked Potentials of Lower Limbs Can Prognosticate Ambulation in Hemiplegic Stroke Patients

    PubMed Central

    2016-01-01

    Objective To examine the association between motor evoked potentials (MEPs) in lower limbs and ambulatory outcomes of hemiplegic stroke patients. Methods Medical records of hemiplegic patients with the first ever stroke who received inpatient rehabilitation from January 2013 to May 2014 were reviewed. Patient who had diabetes, quadriplegia, bilateral lesion, brainstem lesion, severe musculoskeletal problem, and old age over 80 years were excluded. MEPs in lower limbs were measured when they were transferred to the Department of Rehabilitation Medicine. Subjects were categorized into three groups (normal, abnormal, and absent response) according to MEPs findings. Berg Balance Scale (BBS) and Functional Ambulation Category (FAC) at initial and discharge were compared among the three groups by one-way analysis of variance (ANOVA). Correlation was determined using a linear regression model. Results Fifty-eight hemiplegic patients were included. BBS and FAC at discharge were significantly (ANOVA, p<0.001) different according to MEPs findings. In linear regression model of BBS and FAC using stepwise selection, patients' age (p<0.01), BBS at admission (p<0.01), and MEPs (p<0.01) remained significant covariates. In regression assumption model of BBS and FAC at admission, MEPs and gender were significant covariates. Conclusion Initial MEPs of lower limbs can prognosticate the ambulatory outcomes of hemiplegic patients. PMID:27446774

  3. Mechanisms of central motor reorganization in pediatric hemiplegic patients.

    PubMed

    Maegaki, Y; Maeoka, Y; Ishii, S; Shiota, M; Takeuchi, A; Yoshino, K; Takeshita, K

    1997-06-01

    Twenty hemiplegic patients were studied with transcranial magnetic stimulation (TMS). Motor evoked potentials (MEPs) of the biceps brachii (BB) and the abductor pollicis brevis muscles (APB) were recorded on both sides simultaneously. TMS was carried out with an 8-shaped coil over different scalp positions in the intact hemisphere. Bilateral MEPs of BB were elicited in patients with later childhood lesions as well as early lesion, but those of APB were only elicited in the latter (up to 2 years). In patients with prenatal or birth lesion on BB and in all patients on APB, cortical maps of MEP amplitude of paretic and non-paretic sides showed similar distributions. There were no remarkable differences in mean latency between both sides, and correlation coefficients of MEP amplitude between both sides were high in these patients. In patients with postnatal lesion on BB, MEP maps of both sides showed different distributions, ipsilateral MEP latencies were delayed and correlation coefficients were low. We suspect that ipsilateral MEPs after early lesion derive from axonal sprouting both in the proximal and the distal muscles. After postnatal lesion, other mechanisms of ipsilateral motor projection take place in the proximal muscles, but not in the distal ones. PMID:9266555

  4. A novel model incorporating two variability sources for describing motor evoked potentials

    PubMed Central

    Goetz, Stefan M.; Luber, Bruce; Lisanby, Sarah H.; Peterchev, Angel V.

    2014-01-01

    Objective Motor evoked potentials (MEPs) play a pivotal role in transcranial magnetic stimulation (TMS), e.g., for determining the motor threshold and probing cortical excitability. Sampled across the range of stimulation strengths, MEPs outline an input–output (IO) curve, which is often used to characterize the corticospinal tract. More detailed understanding of the signal generation and variability of MEPs would provide insight into the underlying physiology and aid correct statistical treatment of MEP data. Methods A novel regression model is tested using measured IO data of twelve subjects. The model splits MEP variability into two independent contributions, acting on both sides of a strong sigmoidal nonlinearity that represents neural recruitment. Traditional sigmoidal regression with a single variability source after the nonlinearity is used for comparison. Results The distribution of MEP amplitudes varied across different stimulation strengths, violating statistical assumptions in traditional regression models. In contrast to the conventional regression model, the dual variability source model better described the IO characteristics including phenomena such as changing distribution spread and skewness along the IO curve. Conclusions MEP variability is best described by two sources that most likely separate variability in the initial excitation process from effects occurring later on. The new model enables more accurate and sensitive estimation of the IO curve characteristics, enhancing its power as a detection tool, and may apply to other brain stimulation modalities. Furthermore, it extracts new information from the IO data concerning the neural variability—information that has previously been treated as noise. PMID:24794287

  5. Excitatory and inhibitory mechanisms in Wilson's disease: investigation with magnetic motor cortex stimulation.

    PubMed

    Perretti, A; Pellecchia, M T; Lanzillo, B; Campanella, G; Santoro, L

    2001-11-15

    We have evaluated cortical excitability in nine patients affected by Wilson's disease (WD) using transcranial magnetic (TMS) and electric (TES) cortical stimulation and central silent period (CSP) data. A clinical score was derived from the sum of scores assigned to extrapyramidal, pyramidal and cerebellar signs. All patients underwent TMS. Motor evoked potentials (MEPs) from abductor pollicis brevis (APB) and tibialis anterior (TA) muscles were recorded. MEP threshold and amplitude, central motor conduction time (CMCT), CSP threshold, CSP and peripheral silent period (PSP) duration were measured. Three patients also underwent transcranial bifocal electric cortical stimulation (TES) and MEPs were recorded from the APB muscle, and CMCT, MEP threshold and amplitude were measured. TMS MEPs were absent from relaxed muscles in six patients and from contracted muscles in three. CMCT was prolonged in six patients. APB CMCT correlated with clinical score. In three patients in whom TMS revealed abnormal or no MEP, TES MEPs were of normal threshold and amplitude. The CSP threshold was increased in seven patients, and CSP was absent in one. These results suggest an intracortical presynaptic motor dysfunction in WD. PMID:11701150

  6. Characterization of dengue virus 2 growth in megakaryocyte-erythrocyte progenitor cells.

    PubMed

    Clark, Kristina B; Hsiao, Hui-Mien; Bassit, Leda; Crowe, James E; Schinazi, Raymond F; Perng, Guey Chuen; Villinger, Francois

    2016-06-01

    Megakaryocyte-erythrocyte progenitor (MEP) cells are potential in vivo targets of dengue virus (DENV); the virus has been found associated with megakaryocytes ex vivo and platelets during DENV-induced thrombocytopenia. We report here that DENV serotype 2 (DENV2) propagates well in human nondifferentiated MEP cell lines (Meg01 and K562). In comparison to virus propagated in Vero cells, viruses from MEP cell lines had similar structure and buoyant density. However, differences in MEP-DENV2 stability and composition were suggested by distinct protein patterns in western blot analysis. Also, antibody neutralization of envelope domain I/II on MEP-DENV2 was reduced relative to that on Vero-DENV2. Infectious DENV2 was produced at comparable kinetics and magnitude in MEP and Vero cells. However, fewer virion structures appeared in electron micrographs of MEP cells. We propose that DENV2 infects and produces virus efficiently in megakaryocytes and that megakaryocyte impairment might contribute to dengue disease pathogenesis. PMID:27058763

  7. Microextraction in packed sorbent for analysis of antidepressants in human plasma by liquid chromatography and spectrophotometric detection.

    PubMed

    Chaves, Andréa R; Leandro, Fernanda Z; Carris, Juciene A; Queiroz, Maria Eugênia C

    2010-08-01

    A sensitive and reproducible method by microextraction packed sorbent and liquid chromatography with UV detection (MEPS/LC-UV) is described for the determination of new generation antidepressants (sertraline, mirtazapine, fluoxetine, citalopram and paroxetine) in human plasma samples. The MEPS variables, such as sample volume, pH, number of extraction cycles (draw-eject), and desorption conditions (solvent and solvent volume of elution) influenced the MEPS/LC efficiency significantly. Important factors in the optimization of MEPS efficiency, as well as washing steps and carryover effect are discussed. The analyses were carried out using small sample volumes (400 microL), and in a short time period (3 min for the entire sample preparation step). The MEPS/LC-UV method was shown to be linear at concentrations ranging from the limit of quantification (LOQ) to 1000 ng mL(-1). The LOQ values ranged from 10 to 25 ng mL(-1). The inter-day precision of the method presented coefficient of the variation ranging from 1.3% to 8.7%. On the basis of analytical validation, it is shown that the MEPS/LC-UV methodology is adequate for antidepressant analysis, from therapeutic to toxic levels. In order to evaluate the proposed method for clinical use, the MEPS/LC-UV method was applied to analysis of plasma samples from elderly depressed patients. PMID:20630812

  8. Modulation of motor activity by cutaneous input: inhibition of the magnetic motor evoked potential by digital electrical stimulation.

    PubMed

    Clouston, P D; Kiers, L; Menkes, D; Sander, H; Chiappa, K; Cros, D

    1995-04-01

    We examined the inhibitory effect of a brief train of digital (D2) electrical stimuli at 4 times perception threshold on transcranial magnetic motor evoked potentials (MEPs) recorded from abductor pollicis brevis (APB) and flexor carpi radialis (FCR) muscles ipsilateral to the side of D2 stimulation. We compared this to the inhibitory effect of ipsilateral D2 stimulation on averaged rectified EMG recorded at 10% maximum voluntary contraction and on F-responses and H-reflexes recorded from these same muscles. We also compared MEPs recorded following D2 stimulation just above perception threshold to MEPs following higher intensity D2 stimulation. As well, we assessed the effect of preceding D2 stimulation on MEPs recorded from a relaxed versus tonically contracted hand muscle. D2 stimulation elicited a triphasic response of modest MEP facilitation followed by inhibition and further facilitation. The duration and onset of MEP inhibition correlated with those of the initial period of rectified EMG inhibition, however, the magnitude of MEP inhibition was generally less than the magnitude of EMG inhibition, consistent with a greater inhibitory effect of digital afferents on smaller motor neurons. MEPs were not facilitated during the rebound of EMG activity (the E2 period) that usually followed the initial period of EMG inhibition (I1 period). The behavior of H-reflexes and F-responses following ipsilateral D2 stimulation suggested that inhibition of both EMG and MEPs is not mediated via presynaptic inhibition of Ia afferents, and that inhibition is augmented by descending rather than segmental input to spinal motor neurons. Tonic contraction of the target muscle during D2 stimulation decreased the inhibitory effect of the preceding digital stimulus possibly due to recruitment of larger spinal motor neurons less likely to be inhibited by cutaneous input. PMID:7537203

  9. Twenty-five years of maximum-entropy principle

    NASA Astrophysics Data System (ADS)

    Kapur, J. N.

    1983-04-01

    The strengths and weaknesses of the maximum entropy principle (MEP) are examined and some challenging problems that remain outstanding at the end of the first quarter century of the principle are discussed. The original formalism of the MEP is presented and its relationship to statistical mechanics is set forth. The use of MEP for characterizing statistical distributions, in statistical inference, nonlinear spectral analysis, transportation models, population density models, models for brand-switching in marketing and vote-switching in elections is discussed. Its application to finance, insurance, image reconstruction, pattern recognition, operations research and engineering, biology and medicine, and nonparametric density estimation is considered.

  10. Success rate of motor evoked potentials for intraoperative neurophysiologic monitoring: effects of age, lesion location, and preoperative neurologic deficits.

    PubMed

    Chen, Xi; Sterio, Djordje; Ming, Xu; Para, Devaki D; Butusova, Marri; Tong, Teresa; Beric, Aleksandar

    2007-06-01

    Transcranial electrical stimulation with myogenic motor evoked potential (MEP) recording was used for intraoperative neurophysiologic monitoring in 341 consecutive "high-risk" neurosurgical or orthopedic procedures. Overall, the success rate for establishing reliable MEP response was 94.8% for upper extremities and 66.6% for lower extremities. The rate was only 39.1% for lower extremities in patients with preoperative motor deficit and up to 81% in neurologically intact adults. Further analysis demonstrated that extremes of age or the presence of a lesion in the spinal cord and motor deficit contributed to failure in obtaining reliable MEPs. PMID:17545833

  11. Medicaid eligibility workers discuss Medicaid estate planning for nursing home care.

    PubMed

    Walker, L; Gruman, C; Robison, J

    1999-04-01

    This study was part of a multiphase project examining the perceptions of elder law attorneys, certified financial planners, and Medicaid eligibility workers regarding Medicaid estate planning (MEP) for nursing home care. Focus group methodology (5 groups, N = 32 participants) was used to explore the perspectives and experiences of Medicaid eligibility workers, who are responsible for interpreting, administering, and enforcing federal and state regulations. Findings describe factors influencing MEP, enforcement of regulations, and potential policy responses to MEP. Participants identified numerous impediments to effective implementation of current regulations. Recommendations for improved policy include redefinition of spousal assessment policies, other valuable consideration determinations, and penalty period formulas. PMID:10224716

  12. A family-oriented therapy program for youths with substance abuse: long-term outcomes related to relapse and academic or social status

    PubMed Central

    Wang, Liang-Jen; Lu, Shing-Fang; Chong, Mian-Yoon; Chou, Wen-Jiun; Hsieh, Yu-Lian; Tsai, Tung-ning; Chen, Ching; Lee, Yi-Hsuan

    2016-01-01

    Objective The abuse of illegal substances by youths in Taiwan has become a major public health issue. This study explores the outcomes (relapse rate and academic or social status) of a family-oriented therapy program conducted for substance-using youths who were referred by a judge to participate in it. Methods The present study includes 121 participants categorized into three groups: 36 youths underwent a weekly ten-session outpatient motivational enhancement psychotherapy (MEP) group program; 41 youths participated in a program that combined the aforementioned MEP program with an additional weekly ten-session parenting skill training (PST) program for their guardians (MEP + PST group); and 44 adolescents who received standard supervision by the court served as the control group. All participants were followed-up for a maximum of 2 years. Results Of the 121 participants (mean age: 16.1±1.1 years), 33.1% relapsed into substance use during the follow-up period. The probability of relapse did not differ significantly between the MEP group (36.1%) and the control group (40.9%), but the youths in the MEP + PST group (22.0%) were at a lower risk of relapse than the control group participants (adjusted hazard ratio =0.48, 95% confidence interval [CI] =0.21–1.09). By the end of the study follow-up period, participants in both the MEP group and the MEP + PST group were more likely to be attending school (MEP group: adjusted odds ratio [aOR] =6.61, 95% CI =1.60–27.35; MEP + PST group: aOR =8.57, 95% CI =1.94–37.82) or employed (MEP group: aOR =7.75, 95% CI =1.95–30.75; MEP + PST group: aOR =7.27, 95% CI =1.76–29.97), when compared to the control group. Conclusion This study revealed that a family-oriented treatment approach may be a more effective option for preventing youths’ relapsing into substance abuse. In comparison to individuals who received standard supervision by the court, those who received MEP experienced a better school attendance or social

  13. Learning probability distributions from smooth observables and the maximum entropy principle: some remarks

    NASA Astrophysics Data System (ADS)

    Obuchi, Tomoyuki; Monasson, Rémi

    2015-09-01

    The maximum entropy principle (MEP) is a very useful working hypothesis in a wide variety of inference problems, ranging from biological to engineering tasks. To better understand the reasons of the success of MEP, we propose a statistical-mechanical formulation to treat the space of probability distributions constrained by the measures of (experimental) observables. In this paper we first review the results of a detailed analysis of the simplest case of randomly chosen observables. In addition, we investigate by numerical and analytical means the case of smooth observables, which is of practical relevance. Our preliminary results are presented and discussed with respect to the efficiency of the MEP.

  14. QUANTITATIVE COMPARISON OF MOLECULAR ELECTROSTATIC POTENTIALS FOR STRUCTURE-ACTIVITY STUDIES

    EPA Science Inventory

    Further development of an approach for representing, efficiently calculating and comparing discreet 3-dimensional molecular electrostatic potentials using a quantitative similarity index (MEP-SI) based on the Carbo formalism is presented. adial-type (MACRA) grid representation is...

  15. 75 FR 50749 - Manufacturing Extension Partnership Advisory Board

    Federal Register 2010, 2011, 2012, 2013, 2014

    2010-08-17

    ... National Institute of Standards and Technology Manufacturing Extension Partnership Advisory Board AGENCY... announces that the Manufacturing Extension Partnership (MEP) Advisory Board, National Institute of Standards... INFORMATION CONTACT: Karen Lellock, Manufacturing Extension Partnership, National Institute of Standards...

  16. Use and validity of principles of extremum of entropy production in the study of complex systems

    SciTech Connect

    Heitor Reis, A.

    2014-07-15

    It is shown how both the principles of extremum of entropy production, which are often used in the study of complex systems, follow from the maximization of overall system conductivities, under appropriate constraints. In this way, the maximum rate of entropy production (MEP) occurs when all the forces in the system are kept constant. On the other hand, the minimum rate of entropy production (mEP) occurs when all the currents that cross the system are kept constant. A brief discussion on the validity of the application of the mEP and MEP principles in several cases, and in particular to the Earth’s climate is also presented. -- Highlights: •The principles of extremum of entropy production are not first principles. •They result from the maximization of conductivities under appropriate constraints. •The conditions of their validity are set explicitly. •Some long-standing controversies are discussed and clarified.

  17. Master environmental plan for Fort Devens, Massachusetts

    SciTech Connect

    Biang, C.A.; Peters, R.W.; Pearl, R.H.; Tsai, S.Y. . Energy Systems Div.)

    1991-11-01

    Argonne National Laboratory has prepared a master environmental plan (MEP) for Fort Devens, Massachusetts, for the US Army Toxic and Hazardous Materials Agency. The MEP is an assessment based on environmental laws and regulations of both the federal government and the Commonwealth of Massachusetts. The MEP assess the physical and environmental status of 58 potential hazardous waste sites, including 54 study areas (SAs) that pose a potential for releasing contamination into the environment and 4 areas of concern (AOCs) that are known to have substantial contamination. For each SA or AOC, this MEP describes the known history and environment, identifies additional data needs, and proposes possible response actions. Most recommended response actions consist of environmental sampling and monitoring and other characterization studies. 74 refs., 63 figs., 50 tabs.

  18. Scout Missions and Future Exploration of the Martian Poles

    NASA Technical Reports Server (NTRS)

    McBride, K. S.

    2003-01-01

    NASA's Mars Exploration Program, (MEP) complemented by missions in operation by ESA and the Japanese space agency, is revolutionizing the study of Mars as a planet and potential home for life, past, present or future. Within the MEP there are a number of significant opportunities for the study of the Mars polar regions--from the ongoing Mars Global Surveyor and Odyssey missions, the upcoming Mars Reconnaissance Orbiter, and the soon-to-be-defined Mars Science Laboratory. As an internal complement to the Mars missions being developed by JPL for the MEP, Mars Scout investigations can provide substantial future opportunities to study the polar regions of Mars. These relatively small, PI-led missions provide substantial flexibility within the overall MEP, providing the capability to respond to scientific targets of opportunity in Mars science, with special-interest small missions, or to be developed to respond to instrument opportunities for missions developed by international partners.

  19. Eyelid milia en plaque: a treatment challenge with a new CO2 fractional laser.

    PubMed

    Tenna, Stefania; Filoni, Angela; Pagliarello, Calogero; Paradisi, Mauro; Persichetti, Paolo

    2014-01-01

    Milia en plaque (MEP) is an uncommon finding characterized by numerous tiny milia within an erythematous area. Despite its benign and asymptomatic nature, MEP raises cosmetic concerns; moreover, the available treatment modalities for MEP are limited. In view of the few cases described in the literature, no consensus has been reached, with respect to the optimal treatment for MEP, and the choice of therapy should be individualized. We report a case of eyelid milia en plaque successfully treated with a new CO2 fractional laser that is able to ensure superficial ablation of the epidermis remodeling tissue in-depth, with minimal thermal damage and extremely rapid recovery time. The results obtained after only two treatments were good, no scarring or dyschromic changes have been registered. At 1 year, just few recurrent milia were present. PMID:24703259

  20. 32 CFR 728.73 - Applicants for enlistment or reenlistment in the Armed Forces, and applicants for enlistment in...

    Code of Federal Regulations, 2012 CFR

    2012-07-01

    ... Defense Department of Defense (Continued) DEPARTMENT OF THE NAVY PERSONNEL MEDICAL AND DENTAL CARE FOR... at Navy and Marine Corps facilities or MEPS may be furnished emergency medical and dental...

  1. 32 CFR 728.73 - Applicants for enlistment or reenlistment in the Armed Forces, and applicants for enlistment in...

    Code of Federal Regulations, 2013 CFR

    2013-07-01

    ... Defense Department of Defense (Continued) DEPARTMENT OF THE NAVY PERSONNEL MEDICAL AND DENTAL CARE FOR... at Navy and Marine Corps facilities or MEPS may be furnished emergency medical and dental...

  2. 77 FR 20790 - Manufacturing Extension Partnership Advisory Board

    Federal Register 2010, 2011, 2012, 2013, 2014

    2012-04-06

    ... National Institute of Standards and Technology Manufacturing Extension Partnership Advisory Board AGENCY... announces that the Manufacturing Extension Partnership (MEP) Advisory Board, National Institute of Standards... INFORMATION section below. FOR FURTHER INFORMATION CONTACT: Karen Lellock, Manufacturing Extension...

  3. Modulation of amplitude and latency of motor evoked potential by direction of transcranial magnetic stimulation

    NASA Astrophysics Data System (ADS)

    Sato, Aya; Torii, Tetsuya; Iwahashi, Masakuni; Itoh, Yuji; Iramina, Keiji

    2014-05-01

    The present study analyzed the effects of monophasic magnetic stimulation to the motor cortex. The effects of magnetic stimulation were evaluated by analyzing the motor evoked potentials (MEPs). The amplitude and latency of MEPs on the abductor pollicis brevis muscle were used to evaluate the effects of repetitive magnetic stimulation. A figure eight-shaped flat coil was used to stimulate the region over the primary motor cortex. The intensity of magnetic stimulation was 120% of the resting motor threshold, and the frequency of magnetic stimulation was 0.1 Hz. In addition, the direction of the current in the brain was posterior-anterior (PA) or anterior-posterior (AP). The latency of MEP was compared with PA and AP on initial magnetic stimulation. The results demonstrated that a stimulus in the AP direction increased the latency of the MEP by approximately 2.5 ms. MEP amplitude was also compared with PA and AP during 60 magnetic stimulations. The results showed that a stimulus in the PA direction gradually increased the amplitude of the MEP. However, a stimulus in the AP direction did not modulate the MEP amplitude. The average MEP amplitude induced from every 10 magnetic pulses was normalized by the average amplitude of the first 10 stimuli. These results demonstrated that the normalized MEP amplitude increased up to approximately 150%. In terms of pyramidal neuron indirect waves (I waves), magnetic stimulation inducing current flowing backward to the anterior preferentially elicited an I1 wave, and current flowing forward to the posterior elicited an I3 wave. It has been reported that the latency of the I3 wave is approximately 2.5 ms longer than the I1 wave elicitation, so the resulting difference in latency may be caused by this phenomenon. It has also been reported that there is no alteration of MEP amplitude at a frequency of 0.1 Hz. However, this study suggested that the modulation of MEP amplitude depends on stimulation strength and stimulation direction.

  4. Demonstration of facilitatory I wave interaction in the human motor cortex by paired transcranial magnetic stimulation

    PubMed Central

    Ziemann, Ulf; Tergau, Frithjof; Wassermann, Eric M; Wischer, Stephan; Hildebrandt, Jörg; Paulus, Walter

    1998-01-01

    Transcranial magnetic stimulation (TMS) of the human motor cortex results in multiple discharges (D and I waves) in the corticospinal tract. We tested whether these volleys can be explored non-invasively with paired TMS. The intensity of the first stimulus (S1) was set to produce a motor-evoked potential (MEP) of 1 mV in the resting contralateral abductor digiti minimi (ADM) muscle; the second stimulus (S2) was set to 90 % of the resting motor threshold. At interstimulus intervals of 1·1-1·5, 2·3-2·9 and 4·1-4·4 ms the MEP elicited by S1 plus S2 was larger than that produced by S1 alone.Varying the S1 intensity between 70 and 130 % resting motor threshold with S2 held constant at 90 % resting motor threshold showed that the threshold for the first MEP peak was <= 70 % resting motor threshold. The second and third MEP peaks appeared only at higher S1 intensities. The latency of all peaks decreased with increasing S1 intensity.Varying the S2 intensity with S1 held constant to produce a MEP of 1 mV on its own showed that the amplitude of all MEP peaks increased with S2 intensity, but that their timing remained unchanged.Paired TMS in the active ADM (S1 clearly suprathreshold, S2 just above threshold; interstimulus interval, 1 ms) produced strong MEP facilitation. The onset of this facilitation occurred later by about 1·5 ms than the onset of the MEP evoked by S2 alone. No MEP facilitation was seen if the magnetic S2 was replaced by anodal or cathodal transcranial electrical stimulation.It is concluded that the MEP facilitation after paired TMS, at least for the first MEP peak, is due to facilitatory interaction between I waves, and takes place in the motor cortex at or upstream from the corticospinal neurone. PMID:9679173

  5. Real-Time Evaluation of Anterior Choroidal Artery Patency During Aneurysm Clipping

    PubMed Central

    Zhao, David Y; Nayar, Vikram V; Kalhorn, Christopher G; McGrail, Kevin M; Mandir, Allen S; Minahan, Robert E

    2016-01-01

    Inadvertent occlusion of the anterior choroidal artery during aneurysm clipping can cause a disabling stroke in minutes. We evaluate the clinical utility of direct cortical motor evoked potential (MEP) monitoring during aneurysm clipping, as a real-time assessment of arterial patency, prior to performing indocyanine green videoangiography.   Direct cortical MEPs were recorded in seven patients undergoing surgery for aneurysms that involved or abutted the anterior choroidal artery. The aneurysms clipped in those seven patients included four anterior choroidal artery aneurysms and six posterior communicating artery aneurysms. Serial MEP recordings were performed during the intradural dissection, aneurysm exposure, and clip placement. A significant change in MEPs after clip placement would prompt immediate inspection and removal or repositioning of the clip. If the clip placement appeared satisfactory and MEP recordings were stable, then an intraoperative indocyanine green videoangiogram was performed to confirm obliteration of the aneurysm and patency of the arteries.  Seven patients underwent successful clipping of anterior choroidal artery aneurysms and posterior communicating artery aneurysms using direct cortical MEP monitoring, with good clinical and radiographic outcomes. In six patients, no changes in MEP amplitudes were observed following permanent clip placement. In one patient, a profound decrease in MEP amplitude occurred 220 seconds after placement of a permanent clip on a large posterior communicating aneurysm. An inspection revealed that the anterior choroidal artery was kinked. The clip was immediately removed, and the MEP signals returned to baseline shortly thereafter. A clip was then optimally placed, and the patient awoke without neurologic deficit.  Direct cortical MEPs are a useful adjunct to standard electrophysiologic monitoring in aneurysm surgery, particularly when the anterior choroidal artery or lenticulostriate arteries are at

  6. Microwave electric propulsion for orbit transfer applications

    SciTech Connect

    Sercel, J.C.

    1985-01-01

    A Microwave Electric Propulsion (MEP) concept is developed for an unmanned Orbit Transfer Vehicle (OTV). The concept is based on the use of beamed microwave power and on an extrapolation of ion thruster technology. Beamed microwave power transmission is discussed in terms of its relationship to spacecraft propulsion. Characteristics of an MEP OTV are determined and performance is evaluated by comparison to a variety of alternative propulsion systems for the completion of a ten-year mission model.

  7. Estimate of Technical Potential for Minimum Efficiency Performance Standards in 13 Major World Economies

    SciTech Connect

    Letschert, Virginie; Desroches, Louis-Benoit; Ke, Jing; McNeil, Michael

    2012-07-01

    As part of the ongoing effort to estimate the foreseeable impacts of aggressive minimum efficiency performance standards (MEPS) programs in the world’s major economies, Lawrence Berkeley National Laboratory (LBNL) has developed a scenario to analyze the technical potential of MEPS in 13 major economies around the world1 . The “best available technology” (BAT) scenario seeks to determine the maximum potential savings that would result from diffusion of the most efficient available technologies in these major economies.

  8. Influence of electrode site and size on variability of magnetic evoked potentials.

    PubMed

    Dunnewold, R J; van der Kamp, W; van den Brink, A M; Stijl, M; van Dijk, J G

    1998-12-01

    Successive magnetic evoked potentials (MEPs) concern varying motor neurons. We investigated whether this MEP-specific source of variability depends on electrode site and size. Amplitude variability (standard deviation) was largest over the center of the hypothenar muscles. Latencies were longer at distal and proximal sites than at the center site. Large electrodes (10 cm2) did not decrease this source of amplitude variability compared with EEG electrodes, in contrast to other sources of variability. PMID:9843083

  9. Methylosome Protein 50 and PKCδ/p38δ Protein Signaling Control Keratinocyte Proliferation via Opposing Effects on p21Cip1 Gene Expression.

    PubMed

    Saha, Kamalika; Eckert, Richard L

    2015-05-22

    Protein arginine methyltransferase 5 (PRMT5) is a key epigenetic regulator that symmetrically dimethylates arginine residues on histones H3 and H4 to silence gene expression. PRMT5 is frequently observed in a complex with the cofactor methylosome protein 50 (MEP50), which is required for PRMT5 activity. PKCδ/p38δ signaling, a key controller of keratinocyte proliferation and differentiation, increases p21(Cip1) expression to suppress keratinocyte proliferation. We now show that MEP50 enhances keratinocyte proliferation and survival via mechanisms that include silencing of p21(Cip1) expression. This is associated with enhanced PRMT5-MEP50 interaction at the p21(Cip1) promoter and enhanced arginine dimethylation of the promoter-associated histones H3 and H4. It is also associated with a MEP50-dependent reduction in the level of p53, a key controller of p21(Cip1) gene expression. We confirm an important biological role for MEP50 and PRMT5 in regulating keratinocyte proliferation using a stratified epidermal equivalent model that mimics in vivo epidermal keratinocyte differentiation. In this model, PRMT5 or MEP50 knockdown results in reduced keratinocyte proliferation. We further show that PKCδ/p38δ signaling suppresses MEP50 expression, leading to reduced H3/H4 arginine dimethylation at the p21(Cip1) promoter, and that this is associated with enhanced p21(Cip1) expression and reduced cell proliferation. These findings describe an opposing action between PKCδ/p38δ MAPK signaling and PRMT5/MEP50 epigenetic silencing mechanisms in regulating cell proliferation. PMID:25851901

  10. The Spring 2014 Mesoscale Ensemble Prediction System "Dust Offensive"

    NASA Astrophysics Data System (ADS)

    Hunt, E. D.; Adams-Selin, R.; Sartan, J.; Creighton, G.; Kuchera, E.; Keane, J.; Jones, S.

    2014-12-01

    The Mesoscale Ensemble Prediction System (MEPS) at the Air Force Weather Agency (AFWA) is a 10-member ensemble run on a 20-km hemispheric domain and 4-km domains in regions of interest. In the Southwest Asia (SWA) regional domain, dust forecast products from MEPS are of particular interest. Over the past few years, subject matter experts at AFWA have acquired and implemented datasets and developed a soil moisture algorithm that have improved skill scores of dust forecasts in the AFWA GOddard Chemistry Aerosol Radiation and Transport (GOCART) module within the Weather Research and Forecast with Chemistry (WRF-Chem) model. The aforementioned datasets include, but are not limited to, the Desert Research Institute (DRI) dust source region and a dynamic 8-day Leaf Area Index (LAI) vegetation mask. We then tested these individual datasets as ensemble perturbations in all ten MEPS test members during a three-week "dust offensive" test over SWA in spring 2014. Remote sensing specialists at AFWA meticulously documented the locations, duration, and intensity of numerous dust events over the SWA domain during the three weeks. These data were then used for subjective verification of each individual MEPS member and of the individual perturbations within members. Results from the subjective verification showed that individual MEPS members with the DRI dust source region significantly outperformed members that used the standard Ginoux dust source region. The other individual perturbations tested were determined to have a neutral effect (i.e., neither degrading nor improving skill) on individual members during the three-week period. Thus, the DRI dust source region is now used in the majority of MEPS members in both the 4-km SWA domain and the 20-km hemispheric domain. The other individual perturbations are now utilized in a minority of MEPS members. However, additional testing is still needed over other domains to determine if the improved dust forecasting skill has spatial

  11. AKAP150-dependent cooperative TRPV4 channel gating is central to endothelium-dependent vasodilation and is disrupted in hypertension

    PubMed Central

    Sonkusare, Swapnil K.; Dalsgaard, Thomas; Bonev, Adrian D.; Hill-Eubanks, David C.; Kotlikoff, Michael I.; Scott, John D.; Santana, Luis F.; Nelson, Mark T.

    2015-01-01

    Endothelial cell dysfunction, characterized by a diminished response to endothelial cell–dependent vasodilators, is a hallmark of hypertension. TRPV4 channels play a major role in endothelial-dependent vaso-dilation, a function mediated by local Ca2+ influx through clusters of functionally coupled TRPV4 channels rather than by a global increase in endothelial cell Ca2+. We showed that stimulation of muscarinic acetylcholine receptors on endothelial cells of mouse arteries exclusively activated TRPV4 channels that were localized at myoendothelial projections (MEPs), specialized regions of endothelial cells that contact smooth muscle cells. Muscarinic receptor–mediated activation of TRPV4 depended on protein kinase C (PKC) and the PKC-anchoring protein AKAP150, which was concentrated at MEPs. Cooperative opening of clustered TRPV4 channels specifically amplified Ca2+ influx at MEPs. Cooperativity of TRPV4 channels at non-MEP sites was much lower, and cooperativity at MEPs was greatly reduced by chelation of intracellular Ca2+ or AKAP150 knockout, suggesting that Ca2+ entering through adjacent channels underlies the AKAP150-dependent potentiation of TRPV4 activity. In a mouse model of angiotensin II–induced hypertension, MEP localization of AKAP150 was disrupted, muscarinic receptor stimulation did not activate TRPV4 channels, cooperativity among TRPV4 channels at MEPs was weaker, and vasodilation in response to muscarinic receptor stimulation was reduced. Thus, endothelial-dependent dilation of resistance arteries is enabled by MEP-localized AKAP150, which ensures the proximity of PKC to TRPV4 channels and the coupled channel gating necessary for efficient communication from endothelial to smooth muscle cells in arteries. Disruption of this molecular assembly may contribute to altered blood flow in hypertension. PMID:25005230

  12. Preliminary study on safe thresholds for temporary internal carotid artery occlusion in aneurysm surgery based on motor-evoked potential monitoring

    PubMed Central

    Tanabe, Jun; Ishikawa, Tatsuya; Moroi, Junta; Suzuki, Akifumi

    2014-01-01

    Background: The study aims were to clarify safe duration for temporary vessel occlusion of the internal carotid artery (ICA) during aneurysm surgery as exactly as possible. We examined safe time duration (STD), where brain tissue exposed to ischemia will never fall into even the ischemic penumbra using intraoperative motor-evoked potential (MEP). Methods: In 45 patients, temporary occlusion of the ICA was performed with MEP. We measured STD as the duration of temporary vessel occlusion during which MEP changes did not occur. To estimate average STD, we calculated the 95% confidence interval for the population mean from sample data for STD in patients with MEP changes and in patients without changes. Results: In the proximal-control group, 4 of 38 patients (10.5%) developed intraoperative MEP changes. In 4 patients, the time to MEP change (i.e. STD) was 6.0 ± 2.5 min. STD was 3.8 ± 1.6 min in the 34 patients without changes. The average STD was 4.0 ± 0.6 min. In the trap group (proximal and distal flow control), five of seven patients (60.0%) experienced intraoperative MEP changes (STD, 2.3 ± 1.0 min). All patients in the trap group who developed MEP changes showed involvement of the anterior choroidal artery (AchA) in the trapped segment. Average STD was 2.3 ± 1.1 min when trapping involving the AchA. Conclusions: Although the study is preliminary based on the limited number of the patients, the 95% upper confidence limit for average STD was 4.6 min when the ICA was occluded proximal to the aneurysm, 3.4 min when the ICA was trapped involving the AchA. PMID:24818054

  13. Antinociceptive and neuropharmacological activities of methanol extract of Phoenix sylvestris fruit pulp

    PubMed Central

    Shajib, Md. Shafiullah; Akter, Saleha; Ahmed, Tajnin; Imam, Mohammad Zafar

    2015-01-01

    Fruits of Phoenix sylvestris Roxb. (Arecaceae) are used to treat back pain, toothache, headache, arthritis, nervous debility and as sedative. The aim of this study was to evaluate the antinociceptive and neuropharmacological activities of methanol extract of P. sylvestris fruit pulp (MEPS). The antinociceptive activity of MEPS was evaluated by heat-induced (hot plate, tail immersion test) and chemical-induced pain models (acetic acid-induced writhing, formalin-induced nociception, glutamate-induced nociception and paw edema test). The effect of MEPS on central nervous system (CNS) was studied using hole cross test, open field test, sodium thiopental-induced sleeping time and elevated plus maze test. MEPS showed strong, significant and dose-dependent antinociceptive activity in all heat-induced and chemical-induced pain models at all experimental doses. Involvement of opioid receptor mediated analgesia was evident from the reversal of analgesic effect by naloxone. MEPS also showed reduced locomotor activity in both hole cross and open field tests. The increase in sleeping time in sodium thiopental-induced sleeping test and anxiolytic activity in elevated plus maze test were also significant. So, it is evident that MEPS possesses strong central and peripheral antinociceptive activity as well as CNS depressant, sedative and anxiolytic activity. The results justify the ethnomedicinal use of P. sylvestris fruit in different painful conditions and CNS disorders. PMID:26483687

  14. Hyperdynamics boost factor achievable with an ideal bias potential

    DOE PAGESBeta

    Huang, Chen; Perez, Danny; Voter, Arthur F.

    2015-08-20

    Hyperdynamics is a powerful method to significantly extend the time scales amenable to molecular dynamics simulation of infrequent events. One outstanding challenge, however, is the development of the so-called bias potential required by the method. In this work, we design a bias potential using information about all minimum energy pathways (MEPs) out of the current state. While this approach is not suitable for use in an actual hyperdynamics simulation, because the pathways are generally not known in advance, it allows us to show that it is possible to come very close to the theoretical boost limit of hyperdynamics while maintainingmore » high accuracy. We demonstrate this by applying this MEP-based hyperdynamics (MEP-HD) to metallic surface diffusion systems. In most cases, MEP-HD gives boost factors that are orders of magnitude larger than the best existing bias potential, indicating that further development of hyperdynamics bias potentials could have a significant payoff. Lastly, we discuss potential practical uses of MEP-HD, including the possibility of developing MEP-HD into a true hyperdynamics.« less

  15. Hyperdynamics boost factor achievable with an ideal bias potential

    SciTech Connect

    Huang, Chen; Perez, Danny; Voter, Arthur F.

    2015-08-20

    Hyperdynamics is a powerful method to significantly extend the time scales amenable to molecular dynamics simulation of infrequent events. One outstanding challenge, however, is the development of the so-called bias potential required by the method. In this work, we design a bias potential using information about all minimum energy pathways (MEPs) out of the current state. While this approach is not suitable for use in an actual hyperdynamics simulation, because the pathways are generally not known in advance, it allows us to show that it is possible to come very close to the theoretical boost limit of hyperdynamics while maintaining high accuracy. We demonstrate this by applying this MEP-based hyperdynamics (MEP-HD) to metallic surface diffusion systems. In most cases, MEP-HD gives boost factors that are orders of magnitude larger than the best existing bias potential, indicating that further development of hyperdynamics bias potentials could have a significant payoff. Lastly, we discuss potential practical uses of MEP-HD, including the possibility of developing MEP-HD into a true hyperdynamics.

  16. Contact-Mediated Inhibition Between Oligodendrocyte Progenitor Cells and Motor Exit Point Glia Establishes the Spinal Cord Transition Zone

    PubMed Central

    Smith, Cody J.; Morris, Angela D.; Welsh, Taylor G.; Kucenas, Sarah

    2014-01-01

    Rapid conduction of action potentials along motor axons requires that oligodendrocytes and Schwann cells myelinate distinct central and peripheral nervous system (CNS and PNS) domains along the same axon. Despite the importance of this arrangement for nervous system function, the mechanisms that establish and maintain this precise glial segregation at the motor exit point (MEP) transition zone are unknown. Using in vivo time-lapse imaging in zebrafish, we observed that prior to myelination, oligodendrocyte progenitor cells (OPCs) extend processes into the periphery via the MEP and immediately upon contact with spinal motor root glia retract back into the spinal cord. Characterization of the peripheral cell responsible for repelling OPC processes revealed that it was a novel, CNS-derived population of glia we propose calling MEP glia. Ablation of MEP glia resulted in the absence of myelinating glia along spinal motor root axons and an immediate breach of the MEP by OPCs. Taken together, our results identify a novel population of CNS-derived peripheral glia located at the MEP that selectively restrict the migration of OPCs into the periphery via contact-mediated inhibition. PMID:25268888

  17. Electrophysiological assessment of corticorespiratory pathway function in amyotrophic lateral sclerosis.

    PubMed

    Shimizu, Toshio; Komori, Tetsuo; Kugio, Yumiko; Fujimaki, Yumi; Oyanagi, Kiyomitsu; Hayashi, Hideaki

    2010-01-01

    Respiratory muscle paralysis is inevitable in the clinical course of amyotrophic lateral sclerosis (ALS). Our objective was to electrophysiologically assess the function of the phrenic nerve and diaphragm motor cortex in ALS. Phrenic nerve M waves, diaphragm motor evoked potentials (MEPs) induced by transcranial magnetic stimulation, and their clinical correlations were analyzed in 29 ALS patients. The M wave amplitude was significantly lower in patients than in healthy control subjects (p<0.001). The MEP amplitudes both in the expiratory and inspiratory phases were significantly decreased in patients (p<0.01). In particular, 15 patients showed no MEPs in the expiratory phase, six of whom also showed no MEPs in the inspiratory phase. Five of them had no respiratory complaints. There was a weak, non-significant correlation between the inspiratory MEP amplitude and forced vital capacity (p=0.052). We conclude that the loss of MEP might reflect the subclinical involvement of the voluntary respiratory drive from the diaphragm motor cortex, potentially leading to further respiratory deterioration. PMID:19707909

  18. GABAergic mechanisms contributing to categorical amygdala responses to chemosensory signals.

    PubMed

    Westberry, Jenne M; Meredith, Michael

    2016-09-01

    Chemosensory stimuli from conspecific and heterospecific animals, elicit categorically different immediate-early gene response-patterns in medial amygdala in male hamsters and mice. We previously showed that conspecific signals activate posterior (MeP) as well as anterior medial amygdala (MeA), and especially relevant heterospecific signals such as chemosensory stimuli from potential predators also activate MeP in mice. Other heterospecific chemosignals activate MeA, but not MeP. Here we show that male hamster amygdala responds significantly differentially to different conspecific signals, by activating different proportions of cells of different phenotype, possibly leading to differential activation of downstream circuits. Heterospecific signals that fail to activate MeP do activate GABA-immunoreactive cells in the adjacent caudal main intercalated nucleus (mICNc) and elicit selective suppression of MeP cells bearing GABA-Receptors, suggesting GABA inhibition in MeP by GABAergic cells in mICNc. Overall, work presented here suggests that medial amygdala may discriminate between important conspecific social signals, distinguish them from the social signals of other species and convey that information to brain circuits eliciting appropriate social behavior. PMID:27329335

  19. Degradation of methylparaben in water by corona plasma coupled with ozonation.

    PubMed

    Dobrin, D; Magureanu, M; Bradu, C; Mandache, N B; Ionita, P; Parvulescu, V I

    2014-11-01

    The degradation of methylparaben (MeP) in water was investigated using a pulsed corona discharge generated in oxygen, above the liquid. A comparison was made between results obtained in semi-batch corona (SBC) configuration (stationary solution, continuous gas flow) and results obtained in a semi-batch corona with recirculation combined with ozonation (SBCR + O3), where the liquid is continuously circulated between a solution reservoir and the plasma reactor and the effluent gas containing ozone is bubbled through the solution in the reservoir. It was found that MeP was completely degraded after 10-15 min of treatment in both configurations. Oxidation by ozone alone, in the absence of plasma, was a slower process. The energy efficiency for MeP removal (Y MeP) and for mineralization (Y TOC) was significantly higher in the SBCR + O3 configuration (Y MeP = 7.1 g/kWh at 90 % MeP removal and Y TOC = 0.41 g/kWh at 50 % total organic carbon (TOC) removal) than in the SBC configuration (Y MeP = 0.6 g/kWh at 90 % MeP removal and Y TOC = 0.11 g/kWh at 50 % TOC removal). PMID:24801291

  20. Synergy between methylerythritol phosphate pathway and mevalonate pathway for isoprene production in Escherichia coli.

    PubMed

    Yang, Chen; Gao, Xiang; Jiang, Yu; Sun, Bingbing; Gao, Fang; Yang, Sheng

    2016-09-01

    Isoprene, a key building block of synthetic rubber, is currently produced entirely from petrochemical sources. In this work, we engineered both the methylerythritol phosphate (MEP) pathway and the mevalonate (MVA) pathway for isoprene production in E. coli. The synergy between the MEP pathway and the MVA pathway was demonstrated by the production experiment, in which overexpression of both pathways improved the isoprene yield about 20-fold and 3-fold, respectively, compared to overexpression of the MEP pathway or the MVA pathway alone. The (13)C metabolic flux analysis revealed that simultaneous utilization of the two pathways resulted in a 4.8-fold increase in the MEP pathway flux and a 1.5-fold increase in the MVA pathway flux. The synergy of the dual pathway was further verified by quantifying intracellular flux responses of the MEP pathway and the MVA pathway to fosmidomycin treatment and mevalonate supplementation. Our results strongly suggest that coupling of the complementary reducing equivalent demand and ATP requirement plays an important role in the synergy of the dual pathway. Fed-batch cultivation of the engineered strain overexpressing the dual pathway resulted in production of 24.0g/L isoprene with a yield of 0.267g/g of glucose. The synergy of the MEP pathway and the MVA pathway also successfully increased the lycopene productivity in E. coli, which demonstrates that it can be used to improve the production of a broad range of terpenoids in microorganisms. PMID:27174717

  1. Arabidopsis Chlorophyll Biosynthesis: An Essential Balance between the Methylerythritol Phosphate and Tetrapyrrole Pathways[C][W

    PubMed Central

    Kim, Se; Schlicke, Hagen; Van Ree, Kalie; Karvonen, Kristine; Subramaniam, Anant; Richter, Andreas; Grimm, Bernhard; Braam, Janet

    2013-01-01

    Chlorophyll, essential for photosynthesis, is composed of a chlorin ring and a geranylgeranyl diphosphate (GGPP)–derived isoprenoid, which are generated by the tetrapyrrole and methylerythritol phosphate (MEP) biosynthesis pathways, respectively. Although a functional MEP pathway is essential for plant viability, the underlying basis of the requirement has been unclear. We hypothesized that MEP pathway inhibition is lethal because a reduction in GGPP availability results in a stoichiometric imbalance in tetrapyrrolic chlorophyll precursors, which can cause deadly photooxidative stress. Consistent with this hypothesis, lethality of MEP pathway inhibition in Arabidopsis thaliana by fosmidomycin (FSM) is light dependent, and toxicity of MEP pathway inhibition is reduced by genetic and chemical impairment of the tetrapyrrole pathway. In addition, FSM treatment causes a transient accumulation of chlorophyllide and transcripts associated with singlet oxygen-induced stress. Furthermore, exogenous provision of the phytol molecule reduces FSM toxicity when the phytol can be modified for chlorophyll incorporation. These data provide an explanation for FSM toxicity and thereby provide enhanced understanding of the mechanisms of FSM resistance. This insight into MEP pathway inhibition consequences underlines the risk plants undertake to synthesize chlorophyll and suggests the existence of regulation, possibly involving chloroplast-to-nucleus retrograde signaling, that may monitor and maintain balance of chlorophyll precursor synthesis. PMID:24363312

  2. Basic Principles and Recent Trends of Transcranial Motor Evoked Potentials in Intraoperative Neurophysiologic Monitoring

    PubMed Central

    TSUTSUI, Shunji; YAMADA, Hiroshi

    2016-01-01

    Transcranial motor evoked potentials (TcMEPs), which are muscle action potentials elicited by transcranial brain stimulation, have been the most popular method for the last decade to monitor the functional integrity of the motor system during surgery. It was originally difficult to record reliable and reproducible potentials under general anesthesia, especially when inhalation-based anesthetic agents that suppressed the firing of anterior horn neurons were used. Advances in anesthesia, including the introduction of intravenous anesthetic agents, and progress in stimulation techniques, including the use of pulse trains, improved the reliability and reproducibility of TcMEP responses. However, TcMEPs are much smaller in amplitude compared with compound muscle action potentials evoked by maximal peripheral nerve stimulation, and vary from one trial to another in clinical practice, suggesting that only a limited number of spinal motor neurons innervating the target muscle are excited in anesthetized patients. Therefore, reliable interpretation of the critical changes in TcMEPs remains difficult and controversial. Additionally, false negative cases have been occasionally encountered. Recently, several facilitative techniques using central or peripheral stimuli, preceding transcranial electrical stimulation, have been employed to achieve sufficient depolarization of motor neurons and augment TcMEP responses. These techniques might have potentials to improve the reliability of intraoperative motor pathway monitoring using TcMEPs. PMID:26935781

  3. Immediate plasticity in the motor pathways after spinal cord hemisection: implications for transcranial magnetic motor-evoked potentials.

    PubMed

    Fujiki, Minoru; Kobayashi, Hidenori; Inoue, Ryo; Ishii, Keisuke

    2004-06-01

    The present study evaluates motor functional recovery after C2 spinal cord hemisection with or without contralateral brachial root transection, which causes a condition that is similar to the crossed phrenic phenomenon on rats. Descending motor pathways, including the reticulospinal extrapyramidal tract and corticospinal pyramidal tracts, were evaluated by transcranial magnetic motor-evoked potentials (mMEPs) and direct cortical electrical motor-evoked potentials (eMEP), respectively. All MEPs recorded from the left forelimb were abolished immediately after the left C2 hemisection. Left mMEPs recovered dramatically immediately after contralateral right brachial root transection. Corticospinal eMEPs never recovered, regardless of transection. The facilitation of mMEPs in animals that had undergone combined contralateral root transection was well correlated with open-field behavioral motor performance. Both electrophysiological and neurological facilitations were significantly attenuated by the selective serotonin synthesis inhibitor para-chlorophenylalanine (p-CPA). These results suggest that serotonergic reticulospinal fibers located contralateral to hemisection contribute to the behavioral and electrophysiological improvement that immediately follows spinal cord injury (SCI). PMID:15144873

  4. Facilitatory effect of thinking about movement on magnetic motor-evoked potentials.

    PubMed

    Kiers, L; Fernando, B; Tomkins, D

    1997-08-01

    To investigate the facilitatory effect of thinking about movement on motor evoked potential (MEP) amplitude, we recorded MEPs in two test muscles during rest, with the subject thinking about contracting the test muscle but without subsequent contraction, and during 10% maximum voluntary contraction. Stimuli were delivered at 10% above resting motor threshold and at 90-100% stimulator output. H-reflexes, recorded in flexor carpi radialis, were obtained during rest and think conditions. MEP threshold was lower during the think condition (P = 0.004). At both stimulus intensities, median MEP amplitudes and areas were significantly (P < 0.001) larger during the think paradigm compared with rest. This effect was greater at the lower stimulus intensity. There was no significant difference in latency (P = 0.15). In 4/8 subjects, H-reflex amplitudes were mildly facilitated (P < 0.05) during the think condition. We conclude that thinking about movement without detectable EMG activity has a facilitatory effect on magnetic MEPs. The absence of a MEP latency shift between rest and think conditions and absence of a consistent increase in H-reflex amplitude suggests this effect occurs largely at the cortical level. In some subjects, however, an increase in spinal motoneuron excitability may also contribute. PMID:9284233

  5. Ipsilesional motor-evoked potential absence in pediatric hemiparesis impacts tracking accuracy of the less affected hand.

    PubMed

    Cassidy, Jessica M; Carey, James R; Lu, Chiahao; Krach, Linda E; Feyma, Tim; Durfee, William K; Gillick, Bernadette T

    2015-12-01

    This study analyzed the relationship between electrophysiological responses to transcranial magnetic stimulation (TMS), finger tracking accuracy, and volume of neural substrate in children with congenital hemiparesis. Nineteen participants demonstrating an ipsilesional motor-evoked potential (MEP) were compared with eleven participants showing an absent ipsilesional MEP response. Comparisons of finger tracking accuracy from the affected and less affected hands and ipsilesional/contralesional (I/C) volume ratio for the primary motor cortex (M1) and posterior limb of internal capsule (PLIC) were done using two-sample t-tests. Participants showing an ipsilesional MEP response demonstrated superior tracking performance from the less affected hand (p=0.016) and significantly higher I/C volume ratios for M1 (p=0.028) and PLIC (p=0.005) compared to participants without an ipsilesional MEP response. Group differences in finger tracking accuracy from the affected hand were not significant. These results highlight differentiating factors amongst children with congenital hemiparesis showing contrasting MEP responses: less affected hand performance and preserved M1 and PLIC volume. Along with MEP status, these factors pose important clinical implications in pediatric stroke rehabilitation. These findings may also reflect competitive developmental processes associated with the preservation of affected hand function at the expense of some function in the less affected hand. PMID:26426515

  6. Hyperdynamics boost factor achievable with an ideal bias potential

    NASA Astrophysics Data System (ADS)

    Huang, Chen; Perez, Danny; Voter, Arthur F.

    2015-08-01

    Hyperdynamics is a powerful method to significantly extend the time scales amenable to molecular dynamics simulation of infrequent events. One outstanding challenge, however, is the development of the so-called bias potential required by the method. In this work, we design a bias potential using information about all minimum energy pathways (MEPs) out of the current state. While this approach is not suitable for use in an actual hyperdynamics simulation, because the pathways are generally not known in advance, it allows us to show that it is possible to come very close to the theoretical boost limit of hyperdynamics while maintaining high accuracy. We demonstrate this by applying this MEP-based hyperdynamics (MEP-HD) to metallic surface diffusion systems. In most cases, MEP-HD gives boost factors that are orders of magnitude larger than the best existing bias potential, indicating that further development of hyperdynamics bias potentials could have a significant payoff. Finally, we discuss potential practical uses of MEP-HD, including the possibility of developing MEP-HD into a true hyperdynamics.

  7. Repeated tongue lift movement induces neuroplasticity in corticomotor control of tongue and jaw muscles in humans.

    PubMed

    Komoda, Yoshihiro; Iida, Takashi; Kothari, Mohit; Komiyama, Osamu; Baad-Hansen, Lene; Kawara, Misao; Sessle, Barry; Svensson, Peter

    2015-11-19

    This study investigated the effect of repeated tongue lift training (TLT) on the excitability of the corticomotor representation of the human tongue and jaw musculature. Sixteen participants performed three series of TLT for 41 min on each of 5 consecutive days. Each TLT series consisted of two pressure levels (5 kPa and 10 kPa). All participants underwent transcranial magnetic stimulation (TMS) and electromyographic (EMG) recordings of motor evoked potentials (MEPs) in four sessions: (1) before TLT on Day 1 (baseline), (2) after TLT on Day 1, (3) before TLT on Day 5, and (4) after TLT on Day 5. EMG recordings from the left and right tongue dorsum and masseter muscles were made at three pressure levels (5 kPa, 10 kPa, 100% tongue lift), and tongue, masseter, and first dorsal interosseous (FDI) MEPs were measured. There were no significant day-to-day differences in the tongue pressure during maximum voluntary contractions. The amplitudes and thresholds of tongue and masseter MEPs after TLT on Day 5 were respectively higher and lower than before TLT on Day 1 (P<0.005), and there was also a significant increase in tongue and masseter MEP areas; no significant changes occurred in MEP onset latencies. FDI MEP parameters (amplitude, threshold, area, latency) were not significantly different between the four sessions. Our findings suggest that repeated TLT can trigger neuroplasticity reflected in increased excitability of the corticomotor representation of not only the tongue muscles but also the masseter muscles. PMID:26399776

  8. Identification of human erythroid lineage-committed progenitors.

    PubMed

    Mori, Yasuo; Akashi, Koichi; Weissman, Irving L

    2016-05-01

    Elucidating the developmental pathway leading to erythrocytes and being able to isolate their progenitors is crucial to understanding and treating disorders of red cell imbalance such as anemia, myelodysplastic syndrome, and polycythemia vera. Endoglin (CD105) is a key marker for purifying mouse erythroid lineage-committed progenitors (EPs) from bone marrow. Herein, we show that human EPs can also be isolated from adult bone marrow. We identified three subfractions that possessed different expression patterns of CD105 and CD71 within the previously defined human megakaryocyte/erythrocyte progenitor (hMEP; Lineage-CD34(+)CD38(+)IL-3Rα(-)CD45RA(-)) population. Both CD71(-)CD105(-) and CD71(+)CD105(-) MEPs, at least in vitro, retained bipotency for the megakaryocyte (MegK) and erythrocyte (E) lineages, although the latter sub-population had a differentiation potential skewed toward the E-lineage. Notably, the differentiation output of the CD71(+)CD105(+) subset of cells within the MEP population was completely restricted to the E-lineage with the loss of MegK potential; thus, we termed CD71(+)CD105(-) MEPs and CD71(+)CD105(+) cells as E-biased MEPs (E-MEPs) and EPs, respectively. These previously unclassified populations may facilitate understanding of the molecular mechanisms governing human erythroid development and serve as potential therapeutic targets in disorders of the erythroid lineage. PMID:27263782

  9. Utility of magneto-electropolished ternary nitinol alloys for blood contacting applications.

    PubMed

    Pulletikurthi, Chandan; Munroe, Norman; Stewart, Danique; Haider, Waseem; Amruthaluri, Sushma; Rokicki, Ryszard; Dugrot, Manuel; Ramaswamy, Sharan

    2015-10-01

    The thrombogenicity of a biomaterial is mainly dependent on its surface characteristics, which dictates its interactions with blood. Surface properties such as composition, roughness wettability, surface free energy, and morphology will affect an implant material's hemocompatibility. Additionally, in the realm of metallic biomaterials, the specific composition of the alloy and its surface treatment are important factors that will affect the surface properties. The utility of magneto-electropolished (MEP) ternary Nitinol alloys, NiTiTa, and NiTiCr as blood contacting materials was investigated. The hemcompatibility of these alloys were compared to mechanically polished (MP) metallic biomaterial counterparts. In vitro thrombogenicity tests revealed significantly less platelet adherence on ternary MEP Nitinol, especially MEP NiTi10Ta as compared to the MP metals (p < 0.05). The enhanced anti-platelet-adhesive property of MEP NiTi10Ta was in part, attributed to the Ta2 O5 component of the alloy. Furthermore, the formation of a dense and mixed hydrophobic oxide layer during MEP is believed to have inhibited the adhesion of negatively charged platelets. In conclusion, MEP ternary Nitinol alloys can potentially be utilized for blood-contacting devices where, complications resulting from thrombogenicity can be minimized. PMID:25384352

  10. Longitudinal Evaluation of Residual Cortical and Subcortical Motor Evoked Potentials in Spinal Cord Injured Rats.

    PubMed

    Redondo-Castro, Elena; Navarro, Xavier; García-Alías, Guillermo

    2016-05-15

    We have applied transcranial electrical stimulation to rats with spinal cord injury and selectively tested the motor evoked potentials (MEPs) conveyed by descending motor pathways with cortical and subcortical origin. MEPs were elicited by electrical stimulation to the brain and recorded on the tibialis anterior muscles. Stimulation parameters were characterized and changes in MEP responses tested in uninjured rats, in rats with mild or moderate contusion, and in animals with complete transection of the spinal cord. All injuries were located at the T8 vertebral level. Two peaks, termed N1 and N2, were obtained when changing from single pulse stimulation to trains of 9 pulses at 9 Hz. Selective injuries to the brain or spinal cord funiculi evidenced the subcortical origin of N1 and the cortical origin of N2. Animals with mild contusion showed small behavioral deficits and abolished N1 but maintained small amplitude N2 MEPs. Substantial motor deficits developed in rats with moderate contusion, and these rats had completely eliminated N1 and N2 MEPs. Animals with complete cord transection had abolished N1 and N2 and showed severe impairment of locomotion. The results indicate the reliability of MEP testing to longitudinally evaluate over time the degree of impairment of cortical and subcortical spinal pathways after spinal cord injuries of different severity. PMID:26560177

  11. Spread of electrical activity at cortical level after repetitive magnetic stimulation in normal subjects.

    PubMed

    Lorenzano, C; Gilio, F; Inghilleri, M; Conte, A; Fofi, L; Manfredi, M; Berardelli, A

    2002-11-01

    In normal subjects, focal repetitive transcranial magnetic stimulation (rTMS) of the hand motor area evokes muscle potentials (MEPs) from muscles in the hand (target muscles) and the arm (non-target muscles). In this study we investigated the mechanisms underlying the spread of MEPs induced by focal rTMS in non-target muscles. rTMS was delivered with a Magstim stimulator and a figure-of-eight coil placed over the first dorsal interosseus (FDI) motor area of the left hemisphere. Trains of 10 stimuli were given at a suprathreshold intensity (120% of motor threshold) and at frequencies of 5, 10 and 20 Hz at rest. Electromyographic (EMG) activity was recorded simultaneously from the FDI (target muscle) and the contralateral biceps muscle and from the FDI muscle ipsilateral to the side of stimulation (non-target muscle). rTMS delivered in trains to the FDI motor area of the left hemisphere elicited MEPs in the contralateral FDI (target muscle) that gradually increased in amplitude over the course of the train. Focal rTMS trains also induced MEPs in the contralateral biceps (non-target muscle) but did so only after the second or third stimulus; like target-muscle MEPs, in non-target muscle MEPs progressively increased in amplitude during the train. At no frequency did rTMS elicit MEPs in the FDI muscle ipsilateral to the site of stimulation. rTMS left the latency of EMG responses in the FDI and biceps muscles unchanged during the trains of stimuli. The latency of biceps MEPs was longer after rTMS than after a single TMS pulse. In conditioning-test experiments designed to investigate the cortical origin of the spread, a single TMS pulse delivered over the left hemisphere at an interstimulus interval (ISI) of 50, 100 and 150 ms reduced the amplitude of the test MEP evoked by a single TMS pulse delivered over the right hemisphere; and a conditioning rTMS train delivered over the left hemisphere increased the amplitude of the test MEP evoked by a single TMS pulse over the

  12. Comparison of Motor Inhibition in Variants of the Instructed-Delay Choice Reaction Time Task.

    PubMed

    Quoilin, Caroline; Lambert, Julien; Jacob, Benvenuto; Klein, Pierre-Alexandre; Duque, Julie

    2016-01-01

    Using instructed-delay choice reaction time (RT) paradigms, many previous studies have shown that the motor system is transiently inhibited during response preparation: motor-evoked potentials (MEPs) elicited by transcranial magnetic stimulation (TMS) over the primary motor cortex are typically suppressed during the delay period. This effect has been observed in both selected and non-selected effectors, although MEP changes in selected effectors have been more inconsistent across task versions. Here, we compared changes in MEP amplitudes in three different variants of an instructed-delay choice RT task. All variants required participants to choose between left and right index finger movements but the responses were either provided "in the air" (Variant 1), on a regular keyboard (Variant 2), or on a response device designed to control from premature responses (Variant 3). The task variants also differed according to the visual layout (more concrete in Variant 3) and depending on whether participants received a feedback of their performance (absent in Variant 1). Behavior was globally comparable between the three variants of the task although the propensity to respond prematurely was highest in Variant 2 and lowest in Variant 3. MEPs elicited in a non-selected hand were similarly suppressed in the three variants of the task. However, significant differences emerged when considering MEPs elicited in the selected hand: these MEPs were suppressed in Variants 1 and 3 whereas they were often facilitated in Variant 2, especially in the right dominant hand. In conclusion, MEPs elicited in selected muscles seem to be more sensitive to small variations to the task design than those recorded in non-selected effectors, probably because they reflect a complex combination of inhibitory and facilitatory influences on the motor output system. Finally, the use of a standard keyboard seems to be particularly inappropriate because it encourages participants to respond promptly with no

  13. Farnesol-mediated shift in the metabolic origin of prenyl groups used for protein prenylation in plants.

    PubMed

    Huchelmann, Alexandre; Brahim, Mathieu Semir; Gerber, Esther; Tritsch, Denis; Bach, Thomas J; Hemmerlin, Andréa

    2016-08-01

    Little is known about how plant cells regulate the exchange of prenyl diphosphates between the two compartmentalized isoprenoid biosynthesis pathways. Prenylation of proteins is a suitable model to study such interactions between the plastidial methylerythritol phosphate (MEP) and the cytosolic mevalonate (MVA) pathways because prenyl moieties used to modify proteins rely on both origins. Tobacco cells expressing a prenylatable GFP were treated with specific MEP and/or MVA pathways inhibitors to block the formation of prenyl diphosphates and therefore the possibility to modify the proteins. Chemical complementation assays using prenyl alcohol precursors restore the prenylation. Indeed, geranylgeraniol (C20 prenyl alcohol) and to a lesser but significant level C15-farnesol restored the prenylation of a protein bearing a geranylgeranylation CaaX motif, which under standard conditions is modified by a MEP-derived prenyl group. However, the restoration takes place in different ways. While geranylgeraniol operates directly as a metabolic precursor, the C15-prenyl alcohol functions indirectly as a signal that leads to shift the metabolic origin of prenyl groups in modified proteins, here from the plastidial MEP pathway in favor of the cytosolic MVA pathway. Furthermore, farnesol interferes negatively with the MEP pathway in an engineered Escherichia coli strain synthesizing isoprenoids either starting from MVA or from MEP. Following the cellular uptake of a fluorescent analog of farnesol, we showed its close interaction with tobacco plastids and modification of plastid homeostasis. As a consequence, in tobacco farnesol supposedly inhibits the plastidial MEP pathway and activates the cytosolic MVA pathway, leading to the shift in the metabolic origin and thereby acts as a potential regulator of crosstalk between the two pathways. Together, those results suggest a new role for farnesol (or a metabolite thereof) as a central molecule for the regulation of isoprenoid

  14. A microcalorimetric study of molecular interactions between immunoglobulin G and hydrophobic charge-induction ligand.

    PubMed

    Yuan, Xiao-Ming; Lin, Dong-Qiang; Zhang, Qi-Lei; Gao, Dong; Yao, Shan-Jing

    2016-04-22

    Hydrophobic charge-induction chromatography (HCIC) with 4-mercaptoethyl-pyridine (MEP) as the ligand is a novel technology for antibody purification. In this study, isothermal titration calorimetry (ITC) was used to evaluate the molecular interactions between MEP ligand and immunoglobulin G (IgG). Three types of IgG molecules including human IgG (hIgG), bovine IgG (bIgG) and a monoclonal antibody (mAb) were investigated with human serum albumins (HSA) and bovine serum albumin (BSA) as the comparison. The thermodynamic parameters obtained from ITC were compared with the adsorption data. The results indicated that MEP binding to protein at neutral pH was entropy driven and induced by multimodal molecular interactions that was dominated by hydrophobic forces. The interactions between MEP and IgGs were stronger than that of albumins, which resulted in high binding affinity of IgGs. Moreover, the effects of pH and salt addition on MEP-hIgG binding were studied. The change of enthalpy increased obviously with the decrease of pH, which revealed that the electrostatic forces dominated the MEP-hIgG interactions at acidic condition and caused typical charge-induced elution of HCIC. Salt addition influenced both hydrophobic and electrostatic interactions. With the increase of salt concentration, the hydrophobic interactions decreased first and then increased, while the electrostatic interactions showed the opposite trend. This resulted in trade-off between the multimodal interactions, which caused the salt-tolerant property of MEP resin. In general, ITC studies revealed the molecular mechanism of three critical characteristics of HCIC, multimodal interactions, pH-dependent and salt-tolerant properties. PMID:27017449

  15. Comparison of Diffusion Tensor Tractography and Motor Evoked Potentials for the Estimation of Clinical Status in Subacute Stroke

    PubMed Central

    Chun, Kwang-Soo; Lee, Yong-Taek; Park, Jong-Wan; Lee, Joon-Youn; Park, Chul-Hyun

    2016-01-01

    Objective To compare diffusion tensor tractography (DTT) and motor evoked potentials (MEPs) for estimation of clinical status in patients in the subacute stage of stroke. Methods Patients with hemiplegia due to stroke who were evaluated using both DTT and MEPs between May 2012 and April 2015 were recruited. Clinical assessments investigated upper extremity motor and functional status. Motor status was evaluated using Medical Research Council grading and the Fugl-Meyer Assessment of upper limb and hand (FMA-U and FMA-H). Functional status was measured using the Modified Barthel Index (MBI). Patients were classified into subgroups according to DTT findings, MEP presence, fractional anisotropy (FA) value, FA ratio (rFA), and central motor conduction time (CMCT). Correlations of clinical assessments with DTT parameters and MEPs were estimated. Results Fifty-five patients with hemiplegia were recruited. In motor assessments (FMA-U), MEPs had the highest sensitivity and negative predictive value (NPV) as well as the second highest specificity and positive predictive value (PPV). CMCT showed the highest specificity and PPV. Regarding functional status (MBI), FA showed the highest sensitivity and NPV, whereas CMCT had the highest specificity and PPV. Correlation analysis showed that the resting motor threshold (RMT) ratio was strongly associated with motor status of the upper limb, and MEP parameters were not associated with MBI. Conclusion DTT and MEPs could be suitable complementary modalities for analyzing the motor and functional status of patients in the subacute stage of stroke. The RMT ratio was strongly correlated with motor status. PMID:26949679

  16. The Applicability of Intraoperative Neuromonitoring in Patients with Preoperative Motor Weakness during Spine Surgery

    PubMed Central

    Lee, Jae Meen; Kim, Dong Hwan; Kim, Hwan Soo; Choi, Byung Kwan

    2016-01-01

    Objective The purpose of our study is to evaluate the success rate and feasibility of intraoperative neuromonitoring (IONM) focusing on transcranial motor evoked potential (TcMEP) monitoring for patients with preoperative motor weakness in spine surgery. Methods Between November 2011 and December 2013, TcMEP and somatosensory evoked potential (SSEP) monitoring were attempted in 130 consecutive patients undergoing spine surgeries for cervical or thoracic cord lesions. Patients ranged in age from 14 to 81 years (mean±standard deviation, 56.7±14.8 years), and 84 patients were male. The success rates of both SSEP and MEPs monitoring were assessed according to the preoperative Medical Research Council (MRC) and Nurick grades. Results TcMEP was recorded successfully in 0%, 28.6%, 72.3%, and 100% of patients with MRC grades 1, 2, 3, 4, and 5, respectively. SSEP was obtained from 0%, 37.5%, 21.5%, 61.4%, and 85.4% of patients with MRC grades 1, 2, 3, 4, and 5, respectively. TcMEP was recorded successfully in 84% of patients with Nurick grades 1-3 and 26% of patients with Nurick grades 4-5. SSEPs were recorded successfully in 76.3% of patients with Nurick grades 1-3 and 24% of patients with grades 4-5. Conclusion IONM during spine surgery may be useless in patients with MRC grades 1-2, applicable MRC grade 3, and useful MRC grades 4-5. MRC grade 3 is a critical point of indication for application of MEPs. In unmonitorable cases with MRC grade 3, increasing stimulus intensity or facilitation techniques may be considered to improve the usefulness of TcMEP. PMID:27123024

  17. Abnormal surround inhibition does not affect asymptomatic limbs in people with cervical dystonia.

    PubMed

    McDougall, Laura; Kiernan, Dovin; Kiss, Zelma H T; Suchowersky, Oksana; Welsh, Timothy N

    2015-09-14

    Surround inhibition is a neural mechanism hypothesized to facilitate goal-directed action by disinhibiting agonist muscle activity while simultaneously inhibiting antagonist and other uninvolved muscle activity. The present study was designed to investigate if abnormalities in surround inhibition are found in asymptomatic body parts (the hand) of people with focal cervical dystonia (neck). Participants with (n=7) and without (n=17) cervical dystonia completed a protocol in which they abducted their index finger while EMG was recorded from the first dorsal interosseous (agonist) and abductor digiti minimi (uninvolved) muscles. Transcranial magnetic stimulation was delivered over the primary motor cortex at intervals ranging from 0 to 950+ms after the onset of agonist muscle activity. Motor-evoked potential (MEP) amplitudes from both muscles were compared. In control participants, MEPs from the uninvolved muscle were significantly lower than agonist MEPs at intervals from 0 to 480ms. Similarly, in the hands of participants with cervical dystonia - the asymptomatic body part - MEPs from the uninvolved muscle were significantly lower than agonist MEPs from 0 to 175ms. These findings suggest that surround inhibition in people with focal dystonia may be intact in asymptomatic hands. In other words, abnormalities in surround inhibition may be restricted to the dystonic limb. PMID:26219986

  18. Stereospecific suppression of active site mutants by methylphosphonate substituted substrates reveals the stereochemical course of site-specific DNA recombination

    PubMed Central

    Rowley, Paul A.; Kachroo, Aashiq H.; Ma, Chien-Hui; Maciaszek, Anna D.; Guga, Piotr; Jayaram, Makkuni

    2015-01-01

    Tyrosine site-specific recombinases, which promote one class of biologically important phosphoryl transfer reactions in DNA, exemplify active site mechanisms for stabilizing the phosphate transition state. A highly conserved arginine duo (Arg-I; Arg-II) of the recombinase active site plays a crucial role in this function. Cre and Flp recombinase mutants lacking either arginine can be rescued by compensatory charge neutralization of the scissile phosphate via methylphosphonate (MeP) modification. The chemical chirality of MeP, in conjunction with mutant recombinases, reveals the stereochemical contributions of Arg-I and Arg-II. The SP preference of the native reaction is specified primarily by Arg-I. MeP reaction supported by Arg-II is nearly bias-free or RP-biased, depending on the Arg-I substituent. Positional conservation of the arginines does not translate into strict functional conservation. Charge reversal by glutamic acid substitution at Arg-I or Arg-II has opposite effects on Cre and Flp in MeP reactions. In Flp, the base immediately 5′ to the scissile MeP strongly influences the choice between the catalytic tyrosine and water as the nucleophile for strand scission, thus between productive recombination and futile hydrolysis. The recombinase active site embodies the evolutionary optimization of interactions that not only favor the normal reaction but also proscribe antithetical side reactions. PMID:25999343

  19. Can the hydrophilicity of functional monomers affect chemical interaction?

    PubMed

    Feitosa, V P; Ogliari, F A; Van Meerbeek, B; Watson, T F; Yoshihara, K; Ogliari, A O; Sinhoreti, M A; Correr, A B; Cama, G; Sauro, S

    2014-02-01

    The number of carbon atoms and/or ester/polyether groups in spacer chains may influence the interaction of functional monomers with calcium and dentin. The present study assessed the chemical interaction and bond strength of 5 standard-synthesized phosphoric-acid ester functional monomers with different spacer chain characteristics, by atomic absorption spectroscopy (AAS), ATR-FTIR, thin-film x-ray diffraction (TF-XRD), scanning electron microscopy (SEM), and microtensile bond strength (μTBS). The tested functional monomers were 2-MEP (two-carbon spacer chain), 10-MDP (10-carbon), 12-MDDP (12-carbon), MTEP (more hydrophilic polyether spacer chain), and CAP-P (intermediate hydrophilicity ester spacer). The intensity of monomer-calcium salt formation measured by AAS differed in the order of 12-MDDP=10-MDP>CAP-P>MTEP>2-MEP. FTIR and SEM analyses of monomer-treated dentin surfaces showed resistance to rinsing for all monomer-dentin bonds, except with 2-MEP. TF-XRD confirmed the weaker interaction of 2-MEP. Highest µTBS was observed for 12-MDDP and 10-MDP. A shorter spacer chain (2-MEP) of phosphate functional monomers induced formation of unstable monomer-calcium salts, and lower chemical interaction and dentin bond strength. The presence of ester or ether groups within longer spacer carbon chains (CAP-P and MTEP) may affect the hydrophilicity, μTBS, and also the formation of monomer-calcium salts. PMID:24284259

  20. Influence of vegetation in mitigation of methyl parathion runoff.

    PubMed

    Moore, M T; Bennett, E R; Cooper, C M; Smith, S; Farris, J L; Drouillard, K G; Schulz, R

    2006-07-01

    A pesticide runoff event was simulated on two 10 m x 50 m constructed wetlands (one non-vegetated, one vegetated) to evaluate the fate of methyl parathion (MeP) (Penncap-M). Water, sediment, and plant samples were collected at five sites downstream of the inflow for 120 d. Semi-permeable membrane devices (SPMDs) were deployed at each wetland outflow to determine exiting pesticide load. MeP was detected in water at all locations of the non-vegetated wetland (50 m), 30 min post-exposure. MeP was detected 20 m from the vegetated wetland inflow 30 min post-exposure, while after 10d it was detected only at 10 m. MeP was measured only in SPMDs deployed in non-vegetated wetland cells, suggesting detectable levels were not present near the vegetated wetland outflow. Furthermore, mass balance calculations indicated vegetated wetlands were more effective in reducing aqueous loadings of MeP introduced into the wetland systems. This demonstrates the importance of vegetation as sorption sites for pesticides in constructed wetlands. PMID:16314013

  1. Activation and suppression of the trapezius muscle induced by transcranial magnetic stimulation.

    PubMed

    Strenge, H; Jahns, R

    1998-01-01

    Motor evoked potentials (MEPs) and silent periods (SPs) in the trapezius muscle induced by transcranial magnetic stimulation (TMS) were investigated in 15 healthy subjects. Stimuli were applied with a Novametrix Magnetic stimulator using a 14 cm circular coil 4 cm lateral to the vertex on the biauricular line. Surface electrodes were used for simultaneous bilateral electromyographic recordings of the trapezius. TMS invariably induced contralateral MEPs (latency 10.5 +/- 1.3 ms, mean +/- SD), with ipsilateral responses in 53% of the subjects (latency 11.1 +/- 2.5 ms). The mean duration of the SPs was approximately 90 ms on both sides. There were no significant side differences between any of the MEP or SP parameters. To study the influence of subcortical inhibition phenomena TMS induced responses were assessed following electrical mental nerve stimulation with interstimulus intervals (ISI) of 0-100 ms. MEP latencies significantly increased at ISI of 10-100 ms, whereas MEP amplitudes and SPs did not change. These findings may reflect a trigeminal induced exteroceptive suppression of trapezius muscle activity. PMID:9637939

  2. Effects of inhibitors of eicosanoid synthesis in the uterus of ovariectomized rats and rats in natural oestrus: relation with calcium.

    PubMed

    Cantabrana, B; Baamonde, A; Andres-Trelles, F; Hidalgo, A

    1990-01-01

    1. The effects of 4 inhibitors of eicosanoid synthesis (ESIs)--mepacrine (MEP, 10(-5) - 3 x 10(-4) M), nordihydroguaiaretic acid (NDHGA, 10(-6) - 2 x 10(-5) M), indomethacin (IND, 2 x 10(-6) M and 2 x 10(-5) M) and imidazole (IMI, 10(-5) M and 10(-4) M)--on the motility induced by oxytocin (OT, 0.5 and 4 mU/ml) in uterus of rats in natural oestrus and of ovariectomized rats have been studied. 2. MEP, NDHGA and IND, but not IMI, inhibited the motility induced by both concentrations of OT in natural oestrus. Ovariectomy enhanced the effects of all ESIs, except the one of MEP. 3. MEP and NDHGA, but not IND or IMI, inhibited the contractions induced by methacholine (10(-5) M) and prostaglandin F2a (10(-6) M) and relaxed in a dose-dependent way the tonic component of the contractile response to KCl 60 mM (DI50: 6.14 +/- 0.38 and 1.38 +/- 0.29 x 10(-5) M, respectively). 4. CaCl2 (0.1-10 mM) reverted the relaxation of KCl contractions produced by MEP but not by NDHGA. PMID:2298392

  3. Eating tools in hand activate the brain systems for eating action: a transcranial magnetic stimulation study.

    PubMed

    Yamaguchi, Kaori; Nakamura, Kimihiro; Oga, Tatsuhide; Nakajima, Yasoichi

    2014-07-01

    There is increasing neuroimaging evidence suggesting that visually presented tools automatically activate the human sensorimotor system coding learned motor actions relevant to the visual stimuli. Such crossmodal activation may reflect a general functional property of the human motor memory and thus can be operating in other, non-limb effector organs, such as the orofacial system involved in eating. In the present study, we predicted that somatosensory signals produced by eating tools in hand covertly activate the neuromuscular systems involved in eating action. In Experiments 1 and 2, we measured motor evoked response (MEP) of the masseter muscle in normal humans to examine the possible impact of tools in hand (chopsticks and scissors) on the neuromuscular systems during the observation of food stimuli. We found that eating tools (chopsticks) enhanced the masseter MEPs more greatly than other tools (scissors) during the visual recognition of food, although this covert change in motor excitability was not detectable at the behavioral level. In Experiment 3, we further observed that chopsticks overall increased MEPs more greatly than scissors and this tool-driven increase of MEPs was greater when participants viewed food stimuli than when they viewed non-food stimuli. A joint analysis of the three experiments confirmed a significant impact of eating tools on the masseter MEPs during food recognition. Taken together, these results suggest that eating tools in hand exert a category-specific impact on the neuromuscular system for eating. PMID:24835403

  4. Determination of parabens in urine samples by microextraction using packed sorbent and ultra-performance liquid chromatography coupled to tandem mass spectrometry.

    PubMed

    Cristina Jardim, Valeria; de Paula Melo, Lidervan; Soares Domingues, Diego; Costa Queiroz, Maria Eugênia

    2015-01-01

    A simple, sensitive, and selective method using ultra-performance liquid chromatography coupled to tandem mass spectrometry (UPLC-MS/MS) was developed and validated for simultaneous determination of parabens [methyl paraben (MeP), ethyl paraben (EtP), propyl paraben (PrP), butyl paraben (BuP), and benzyl paraben (BzP)] in human urine samples. After microextraction by packed sorbent (MEPS) using a C18 phase, the parabens were separated on a Kinetex C18 column (100 mm × 2.1 mm × 1.7 μm) within 4.6 min using isocratic elution. These compounds were detected on a triple quadrupole tandem mass spectrometer using the multiple reactions monitoring (MRM) mode via an electrospray ionization source operating in the negative ionization mode. Important factors that influence MEPS performance were evaluated, such as the sample pH, draw-eject sample volume, clean-up step, and desorption conditions. The proposed MEPS/UPLC-MS/MS method presented a linear range from 0.5 ng mL(-1) (limit of quantification - LOQ) to 50 ng mL(-1), and interassay precision with coefficients of variation lower than 15%, and relative standard error values of the accuracy ranged from -8.8% to 15%. The MEPS/UPLC-MS/MS method was applied successfully to determine parabens in urine samples from 30 postpartum volunteers, enabling assessment of human exposure to these compounds. PMID:25463195

  5. Maximal respiratory static pressures in patients with different stages of COPD severity

    PubMed Central

    Terzano, Claudio; Ceccarelli, Daniela; Conti, Vittoria; Graziani, Elda; Ricci, Alberto; Petroianni, Angelo

    2008-01-01

    Background In this study, we analyzed maximal inspiratory pressure (MIP) and maximal expiratory pressure (MEP) values in a stable COPD population compared with normal subjects. We evaluated the possible correlation between functional maximal respiratory static pressures and functional and anthropometric parameters at different stages of COPD. Furthermore, we considered the possible correlation between airway obstruction and MIP and MEP values. Subject and methods 110 patients with stable COPD and 21 age-matched healthy subjects were enrolled in this study. Patients were subdivided according to GOLD guidelines: 31 mild, 39 moderate and 28 severe. Results Both MIP and MEP were lower in patients with severe airway impairment than in normal subjects. Moreover, we found a correlation between respiratory muscle function and some functional and anthropometric parameters: FEV1 (forced expiratory volume in one second), FVC (forced vital capacity), PEF (peak expiratory flow), TLC (total lung capacity) and height. MIP and MEP values were lower in patients with severe impairment than in patients with a slight reduction of FEV1. Conclusion The measurement of MIP and MEP indicates the state of respiratory muscles, thus providing clinicians with a further and helpful tool in monitoring the evolution of COPD. PMID:18208602

  6. The effects of propofol on rat transcranial magnetic motor evoked potentials.

    PubMed

    Fishback, A S; Shields, C B; Linden, R D; Zhang, Y P; Burke, D

    1995-11-01

    INTRAOPERATIVE MONITORING OF motor evoked potentials (MEPs) may become a valuable test of spinal cord function during surgery. Unfortunately, MEP responses are affected by most common anesthetics. We studied the effect of intravenous propofol on transcranial magnetic MEPs (tcMMEPs) in the rat. Baseline tcMMEPs were recorded before administration of the drug. Each rat then received three induction doses of propofol, 10, 5, and 5 mg/kg (totaling 10, 15, and 20 mg/kg) and three successive 20-minute infusion doses at rates of 10, 20, and 40 mg/kg/h, respectively. An MEP intensity series was performed after each induction dose, during each infusion, and during a 20-minute recovery period. tcMMEPs recorded during the induction period demonstrated a significant, dose-dependent increase in onset latency and a marked decrease in amplitude. Infusion tcMMEPs displayed increased onset latencies but demonstrated a significant change in amplitudes only after the largest infusion dose. The MEPs approached baseline levels after discontinuation of the propofol. This study demonstrates that tcMMEPs can be successfully recorded from the rat during propofol anesthesia. PMID:8559347

  7. Different corticospinal control between discrete and rhythmic movement of the ankle

    PubMed Central

    Goto, Yumeno; Jono, Yasutomo; Hatanaka, Ryota; Nomura, Yoshifumi; Tani, Keisuke; Chujo, Yuta; Hiraoka, Koichi

    2014-01-01

    We investigated differences in corticospinal and spinal control between discrete and rhythmic ankle movements. Motor evoked potentials (MEPs) in the tibialis anterior and soleus muscles and soleus H-reflex were elicited in the middle of the plantar flexion phase during discrete ankle movement or in the initial or later cycles of rhythmic ankle movement. The H-reflex was evoked at an intensity eliciting a small M-wave and MEPs were elicited at an intensity of 1.2 times the motor threshold of the soleus MEPs. Only trials in which background EMG level, ankle angle, and ankle velocity were similar among the movement conditions were included for data analysis. In addition, only trials with a similar M-wave were included for data analysis in the experiment evoking H-reflexes. Results showed that H reflex and MEP amplitudes in the soleus muscle during discrete movement were not significantly different from those during rhythmic movement. MEP amplitude in the tibialis anterior muscle during the later cycles of rhythmic movement was significantly larger than that during the initial cycle of the rhythmic movement or during discrete movement. Higher corticospinal excitability in the tibialis anterior muscle during the later cycles of rhythmic movement may reflect changes in corticospinal control from the initial cycle to the later cycles of rhythmic movement. PMID:25126066

  8. Effects of ketamine and propofol on motor evoked potentials elicited by intracranial microstimulation during deep brain stimulation

    PubMed Central

    Furmaga, Havan; Park, Hyun-Joo; Cooperrider, Jessica; Baker, Kenneth B.; Johnson, Matthew; Gale, John T.; Machado, Andre G.

    2014-01-01

    Few preclinical or clinical studies have evaluated the effect of anesthetics on motor evoked potentials (MEPs), either alone or in the presence of conditioning stimuli such as deep brain stimulation (DBS). In this study we evaluated the effects of two commonly used anesthetic agents, propofol and ketamine (KET), on MEPs elicited by intra-cortical microstimulation of the motor cortex in a rodent model with and without DBS of the dentatothalamocortical (DTC) pathway. The effects of propofol anesthesia on MEP amplitudes during DTC DBS were found to be highly dose dependent. Standard, but not high, dose propofol potentiated the facilitatory effects of 30 Hz DTC DBS on MEPs. This facilitation was sustained and phase-dependent indicating that, compared to high dose propofol, standard dose propofol has a beta-band excitatory effect on cortical networks. In contrast, KET anesthetic demonstrated a monotonic relationship with increasing frequencies of stimulation, such that the highest frequency of stimulation resulted in the greatest MEP amplitude. KET also showed phase dependency but less pronounced than standard dose propofol. The results underscore the importance of better understanding the complex effects of anesthetics on cortical networks and exogenous stimuli. Choice of anesthetic agents and dosing may significantly confound or even skew research outcomes, including experimentation in novel DBS indications and paradigms. PMID:24904312

  9. Microvascular decompression for glossopharyngeal neuralgia using intraoperative neurophysiological monitoring: Technical case report

    PubMed Central

    Motoyama, Yasushi; Nakagawa, Ichiro; Takatani, Tsunenori; Park, Hun-Soo; Kotani, Yukiko; Tanaka, Yoshitaka; Gurung, Pritam; Park, Young-Soo; Nakase, Hiroyuki

    2016-01-01

    Background: Glossopharyngeal neuralgia (GN) is a rare functional disorder representing around 1% of cases of trigeminal neuralgia. Lancinating throat and ear pain while swallowing are the typical manifestations, and are initially treated using anticonvulsants such as carbamazepine. Medically refractory GN is treated surgically. Microvascular decompression (MVD) is reportedly effective against GN, superseding rhizotomy and tractotomy. Methods: We encountered three patients with medically refractory GN who underwent MVD using intraoperative neurophysiological monitoring (IONM). The offending vessels were the posterior inferior cerebellar arteries, which were confirmed intraoperatively via a transcondylar fossa approach to be affecting the root exit zones of the glossopharyngeal and vagus nerves. As IONM, facial motor-evoked potentials (MEPs) and brainstem auditory-evoked potentials were monitored during microsurgery in all three patients. Pharyngeal and vagal MEPs were added for two patients to avoid postoperative dysphagia. Results: GN disappeared immediately after surgery with complete preservation of hearing acuity and facial nerve function. Transient mild swallowing disturbance was observed in 1 patient without pharyngeal or vagal MEPs, whereas the remaining two patients with pharyngeal and vagal MEPs demonstrated no postoperative dysphagia. Conclusion: Although control of severe pain is expected in surgical intervention for GN, lower cranial nerves are easily damaged because of their fragility, even in MVD. IONM including pharyngeal and vagal MEPs appears very useful for avoiding postoperative sequelae during MVD for GN. PMID:26862458

  10. Effect of Paired-Pulse Electrical Stimulation on the Activity of Cortical Circuits

    PubMed Central

    Saito, Kei; Onishi, Hideaki; Miyaguchi, Shota; Kotan, Shinichi; Fujimoto, Shuhei

    2015-01-01

    Objective: We investigated the transient effect of short-duration paired-pulse electrical stimulation (ppES) on corticospinal excitability and the after-effect of long-duration ppES on excitability, short-latency afferent inhibition (SAI), and afferent facilitation (AF). Methods: A total of 28 healthy subjects participated in two different experiments. In Experiment 1, motor-evoked potentials (MEPs) were measured in the abductor pollicis brevis (APB) and abductor digiti minimi (ADM) muscles before and immediately after short-duration ppES (5 s) at various inter-pulse intervals (2, 3, 4, 5, 6, 7, 10, 15, 20, and 30 ms). In Experiment 2, MEPs, SAI, and AF were measured before, immediately, and 20 and 40 min after long-duration ppES (20 min, inter-pulse interval of 5 and 15 ms) and peripheral electrical stimulation (20 min, 10 and 20 Hz). Results: Short-duration ppES with inter-pulse intervals of 5 and 20 ms significantly increased MEP measured in APB but not in ADM. Long-duration ppES with an inter-pulse interval of 5 ms significantly decreased SAI but not MEPs in APB. In contrast, long-duration ppES did not affect ADM. Conclusion: The afferent inputs induced by ppES-5 ms were effective for transiently increasing MEP and sustaining SAI reduction. PMID:26733847

  11. Verification and intercomparison of mesoscale ensemble prediction systems in the Beijing 2008 Olympics Research and Development Project

    NASA Astrophysics Data System (ADS)

    Kunii, Masaru; Saito, Kazuo; Seko, Hiromu; Hara, Masahiro; Hara, Tabito; Yamaguchi, Munehiko; Gong, Jiandong; Charron, Martin; Du, Jun; Wang, Yong; Chen, Dehui

    2011-05-01

    During the period around the Beijing 2008 Olympic Games, the Beijing 2008 Olympics Research and Development Project (B08RDP) was conducted as part of the World Weather Research Program short-range weather forecasting research project. Mesoscale ensemble prediction (MEP) experiments were carried out by six organizations in near-real time, in order to share their experiences in the development of MEP systems. The purpose of this study is to objectively verify these experiments and to clarify the problems associated with the current MEP systems through the same experiences. Verification was performed using the MEP outputs interpolated into a common verification domain with a horizontal resolution of 15 km. For all systems, the ensemble spreads grew as the forecast time increased, and the ensemble mean improved the forecast errors compared with individual control forecasts in the verification against the analysis fields. However, each system exhibited individual characteristics according to the MEP method. Some participants used physical perturbation methods. The significance of these methods was confirmed by the verification. However, the mean error (ME) of the ensemble forecast in some systems was worse than that of the individual control forecast. This result suggests that it is necessary to pay careful attention to physical perturbations.

  12. Analogous corticocortical inhibition and facilitation in ipsilateral and contralateral human motor cortex representations of the tongue.

    PubMed

    Muellbacher, W; Boroojerdi, B; Ziemann, U; Hallett, M

    2001-11-01

    How the human brain controls activation of the ipsilateral part of midline muscles is unknown. We studied corticospinal and corticocortical network excitability of both ipsilateral and contralateral motor representations of the tongue to determine whether they are under analogous or disparate inhibitory and facilitatory corticocortical control. Motor evoked potentials (MEPs) to unilateral focal transcranial magnetic stimulation (TMS) of the tongue primary motor cortex were recorded simultaneously from the ipsilateral and contralateral lingual muscles. Single-pulse TMS was used to assess motor threshold (MT) and MEP recruitment. Paired-pulse TMS was used to study intracortical inhibition (ICI) and intracortical facilitation (ICF) at various interstimulus intervals (ISIs) between the conditioning stimulus (CS) and the test stimulus (TS), and at different CS and TS intensities, respectively. Focal TMS invariably produced MEPs in both ipsilateral and contralateral lingual muscles. MT was lower and MEP recruitment was steeper when recorded from the contralateral muscle group. ICI and ICF were identical in the ipsilateral and contralateral representations, with inhibition occurring at short ISIs (2 and 3 ms) and facilitation occurring at longer ISIs (10 and 15 ms). Moreover, changing one stimulus parameter regularly produced analogous changes in MEP size bilaterally, revealing strong linear correlations between ipsilateral and contralateral ICI and ICF (P < 0.0001). These findings indicate that the ipsilateral and contralateral representations of the tongue are under analogous inhibitory and facilitatory control, possibly by a common intracortical network. PMID:11779968

  13. Differential activation of ammonium transporters during the accumulation of ammonia by Colletotrichum gloeosporioides and its effect on appressoria formation and pathogenicity.

    PubMed

    Shnaiderman, Chen; Miyara, Itay; Kobiler, Ilana; Sherman, Amir; Prusky, Dov

    2013-03-01

    Ammonium secreted by the post-harvest pathogen Colletotrichum gloeosporioides during host colonization accumulates in the host environment due to enhanced fungal nitrogen metabolism. Two types of ammonium transporter-encoding genes, AMET and MEP, are expressed during pathogenicity. Gene disruption of AMET, a gene modulating ammonia secretion, showed twofold reduced ammonia secretion and 45% less colonization on avocado fruit, suggesting a contribution to pathogenicity. MEPB, a gene modulating ammonium transport, is expressed by C. gloeosporioides during pathogenicity and starvation conditions in culture. Gene disruption of MEPB, the most highly expressed gene of the MEP family, resulted in twofold overexpression of MEPA and MEPC but reduced colonization, suggesting MEPB expression's contribution to pathogenicity. Analysis of internal and external ammonia accumulation by ΔmepB strains in mycelia and germinated spores showed rapid uptake and accumulation, and reduced secretion of ammonia in the mutant versus wild-type (WT) strains. Ammonia uptake by the WT germinating spores but not by the ΔmepB strain with compromised ammonium transport activated cAMP and transcription of PKA subunits PKAR and PKA2. ΔmepB mutants showed 75% less appressorium formation and colonization than the WT, which was partially restored by 10 mM exogenous ammonia. Thus, whereas both AMET and MEPB genes modulate ammonia secretion, only MEPB contributes to ammonia accumulation by mycelia and germinating spores that activate the cAMP pathways, inducing the morphogenetic processes contributing to C. gloeosporioides pathogenicity. PMID:23387470

  14. Soil Moisture and Vegetation Controls on Surface Energy Balance Using the Maximum Entropy Production Model of Evapotranspiration

    NASA Astrophysics Data System (ADS)

    Wang, J.; Parolari, A.; Huang, S. Y.

    2014-12-01

    The objective of this study is to formulate and test plant water stress parameterizations for the recently proposed maximum entropy production (MEP) model of evapotranspiration (ET) over vegetated surfaces. . The MEP model of ET is a parsimonious alternative to existing land surface parameterizations of surface energy fluxes from net radiation, temperature, humidity, and a small number of parameters. The MEP model was previously tested for vegetated surfaces under well-watered and dry, dormant conditions, when the surface energy balance is relatively insensitive to plant physiological activity. Under water stressed conditions, however, the plant water stress response strongly affects the surface energy balance. This effect occurs through plant physiological adjustments that reduce ET to maintain leaf turgor pressure as soil moisture is depleted during drought. To improve MEP model of ET predictions under water stress conditions, the model was modified to incorporate this plant-mediated feedback between soil moisture and ET. We compare MEP model predictions to observations under a range of field conditions, including bare soil, grassland, and forest. The results indicate a water stress function that combines the soil water potential in the surface soil layer with the atmospheric humidity successfully reproduces observed ET decreases during drought. In addition to its utility as a modeling tool, the calibrated water stress functions also provide a means to infer ecosystem influence on the land surface state. Challenges associated with sampling model input data (i.e., net radiation, surface temperature, and surface humidity) are also discussed.

  15. bcc-to-hcp transformation pathways for iron versus hydrostatic pressure: Coupled shuffle and shear modes

    NASA Astrophysics Data System (ADS)

    Liu, J. B.; Johnson, D. D.

    2009-04-01

    Using density-functional theory, we calculate the potential-energy surface (PES), minimum-energy pathway (MEP), and transition state (TS) versus hydrostatic pressure σhyd for the reconstructive transformation in Fe from body-centered cubic (bcc) to hexagonal closed-packed (hcp). At fixed σhyd , the PES is described by coupled shear (γ) and shuffle (η) modes and is determined from structurally minimized hcp-bcc energy differences at a set of (η,γ) . We fit the PES using symmetry-adapted polynomials, permitting the MEP to be found analytically. The MEP is continuous and fully explains the transformation and its associated magnetization and volume discontinuity at TS. We show that σhyd (while not able to induce shear) dramatically alters the MEP to drive reconstruction by a shuffle-only mode at ≤30GPa , as observed. Finally, we relate our polynomial-based results to Landau and nudge-elastic-band approaches and show they yield incorrect MEP in general.

  16. Cortical brain states and corticospinal synchronization influence TMS-evoked motor potentials.

    PubMed

    Keil, Julian; Timm, Jana; Sanmiguel, Iria; Schulz, Hannah; Obleser, Jonas; Schönwiesner, Marc

    2014-02-01

    Transcranial magnetic stimulation (TMS) influences cortical processes. Recent findings indicate, however, that, in turn, the efficacy of TMS depends on the state of ongoing cortical oscillations. Whereas power and phase of electromyographic (EMG) activity recorded from the hand muscles as well as neural synchrony between cortex and hand muscles are known to influence the effect of TMS, to date, no study has shown an influence of the phase of cortical oscillations during wakefulness. We applied single-pulse TMS over the motor cortex and recorded motor-evoked potentials along with the electroencephalogram (EEG) and EMG. We correlated phase and power of ongoing EEG and EMG signals with the motor-evoked potential (MEP) amplitude. We also investigated the functional connectivity between cortical and hand muscle activity (corticomuscular coherence) with the MEP amplitude. EEG and EMG power and phase in a frequency band around 18 Hz correlated with the MEP amplitude. High beta-band (∼34 Hz) corticomuscular coherence exhibited a positive linear relationship with the MEP amplitude, indicating that strong synchrony between cortex and hand muscles at the moment when TMS is applied entails large MEPs. Improving upon previous studies, we demonstrate a clear dependence of TMS-induced motor effects on the state of ongoing EEG phase and power fluctuations. We conclude that not only the sampling of incoming information but also the susceptibility of cortical communication flow depends cyclically on neural phase. PMID:24198325

  17. Stereospecific suppression of active site mutants by methylphosphonate substituted substrates reveals the stereochemical course of site-specific DNA recombination.

    PubMed

    Rowley, Paul A; Kachroo, Aashiq H; Ma, Chien-Hui; Maciaszek, Anna D; Guga, Piotr; Jayaram, Makkuni

    2015-07-13

    Tyrosine site-specific recombinases, which promote one class of biologically important phosphoryl transfer reactions in DNA, exemplify active site mechanisms for stabilizing the phosphate transition state. A highly conserved arginine duo (Arg-I; Arg-II) of the recombinase active site plays a crucial role in this function. Cre and Flp recombinase mutants lacking either arginine can be rescued by compensatory charge neutralization of the scissile phosphate via methylphosphonate (MeP) modification. The chemical chirality of MeP, in conjunction with mutant recombinases, reveals the stereochemical contributions of Arg-I and Arg-II. The SP preference of the native reaction is specified primarily by Arg-I. MeP reaction supported by Arg-II is nearly bias-free or RP-biased, depending on the Arg-I substituent. Positional conservation of the arginines does not translate into strict functional conservation. Charge reversal by glutamic acid substitution at Arg-I or Arg-II has opposite effects on Cre and Flp in MeP reactions. In Flp, the base immediately 5' to the scissile MeP strongly influences the choice between the catalytic tyrosine and water as the nucleophile for strand scission, thus between productive recombination and futile hydrolysis. The recombinase active site embodies the evolutionary optimization of interactions that not only favor the normal reaction but also proscribe antithetical side reactions. PMID:25999343

  18. Different Stimulation Frequencies Alter Synchronous Fluctuations in Motor Evoked Potential Amplitude of Intrinsic Hand Muscles—a TMS Study

    PubMed Central

    Sale, Martin V.; Rogasch, Nigel C.; Nordstrom, Michael A.

    2016-01-01

    The amplitude of motor-evoked potentials (MEPs) elicited with transcranial magnetic stimulation (TMS) varies from trial-to-trial. Synchronous oscillations in cortical neuronal excitability contribute to this variability, however it is not known how different frequencies of stimulation influence MEP variability, and whether these oscillations are rhythmic or aperiodic. We stimulated the motor cortex with TMS at different regular (i.e., rhythmic) rates, and compared this with pseudo-random (aperiodic) timing. In 18 subjects, TMS was applied at three regular frequencies (0.05 Hz, 0.2 Hz, 1 Hz) and one aperiodic frequency (mean 0.2 Hz). MEPs (n = 50) were recorded from three intrinsic hand muscles of the left hand with different functional and anatomical relations. MEP amplitude correlation was highest for the functionally related muscle pair, less for the anatomically related muscle pair and least for the functionally- and anatomically-unrelated muscle pair. MEP correlations were greatest with 1 Hz, and least for stimulation at 0.05 Hz. Corticospinal neuron synchrony is higher with shorter TMS intervals. Further, corticospinal neuron synchrony is similar irrespective of whether the stimulation is periodic or aperiodic. These findings suggest TMS frequency is a crucial consideration for studies using TMS to probe correlated activity between muscle pairs. PMID:27014031

  19. Highlights of Magnetoelectropolishing

    NASA Astrophysics Data System (ADS)

    Hryniewicz, Tadeusz; Rokosz, Krzysztof

    2014-05-01

    The research work has been concerned on the studies and development of electrochemical polishing in the magnetic field (MEP) in comparison with the standard electropolishing methods performed without stirring (EP) and the process with the forced electrolyte mixing (MIX). Advanced techniques were used in the studies, to measure the effects of the surface treatment. They are as follows: scanning electron microscopy (SEM/EDS), X-ray Photoelectron Spectroscopy (XPS), Secondary Ion Mass Spectroscopy (SIMS), surface roughness, and nanoindentation measurements. For the corrosion studies, the electrochemical methods were used, such as: open circuit potential OCP, potentiodynamic curves PC, and electrochemical impedance spectroscopy EIS measurements. The MEP process is advised mainly due to the opportunity to enrich the surface layer with the chromium compounds (austenitic stainless steels), or titanium compounds (CP Ti Grade 2, Nitinol). The surface layers of biomaterials after MEP process contain much lower amount of carcinogenic compounds, like chromium VI oxidation stage (Cr6+) (austenitic stainless steels) and nickel compounds (austenitic stainless steel & Nitinol) versus those ones obtained after EP & MIX treatments. It is interesting that apart from the significant modification of the surface layer obtained after MEP, also mechanical properties, such as nanohardness, modulus of elasticity, and mechanical resistance to bending and torsion, undergo considerable advantageous changes. All they make the MEP process very promising for application in many clean industries, such as medical equipment and devices, electronics, food industry, etc.

  20. Inexpensive Designer Antigen for Anti-HIV Antibody Detection with High Sensitivity and Specificity ▿

    PubMed Central

    Talha, Sheikh M.; Salminen, Teppo; Chugh, Deepti A.; Swaminathan, Sathyamangalam; Soukka, Tero; Pettersson, Kim; Khanna, Navin

    2010-01-01

    A novel recombinant multiepitope protein (MEP) has been designed that consists of four linear, immunodominant, and phylogenetically conserved epitopes, taken from human immunodeficiency virus (HIV)-encoded antigens that are used in many third-generation immunoassay kits. This HIV-MEP has been evaluated for its diagnostic potential in the detection of anti-HIV antibodies in human sera. A synthetic MEP gene encoding these epitopes, joined by flexible peptide linkers in a single open reading frame, was designed and overexpressed in Escherichia coli. The recombinant HIV-MEP was purified using a single affinity step, yielding >20 mg pure protein/liter culture, and used as the coating antigen in an in-house immunoassay. Bound anti-HIV antibodies were detected by highly sensitive time-resolved fluorometry, using europium(III) chelate-labeled anti-human antibody. The sensitivity and specificity of the HIV-MEP were evaluated using Boston Biomedica worldwide HIV performance, HIV seroconversion, and viral coinfection panels and were found to be comparable with those of commercially available anti-HIV enzyme immunoassay (EIA) kits. The careful choice of epitopes, high epitope density, and an E. coli-based expression system, coupled with a simple purification protocol and the use of europium(III) chelate-labeled tracer, provide the capability for the development of an inexpensive diagnostic test with high degrees of sensitivity and specificity. PMID:20089793

  1. Combining De Ley-Doudoroff and methylerythritol phosphate pathways for enhanced isoprene biosynthesis from D-galactose.

    PubMed

    Ramos, Kristine Rose M; Valdehuesa, Kris Niño G; Liu, Huaiwei; Nisola, Grace M; Lee, Won-Keun; Chung, Wook-Jin

    2014-12-01

    An engineered Escherichia coli strain was developed for enhanced isoprene production using D-galactose as substrate. Isoprene is a valuable compound that can be biosynthetically produced from pyruvate and glyceraldehyde-3-phosphate (G3P) through the methylerythritol phosphate pathway (MEP). The Leloir and De Ley-Doudoroff (DD) pathways are known existing routes in E. coli that can supply the MEP precursors from D-galactose. The DD pathway was selected as it is capable of supplying equimolar amounts of pyruvate and G3P simultaneously. To exclusively direct D-galactose toward the DD pathway, an E. coli ΔgalK strain with blocked Leloir pathway was used as the host. To obtain a fully functional DD pathway, a dehydrogenase encoding gene (gld) was recruited from Pseudomonas syringae to catalyze D-galactose conversion to D-galactonate. Overexpressions of endogenous genes known as MEP bottlenecks, and a heterologous gene, were conducted to enhance and enable isoprene production, respectively. Growth test confirmed a functional DD pathway concomitant with equimolar generation of pyruvate and G3P, in contrast to the wild-type strain where G3P was limiting. Finally, the engineered strain with combined DD-MEP pathway exhibited the highest isoprene production. This suggests that the equimolar pyruvate and G3P pools resulted in a more efficient carbon flux toward isoprene production. This strategy provides a new platform for developing improved isoprenoid producing strains through the combined DD-MEP pathway. PMID:24928200

  2. Nudged-elastic band method with two climbing images: Finding transition states in complex energy landscapes

    SciTech Connect

    Zarkevich, Nikolai A.; Johnson, Duane D.

    2015-01-09

    The nudged-elastic band (NEB) method is modified with concomitant two climbing images (C2-NEB) to find a transition state (TS) in complex energy landscapes, such as those with a serpentine minimal energy path (MEP). If a single climbing image (C1-NEB) successfully finds the TS, then C2-NEB finds it too. Improved stability of C2-NEB makes it suitable for more complex cases, where C1-NEB misses the TS because the MEP and NEB directions near the saddle point are different. Generally, C2-NEB not only finds the TS, but guarantees, by construction, that the climbing images approach it from the opposite sides along the MEP. In addition, C2-NEB provides an accuracy estimate from the three images: the highest-energy one and its climbing neighbors. C2-NEB is suitable for fixed-cell NEB and the generalized solid-state NEB.

  3. Nudged-elastic band method with two climbing images: Finding transition states in complex energy landscapes

    DOE PAGESBeta

    Zarkevich, Nikolai A.; Johnson, Duane D.

    2015-01-09

    The nudged-elastic band (NEB) method is modified with concomitant two climbing images (C2-NEB) to find a transition state (TS) in complex energy landscapes, such as those with a serpentine minimal energy path (MEP). If a single climbing image (C1-NEB) successfully finds the TS, then C2-NEB finds it too. Improved stability of C2-NEB makes it suitable for more complex cases, where C1-NEB misses the TS because the MEP and NEB directions near the saddle point are different. Generally, C2-NEB not only finds the TS, but guarantees, by construction, that the climbing images approach it from the opposite sides along the MEP.more » In addition, C2-NEB provides an accuracy estimate from the three images: the highest-energy one and its climbing neighbors. C2-NEB is suitable for fixed-cell NEB and the generalized solid-state NEB.« less

  4. The Thermodynamics of Marine Biogeochemical Cycles: Lotka Revisited

    NASA Astrophysics Data System (ADS)

    Vallino, Joseph J.; Algar, Christopher K.

    2016-01-01

    Nearly 100 years ago, Alfred Lotka published two short but insightful papers describing how ecosystems may organize. Principally, Lotka argued that ecosystems will grow in size and that their cycles will spin faster via predation and nutrient recycling so as to capture all available energy, and that evolution and natural selection are the mechanisms by which this occurs and progresses. Lotka's ideas have often been associated with the maximum power principle, but they are more consistent with recent developments in nonequilibrium thermodynamics, which assert that complex systems will organize toward maximum entropy production (MEP). In this review, we explore Lotka's hypothesis within the context of the MEP principle, as well as how this principle can be used to improve marine biogeochemistry models. We need to develop the equivalent of a climate model, as opposed to a weather model, to understand marine biogeochemistry on longer timescales, and adoption of the MEP principle can help create such models.

  5. Development and Characterization of Healable Carbon Fiber Composites with a Reversibly Cross Linked Polymer

    SciTech Connect

    Ghezzo, Fabrizia; Smith, David R.; Starr, Tatiana N.; Perram, Timothy; Starr, Anthony F.; Darlington, Thomas K.; Baldwin, Richard K.; Oldenburg, Steven J.

    2010-10-18

    Carbon fiber reinforced polymer (CFRP) laminates with remendable cross-linked polymeric matrices were fabricated using a modified resin transfer mold (RTM) technique. The healable composite resin, bis-maleimide tetrafuran (2MEP4F), was synthesized by mixing two monomers, furan (4F) and maleimide (2MEP), at elevated temperatures. The fast kinetic rate of the reaction of polymer constituents requires a fast injection of the healable resin into the carbon fiber preform. The polymer viscosity as a function of time and temperature was experimentally quantified in order to optimize the fabrication of the composite material and to guarantee a uniform flow of the resin through the reinforcement. The method was validated by characterizing the thermo-mechanical properties of the polymerized 2MEP4F. Additionally, the thermo-mechanical properties of the remendable CFRP material were studied.

  6. Minimum number of trials required for within- and between-session reliability of TMS measures of corticospinal excitability.

    PubMed

    Goldsworthy, M R; Hordacre, B; Ridding, M C

    2016-04-21

    Transcranial magnetic stimulation (TMS)-elicited motor-evoked potentials (MEPs) exhibit considerable trial-to-trial variability, potentially reducing the sensitivity and reproducibility of this measure. While increasing the number of trials will improve accuracy, prolonged recording blocks are not always feasible. In this study, we investigated the minimum number of trials required to provide a measure of human corticospinal excitability that is stable both within and between sessions. Single-pulse TMS was applied to the left primary motor cortex, and MEPs were recorded from the right first dorsal interosseous muscle. Approximately 20-30 trials were required to provide a stable measure of MEP amplitude with high within- and between-session reliability. Extending the number of trials beyond 30 provided no additional benefit. Collecting 30 trials may be optimal for reliably estimating corticospinal excitability using TMS. These findings may have significant implications for using TMS to measure corticospinal excitability in both basic and clinical research settings. PMID:26872998

  7. Evolution of Electrogenic Ammonium Transporters (AMTs)

    PubMed Central

    McDonald, Tami R.; Ward, John M.

    2016-01-01

    The ammonium transporter gene family consists of three main clades, AMT, MEP, and Rh. The evolutionary history of the AMT/MEP/Rh gene family is characterized by multiple horizontal gene transfer events, gene family expansion and contraction, and gene loss; thus the gene tree for this family of transporters is unlike the organismal tree. The genomes of angiosperms contain genes for both electrogenic and electroneutral ammonium transporters, but it is not clear how far back in the land plant lineage electrogenic ammonium transporters occur. Here, we place Marchantia polymorpha ammonium transporters in the AMT/MEP/Rh phylogeny and we show that AMTs from the liverwort M. polymorpha are electrogenic. This information suggests that electrogenic ammonium transport evolved at least as early as the divergence of bryophytes in the land plant lineage. PMID:27066024

  8. [Detection of short tandem repeat (STR) polymorphisms by microchip electrophoresis for individual identification of cattle].

    PubMed

    Yamaguchi, Akihiro; Shimizu, Kaori; Mishima, Takashi; Hattori, Hideki; Katsuda, Shin-ichi; Sato, Hidetaka; Ueda, Nobuo; Sato, Noriyuki

    2006-12-01

    A simple and rapid detection of short tandem repeat (STR) markers was studied as a screening test for individual identification of cattle. DNAs were extracted from eight commercial beef samples by a proteinase K-boil method followed by purification with 2-propanol precipitation. Five STR markers, known to be highly polymorphic, were amplified by PCR and analyzed both by a conventional sequencing analysis (SEQ) and by a proposed microchip electrophoresis (MEP). Every marker revealed high polymorphism, such as 5-9 alleles in SEQ analysis, and 4-6 alleles in MEP analysis. This simple and rapid MEP analysis is expected to be an effective screening tool with use of confirmatory SEQ analysis. PMID:17228791

  9. Effect of observation combined with motor imagery of a skilled hand-motor task on motor cortical excitability: difference between novice and expert.

    PubMed

    Tsukazaki, Izumi; Uehara, Kazumasa; Morishita, Takuya; Ninomiya, Masato; Funase, Kozo

    2012-06-19

    We examined the effects of observation combined with motor imagery (MI) of a skilled hand-motor task on motor cortex excitability, which was assessed by transcranial magnetic stimulation (TMS). Novices and experts at 3-ball cascade juggling (3BCJ) participated in this study. In one trial, the subjects observed a video clip of 3BCJ while imagining performing it. In addition, the subjects also imagined performing 3BCJ without video clip observation. Motor evoked potentials (MEPs) were recorded from the hand muscles that were activated by the task during each trial. In the novices, the MEP amplitude was significantly increased by video clip observation combined with MI. In contrast, MI without video clip observation significantly increased the MEP amplitude of the experts. These results suggest that action observation of 3BCJ increases the ability of novices to make their MI performing the task. Meanwhile, experts use their own motor program to recall their MI of the task. PMID:22580208

  10. Motor-Evoked Potential Confirmation of Functional Improvement by Transplanted Bone Marrow Mesenchymal Stem Cell in the Ischemic Rat Brain

    PubMed Central

    Jang, Dong-Kyu; Park, Sang-In; Han, Young-Min; Jang, Kyung-Sool; Park, Moon-Seo; Chung, Young-An; Kim, Min-Wook; Maeng, Lee-So; Huh, Pil-Woo; Yoo, Do-Sung; Jung, Seong-Whan

    2011-01-01

    This study investigated the effect of bone marrow mesenchymal stem cells (BMSCs) on the motor pathway in the transient ischemic rat brain that were transplanted through the carotid artery, measuring motor-evoked potential (MEP) in the four limbs muscle and the atlantooccipital membrane, which was elicited after monopolar and bipolar transcortical stimulation. After monopolar stimulation, the latency of MEP was significantly prolonged, and the amplitude was less reduced in the BMSC group in comparison with the control group (P < .05). MEPs induced by bipolar stimulation in the left forelimb could be measured in 40% of the BMSC group and the I wave that was not detected in the control group was also detected in 40% of the BMSC group. Our preliminary results imply that BMSCs transplanted to the ischemic rat brain mediate effects on the functional recovery of the cerebral motor cortex and the motor pathway. PMID:21772790

  11. Methanolic extract of Pterocarpus santalinus induces apoptosis in HeLa cells.

    PubMed

    Kwon, H J; Hong, Y K; Kim, K H; Han, C H; Cho, S H; Choi, J S; Kim, Byung-Woo

    2006-04-21

    Ptercarpus santalinus (Fabaceae) has been used as a folk remedy in Korea, and it has been shown to exhibit antiinflammations, antiulcers and anticancer effects. In this study, therefore, we report the cytotoxic activity and the mechanism of cell death exhibited by the methanol extract of Ptercarpus santalinus (MEPS) against human cervical adenocarcinoma cell line, HeLa. Treatment of HeLa cells with various concentrations of MEPS resulted in growth inhibition and induction of apoptosis in a dose-dependent manner as determined by cell viability, chromatin condensation, DNA fragmentation and sub-G1 phase accumulation. In Western blot analysis, apoptosis in the HeLa cells was associated with the release of cytochrome C from mitochondria into the cytosol, activation of caspases-3, -8, -9 and proteolytic cleavage of PARP. These results suggest that MEPS exhibits antiproliferative effect on HeLa cells via apoptosis, and it may be a potential candidate in field of anticancer drug discovery. PMID:16326057

  12. Terpenoids and Their Biosynthesis in Cyanobacteria

    PubMed Central

    Pattanaik, Bagmi; Lindberg, Pia

    2015-01-01

    Terpenoids, or isoprenoids, are a family of compounds with great structural diversity which are essential for all living organisms. In cyanobacteria, they are synthesized from the methylerythritol-phosphate (MEP) pathway, using glyceraldehyde 3-phosphate and pyruvate produced by photosynthesis as substrates. The products of the MEP pathway are the isomeric five-carbon compounds isopentenyl diphosphate and dimethylallyl diphosphate, which in turn form the basic building blocks for formation of all terpenoids. Many terpenoid compounds have useful properties and are of interest in the fields of pharmaceuticals and nutrition, and even potentially as future biofuels. The MEP pathway, its function and regulation, and the subsequent formation of terpenoids have not been fully elucidated in cyanobacteria, despite its relevance for biotechnological applications. In this review, we summarize the present knowledge about cyanobacterial terpenoid biosynthesis, both regarding the native metabolism and regarding metabolic engineering of cyanobacteria for heterologous production of non-native terpenoids. PMID:25615610

  13. Effect of transcranial magnetic stimulation on force of finger pinch

    NASA Astrophysics Data System (ADS)

    Odagaki, Masato; Fukuda, Hiroshi; Hiwaki, Osamu

    2009-04-01

    Transcranial magnetic stimulation (TMS) is used to explore many aspects of brain function, and to treat neurological disorders. Cortical motor neuronal activation by TMS over the primary motor cortex (M1) produces efferent signals that pass through the corticospinal tracts. Motor-evoked potentials (MEPs) are observed in muscles innervated by the stimulated motor cortex. TMS can cause a silent period (SP) following MEP in voluntary electromyography (EMG). The present study examined the effects of TMS eliciting MEP and SP on the force of pinching using two fingers. Subjects pinched a wooden block with the thumb and index finger. TMS was applied to M1 during the pinch task. EMG of first dorsal interosseous muscles and pinch forces were measured. Force output increased after the TMS, and then oscillated. The results indicated that the motor control system to keep isotonic forces of the muscles participated in the finger pinch was disrupted by the TMS.

  14. Development of a NASA 2018 Mars Landed Mission Concept

    NASA Technical Reports Server (NTRS)

    Wilson, M. G.; Salvo, C. G.; Abilleira, F.; Sengstacken, A. J.; Allwood, A. G.; Backes, P. G.; Lindemann, R. A.; Jordan, J. F.

    2010-01-01

    Fundamental to NASA's Mars Exploration Program (MEP) is an ongoing development of an integrated and coordinated set of possible future candidate missions that meet fundamental science and programmatic objectives of NASA and the Mars scientific community. In the current planning horizon of the NASA MEP, a landed mobile surface exploration mission launching in the 2018 Mars launch opportunity exists as a candidate project to meet MEP in situ science and exploration objectives. This paper describes the proposed mission science objectives and the mission implementation concept developed for the 2018 opportunity. As currently envisioned, this mission concept seeks to explore a yet-to-be-selected site with high preservation potential for physical and chemical biosignatures, evaluate paleoenvironmental conditions, characterize the potential for preservation of biosignatures, and access multiple sequences of geological units in a search for evidence of past life and/or prebiotic chemistry at a site on Mars.

  15. Evolution of Electrogenic Ammonium Transporters (AMTs).

    PubMed

    McDonald, Tami R; Ward, John M

    2016-01-01

    The ammonium transporter gene family consists of three main clades, AMT, MEP, and Rh. The evolutionary history of the AMT/MEP/Rh gene family is characterized by multiple horizontal gene transfer events, gene family expansion and contraction, and gene loss; thus the gene tree for this family of transporters is unlike the organismal tree. The genomes of angiosperms contain genes for both electrogenic and electroneutral ammonium transporters, but it is not clear how far back in the land plant lineage electrogenic ammonium transporters occur. Here, we place Marchantia polymorpha ammonium transporters in the AMT/MEP/Rh phylogeny and we show that AMTs from the liverwort M. polymorpha are electrogenic. This information suggests that electrogenic ammonium transport evolved at least as early as the divergence of bryophytes in the land plant lineage. PMID:27066024

  16. Milia En Plaque as a Distinct Follicular Hamartoma With Cystic Trichoepitheliomatous Features

    PubMed Central

    Hashimoto, Akira; Yamasaki, Kenshi; Aiba, Setsuya

    2016-01-01

    Abstract: Milia en plaque (MEP) is an uncommon disorder characterized by an erythematous plaque containing numerous milia. The pathogenesis of MEP is not clear. The authors report a man with an erythematous plaque on the right retroauricular area, containing numerous white-yellow cysts varying in size. Histological examination showed that multiple cystic structures at various levels of the dermis that were lined by stratified squamous epithelium and contained keratinous material—these findings were consistent with the diagnosis of multiple milia. In addition to epidermal cysts, however, the lesion consisted of a branched proliferation of pale-staining keratinocytes lined with basal keratinocytes budding from the overlying epidermis. Moreover, some cysts were formed within the branched epithelial proliferation, had thicker cyst walls than the ordinary milium, or had irregular or branched projections toward the surrounding dermis. From these findings, the authors conclude that MEP is a distinct follicular hamartoma with cystic trichoepitheliomatous features. PMID:26381115

  17. Removing External Degrees of Freedom from Transition-State Search Methods using Quaternions.

    PubMed

    Melander, Marko; Laasonen, Kari; Jónsson, Hannes

    2015-03-10

    In finite systems, such as nanoparticles and gas-phase molecules, calculations of minimum energy paths (MEPs) connecting initial and final states of transitions as well as searches for saddle points are complicated by the presence of external degrees of freedom, such as overall translation and rotation. A method based on quaternion algebra for removing the external degrees of freedom is described here and applied in calculations using two commonly used methods: the nudged elastic band (NEB) method for MEPs and the DIMER method for finding the minimum mode in minimum mode following searches of first-order saddle points. With the quaternion approach, fewer images in the NEB are needed to represent MEPs accurately. In both NEB and DIMER calculations of finite systems, the number of iterations required to reach convergence is significantly reduced. The algorithms have been implemented in the Atomic Simulation Environment (ASE) open source software. PMID:26579757

  18. Maximum entropy decomposition of flux distribution at steady state to elementary modes.

    PubMed

    Zhao, Quanyu; Kurata, Hiroyuki

    2009-01-01

    Enzyme Control Flux (ECF) is a method of correlating enzyme activity and flux distribution. The advantage of ECF is that the measurement integrates proteome data with metabolic flux analysis through Elementary Modes (EMs). But there are a few methods of effectively determining the Elementary Mode Coefficient (EMC) in cases where no objective biological function is available. Therefore, we proposed a new algorithm implementing the maximum entropy principle (MEP) as an objective function for estimating the EMC. To demonstrate the feasibility of using the MEP in this way, we compared it with Linear Programming and Quadratic Programming for modeling the metabolic networks of Chinese Hamster Ovary, Escherichia coli, and Saccharomyces cerevisiae cells. The use of the MEP presents the most plausible distribution of EMCs in the absence of any biological hypotheses describing the physiological state of cells, thereby enhancing the prediction accuracy of the flux distribution in various mutants. PMID:19147116

  19. Astacin Proteases Cleave Dentin Sialophosphoprotein (Dspp) to Generate Dentin Phosphoprotein (Dpp)

    PubMed Central

    Tsuchiya, Shuhei; Simmer, James P; Hu, Jan C-C; Richardson, Amelia S; Yamakoshi, Fumiko; Yamakoshi, Yasuo

    2011-01-01

    Dentin sialophosphoprotein (Dspp) is critical for proper dentin biomineralization because genetic defects in DSPP cause dentin dysplasia type II and dentinogenesis imperfecta types II and III. Dspp is processed by proteases into smaller subunits; the initial cleavage releases dentin phosphoprotein (Dpp). We incubated fluorescence resonance energy transfer (FRET) peptides containing the amino acid context of the Dpp cleavage site (YEFDGKSMQGDDPN, designated Dspp-FRET) or a mutant version of that context (YEFDGKSIEGDDPN, designated mutDspp-FRET) with BMP-1, MEP1A, MEP1B, MMP-2, MMP-8, MMP-9, MT1-MMP, MT3-MMP, Klk4, MMP-20, plasmin, or porcine Dpp and characterized the peptide cleavage products. Only BMP-1, MEP1A, and MEP1B cleaved Dspp-FRET at the G–D peptide bond that releases Dpp from Dspp in vivo. We isolated Dspp proteoglycan from dentin power and incubated it with the three enzymes that cleaved Dspp-FRET at the G–D bond. In each case, the released Dpp domain was isolated, and its N-terminus was characterized by Edman degradation. BMP-1 and MEP1A both cleaved native Dspp at the correct site to generate Dpp, making both these enzymes prime candidates for the protease that cleaves Dspp in vivo. MEP1B was able to degrade Dpp when the Dpp was at sufficiently high concentration to deplete free calcium ion concentration. Immunohistochemistry of developing porcine molars demonstrated that astacins are expressed by odontoblasts, a result that is consistent with RT-PCR analyses. We conclude that during odontogenesis, astacins in the predentin matrix cleave Dspp before the DDPN sequence at the N-terminus of Dpp to release Dpp from the parent Dspp protein. © 2011 American Society for Bone and Mineral Research. PMID:20687161

  20. Brain-robot interface driven plasticity: Distributed modulation of corticospinal excitability.

    PubMed

    Kraus, Dominic; Naros, Georgios; Bauer, Robert; Leão, Maria Teresa; Ziemann, Ulf; Gharabaghi, Alireza

    2016-01-15

    Brain-robot interfaces (BRI) are studied as novel interventions to facilitate functional restoration in patients with severe and persistent motor deficits following stroke. They bridge the impaired connection in the sensorimotor loop by providing brain-state dependent proprioceptive feedback with orthotic devices attached to the hand or arm of the patients. The underlying neurophysiology of this BRI neuromodulation is still largely unknown. We investigated changes of corticospinal excitability with transcranial magnetic stimulation in thirteen right-handed healthy subjects who performed 40min of kinesthetic motor imagery receiving proprioceptive feedback with a robotic orthosis attached to the left hand contingent to event-related desynchronization of the right sensorimotor cortex in the β-band (16-22Hz). Neural correlates of this BRI intervention were probed by acquiring the stimulus-response curve (SRC) of both motor evoked potential (MEP) peak-to-peak amplitudes and areas under the curve. In addition, a motor mapping was obtained. The specificity of the effects was studied by comparing two neighboring hand muscles, one BRI-trained and one control muscle. Robust changes of MEP amplitude but not MEP area occurred following the BRI intervention, but only in the BRI-trained muscle. The steep part of the SRC showed an MEP increase, while the plateau of the SRC showed an MEP decrease. MEP mapping revealed a distributed pattern with a decrease of excitability in the hand area of the primary motor cortex, which controlled the BRI, but an increase of excitability in the surrounding somatosensory and premotor cortex. In conclusion, the BRI intervention induced a complex pattern of modulated corticospinal excitability, which may boost subsequent motor learning during physiotherapy. PMID:26505298

  1. Optimization of the IPP Precursor Supply for the Production of Lycopene, Decaprenoxanthin and Astaxanthin by Corynebacterium glutamicum

    PubMed Central

    Heider, Sabine A. E.; Wolf, Natalie; Hofemeier, Arne; Peters-Wendisch, Petra; Wendisch, Volker F.

    2014-01-01

    The biotechnologically relevant bacterium Corynebacterium glutamicum, currently used for the million ton-scale production of amino acids for the food and feed industries, is pigmented due to synthesis of the rare cyclic C50 carotenoid decaprenoxanthin and its glucosides. The precursors of carotenoid biosynthesis, isopenthenyl pyrophosphate (IPP) and its isomer dimethylallyl pyrophosphate, are synthesized in this organism via the methylerythritol phosphate (MEP) or non-mevalonate pathway. Terminal pathway engineering in recombinant C. glutamicum permitted the production of various non-native C50 and C40 carotenoids. Here, the role of engineering isoprenoid precursor supply for lycopene production by C. glutamicum was characterized. Overexpression of dxs encoding the enzyme that catalyzes the first committed step of the MEP-pathway by chromosomal promoter exchange in a prophage-cured, genome-reduced C. glutamicum strain improved lycopene formation. Similarly, an increased IPP supply was achieved by chromosomal integration of two artificial operons comprising MEP pathway genes under the control of a constitutive promoter. Combined overexpression of dxs and the other six MEP pathways genes in C. glutamicum strain LYC3-MEP was not synergistic with respect to improving lycopene accumulation. Based on C. glutamicum strain LYC3-MEP, astaxanthin could be produced in the milligrams per gram cell dry weight range when the endogenous genes crtE, crtB, and crtI for conversion of geranylgeranyl pyrophosphate to lycopene were coexpressed with the genes for lycopene cyclase and β-carotene hydroxylase from Pantoea ananatis and carotene C(4) oxygenase from Brevundimonas aurantiaca. PMID:25191655

  2. Intraoperative Neurophysiologic Monitoring (INM) in scoliosis surgery.

    PubMed

    Pankowski, Rafal; Dziegiel, Krzysztof; Roclawski, Marek; Smoczynski, Andrzej; Ceynowa, Marcin; Kloc, Wojciech; Wasilewski, Wojciech; Jende, Piotr; Liczbik, Wieslaw; Beldzinski, Piotr; Libionka, Witold; Pierzak, Olaf; Adamski, Stanislaw; Niedbala, Miroslaw

    2012-01-01

    Even among skilled spinal deformity surgeons, neurologic deficits are inherent potential complications of spine surgery. The aim was to assess the meaning of changes and to evaluate the critical rates of Somatosensory Evoked Potentials (SEP) and Motor Evoked Potentials (MEP) for Neurologic Deficit (ND) occurrence associated with scoliosis surgery. A Group of 30 patients with idiopathic scoliosis treated surgically by posterior correction and stabilisation were included. Patients were matched by age, sex, aetiology, Cobb angle, and surgical criteria. Data on three planar scoliosis correction and concomitant (INM) alarms were compared. Radiographic assessment was performed from radiographs taken before surgery and just after it. The (INM) was performed with the use of ISSIS (Inomed) in every patients the same fashion. The average thoracic curve correction was 69.7% and lumbar 69.8%. The average preoperative Apical Vertebral Rotation was 23.5° for thoracic and 27.9° for lumbar curves and postoperatively 10.9° and 14.3° respectively. There was a significant variability of SEP during surgery with only 7 (23%) patients with stable SEP. 15(50%) patients had a decrease of SEP below 50% and 8(27%) had severe decrease of SEP below 50% what caused us to stop surgery or to decrease correction of curves. There was a MEP decrease in 11(37%) patients and in 6 (20%) directly after correction up to 50% of normal value. In 5 of 30 (17%) patients there was a significant decrease of MEP below 50% and we immediately released the implant. The SEP decrease up to 50% without any MEP change did not influenced the outcome. There was no correlation between flexibility and correction of the curve and SEP and MEP decrease. The safe level for MEP was not determined but its meaning for the outcome was more important than SEP value. The need of (INM) during scoliosis surgery to avoid (ND) was confirmed. PMID:22744519

  3. Transcutaneous spinal direct current stimulation modulates human corticospinal system excitability.

    PubMed

    Bocci, Tommaso; Marceglia, Sara; Vergari, Maurizio; Cognetto, Valeria; Cogiamanian, Filippo; Sartucci, Ferdinando; Priori, Alberto

    2015-07-01

    This study aimed to assess the effects of thoracic anodal and cathodal transcutaneous spinal direct current stimulation (tsDCS) on upper and lower limb corticospinal excitability. Although there have been studies assessing how thoracic tsDCS influences the spinal ascending tract and reflexes, none has assessed the effects of this technique over upper and lower limb corticomotor neuronal connections. In 14 healthy subjects we recorded motor evoked potentials (MEPs) elicited by transcranial magnetic stimulation (TMS) from abductor hallucis (AH) and hand abductor digiti minimi (ADM) muscles before (baseline) and at different time points (0 and 30 min) after anodal or cathodal tsDCS (2.5 mA, 20 min, T9-T11 level). In 8 of the 14 subjects we also tested the soleus H reflex and the F waves from AH and ADM before and after tsDCS. Both anodal and cathodal tsDCS left the upper limb MEPs and F wave unchanged. Conversely, while leaving lower limb H reflex unchanged, they oppositely affected lower limb MEPs: whereas anodal tsDCS increased resting motor threshold [(mean ± SE) 107.33 ± 3.3% increase immediately after tsDCS and 108.37 ± 3.2% increase 30 min after tsDCS compared with baseline] and had no effects on MEP area and latency, cathodal tsDCS increased MEP area (139.71 ± 12.9% increase immediately after tsDCS and 132.74 ± 22.0% increase 30 min after tsDCS compared with baseline) without affecting resting motor threshold and MEP latency. Our results show that tsDCS induces polarity-specific changes in corticospinal excitability that last for >30 min after tsDCS offset and selectively affect responses in lower limb muscles innervated by lumbar and sacral motor neurons. PMID:25925328

  4. Surgical limitations in convexity meningiomas en-plaque: Is radical resection necessary?

    PubMed

    Yao, Amy; Sarkiss, Christopher A; Lee, James; Zarzour, Hekmat K; Shrivastava, Raj K

    2016-05-01

    Meningiomas-en-plaque (MEP) comprise 2.5% of all meningiomas. While they typically arise in the sphenoid wing, convexity MEP are comparatively rare and are often confused with meningeal sarcoidosis, osteoma, tuberculoma, or fibrous dysplasia, with very little information published in the literature. We conducted a literature review on PubMed of English-only articles using a keyword search. All studies that described reports of convexity MEP were reviewed for patient demographics, presenting symptoms, radiological reports, surgical management, recurrence rates, histopathological presentation, post-operative complications, and follow-up. This resulted in 12 papers comprising 22 cases of convexity MEP. Seventeen (77%) of the 22 patients were female with an average age of 53.2years. Intitial presenting symptoms included headache in 12/20 (60%), hemiparesis in 5/20 (25%), and visual symptoms in 1/20 (5%). Of the 14 patients who underwent surgical resection, only four were reported as gross total resection. Twelve reports had associated pathology reports, with all 12 tumors graded as World Health Organization Grade I. Convexity MEP, while rare, present a challenge with regard to correct diagnosis and subsequent resection. The easier accessibility of these meningiomas predicts higher surgical success rates and incidence of total resection, though care must be taken to ensure gross total removal of tumor, dural attachments, and any overlying hyperostotic bone. Though hyperostosis is frequently observed with this variant of meningioma, it is neither exclusive nor wholly indicative of MEP. Due to its rarity in both clinical practice and the literature, further studies are warranted to identify modern imaging means to correctly diagnose this condition. PMID:26778515

  5. Maggot excretion products from the blowfly Lucilia sericata contain contact phase/intrinsic pathway-like proteases with procoagulant functions.

    PubMed

    Kahl, M; Gökçen, A; Fischer, S; Bäumer, M; Wiesner, J; Lochnit, G; Wygrecka, M; Vilcinskas, A; Preissner, K T

    2015-08-01

    For centuries, maggots have been used for the treatment of wounds by a variety of ancient cultures, as part of their traditional medicine. With increasing appearance of antimicrobial resistance and in association with diabetic ulcers, maggot therapy was revisited in the 1980s. Three mechanisms by which sterile maggots of the green bottle fly Lucilia sericata may improve healing of chronic wounds have been proposed: Biosurgical debridement, disinfecting properties, and stimulation of the wound healing process. However, the influence of maggot excretion products (MEP) on blood coagulation as part of the wound healing process has not been studied in detail. Here, we demonstrate that specific MEP-derived serine proteases from Lucilia sericata induce clotting of human plasma and whole blood, particularly by activating contact phase proteins factor XII and kininogen as well as factor IX, thereby providing kallikrein-bypassing and factor XIa-like activities, both in plasma and in isolated systems. In plasma samples deficient in contact phase proteins, MEP restored full clotting activity, whereas in plasma deficient in either factor VII, IX, X or II no effect was seen. The observed procoagulant/intrinsic pathway-like activity was mediated by (chymo-) trypsin-like proteases in total MEP, which were significantly blocked by C1-esterase inhibitor or other contact phase-specific protease inhibitors. No significant influence of MEP on platelet activation or fibrinolysis was noted. Together, MEP provides contact phase bypassing procoagulant activity and thereby induces blood clotting in the context of wound healing. Further characterisation of the active serine protease(s) may offer new perspectives for biosurgical treatment of chronic wounds. PMID:25948398

  6. Abnormal motor cortex excitability during linguistic tasks in adductor-type spasmodic dysphonia.

    PubMed

    Suppa, A; Marsili, L; Giovannelli, F; Di Stasio, F; Rocchi, L; Upadhyay, N; Ruoppolo, G; Cincotta, M; Berardelli, A

    2015-08-01

    In healthy subjects (HS), transcranial magnetic stimulation (TMS) applied during 'linguistic' tasks discloses excitability changes in the dominant hemisphere primary motor cortex (M1). We investigated 'linguistic' task-related cortical excitability modulation in patients with adductor-type spasmodic dysphonia (ASD), a speech-related focal dystonia. We studied 10 ASD patients and 10 HS. Speech examination included voice cepstral analysis. We investigated the dominant/non-dominant M1 excitability at baseline, during 'linguistic' (reading aloud/silent reading/producing simple phonation) and 'non-linguistic' tasks (looking at non-letter strings/producing oral movements). Motor evoked potentials (MEPs) were recorded from the contralateral hand muscles. We measured the cortical silent period (CSP) length and tested MEPs in HS and patients performing the 'linguistic' tasks with different voice intensities. We also examined MEPs in HS and ASD during hand-related 'action-verb' observation. Patients were studied under and not-under botulinum neurotoxin-type A (BoNT-A). In HS, TMS over the dominant M1 elicited larger MEPs during 'reading aloud' than during the other 'linguistic'/'non-linguistic' tasks. Conversely, in ASD, TMS over the dominant M1 elicited increased-amplitude MEPs during 'reading aloud' and 'syllabic phonation' tasks. CSP length was shorter in ASD than in HS and remained unchanged in both groups performing 'linguistic'/'non-linguistic' tasks. In HS and ASD, 'linguistic' task-related excitability changes were present regardless of the different voice intensities. During hand-related 'action-verb' observation, MEPs decreased in HS, whereas in ASD they increased. In ASD, BoNT-A improved speech, as demonstrated by cepstral analysis and restored the TMS abnormalities. ASD reflects dominant hemisphere excitability changes related to 'linguistic' tasks; BoNT-A returns these excitability changes to normal. PMID:26061279

  7. Bilateral sensory deprivation of trigeminal afferent fibres on corticomotor control of human tongue musculature: a preliminary study.

    PubMed

    Kothari, M; Baad-Hansen, L; Svensson, P

    2016-09-01

    Transcranial magnetic stimulation (TMS) has demonstrated changes in motor evoked potentials (MEPs) in human limb muscles following modulation of sensory afferent inputs. The aim of this study was to determine whether bilateral local anaesthesia (LA) of the lingual nerve affects the excitability of the tongue motor cortex (MI) as measured by TMS. The effect on MEPs after bilateral LA of the lingual nerve was studied, while the first dorsal interosseous (FDI) muscle served as a control in ten healthy participants. MEPs were measured on the right side of the tongue dorsum in four different conditions: (i) immediately prior to anaesthesia (baseline), (ii) during bilateral LA block of the lingual nerve, (iii) after anaesthesia had subjectively subsided (recovery) and (iv) 3 h after bilateral lingual block injection. MEPs were assessed using stimulus-response curves in steps of 10% of motor threshold (T). Eight stimuli were given at each stimulus level. The amplitudes of the tongue MEPs were significantly influenced by the stimulus intensity (P < 0·001) but not by condition (P = 0·186). However, post hoc tests showed that MEPS were statistically significantly higher during bilateral LA block condition compared with baseline at T + 40%, T + 50% and T + 60% (P < 0·028) and also compared with recovery at T + 60% (P = 0·010) as well as at 3 h after injection at T + 50% and T + 60% (P < 0·029). Bilateral LA block of the lingual nerve seems to be associated with a facilitation of the corticomotor pathways related to the tongue musculature. PMID:27265155

  8. The response to paired motor cortical stimuli is abolished at a spinal level during human muscle fatigue

    PubMed Central

    McNeil, Chris J; Martin, Peter G; Gandevia, Simon C; Taylor, Janet L

    2009-01-01

    During maximal exercise, supraspinal fatigue contributes significantly to the decline in muscle performance but little is known about intracortical inhibition during such contractions. Long-interval inhibition is produced by a conditioning motor cortical stimulus delivered via transcranial magnetic stimulation (TMS) 50–200 ms prior to a second test stimulus. We aimed to delineate changes in this inhibition during a sustained maximal voluntary contraction (MVC). Eight subjects performed a 2 min MVC of elbow flexors. Single test and paired (conditioning–test interval of 100 ms) stimuli were delivered via TMS over the motor cortex every 7–8 s throughout the effort and during intermittent MVCs in the recovery period. To determine the role of spinal mechanisms, the protocol was repeated but the TMS test stimulus was replaced by cervicomedullary stimulation which activates the corticospinal tract. TMS motor evoked potentials (MEPs) and cervicomedullary motor evoked potentials (CMEPs) were recorded from biceps brachii. Unconditioned MEPs increased progressively with fatigue, whereas CMEPs increased initially but returned to the control value in the final 40 s of contraction. In contrast, both conditioned MEPs and CMEPs decreased rapidly with fatigue and were virtually abolished within 30 s. In recovery, unconditioned responses required <30 s but conditioned MEPs and CMEPs required ∼90 s to return to control levels. Thus, long-interval inhibition increased markedly as fatigue progressed. Contrary to expectations, subcortically evoked CMEPs were inhibited as much as MEPs. This new phenomenon was also observed in the first dorsal interosseous muscle. Tested with a high intensity conditioning stimulus during a fatiguing maximal effort, long-interval inhibition of MEPs was increased primarily by spinal rather than motor cortical mechanisms. The spinal mechanisms exposed here may contribute to the development of central fatigue in human muscles. PMID:19805743

  9. High-dose ketamine hydrochloride maintains somatosensory and magnetic motor evoked potentials in primates.

    PubMed

    Ghaly, R F; Ham, J H; Lee, J J

    2001-12-01

    Monitoring the descending neural motor volleys (MEPs), in comparison to muscle action potentials, allows sensitive motor assessment under anesthesia irrespective of the use of muscular blockade and status of skeletal musculature. Ketamine hydrochloride (KH) had preserved muscle MEPs on a pre-established primate model. The present work examines the effect of incremental hypnotic KH dosages thoracic neural on somatosensory (SEP) and MEPs recorded epidurally in response to transcranial magnetic stimulation (TMS). Through a small thoracic T11-T12 laminotomy, an insulated double bipolar electrode was inserted epidurally and cephalad in seven cynomolgus monkeys. Thoracic spinal TMS-MEPs, and SEPs, were tested against graded increase of KH doses (0.01, 0.018, 0.032, 0.056, 0.1, and 0.18 mg kg(-1) min(-1) i.v. infusion). The direct (D-) and indirect (I-) epidural MEP peaks were well-defined under sole KH infusion. The waveforms were consistent at various dosages. At the highest cumulative dose (0.18 mg kg(-1) min(-1), total 6.5 mg kg(-1) over 150 min), I5 was host and I3 and I4 latencies were delayed. The scalp and spinal SEP showed no significant change. Recording of both neural D- and I- MEPs and SEPs is feasible under high sole i.v. KH. It is the first agent to maintain up to four later I1 peaks. The reproducibility of both modalities is unquestionable under KH-based deep anesthesia. This reflects the maintenance of state of neural excitability under KH. PMID:11760882

  10. Maximum Entropy Production Modeling of Evapotranspiration Partitioning on Heterogeneous Terrain and Canopy Cover: advantages and limitations.

    NASA Astrophysics Data System (ADS)

    Gutierrez-Jurado, H. A.; Guan, H.; Wang, J.; Wang, H.; Bras, R. L.; Simmons, C. T.

    2015-12-01

    Quantification of evapotranspiration (ET) and its partition over regions of heterogeneous topography and canopy poses a challenge using traditional approaches. In this study, we report the results of a novel field experiment design guided by the Maximum Entropy Production model of ET (MEP-ET), formulated for estimating evaporation and transpiration from homogeneous soil and canopy. A catchment with complex terrain and patchy vegetation in South Australia was instrumented to measure temperature, humidity and net radiation at soil and canopy surfaces. Performance of the MEP-ET model to quantify transpiration and soil evaporation was evaluated during wet and dry conditions with independently and directly measured transpiration from sapflow and soil evaporation using the Bowen Ratio Energy Balance (BREB). MEP-ET transpiration shows remarkable agreement with that obtained through sapflow measurements during wet conditions, but consistently overestimates the flux during dry periods. However, an additional term introduced to the original MEP-ET model accounting for higher stomatal regulation during dry spells, based on differences between leaf and air vapor pressure deficits and temperatures, significantly improves the model performance. On the other hand, MEP-ET soil evaporation is in good agreement with that from BREB regardless of moisture conditions. The experimental design allows a plot and tree scale quantification of evaporation and transpiration respectively. This study confirms for the first time that the MEP-ET originally developed for homogeneous open bare soil and closed canopy can be used for modeling ET over heterogeneous land surfaces. Furthermore, we show that with the addition of an empirical function simulating the plants ability to regulate transpiration, and based on the same measurements of temperature and humidity, the method can produce reliable estimates of ET during both wet and dry conditions without compromising its parsimony.

  11. Theory of using magnetic deflections to combine charged particle beams.

    SciTech Connect

    Doyle, Barney Lee; Steckbeck, Mackenzie K.

    2014-09-01

    Several radiation effects projects in the Ion Beam Lab (IBL) have recently required two disparate charged particle beams to simultaneously strike a single sample through a single port of the target chamber. Because these beams have vastly different mass- energy products (MEP), the low MEP beam requires a large angle of deflection toward the sample by a bending electromagnet. A second electromagnet located further upstream provides a means to compensate for the small angle deflection experienced by the high MEP beam during its path through the bending magnet. This paper derives the equations used to select the magnetic fields required by these two magnets to achieve uniting both beams at the target sample. A simple result was obtained when the separation of the two magnets was equivalent to the distance from the bending magnet to the sample, and the equations is given by: , 1 2 c s c s r B B r where and are the magnetic fields in the steering and bending magnet and is s B c B c s r r the ratio of the radii of the bending magnet to that of the steering magnet. This result is not dependent upon the parameters of the high MEP beam, i.e. energy, mass, charge state. Therefore, once the field of the bending magnet is set for the low MEP beam, and the field in the steering magnet is set as indicted in the equation, the trajectory path of any high MEP beam will be directed into the sample. (page intentionally left blank)

  12. Interaction of transcutaneous spinal stimulation and transcranial magnetic stimulation in human leg muscles.

    PubMed

    Roy, François D; Bosgra, Dillen; Stein, Richard B

    2014-06-01

    Transcutaneous spinal stimulation is a noninvasive method that can activate dorsal and/or ventral roots depending on the location and intensity of stimulation. Reflex root-evoked potentials (REPs) were studied in muscles that traditionally evoke large (soleus) and small H-reflexes (tibialis anterior), as well as muscles where H-reflexes are difficult to study (hamstrings). This study characterizes the interaction of the REP and the motor-evoked potential (MEP). Transcranial magnetic stimulation (TMS) delivered 11-25 ms before spinal stimulation resulted in more than linear summation of the two responses. Because of overlap, the modulation was quantified after subtracting the contribution of the conditioning MEP or REP. At rest, the mean-rectified soleus response was facilitated by up to ~250 μV (21-times the MEP or 161% of the REP). The increases were more reliable during a voluntary contraction (up to ~300 μV, 517% of the MEP or 181% of the REP). At the 13-ms interval, the mean-rectified response in the pre-contracted hamstrings was increased by 227% of the MEP or 300% of the REP. In some subjects, TMS could also eliminate the post-activation depression produced using two spinal stimuli, confirming that the interaction can extend to presynaptic spinal neurons. The spatiotemporal facilitation in tibialis anterior was not significant. However, the large MEP was facilitated when the spinal stimulus preceded TMS by 100-150 ms, presumably because of rebound excitation. These strong interactions may be important for inducing motor plasticity and improved training procedures for recovery after neurological damage. PMID:24531641

  13. Baroreceptor reflex during forced expiratory maneuvers in individuals with chronic spinal cord injury.

    PubMed

    Legg Ditterline, Bonnie E; Aslan, Sevda C; Randall, David C; Harkema, Susan J; Ovechkin, Alexander V

    2016-07-15

    Pulmonary and cardiovascular dysfunctions are leading causes of morbidity and mortality in patients with chronic Spinal Cord Injury (SCI). Impaired respiratory motor function and decreased Baroreflex Sensitivity (BS) are predictors for the development of cardiopulmonary disease. This observational case-controlled clinical study was undertaken to investigate if respiratory motor control deficits in individuals with SCI affect their ability to perform the Valsalva maneuver, and to determine if a sustained Maximum Expiratory Pressure (MEP) effort can serve as an acceptable maneuver for determination of the BS in the event that the Valsalva maneuver cannot be performed. The BS outcomes (ms/mmHg) were obtained using continuous beat-to-beat arterial blood pressure (BP) and heart rate (HR) recordings during Valsalva or MEP maneuvers in thirty nine individuals with chronic C3-T12 SCI. Twenty one participants (54%) reported signs of intolerance during the Valsalva maneuver and only 15 individuals (39%) were able to complete this task. Cervical level of injury was a significant risk factor (p=0.001) for failing to complete the Valsalva maneuver, and motor-complete injury was a significant risk factor for symptoms of intolerance (p=0.04). Twenty eight participants (72%) were able to perform the MEP maneuver; the other 11 participants failed to exceed the standard airway pressure threshold of 27cm H2O. Neither level nor completeness of injury were significant risk factors for failure of MEP maneuver. When the required airway pressure was sustained, there were no significant differences between BS outcomes obtained during Valsalva and MEP maneuvers. The results of this study indicate that individuals with high-level and motor-complete SCI are at increased risk of not completing the Valsalva maneuver and that baroreflex-mediated responses can be evaluated by using sustained MEP maneuver when the Valsalva maneuver cannot be performed. PMID:27137412

  14. Differences in Supraspinal and Spinal Excitability during Various Force Outputs of the Biceps Brachii in Chronic- and Non-Resistance Trained Individuals

    PubMed Central

    Pearcey, Gregory E. P.; Power, Kevin E.; Button, Duane C.

    2014-01-01

    Motor evoked potentials (MEP) and cervicomedullary evoked potentials (CMEP) may help determine the corticospinal adaptations underlying chronic resistance training-induced increases in voluntary force production. The purpose of the study was to determine the effect of chronic resistance training on corticospinal excitability (CE) of the biceps brachii during elbow flexion contractions at various intensities and the CNS site (i.e. supraspinal or spinal) predominantly responsible for any training-induced differences in CE. Fifteen male subjects were divided into two groups: 1) chronic resistance-trained (RT), (n = 8) and 2) non-RT, (n = 7). Each group performed four sets of ∼5 s elbow flexion contractions of the dominant arm at 10 target forces (from 10%–100% MVC). During each contraction, subjects received 1) transcranial magnetic stimulation, 2) transmastoid electrical stimulation and 3) brachial plexus electrical stimulation, to determine MEP, CMEP and compound muscle action potential (Mmax) amplitudes, respectively, of the biceps brachii. All MEP and CMEP amplitudes were normalized to Mmax. MEP amplitudes were similar in both groups up to 50% MVC, however, beyond 50% MVC, MEP amplitudes were lower in the chronic RT group (p<0.05). CMEP amplitudes recorded from 10–100% MVC were similar for both groups. The ratio of MEP amplitude/absolute force and CMEP amplitude/absolute force were reduced (p<0.012) at all contraction intensities from 10–100% MVC in the chronic-RT compared to the non-RT group. In conclusion, chronic resistance training alters supraspinal and spinal excitability. However, adaptations in the spinal cord (i.e. motoneurone) seem to have a greater influence on the altered CE. PMID:24875495

  15. Hemocompatibility Improvement of Chromium-Bearing Bare-Metal Stent Platform After Magnetoelectropolishing

    NASA Astrophysics Data System (ADS)

    Rokicki, Ryszard; Haider, Waseem; Maffi, Shivani Kaushal

    2015-01-01

    Research was undertaken to determine the influence of the increased content of chromium in the outermost passive layer of magneto-electrochemically refined Co-Cr alloy L-605 surface on its hemocompatibility. The chemistry, roughness, surface energy, and wettability of conventionally electropolished (EP) and magnetoelectropolished (MEP) samples were studied with x-ray photoelectron spectroscopy (XPS), open circuit potential, atomic force microscopy, and contact angle meter. In vitro hemocompatibility of tested material surfaces was assessed using two important indicators of vascular responses to biomaterial, namely endothelialization and platelets adhesion. The endothelialization was assessed by seeding and incubating samples with human umbilical vein endothelial cells (HUVEC) for 3 days before counting and observing them under a fluorescent microscope. The platelet (rich plasma blood) adhesion and activation test on EP and MEP L-605 alloy surfaces was assessed using a laser scanning confocal microscope. The XPS analysis of MEP samples showed significant enrichment of the passive layer with Cr and O when compared with the EP one. The amount of other elements in the passive layer did not show a significant difference between EP and MEP treatments. The adhesion of HUVEC cells shows remarkable affinity to surfaces enriched in Cr (MEP) with almost 100% confluency. In addition, the number of platelets that adhered to standard EP surfaces was higher compared to the MEP surface. The present study shows that the chromium-enriched surface of cobalt-chromium alloy L-605 by the magnetoelectropolishing process tremendously improves surface hemocompatibility with regard to stent functionality by enhanced endothelialization and lower platelet adhesion and should be taken under consideration as an alternative surface of biodegradable polymer drug-eluting stents, polymer-free drug-eluting stents as well as bare-metal stents.

  16. Radial scan of the molecular electrostatic potential of RNA double helices: an application to the enzyme-tRNA recognition.

    PubMed

    Marín, Ray M; Agudelo, William A; Daza C, Edgar E

    2008-10-01

    We introduced a method to characterize quantitatively the molecular electrostatic potential (MEP) of the minor and major grooves of base pairs located at nucleic acid double helices. By means of a radial MEP scan, we obtained a n-tuple of potential values corresponding to each groove, which can be analyzed by plotting the MEP values as a function of the angle in the radial scan. We studied base pairs of two different tRNAs, relevant in the recognition process with their cognate aminoacyl tRNA synthetases (aaRSs), in order to correlate their electrostatic behavior with the corresponding aminoacylation activity. We analyzed the first three base pairs of the Escherichia coli tRNA(Ala) acceptor stem, finding several cases where the MEP profiles obtained from the plots are in agreement with the reported aminoacylation activities. Additionally, a non-hierarchical clustering performed over the MEP n-tuples resulted in meaningful classifications that correlate with the activity and with the predicted stereochemistry of the reaction. We also studied the first two base pairs of the E. coli tRNA(Thr) acceptor stem but constraining the analysis to the angle intervals that seem relevant for the binding sites of the enzyme. These intervals were deduced from the ThrRS-tRNA(Thr) complex crystal structure. In this case, we also found a good agreement between the MEP profiles and the activity, supporting the idea that the tRNA identity elements function is to allow an optimal electrostatic complementarity between the aminoacyl-tRNA synthetase and the tRNA. PMID:18586541

  17. Evaluation of optimal electrode configurations for epidural spinal cord stimulation in cervical spinal cord injured rats

    PubMed Central

    Alam, Monzurul; Garcia-Alias, Guillermo; Shah, Prithvi K.; Gerasimenko, Yury; Zhong, Hui; Roy, Roland R.; Edgerton, V. Reggie

    2015-01-01

    Background Epidural spinal cord stimulation is a promising technique for modulating the level of excitability and reactivation of dormant spinal neuronal circuits after spinal cord injury (SCI). We examined the ability of chronically implanted epidural stimulation electrodes within the cervical spinal cord to (1) directly elicit spinal motor evoked potentials (sMEPs) in forelimb muscles and (2) determine whether these sMEPs can serve as a biomarker of forelimb motor function after SCI. New method We implanted EMG electrodes in forelimb muscles and epidural stimulation electrodes at C6 and C8 in adult rats. After recovering from a dorsal funiculi crush (C4), rats were tested with different stimulation configurations and current intensities to elicit sMEPs and determined forelimb grip strength. Results: sMEPs were evoked in all muscles tested and their characteristics were dependent on electrode configurations and current intensities. C6(−) stimulation elicited more robust sMEPs than stimulation at C8(−). Stimulating C6 and C8 simultaneously produced better muscle recruitment and higher grip strengths than stimulation at one site. Comparison with existing method(s) Classical method to select the most optimal stimulation configuration is to empirically test each combination individually for every subject and relate to functional improvements. This approach is impractical, requiring extensively long experimental time to determine the more effective stimulation parameters. Our proposed method is fast and physiologically sound. Conclusions Results suggest that sMEPs from forelimb muscles can be useful biomarkers for identifying optimal parameters for epidural stimulation of the cervical spinal cord after SCI. PMID:25791014

  18. Urinary excretion and daily intake rates of diethyl phthalate in the general Canadian population.

    PubMed

    Saravanabhavan, Gurusankar; Walker, Mike; Guay, Mireille; Aylward, Lesa

    2014-12-01

    We have analyzed the trends in the body-weight-adjusted urinary monoethyl phthalate (MEP) concentrations and the diethyl ethyl phthalate (DEP) daily intake estimates in the general Canadian population (aged 6-49 years) using the Canadian Health Measures Survey 2007-2009 dataset. The creatinine correction approach, as well as the urine volume approach in a simple one compartment model were used to calculate the daily urinary MEP excretion rates and DEP intake rates in individual survey participants. Using multiple regression models, we have estimated least square geometric means (LSGMs) of body-weight-adjusted MEP concentration, daily excretion and intake rates among different age groups and sex. We observed that body weight affects the trends in the MEP concentrations significantly among children (aged 6-11 years), adolescents (aged 12-19 years) and adults (aged 20-49 years). The body-weight-adjusted MEP concentrations in children were significantly higher than those in adults. On the other hand the DEP daily intakes in children were significantly lower than those in adults. We did not observe any differences in the DEP daily intake rates between males and females. Although the urinary MEP concentrations are correlated well with DEP daily intake estimates in the overall population, one should be cautious when directly using the urinary concentrations to compare the intake trends in the sub-populations (e.g. children vs. adults) as these trends are governed by additional physiological factors. The DEP daily intake calculated using the creatinine approach and that using the urine volume approach were similar to each other. The estimated geometric mean and 95th percentile of DEP daily intake in the general Canadian population are 2 and 20 μg/kg-bw/day, respectively. These daily intake estimates are significantly lower than the US Environmental Protection Agency's oral reference dose of 800 μg/kg-bw/day. PMID:25217994

  19. Transpinal and Transcortical Stimulation Alter Corticospinal Excitability and Increase Spinal Output

    PubMed Central

    Knikou, Maria

    2014-01-01

    The objective of this study was to assess changes in corticospinal excitability and spinal output following noninvasive transpinal and transcortical stimulation in humans. The size of the motor evoked potentials (MEPs), induced by transcranial magnetic stimulation (TMS) and recorded from the right plantar flexor and extensor muscles, was assessed following transcutaneous electric stimulation of the spine (tsESS) over the thoracolumbar region at conditioning-test (C-T) intervals that ranged from negative 50 to positive 50 ms. The size of the transpinal evoked potentials (TEPs), induced by tsESS and recorded from the right and left plantar flexor and extensor muscles, was assessed following TMS over the left primary motor cortex at 0.7 and at 1.1× MEP resting threshold at C-T intervals that ranged from negative 50 to positive 50 ms. The recruitment curves of MEPs and TEPs had a similar shape, and statistically significant differences between the sigmoid function parameters of MEPs and TEPs were not found. Anodal tsESS resulted in early MEP depression followed by long-latency MEP facilitation of both ankle plantar flexors and extensors. TEPs of ankle plantar flexors and extensors were increased regardless TMS intensity level. Subthreshold and suprathreshold TMS induced short-latency TEP facilitation that was larger in the TEPs ipsilateral to TMS. Noninvasive transpinal stimulation affected ipsilateral and contralateral actions of corticospinal neurons, while corticocortical and corticospinal descending volleys increased TEPs in both limbs. Transpinal and transcortical stimulation is a noninvasive neuromodulation method that alters corticospinal excitability and increases motor output of multiple spinal segments in humans. PMID:25007330

  20. Offsite Source Recovery Program (OSRP) Workshop Module: Tianjin, China, July 16-July 17, 2012

    SciTech Connect

    Houlton, Robert J.

    2012-07-11

    Recovering and disposal of radioactive sources that are no longer in service in their intended capacity is an area of high concern Globally. A joint effort to recover and dispose of such sources was formed between the US Department of Energy and the Chinese Ministry of Environmental Protection (MEP), in preparation for the 2008 Beijing Olympics. LANL involvement in this agreement continues today under the DOE-Global Threat Reduction Initiative (GTRI) program. LANL will be presenting overview information on their Offsite Source Recovery (OSRP) and Source Disposal programs, in a workshop for the Ministry of Environmental Protection (MEP) at Tianjin, China, on July 16 and 17, 2012.

  1. Surgery for intramedullary spinal cord tumors: the role of intraoperative (neurophysiological) monitoring.

    PubMed

    Sala, Francesco; Bricolo, Albino; Faccioli, Franco; Lanteri, Paola; Gerosa, Massimo

    2007-11-01

    In spite of advancements in neuro-imaging and microsurgical techniques, surgery for intramedullary spinal cord tumors (ISCT) remains a challenging task. The rationale for using intraoperative neurophysiological monitoring (IOM) is in keeping with the goal of maximizing tumor resection and minimizing neurological morbidity. For many years, before the advent of motor evoked potentials (MEPs), only somatosensory evoked potentials (SEPs) were monitored. However, SEPs are not aimed to reflect the functional integrity of motor pathways and, nowadays, the combined used of SEPs and MEPs in ISCT surgery is almost mandatory because of the possibility to selectively injury either the somatosensory or the motor pathways. This paper is aimed to review our perspective in the field of IOM during ISCT surgery and to discuss it in the light of other intraoperative neurophysiologic strategies that have recently appeared in the literature with regards to ISCT surgery. Besides standard cortical SEP monitoring after peripheral stimulation, both muscle (mMEPs) and epidural MEPs (D-wave) are monitored after transcranial electrical stimulation (TES). Given the dorsal approach to the spinal cord, SEPs must be monitored continuously during the incision of the dorsal midline. When the surgeon starts to work on the cleavage plane between tumor and spinal cord, attention must be paid to MEPs. During tumor removal, we alternatively monitor D-wave and mMEPs, sustaining the stimulation during the most critical steps of the procedure. D-waves, obtained through a single pulse TES technique, allow a semi-quantitative assessment of the functional integrity of the cortico-spinal tracts and represent the strongest predictor of motor outcome. Whenever evoked potentials deteriorate, temporarily stop surgery, warm saline irrigation and improved blood perfusion have proved useful for promoting recovery, Most of intraoperative neurophysiological derangements are reversible and therefore IOM is able to

  2. The two-box model of climate: limitations and applications to planetary habitability and maximum entropy production studies.

    PubMed

    Lorenz, Ralph D

    2010-05-12

    The 'two-box model' of planetary climate is discussed. This model has been used to demonstrate consistency of the equator-pole temperature gradient on Earth, Mars and Titan with what would be predicted from a principle of maximum entropy production (MEP). While useful for exposition and for generating first-order estimates of planetary heat transports, it has too low a resolution to investigate climate systems with strong feedbacks. A two-box MEP model agrees well with the observed day : night temperature contrast observed on the extrasolar planet HD 189733b. PMID:20368253

  3. Comparison of Test Procedures and Energy Efficiency Criteria in Selected International Standards & Labeling Programs for Copy Machines, External Power Supplies, LED Displays, Residential Gas Cooktops and Televisions

    SciTech Connect

    Zheng, Nina; Zhou, Nan; Fridley, David

    2012-03-01

    This report presents a technical review of international minimum energy performance standards (MEPS), voluntary and mandatory energy efficiency labels and test procedures for five products being considered for new or revised MEPS in China: copy machines, external power supply, LED displays, residential gas cooktops and flat-screen televisions. For each product, an overview of the scope of existing international standards and labeling programs, energy values and energy performance metrics and description and detailed summary table of criteria and procedures in major test standards are presented.

  4. Safe transcranial electric stimulation motor evoked potential monitoring during posterior spinal fusion in two patients with cochlear implants.

    PubMed

    Yellin, Joseph L; Wiggins, Cheryl R; Franco, Alier J; Sankar, Wudbhav N

    2016-08-01

    Transcranial electric stimulation (TES) motor evoked potentials (MEPs) have become a regular part of intraoperative neurophysiologic monitoring (IONM) for posterior spinal fusion (PSF) surgery. Almost all of the relative contraindications to TES have come and gone. One exception is in the case of patients with a cochlear implant (CI). Herein we illustrate two cases of pediatric patients with CIs who underwent PSF using TES MEPs as part of IONM. In both instances the patients displayed no untoward effects from TES, and post-operatively both CIs were intact and functioning as they were prior to surgery. PMID:26103915

  5. Confronting Regulatory Cost and Quality Expectations. An Exploration of Technical Change in Minimum Efficiency Performance Standards

    SciTech Connect

    Taylor, Margaret; Spurlock, C. Anna; Yang, Hung-Chia

    2015-09-21

    The dual purpose of this project was to contribute to basic knowledge about the interaction between regulation and innovation and to inform the cost and benefit expectations related to technical change which are embedded in the rulemaking process of an important area of national regulation. The area of regulation focused on here is minimum efficiency performance standards (MEPS) for appliances and other energy-using products. Relevant both to U.S. climate policy and energy policy for buildings, MEPS remove certain product models from the market that do not meet specified efficiency thresholds.

  6. Early detection of pedicle screw-related spinal cord injury by continuous intraoperative neurophysiological monitoring (IONM).

    PubMed

    Jahangiri, Faisal R; Sheryar, Muhammad; Al Behairy, Yaser

    2014-12-01

    Pedicle screw placement has a high risk of damaging the motor and sensory pathways due to the close proximity to the spinal cord and nerve roots. Early detection and prevention of injury can be achieved by utilizing Somatosensory Evoked Potentials (SSEP) and Transcranial electrical Motor Evoked Potentials (TCeMEP) during a scoliosis surgery. A 19-year-old female presented for correction of scoliosis. After intubation, electrodes were placed for upper and lower SSEPs, EMGs and TCeMEPs. Total intravenous anesthesia was used. Baseline SSEP and TCeMEP responses were present in all limbs. Eight pedicle screws were placed. After placing the last screw, TCeMEP signals were lost bilaterally in lower extremities. Surgery was paused. After removing all the screws TCeMEP responses returned to baseline in left lower limb but remained absent in right lower limb. A wake-up test was performed which was positive in her right leg. Blood pressure was increased and bolus of steroids was given. There was no improvement in right lower limb TCeMEP responses. Surgeon was advised to stop the surgery and proceed for MRI and follow-up. SSEP signals remained stable in all four-extremities. The surgical correction was cancelled. MRI revealed intramedullary spinal cord ischemic changes at T11. After extubation, patient was unable to move her right lower extremity with flaccid paralysis. She also complained about severe burning in her left lower extremity. The patient was taken for rehabilitation exercises. One week post-op, she was moving hip flexors and two weeks later had afull motor function, bilaterally. Real-time IONM was useful in early identification of spinal cord injury. Significant changes were seen in TCeMEP, without any change in SSEP. We highly recommend utilizing continuous TCeMEP and SSEP monitoring during pedicle screw placement for prevention of injury to the spinal cord. In this case, the patient would have been paralyzed post-operatively without the use of IONM. PMID

  7. The two-box model of climate: limitations and applications to planetary habitability and maximum entropy production studies

    PubMed Central

    Lorenz, Ralph D.

    2010-01-01

    The ‘two-box model’ of planetary climate is discussed. This model has been used to demonstrate consistency of the equator–pole temperature gradient on Earth, Mars and Titan with what would be predicted from a principle of maximum entropy production (MEP). While useful for exposition and for generating first-order estimates of planetary heat transports, it has too low a resolution to investigate climate systems with strong feedbacks. A two-box MEP model agrees well with the observed day : night temperature contrast observed on the extrasolar planet HD 189733b. PMID:20368253

  8. Lipophilic prodrugs of FR900098 are antimicrobial against Francisella novicida in vivo and in vitro and show GlpT independent efficacy.

    PubMed

    McKenney, Elizabeth S; Sargent, Michelle; Khan, Hameed; Uh, Eugene; Jackson, Emily R; San Jose, Géraldine; Couch, Robin D; Dowd, Cynthia S; van Hoek, Monique L

    2012-01-01

    Bacteria, plants, and algae produce isoprenoids through the methylerythritol phosphate (MEP) pathway, an attractive pathway for antimicrobial drug development as it is present in prokaryotes and some lower eukaryotes but absent from human cells. The first committed step of the MEP pathway is catalyzed by 1-deoxy-D-xylulose 5-phosphate reductoisomerase (DXR/MEP synthase). MEP pathway genes have been identified in many biothreat agents, including Francisella, Brucella, Bacillus, Burkholderia, and Yersinia. The importance of the MEP pathway to Francisella is demonstrated by the fact that MEP pathway mutations are lethal. We have previously established that fosmidomycin inhibits purified MEP synthase (DXR) from F. tularensis LVS. FR900098, the acetyl derivative of fosmidomycin, was found to inhibit the activity of purified DXR from F. tularensis LVS (IC(50)=230 nM). Fosmidomycin and FR900098 are effective against purified DXR from Mycobacterium tuberculosis as well, but have no effect on whole cells because the compounds are too polar to penetrate the thick cell wall. Fosmidomycin requires the GlpT transporter to enter cells, and this is absent in some pathogens, including M. tuberculosis. In this study, we have identified the GlpT homologs in F. novicida and tested transposon insertion mutants of glpT. We showed that FR900098 also requires GlpT for full activity against F. novicida. Thus, we synthesized several FR900098 prodrugs that have lipophilic groups to facilitate their passage through the bacterial cell wall and bypass the requirement for the GlpT transporter. One compound, that we termed "compound 1," was found to have GlpT-independent antimicrobial activity. We tested the ability of this best performing prodrug to inhibit F. novicida intracellular infection of eukaryotic cell lines and the caterpillar Galleria mellonella as an in vivo infection model. As a lipophilic GlpT-independent DXR inhibitor, compound 1 has the potential to be a broad

  9. Spinal inhibition of descending command to soleus motoneurons is removed prior to dorsiflexion

    PubMed Central

    Geertsen, Svend S; van de Ruit, Mark; Grey, Michael J; Nielsen, Jens B

    2011-01-01

    Abstract It has recently been demonstrated that soleus motor-evoked potentials (MEPs) are facilitated prior to the onset of dorsiflexion. The purpose of this study was to examine if this could be explained by removal of spinal inhibition of the descending command to soleus motoneurons. To test this, we investigated how afferent inputs from the tibialis anterior muscle modulate the corticospinal activation of soleus spinal motoneurons at rest, during static contraction and prior to movement. MEPs activated by transcranial magnetic stimulation (TMS) and Hoffmann reflexes (H-reflexes), activated by electrical stimulation of the posterior tibial nerve (PTN), were conditioned by prior stimulation of the common peroneal nerve (CPN) at a variety of conditioning–test (CT) intervals. MEPs in the precontracted soleus muscle were inhibited when the TMS pulse was preceded by CPN stimulation with a CT interval of 35 ms, and they were facilitated for CT intervals of 50–55 ms. A similar inhibition of the soleus H-reflex was not observed. To investigate which descending pathways might be responsible for the afferent-evoked inhibition and facilitation, we examined the effect of CPN stimulation on short-latency facilitation (SLF) and long-latency facilitation (LLF) of the soleus H-reflex induced by a subthreshold TMS pulse at different CT intervals. SLF is known to reflect the excitability of the fastest conducting, corticomotoneuronal cells whereas LLF is believed to be caused by more indirect descending pathways. At CT intervals of 40–45 ms, the LLF was significantly more inhibited compared to the SLF when taking the effect on the H-reflex into account. Finally, we investigated how the CPN-induced inhibition and facilitation of the soleus MEP were modulated prior to dorsiflexion. Whereas the late facilitation (CT interval: 55 ms) was similar prior to dorsiflexion and at rest, no inhibition could be evoked at the earlier latency (CT interval: 35 ms) prior to onset of

  10. Fine Tuned Modulation of the Motor System by Adjectives Expressing Positive and Negative Properties

    ERIC Educational Resources Information Center

    Gough, P. M.; Campione, G. C.; Buccino, G.

    2013-01-01

    Using transcranial magnetic stimulation (TMS), motor evoked potentials (MEPs) were recorded from two antagonistic muscles, the first dorsal interosseus (FDI) of the hand and the extensor communis digitorum (EC) of the forearm. FDI is involved in grasping actions and EC in releasing. TMS pulses were delivered while participants were reading…

  11. Corticospinal Excitability during the Observation of Social Behavior

    ERIC Educational Resources Information Center

    Bucchioni, Giulia; Cavallo, Andrea; Ippolito, Davide; Marton, Gianluca; Castiello, Umberto

    2013-01-01

    Evidence suggests that the observation of an action induces in the observers an enhancement of motor evoked potentials (MEPs) recorded by the observer's muscles corresponding to those involved in the observed action. Although this is a well-studied phenomenon, it remains still unclear how the viewer's motor facilitation is influenced by the social…

  12. Diabetes among Adults with Cognitive Limitations Compared to Individuals with No Cognitive Disabilities

    ERIC Educational Resources Information Center

    Reichard, Amanda; Stolzle, Hayley

    2011-01-01

    Using a retrospective analysis of data from the 2006 Medical Expenditures Panel Survey (MEPS), we assessed the health status of working-age adults with cognitive limitations in comparison to adults with no disability (unweighted N = 27,116; weighted N = 240,343,457). Adults with cognitive limitations had a significantly higher prevalence of…

  13. Microextraction by packed sorbent-high-pressure liquid chromatographic-ultra violet analysis of endocrine disruptor pesticides in various matrices.

    PubMed

    Kaur, Manpreet; Rani, Susheela; Malik, Ashok Kumar; Aulakh, Jatinder Singh

    2014-10-01

    Microextraction by a packed sorbent (MEPS) is the miniaturized version of solid-phase extraction whereby sample volumes as small as 10 μL can be used. A syringe (100-250 μL) is used in MEPS technique, which generally contains 4 mg of solid packing material inserted as a plug. The sample preparation occurs on the surface of this bed which can be modified to provide varied sampling conditions. In the present work, MEPS has been employed as a sample preparation technique for the analysis of endocrine disruptor (ED) and suspected ED pesticides in biological and environmental samples. The pesticides aldicarb, dimethoate, propazine and terbutryn have been successfully separated by high performance liquid chromatography-ultra violet (HPLC-UV) system with acetonitrile/water as the mobile phase in the ratio 60/40. Several factors affecting the performance of MEPS technique such as the number of extraction cycles, type of washing and elution solvent were optimized. This method has been applied to the analysis of these pesticides in urine, soil and tap water samples with good recoveries in the range of 81.4-97.8%. The detection limit ranged between 0.05 and 0.6 ng mL for the analyzed pesticides. PMID:24046162

  14. Reference Range of Respiratory Muscle Strength and Its Clinical Application in Amyotrophic Lateral Sclerosis: A Single-Center Study

    PubMed Central

    Park, Kee Hong; Kim, Rock Bum; Yang, Jiwon; Oh, Jung-Hwan; Park, Su-Yeon; Kim, Dong-Gun; Shin, Je-Young

    2016-01-01

    Background and Purpose Evaluating respiratory function is important in neuromuscular diseases. This study explored the reference ranges of the maximal inspiratory pressure (MIP), maximal expiratory pressure (MEP), and sniff nasal inspiratory pressure (SNIP) in healthy adults, and applied them to amyotrophic lateral sclerosis (ALS) patients. Methods MIP, MEP, and SNIP were measured in 67 healthy volunteers aged from 21 to 82 years. Reference ranges were evaluated by multivariate regression analysis using the generalized additive modeling of location, scale, and shape method. Thirty-six ALS patients were reviewed retrospectively, and abnormal values of MIP, MEP, and SNIP were determined according to the reference ranges. Results MIP, MEP, and SNIP were abnormal in 57.1%, 51.4%, and 25.7% of the ALS patients, respectively. MIP and SNIP were significantly correlated with the degree of restrictive pattern and respiratory symptoms. The ALS Functional Rating Scale-Revised score was correlated with SNIP. Conclusions This study has provided the reference range of respiratory muscle strength in healthy adults. This range is suitable for evaluating respiratory function in ALS patients. PMID:27449914

  15. Homeostatic metaplasticity of the motor cortex is altered during headache-free intervals in migraine with aura.

    PubMed

    Antal, Andrea; Lang, Nicolas; Boros, Klara; Nitsche, Michael; Siebner, Hartwig R; Paulus, Walter

    2008-11-01

    Preconditioning of the human primary motor cortex (M1) with transcranial direct current stimulation (tDCS) can shape the magnitude and direction of excitability changes induced by a subsequent session of repetitive transcranial magnetic stimulation (rTMS). Here, we examined this form of metaplasticity in migraine patients with visual aura and healthy controls. In both groups, facilitatory preconditioning of left M1 with anodal tDCS increased the mean amplitudes of motor-evoked potentials (MEPs) elicited in the contralateral hand, whereas inhibitory preconditioning with cathodal tDCS produced a decrease in amplitude. Following cathodal tDCS, a short train of low-intensity 5-Hz rTMS antagonized the suppression of the mean MEP amplitude in both groups. In contrast, the homeostatic effects of 5-Hz rTMS differed between groups when rTMS was given after anodal tDCS. In controls 5-Hz rTMS induced a marked decrease in MEP amplitudes, whereas in migraineurs rTMS induced only a modest decrease in MEP amplitudes, which were still facilitated after rTMS when compared with baseline amplitudes. These findings indicate that short-term homeostatic plasticity is altered in patients with visual aura between the attacks. PMID:18372292

  16. Keeping Up with Our Nation's Migrant Students. A Report on the Migrant Student Record Transfer System (MSRTS).

    ERIC Educational Resources Information Center

    National Commission on Migrant Education, Bethesda, MD.

    This report evaluates the Migrant Student Record Transfer System (MSRTS), a national computer system of records on migrant children, that was voluntarily created in 1969 by state directors of the Migrant Education Program (MEP). As the Federal Government instituted policy changes, MSRTS became the primary source of data for federal and state…

  17. Thermoresponsive Agarose Based Microparticles for Antibody Separation.

    PubMed

    Ooi, Huey Wen; Ketterer, Benedikt; Trouillet, Vanessa; Franzreb, Matthias; Barner-Kowollik, Christopher

    2016-01-11

    We report the development of thermoresponsive 4-mercaptoethylpyridine (MEP)-based chromatographic microsphere based resins for antibody separation that show switchable release abilities by adsorbing immunoglobulins at 40 °C and releasing the proteins at 5 °C. The thermoswitchable release properties were introduced to the porous resins by the grafting of linear poly(N-isopropylacrylamide) (PNIPAM) chains synthesized via reversible addition-fragmentation chain transfer (RAFT) polymerization, which were modified to possess MEP end functionalities. Adsorption of γ-globulins as a model antibody on the shortest PNIPAM-MEP (3 kDa) grafted microparticles display binding capacities of up to 20 g L(-1) at 40 °C and a significant decrease in binding capacity to less than 2.5 g L(-1) at 5 °C. By switching the temperature to 5 °C, the release of bound γ-globulins is shown to be as high as 90%. The effects of polymer chain length on the binding capacity are studied in detail and found to be critical as they influence the density of MEP functionalities on the particle surfaces. PMID:26626821

  18. Usefulness of the Combined Motor Evoked and Somatosensory Evoked Potentials for the Predictive Index of Functional Recovery After Primary Pontine Hemorrhage

    PubMed Central

    Seong, Jin Wan; Kim, Min Ho; Shin, Hyo Keong; Lee, Han Do; Park, Jun Bum

    2014-01-01

    Objective To investigate the predictive index of functional recovery after primary pontine hemorrhage (PPH) using the combined motor evoked potential (MEP) and somatosensory evoked potential (SEP) in comparison to the hematoma volume and transverse diameter measured with computerized tomography. Methods Patients (n=14) with PPH were divided into good- and poor-outcome groups according to the modified Rankin Score (mRS). We evaluated clinical manifestations, radiological characteristics, and the combined MEP and SEP responses. The summed MEP and SEP (EP sum) was compared to the hematoma volume and transverse diameter predictive index of global disability, gait ability, and trunk stability in sitting posture. Results All measures of functional status and radiological parameters of the good-outcome group were significantly better than those of the poor-outcome group. The EP sum showed the highest value for the mRS and functional ambulatory category, and transverse diameter showed the highest value for "sitting-unsupported" of Berg Balance Scale. Conclusion The combined MEP and SEP is a reliable and useful tool for functional recovery after PPH. PMID:24639921

  19. Training-induced modifications of corticospinal reactivity in severely affected stroke survivors.

    PubMed

    Barker, Ruth N; Brauer, Sandra G; Barry, Benjamin K; Gill, Toby J; Carson, Richard G

    2012-08-01

    When permitted access to the appropriate forms of rehabilitation, many severely affected stroke survivors demonstrate a capacity for upper limb functional recovery well in excess of that formerly considered possible. Yet, the mechanisms through which improvements in arm function occur in such profoundly impaired individuals remain poorly understood. An exploratory study was undertaken to investigate the capacity for brain plasticity and functional adaptation, in response to 12-h training of reaching using the SMART Arm device, in a group of severely affected stroke survivors with chronic upper limb paresis. Twenty-eight stroke survivors were enroled. Eleven healthy adults provided normative data. To assess the integrity of ipsilateral and contralateral corticospinal pathways, transcranial magnetic stimulation was applied to evoke responses in triceps brachii during an elbow extension task. When present, contralateral motor-evoked potentials (MEPs) were delayed and reduced in amplitude compared to those obtained in healthy adults. Following training, contralateral responses were more prevalent and their average onset latency was reduced. There were no reliable changes in ipsilateral MEPs. Stroke survivors who exhibited contralateral MEPs prior to training achieved higher levels of arm function and exhibited greater improvements in performance than those who did not initially exhibit contralateral responses. Furthermore, decreases in the onset latency of contralateral MEPs were positively related to improvements in arm function. Our findings demonstrate that when severely impaired stroke survivors are provided with an appropriate rehabilitation modality, modifications of corticospinal reactivity occur in association with sustained improvements in upper limb function. PMID:22777103

  20. Uninsured Rural Families

    ERIC Educational Resources Information Center

    Ziller, Erika C.; Coburn, Andrew F.; Anderson, Nathaniel J.; Loux, Stephenie L.

    2008-01-01

    Context: Although research shows higher uninsured rates among rural versus urban individuals, prior studies are limited because they do not examine coverage across entire rural families. Purpose: This study uses the Medical Expenditure Panel Survey (MEPS) to compare rural and urban insurance coverage within families, to inform the design of…

  1. Maximum entropy production in environmental and ecological systems.

    PubMed

    Kleidon, Axel; Malhi, Yadvinder; Cox, Peter M

    2010-05-12

    The coupled biosphere-atmosphere system entails a vast range of processes at different scales, from ecosystem exchange fluxes of energy, water and carbon to the processes that drive global biogeochemical cycles, atmospheric composition and, ultimately, the planetary energy balance. These processes are generally complex with numerous interactions and feedbacks, and they are irreversible in their nature, thereby producing entropy. The proposed principle of maximum entropy production (MEP), based on statistical mechanics and information theory, states that thermodynamic processes far from thermodynamic equilibrium will adapt to steady states at which they dissipate energy and produce entropy at the maximum possible rate. This issue focuses on the latest development of applications of MEP to the biosphere-atmosphere system including aspects of the atmospheric circulation, the role of clouds, hydrology, vegetation effects, ecosystem exchange of energy and mass, biogeochemical interactions and the Gaia hypothesis. The examples shown in this special issue demonstrate the potential of MEP to contribute to improved understanding and modelling of the biosphere and the wider Earth system, and also explore limitations and constraints to the application of the MEP principle. PMID:20368247

  2. Maximum Entropy Production and the Evolution of the Biotic Carbon Cycle

    NASA Astrophysics Data System (ADS)

    Kleidon, A.

    2003-12-01

    The MEP hypothesis states that diabatic processes with sufficient degrees of freedom maintain states at which the rate of entropy production is maximized. A common example in climatology is the application of MEP to poleward heat transport, which leads to predicted equator-pole temperature gradients that are consistent with observations. Here the MEP hypothesis is applied to biotic activity as a diabatic process which affects the atmospheric concentration of carbon dioxide (pCO2) and therefore the strength of the Earth's greenhouse effect. It is first shown with a conceptual climate model that there should be a minimum planetary albedo for which entropy production associated with absorption of solar radiation would be at a maximum as a consequence of the competing effects of surface temperature on the extent of snow cover and convective cloud cover. When pCO2 is simulated by a simple carbon cycle model, it is then shown that the application of MEP to biotic activity leads to an insensitivity of simulated surface temperature to long-term changes in solar luminosity. These predicted changes are consistent with the general suggested pattern of Earth system evolution (decreased greenhouse strength and roughly constant surface temperature through time) and share similarity with the Gaia hypothesis.

  3. Changes in corticomotor excitability after fatiguing muscle contractions.

    PubMed

    Sacco, P; Thickbroom, G W; Byrnes, M L; Mastaglia, F L

    2000-12-01

    To investigate whether the type and duration of activity influences corticomotor excitability following fatiguing exercise, we compared motor evoked potential (MEP) responses of the biceps brachii to transcranial magnetic stimulation (TMS) during recovery from two different exercise regimens. Responses were recorded in both the resting state and during a weak contraction. Ten subjects performed a 60-s maximal voluntary contraction (MVC) and, on a subsequent occasion, a sustained 20% MVC to the point of exhaustion. Resting MEP amplitude declined following maximal and submaximal protocols, reaching 34% and 31% of pre-exercise means, respectively (P < 0.001 for both). In contrast, mean facilitated MEP amplitude showed a smaller and more transient decrement following the sustained submaximal effort (64%; P < 0.05), but not the 60-s MVC. Abolition of the postexercise depression in resting MEP amplitude by a weak tonic contraction indicates that decreases in excitability at the spinal level contribute to the reduced corticomotor excitability observed after fatiguing exercise. PMID:11102907

  4. Determination of organic priority pollutants and emerging compounds in wastewater and snow samples using multiresidue protocols on the basis of microextraction by packed sorbents coupled to large volume injection gas chromatography-mass spectrometry analysis.

    PubMed

    Prieto, A; Schrader, S; Moeder, M

    2010-09-17

    This paper describes the development and validation of a new procedure for the simultaneous determination of 41 multi-class priority and emerging organic pollutants in water samples using microextraction by packed sorbent (MEPS) followed by large volume injection-gas chromatography-mass spectrometry (LVI-GC-MS). Apart from method parameter optimization the influence of humic acids as matrix components on the extraction efficiency of MEPS procedure was also evaluated. The list of target compounds includes polycyclic aromatic hydrocarbons (PAHs), polychlorinated biphenyls (PCBs), phthalate esters (PEs), nonylphenols (NPs), bisphenol A (BPA) and selected steroid hormones. The performance of the new at-line microextraction-LVI-GC-MS protocol was compared to standard solid-phase extraction (SPE) and LVI-GC-MS analysis. LODs for 100 mL samples (SPE) ranged from 0.2 to 736 ng L(-1) were obtained. LODs for 800 microL of sample (MEPS) were between 0.2 and 266 ng L(-1). In the case of MEPS methodology even a sample volume of only 800 microL allowed to detect the target compounds. These results demonstrate the high sensitivity of both procedures which permitted to obtain good recoveries (>75%) for all cases. The precision of the methods, calculated as relative standard deviation (RSD) was below 21% for all compounds and both methodologies. Finally, the developed methods were applied to the determination of target analytes in various samples, including snow and wastewater. PMID:20719318

  5. 78 FR 17210 - Agency Information Collection Activities; Proposed Collection; Comment Request

    Federal Register 2010, 2011, 2012, 2013, 2014

    2013-03-20

    ... the AHRQ Reports Clearance Officer. FOR FURTHER INFORMATION CONTACT: Doris Lefkowitz, AHRQ Reports Clearance Officer, (301) 427-1477, or by email at doris.lefkowitz@AHRQ.hhs.gov . SUPPLEMENTARY INFORMATION...-0935-001 . The current MEPS-IC clearance noted the possibility of making changes to the 2013...

  6. 78 FR 40084 - Proposed Requirement-Migrant Education Program Consortium Incentive Grant Program

    Federal Register 2010, 2011, 2012, 2013, 2014

    2013-07-03

    ... Register on March 3, 2004 (69 FR 10110) (2004 Notice), and we have used these final requirements for CIG... final priority for the MEP CIG program in the Federal Register on March 12, 2008 (73 FR 13217), in which... From the Federal Register Online via the Government Publishing Office DEPARTMENT OF EDUCATION...

  7. 40 CFR 122.34 - As an operator of a regulated small MS4, what will my NPDES MS4 storm water permit require?

    Code of Federal Regulations, 2011 CFR

    2011-07-01

    ... practicable (MEP), to protect water quality, and to satisfy the appropriate water quality requirements of the... water quality. Implementation of best management practices consistent with the provisions of the storm... (as defined at 40 CFR 35.2005(20)), uncontaminated pumped ground water, discharges from potable...

  8. 40 CFR 122.34 - As an operator of a regulated small MS4, what will my NPDES MS4 storm water permit require?

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ... practicable (MEP), to protect water quality, and to satisfy the appropriate water quality requirements of the... water quality. Implementation of best management practices consistent with the provisions of the storm... (as defined at 40 CFR 35.2005(20)), uncontaminated pumped ground water, discharges from potable...

  9. 40 CFR 122.34 - As an operator of a regulated small MS4, what will my NPDES MS4 storm water permit require?

    Code of Federal Regulations, 2012 CFR

    2012-07-01

    ... practicable (MEP), to protect water quality, and to satisfy the appropriate water quality requirements of the... water quality. Implementation of best management practices consistent with the provisions of the storm... (as defined at 40 CFR 35.2005(20)), uncontaminated pumped ground water, discharges from potable...

  10. 40 CFR 122.34 - As an operator of a regulated small MS4, what will my NPDES MS4 storm water permit require?

    Code of Federal Regulations, 2013 CFR

    2013-07-01

    ... practicable (MEP), to protect water quality, and to satisfy the appropriate water quality requirements of the... water quality. Implementation of best management practices consistent with the provisions of the storm... (as defined at 40 CFR 35.2005(20)), uncontaminated pumped ground water, discharges from potable...

  11. 40 CFR 122.34 - As an operator of a regulated small MS4, what will my NPDES MS4 storm water permit require?

    Code of Federal Regulations, 2014 CFR

    2014-07-01

    ... practicable (MEP), to protect water quality, and to satisfy the appropriate water quality requirements of the... water quality. Implementation of best management practices consistent with the provisions of the storm... (as defined at 40 CFR 35.2005(20)), uncontaminated pumped ground water, discharges from potable...

  12. Essential involvement of the NMDA receptor in ethanol preconditioning-dependent neuroprotection from amyloid-betain vitro.

    PubMed

    Mitchell, Robert M; Neafsey, Edward J; Collins, Michael A

    2009-10-01

    In several epidemiological studies, moderate ethanol consumption has been associated with reduced risks of cognitive decline or Alzheimer's dementia. Of potential relevance is that brain cultures preconditioned with moderate ethanol concentrations are resistant to neurotoxic Alzheimer's amyloid-beta (Abeta) peptides. Using rat cerebellar mixed cultures we investigated whether certain membrane receptors were early 'sensors' in moderate ethanol preconditioning (MEP). In a 6-day MEP protocol (30 mM ethanol), neuroprotection from Abeta25-35 was undiminished by antagonism during the first 3 days of either adenosine A(1) or Galpha(i/o) protein-coupled receptors. However, similar cotreatment with memantine or DL-2-amino-5-phosphono-pentanoic acid (AP-5), antagonists of NMDA receptors (NMDAR), abolished neuroprotection, indicating key early involvement of this ionotropic glutamate receptor. Also in these cultures, directly activating NMDAR using subexcitotoxic NMDA preconditioning prevented Abeta neurotoxicity. By day 2 of MEP, we observed increased levels of NMDAR subunits NR1, NR2B, and NR2C that persisted through day 6. Interestingly, memantine co-exposure blocked elevations in the obligatory NR1 subunit. Furthermore, 2 days of MEP significantly increased two indicators of synaptic NMDAR localization, NR2B phospho-Tyr1472, and post-synaptic density 95 scaffolding protein. The results indicate that ethanol preconditioning-dependent neuroprotection is associated with early increases in NR subunits concomitant with enhancement of synaptic localization and activity of NMDAR. PMID:19694907

  13. 78 FR 17917 - Medical Waivers for Merchant Mariner Credential Applicants With a History of Seizure Disorders

    Federal Register 2010, 2011, 2012, 2013, 2014

    2013-03-25

    ... Federal Register (73 FR 3316). Background and Purpose Coast Guard regulations in 46 CFR 10.215 contain the... drive a commercial motor vehicle. The MEP recommended that an individual with a history of epilepsy (a... be considered for a waiver. (1) Mariners with a history of epilepsy or seizure disorder may...

  14. In situ derivatization combined to automated microextraction by packed sorbents for the determination of chlorophenols in soil samples by gas chromatography mass spectrometry.

    PubMed

    González Paredes, Rosa María; García Pinto, Carmelo; Pérez Pavón, José Luis; Moreno Cordero, Bernardo

    2014-09-12

    A method based on the coupling of in situ extraction and derivatization of chlorophenols (CPs) (2-chlorophenol, 4-chloro-3-methylphenol, 2,4-dichlorophenol, and 2,4,6-trichlorophenol) from soils, accomplishing their preconcentration by means of automated microextraction by packed sorbent (MEPS), is proposed. After extraction and acylation of the chlorophenols in aqueous medium, the liquid phase obtained is subjected to the MEPS procedure. The QuEChERS (Quick, Easy, Cheap, Effective, Rugged and Safe) and MEPS techniques were compared and the results confirmed the preconcentration carried out with MEPS. The existence of a matrix effect was checked and the analytical characteristics of the method were determined in a soil sample. The method provided good linearity (from 1 to 12μgkg(-1)), together with good repeatability and reproducibility values (RSD equal to or less than 10%). The limits of detection were in the 0.118-0.894μgkg(-1) range. A certified reference material was applied to validate the proposed methodology. PMID:25113872

  15. ACUTE TOXICITY OF METHYL-PARATHION IN WETLAND MESOCOSMS: INFLUENCE OF AQUATIC PLANTS

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The acute toxicity of methyl-parathion (MeP) introduced into constructed wetlands for the purpose of assessing the importance of emergent vegetation was tested using Hyalella azecta (Crustacea: Amphipoda). A vegetated (90% cover, mainly Juncus effuses) and a non-vegetated wetland (each with a water...

  16. Transcranial Magnetic Stimulation and Diffusion Tensor Tractography for Evaluating Ambulation after Stroke

    PubMed Central

    Kim, Bo-Ram; Moon, Won-Jin; Kim, Hyuntae; Jung, Eunhwa; Lee, Jongmin

    2016-01-01

    Background and Purpose We aimed to investigate the usefulness of combining transcranial magnetic stimulation (TMS) and diffusion tensor tractography (DTT) to evaluate corticospinal tract (CST) integrity and subsequently predict ambulatory function after middle cerebral artery (MCA) stroke. Methods Forty-three patients with first MCA stroke underwent TMS and DTT to evaluate CST integrity. Patients were classified into four groups according to the presence of motor-evoked potentials (MEPs) obtained from the tibialis anterior muscle and CST integrity. Motor impairment and functional status were assessed using the Fugl-Meyer Assessment, Functional Ambulation Category, and Korean modified Barthel Index, both at the time of admission and after 4 weeks of rehabilitation. Results Patients with the presence of both measurable MEPs and a preserved CST showed better motor recovery and ambulatory function than other groups at the 4-week follow-up. Intact CSTs were not visualized in patients without detectable MEPs. Among the patients displaying MEPs, those with preserved CSTs showed better recovery of paretic lower extremities. Conclusions Combined assessment using TMS and DTT to evaluate CST integrity confers advantages in predicting motor and ambulation recovery in patients with MCA stroke. PMID:27283282

  17. Paired-Pulse TMS and Fine-Wire Recordings Reveal Short-Interval Intracortical Inhibition and Facilitation of Deep Multifidus Muscle Fascicles

    PubMed Central

    Massé-Alarie, Hugo; Elgueta Cancino, Edith; Schneider, Cyril; Hodges, Paul

    2016-01-01

    Objective Paired-pulse transcranial magnetic stimulation (ppTMS) is used to probe inhibitory and excitatory networks within the primary motor cortex (M1). These mechanisms are identified for limb muscles but it is unclear whether they share properties with trunk muscles. The aim was to determine whether it was possible to test the intracortical inhibition and facilitation of the deep multifidus muscle fascicles (DM) and at which inter-stimulus intervals (ISI). Methods In ten pain-free individuals, TMS was applied over M1 and motor evoked potentials (MEP) were recorded using fine-wire electrodes in DM. MEPs were conditioned with subthreshold stimuli at ISIs of 1 to 12 ms to test short-interval intracortical inhibition (SICI) and at 15 ms for long-interval intracortical facilitation. Short-interval facilitation (SICF) was tested using 1-ms ISI. Results SICI of DM was consistently obtained with ISI of 1-, 3-, 4- and 12-ms. Facilitation of DM MEP was only identified using SICF paradigm. Conclusions A similar pattern of MEP modulation with ISI changes for deep trunk and limb muscles implies that M1 networks share some functional properties. Significance The ppTMS paradigm presents a potential to determine how M1 inhibitory and excitatory mechanisms participate in brain re-organization in back pain that affects control of trunk muscles. PMID:27509086

  18. The Legacy of the Microelectronics Education Programme.

    ERIC Educational Resources Information Center

    Thorne, Michael

    1987-01-01

    Describes the Microelectronics Education Programme (MEP), a plan developed to help British secondary school students learn about microcomputers and the role of technology in society, and its successor, the Microelectronics Support Unit (MESU). Highlights include curriculum development, teacher training, computer assisted instruction and the…

  19. Corticospinal excitability modulation in resting digit muscles during cyclical movement of the digits of the ipsilateral limb.

    PubMed

    Muraoka, Tetsuro; Sakamoto, Masanori; Mizuguchi, Nobuaki; Nakagawa, Kento; Kanosue, Kazuyuki

    2015-01-01

    We investigated how corticospinal excitability of the resting digit muscles was modulated by the digit movement in the ipsilateral limb. Subjects performed cyclical extension-flexion movements of either the right toes or fingers. To determine whether corticospinal excitability of the resting digit muscles was modulated on the basis of movement direction or action coupling between ipsilateral digits, the right forearm was maintained in either the pronated or supinated position. During the movement, the motor evoked potential (MEP) elicited by transcranial magnetic stimulation (TMS) was measured from either the resting right finger extensor and flexor, or toe extensor and flexor. For both finger and toe muscles, independent of forearm position, MEP amplitude of the flexor was greater during ipsilateral digit flexion as compared to extension, and MEP amplitude of the extensor was greater during ipsilateral digit extension as compared to flexion. An exception was that MEP amplitude of the toe flexor with the supinated forearm did not differ between during finger extension and flexion. These findings suggest that digit movement modulates corticospinal excitability of the digits of the ipsilateral limb such that the same action is preferred. Our results provide evidence for a better understanding of neural interactions between ipsilateral limbs, and may thus contribute to neurorehabilitation after a stroke or incomplete spinal cord injury. PMID:26582985

  20. Usefulness of magnetic motor evoked potentials in the surgical treatment of hemiplegic patients with intractable epilepsy.

    PubMed

    Kamida, Tohru; Baba, Hiroshi; Ono, Kenji; Yonekura, Masato; Fujiki, Minoru; Kobayashi, Hidenori

    2003-09-01

    Five hemiplegic patients with intractable epilepsy were studied with transcranial magnetic stimulation (TMS) before and after various surgical treatments. These patients had unilateral widespread cerebral lesions acquired at various times, including congenital, infantile and childhood injury. Motor evoked potentials (MEPs) of the abductor pollicis brevis (APB) muscles were simultaneously recorded on both sides following TMS of the motor cortex in the respective hemisphere using a figure-8 or circular coil. In all patients with congenital disease, the abolition of motor function in the affected hemisphere was estimated by magnetic MEPs, and the hemiplegia did not deteriorate after functional hemispherectomy (HS) was performed in two of them. In two patients with acquired disease, HS was not performed because it was shown by magnetic maps that the motor function in the affected hemisphere remained. Furthermore, it was shown by electric MEPs using subdural electrodes that a patient who had had encephalitis in early childhood had a reorganised motor area in the parietal cortex of the affected hemisphere. The present findings indicate that magnetic MEPs are a very useful non-invasive method of assessing whether the motor area in the affected hemisphere can be resected in hemiplegic patients with intractable epilepsy. PMID:12915083

  1. Effect of fatiguing maximal voluntary contraction on excitatory and inhibitory responses elicited by transcranial magnetic motor cortex stimulation.

    PubMed

    McKay, W B; Stokic, D S; Sherwood, A M; Vrbova, G; Dimitrijevic, M R

    1996-08-01

    Vertex transcranial magnetic stimulation (TMS) elicited tibialis anterior motor evoked potentials (MEPs) and silent periods (SPs) that were recorded during and following isometric maximal volitional contraction (MVC). During MVC in 6 healthy subjects, MEP amplitudes in the exercised muscle showed an increasing trend from an initial value of 4539 +/- 809 muV (mean +/- SE) to 550 +/- 908 muV (P < 0.13) while force and EMG decreased (P < 0.01). Also, SP duration increased from 165 +/- 37 ms to 231 +/- 32 ms (P < 0.01). Thus, during a fatiguing MVC both excitatory and inhibitory TMS-induced responses increased. TMS delivered during repeated brief 10% MVC contractions before and after a fatiguing MVC in 5 subjects, showed no change in MEP amplitude but SP duration was prolonged after MVC. This SP prolongation was focal to the exercised muscle. Silent periods recorded after pyramidal tract stimulation were unchanged following the MVC. These results suggest that MEP and SP might have common sources of facilitation during an MVC and that inhibitory mechanisms remain focally augmented following a fatiguing MVC. PMID:8756168

  2. The syndrome of delayed posthemiplegic hemidystonia, hemiatrophy, and partial seizure: clinical, neuroimaging, and motor-evoked potential studies.

    PubMed

    Thajeb, P

    1996-08-01

    Magnetic motor-evoked potential (MEP) study of patients with the syndrome of delayed posthemiplegic hemidystonia, hemiatrophy, and partial or hemi-seizures ('4-hemi' syndrome) has not been described. Among 35 patients investigated for posthemiplegic movement disorders from February 1988 to January 1995, seven showed '4-hemi' syndrome. Clinical work-up, magnetic resonance images (MRI) and/or computed tomography (CT) were performed in all. Transcranial MEP studies were done in five patients. The remote causes of '4-hemi' syndrome were neonatal stroke, trauma, and encephalitis in infancy. The dystonia may occur as long as a decade after the initial insult. MRI or CT showed destructive lesion in the contralateral putamen (five patients), caudate (four), thalamus (five), and atrophy of the contralateral hemisphere (five). Other associations were porencephalic cyst. Wallerian degeneration, and asymmetric compensatory ventriculomegaly. MEP showed abnormalities in the affected upper limbs in four of five patients. The abnormalities were reduced amplitude of the compound muscle action potential following cortical stimulations with or without temporal dispersion, and with or without prolongation of its latency. The peripheral motor conductions following cervical stimulations were normal. MEP abnormalities may not be related to the hemiatrophy and the size of brain lesion per se. The hemidystonia is static after the second decade of life, and it is often difficult to treat. PMID:8884090

  3. Inhibitory mechanisms following electrical stimulation of tendon and cutaneous afferents in the lower limb.

    PubMed

    Khan, Serajul I; Burne, John A

    2010-01-13

    Electrical stimulation of the Achilles tendon (TES) produced strong reflex depression (duration>250 ms) of a small background contraction in both heads of gastrocnemius (GA) via large diameter electrodes localized to the tendon. The inhibitory responses were produced without electrical (M wave) or mechanical (muscle twitch) signs of direct muscle stimulation. In this study, the contribution of presynaptic and postsynaptic mechanisms to the depression was investigated by studying conditioning effects of tendon afferent stimulation on the mechanical tendon reflex (TR) and magnetic motor evoked potential (MEP). TES completely inhibited the TR over an ISI of 300 ms that commenced before and continued during and after the period of voluntary EMG depression. Tendon afferent conditioning stimuli also partially inhibited the MEP, but over a short time course confined to the period of voluntary EMG depression. The strength and extended time course of tendon afferent conditioning of the TR and its failure to produce a similar depression of the MEP are consistent with a mechanism involving presynaptic inhibition of Ia terminals. Cutaneous (sural nerve) afferent conditioning partially inhibited the TR and MEP over a short time course (ISI <100 ms) resembling the inhibition seen in the voluntary EMG. This was consistent with the postsynaptic origin of cutaneous inhibition of the motoneurons. PMID:19850015

  4. The silent period after transcranial magnetic stimulation is of exclusive cortical origin: evidence from isolated cortical ischemic lesions in man.

    PubMed

    Schnitzler, A; Benecke, R

    1994-10-10

    In two representative patients suffering from focal isolated ischemic lesions of the arm-associated (patient 1) or leg-associated (patient 2) primary motor cortex excitatory responses (motor evoked potentials, MEPs) and inhibitory phenomena (silent period, SP) following transcranial magnetic motor cortex stimulation (TMS) are demonstrated. Furthermore, supramaximal peripheral nerve stimulations for testing spinal inhibitory actions were performed. Results were compared to a control group of 12 normal subjects. In patient 1, SP induced by TMS in the clinically affected left extensor carpi radialis muscle (ECR) was lacking in the presence of an only marginally reduced MEP and a normal spinal silent period in this muscle. Normal MEPs and SP durations were observed in the right ECR and in the first dorsal interosseus (FDI) and anterior tibial (TA) muscles on both sides. Similarly, in patient 2 a loss of SP induced by TMS in the clinically affected right TA was observed with normal SP durations in the left TA and both FDI muscles and normal MEP amplitudes in all muscles studied. It is concluded that both early and late phases of SP induced by TMS are of cortical origin and generated in the primary motor cortex. PMID:7877758

  5. The site of impulse generation in transcranial magnetic stimulation of the facial nerve.

    PubMed

    Rimpiläinen, I; Pyykkö, I; Blomstedt, G; Kuurne, T; Karma, P

    1993-05-01

    The facial nerve can be stimulated in its intracranial course through transcranial magnetic stimulation (TMS). We studied the site of impulse generation produced by TMS by comparing the latencies of the muscle evoked potentials (MEPs) elicited with TMS and intracranial electrical stimulation (IES) of the facial nerve during neurosurgical posterior fossa procedures. In a series of 25 patients, the mean latency of the TMS elicited MEPs, recorded in the orbicularis oris muscle, was 5.0 ms (SD 0.58). Also IES of the distal part of the facial nerve in the internal acoustic meatus showed a mean latency of 5.0 ms (SD 0.68). Proximal IES in the root entry zone of the facial nerve, and intermediate IES between root entry zone and meatus, produced MEPs with significantly longer latencies compared to TMS and distal IES (p < 0.05). The findings suggest that the TMS induced facial nerve activation, leading to a MEP response, takes place within the internal acoustic meatus. PMID:8517138

  6. Notch Stimulates Both Self-Renewal and Lineage Plasticity in a Subset of Murine CD9High Committed Megakaryocytic Progenitors

    PubMed Central

    Chaabouni, Azza; Chazaud, Bénédicte; Morlé, François

    2016-01-01

    This study aimed at reinvestigating the controversial contribution of Notch signaling to megakaryocytic lineage development. For that purpose, we combined colony assays and single cells progeny analyses of purified megakaryocyte-erythroid progenitors (MEP) after short-term cultures on recombinant Notch ligand rDLL1. We showed that Notch activation stimulated the SCF-dependent and preferential amplification of Kit+ erythroid and bipotent progenitors while favoring commitment towards the erythroid at the expense of megakaryocytic lineage. Interestingly, we also identified a CD9High MEP subset that spontaneously generated almost exclusively megakaryocytic progeny mainly composed of single megakaryocytes. We showed that Notch activation decreased the extent of polyploidization and maturation of megakaryocytes, increased the size of megakaryocytic colonies and surprisingly restored the generation of erythroid and mixed colonies by this CD9High MEP subset. Importantly, the size increase of megakaryocytic colonies occurred at the expense of the production of single megakaryocytes and the restoration of colonies of alternative lineages occurred at the expense of the whole megakaryocytic progeny. Altogether, these results indicate that Notch activation is able to extend the number of divisions of MK-committed CD9High MEPs before terminal maturation while allowing a fraction of them to generate alternative lineages. This unexpected plasticity of MK-committed progenitors revealed upon Notch activation helps to better understand the functional promiscuity between megakaryocytic lineage and hematopoietic stem cells. PMID:27089435

  7. Monitoring transcranial direct current stimulation induced changes in cortical excitability during the serial reaction time task.

    PubMed

    Ambrus, Géza Gergely; Chaieb, Leila; Stilling, Roman; Rothkegel, Holger; Antal, Andrea; Paulus, Walter

    2016-03-11

    The measurement of the motor evoked potential (MEP) amplitudes using single pulse transcranial magnetic stimulation (TMS) is a common method to observe changes in motor cortical excitability. The level of cortical excitability has been shown to change during motor learning. Conversely, motor learning can be improved by using anodal transcranial direct current stimulation (tDCS). In the present study, we aimed to monitor cortical excitability changes during an implicit motor learning paradigm, a version of the serial reaction time task (SRTT). Responses from the first dorsal interosseous (FDI) and forearm flexor (FLEX) muscles were recorded before, during and after the performance of the SRTT. Online measurements were combined with anodal, cathodal or sham tDCS for the duration of the SRTT. Negative correlations between the amplitude of online FDI MEPs and SRTT reaction times (RTs) were observed across the learning blocks in the cathodal condition (higher average MEP amplitudes associated with lower RTs) but no significant differences in the anodal and sham conditions. tDCS did not have an impact on SRTT performance, as would be predicted based on previous studies. The offline before-after SRTT MEP amplitudes showed an increase after anodal and a tendency to decrease after cathodal stimulation, but these changes were not significant. The combination of different interventions during tDCS might result in reduced efficacy of the stimulation that in future studies need further attention. PMID:26826607

  8. Late cortical disinhibition in relaxed versus active hand muscles.

    PubMed

    Caux-Dedeystère, A; Derambure, P; Devanne, H

    2015-07-01

    Recent research suggests that long-interval intracortical inhibition (LICI) is followed by a transitory period of late cortical disinhibition (LCD) that can even lead to a net increase in cortical excitability. The relationship between LICI/LCD and voluntary drive remains poorly understood. Our study aims at investigating the influence of index abduction on LICI and LCD in an actively engaged muscle and a neighboring muscle, while varying the intensity of the conditioning stimulus (CS). Motor-evoked potentials (MEPs) were recorded from the first dorsal interosseus (FDI) and abductor digiti minimi (ADM) muscles in 13 subjects. Paired-pulses were delivered with 10 different interstimulus intervals (ranging from 60 to 290 ms). Whatever the condition (relaxed or active FDI), the test stimulus was set to evoke an MEP of 1mV. The time course of conditioned MEP amplitude was compared for relaxed and active conditions when the CS intensity was set to (i) 130% of the rest motor threshold (RMT) or (ii) to evoke the same size of MEP under both conditions. LICI lasted longer (i.e. disinhibition occurred later) at rest than during abduction when evoked either by similar or matched conditioning stimuli. No post-LICI facilitation was observed at rest - even when the CS intensity was set to 160% RMT. In contrast, long-interval intracortical facilitation (LICF) was observed in the quiescent ADM when FDI was active. LICF may then be associated with voluntary activity albeit with lack of topographic specificity. PMID:25888934

  9. Effect of tactile stimulation on primary motor cortex excitability during action observation combined with motor imagery.

    PubMed

    Tanaka, Megumi; Kubota, Shinji; Onmyoji, Yusuke; Hirano, Masato; Uehara, Kazumasa; Morishita, Takuya; Funase, Kozo

    2015-07-23

    We aimed to investigate the effects of the tactile stimulation to an observer's fingertips at the moment that they saw an object being pinched by another person on the excitability of observer's primary motor cortex (M1) using transcranial magnetic stimulation (TMS). In addition, the above effects were also examined during action observation combined with the motor imagery. Motor evoked potentials (MEP) were evoked from the subjects' right first dorsal interosseous (FDI) and abductor digiti minimi (ADM) muscles. Electrical stimulation (ES) inducing tactile sensation was delivered to the subjects' first and second fingertips at the moment of pinching action performed by another person. Although neither the ES nor action observation alone had significant effects on the MEP amplitude of the FDI or ADM, the FDI MEP amplitude which acts as the prime mover during pinching was reduced when ES and action observation were combined; however, no such changes were seen in the ADM. Conversely, that reduced FDI MEP amplitude was increased during the motor imagery. These results indicated that the M1 excitability during the action observation of pinching action combined with motor imagery could be enhanced by the tactile stimulation delivered to the observer's fingertips at the moment corresponding to the pinching being observed. PMID:26033185

  10. Corticospinal excitability modulation in resting digit muscles during cyclical movement of the digits of the ipsilateral limb

    PubMed Central

    Muraoka, Tetsuro; Sakamoto, Masanori; Mizuguchi, Nobuaki; Nakagawa, Kento; Kanosue, Kazuyuki

    2015-01-01

    We investigated how corticospinal excitability of the resting digit muscles was modulated by the digit movement in the ipsilateral limb. Subjects performed cyclical extension-flexion movements of either the right toes or fingers. To determine whether corticospinal excitability of the resting digit muscles was modulated on the basis of movement direction or action coupling between ipsilateral digits, the right forearm was maintained in either the pronated or supinated position. During the movement, the motor evoked potential (MEP) elicited by transcranial magnetic stimulation (TMS) was measured from either the resting right finger extensor and flexor, or toe extensor and flexor. For both finger and toe muscles, independent of forearm position, MEP amplitude of the flexor was greater during ipsilateral digit flexion as compared to extension, and MEP amplitude of the extensor was greater during ipsilateral digit extension as compared to flexion. An exception was that MEP amplitude of the toe flexor with the supinated forearm did not differ between during finger extension and flexion. These findings suggest that digit movement modulates corticospinal excitability of the digits of the ipsilateral limb such that the same action is preferred. Our results provide evidence for a better understanding of neural interactions between ipsilateral limbs, and may thus contribute to neurorehabilitation after a stroke or incomplete spinal cord injury. PMID:26582985

  11. 77 FR 55811 - Manufacturing Extension Partnership Advisory Board On-line Open Meeting

    Federal Register 2010, 2011, 2012, 2013, 2014

    2012-09-11

    ... National Institute of Standards and Technology Manufacturing Extension Partnership Advisory Board On-line... Manufacturing Extension Partnership (MEP) Advisory Board will hold an open, on-line meeting via webcast on... their remote location. Questions regarding the on-line meeting should be sent to the...

  12. 77 FR 58386 - Agency Information Collection Activities: Proposed Collection; Comment Request

    Federal Register 2010, 2011, 2012, 2013, 2014

    2012-09-20

    ... From the Federal Register Online via the Government Publishing Office DEPARTMENT OF HEALTH AND... health services researchers to analyze health care use, expenses, and health policy. Major changes continue to take place in the health care delivery system. The MEPS is needed to provide information...

  13. 77 FR 35399 - Proposed Collection; Comment Request

    Federal Register 2010, 2011, 2012, 2013, 2014

    2012-06-13

    ... From the Federal Register Online via the Government Publishing Office ] DEPARTMENT OF HEALTH AND... health services researchers to analyze health care use, expenses and health policy. Major changes continue to take place in the health care delivery system. The MEPS is needed to provide information...

  14. Characterization of the Interaction Between Endostatin Short Peptide and VEGF Receptor 3

    PubMed Central

    Han, Kyu-Yeon; Azar, Dimitri T.; Sabri, Abdellah; Lee, Hyun; Jain, Sandeep; Lee, Bao-Shiang; Chang, Jin-Hong

    2013-01-01

    Corneal angiogenesis and lymphangiogenesis are induced by vascular endothelial growth factors (VEGFs) signaling through its receptors VEGFR-1, -2, and -3. Endostatin is a peptide antagonist of these receptors that causes inhibition of bFGF-induced corneal angiogenesis and lymphangiogenesis. Here we show that binding of VEGF-C and endostatin to recombinant VEGFR-3 is competitive. Alignments of the primary amino acid sequences of VEGF-C and the C-terminal endostatin peptide (mEP: LEQKAASCHNSYIVLCIENSFMTSFSK) identified two conserved cysteine residues separated by seven amino acids. Peptides of VEGF-C and mEP containing these conserved residues bound toVEGFR-3. However, substitution of alanine for either of the cysteines in the mEP peptide perturbed the secondary structure, and this mutated peptide was unable to bind to VEGFR-3. Analysis by surface plasmon resonance demonstrated that the binding of the mEP peptide for recombinant VEGFR-3 had a Ka of 1.41 × 107M−1s−1, Kd of 0.6718 s−1, and a KD of 4.78 10−8M. Characterization of the mechanism of endostatin binding to VEGFR-3 may lead to the development of novel therapies for lymphangiogenesis-related disorders, such as transplant rejection, lymphedema, and cancer metastasis. PMID:22512651

  15. Motor excitability during movement preparation in Tourette syndrome

    PubMed Central

    Draper, Amelia; Jude, Lucinda; Jackson, Georgina M; Jackson, Stephen R

    2015-01-01

    Tourette syndrome (TS) is a neurodevelopmental disorder characterized by the occurrence of motor and vocal tics. TS has been linked to the impaired operation of cortical-striatal-thalamic-cortical circuits that give rise to hyper-excitability of cortical motor areas, which may be exacerbated by dysfunctional intra-cortical inhibitory mechanisms. That said, many individuals gain control over their tics during adolescence and it has been suggested that this increased control arises as a result of the development of mechanisms that operate to suppress corticospinal excitability (CSE) ahead of volitional movements. Here we used single-pulse transcranial magnetic stimulation (TMS) in conjunction with a manual Go/NoGo task to investigate alterations in CSE ahead of volitional movements in a group of adolescents with TS (N = 10). Our study demonstrated that CSE, as measured by TMS-induced motor-evoked potentials (MEPs), was significantly reduced in the TS group in the period immediately preceding a finger movement. More specifically, we show that individuals with TS, unlike their age-matched controls, do not exhibit the predicted increase in mean MEP amplitude and decrease in MEP variability that immediately precede the execution of volitional movements in typically developing young adults. Finally, we report that the magnitude of the rise in MEP amplitude across the movement preparation period in TS is significantly negatively correlated with clinical measures of motor tic severity, suggesting that individuals with severe motor tics are least able to modulate motor cortical excitability. PMID:24283505

  16. Optimisation by response surface methodology of microextraction by packed sorbent of non steroidal anti-inflammatory drugs and ultra-high performance liquid chromatography analysis of dialyzed samples.

    PubMed

    D'Archivio, Angelo Antonio; Maggi, Maria Anna; Ruggieri, Fabrizio; Carlucci, Maura; Ferrone, Vincenzo; Carlucci, Giuseppe

    2016-06-01

    A procedure based on microextraction by packed sorbent (MEPS) followed by ultra-high performance liquid chromatography (UHPLC) with photodiode array (PDA) detection has been developed for the analysis of seven selected non steroidal anti-inflammatory drugs (NSAIDs) in human dialysates. The influence on MEPS efficiency of pH of the sample, pH of the washing solvent and methanol content in the hydro-alcoholic elution mixture has been investigated by response surface methodology based on a Box-Behnken design of experiments. Among the above factors, pH of sample is the variable that mostly influences MEPS recovery. UHPLC separation of the NSAIDs was completed within less than 4min under isocratic elution conditions on a Fortis SpeedCore C18 column (150×4.6mm I.D., 2.6μm) using acetonitrile-phosphate buffer as the mobile phase. Calibration curves of the NSAIDs were linear over the concentration range 0.025-15μg/mL, with correlation coefficients≥0.998. Intra- and inter-assay relative standard deviations were <8% and recovery values ranged from 94% to 100% for the quality control samples. The results reveal that the developed MEPS/PDA-UHPLC method exhibits a good accuracy and precision and is well suited for the rapid analysis of human dialysate from patients treated with the selected NSAIDs. PMID:27017570

  17. 32 CFR 1624.9 - Induction into the Armed Forces.

    Code of Federal Regulations, 2012 CFR

    2012-07-01

    ... 32 National Defense 6 2012-07-01 2012-07-01 false Induction into the Armed Forces. 1624.9 Section... INDUCTIONS § 1624.9 Induction into the Armed Forces. Registrants in classes 1-A and 1-A-0, who have been... inducted at the MEPS into the Armed Forces....

  18. 32 CFR 1624.9 - Induction into the Armed Forces.

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ... 32 National Defense 6 2010-07-01 2010-07-01 false Induction into the Armed Forces. 1624.9 Section... INDUCTIONS § 1624.9 Induction into the Armed Forces. Registrants in classes 1-A and 1-A-0, who have been... inducted at the MEPS into the Armed Forces....

  19. 32 CFR 1624.9 - Induction into the Armed Forces.

    Code of Federal Regulations, 2014 CFR

    2014-07-01

    ... 32 National Defense 6 2014-07-01 2014-07-01 false Induction into the Armed Forces. 1624.9 Section... INDUCTIONS § 1624.9 Induction into the Armed Forces. Registrants in classes 1-A and 1-A-0, who have been... inducted at the MEPS into the Armed Forces....

  20. 32 CFR 1624.9 - Induction into the Armed Forces.

    Code of Federal Regulations, 2011 CFR

    2011-07-01

    ... 32 National Defense 6 2011-07-01 2011-07-01 false Induction into the Armed Forces. 1624.9 Section... INDUCTIONS § 1624.9 Induction into the Armed Forces. Registrants in classes 1-A and 1-A-0, who have been... inducted at the MEPS into the Armed Forces....

  1. 32 CFR 1624.9 - Induction into the Armed Forces.

    Code of Federal Regulations, 2013 CFR

    2013-07-01

    ... 32 National Defense 6 2013-07-01 2013-07-01 false Induction into the Armed Forces. 1624.9 Section... INDUCTIONS § 1624.9 Induction into the Armed Forces. Registrants in classes 1-A and 1-A-0, who have been... inducted at the MEPS into the Armed Forces....

  2. Tax Subsidies for Employer-Sponsored Health Insurance: Updated Microsimulation Estimates and Sensitivity to Alternative Incidence Assumptions

    PubMed Central

    Miller, G Edward; Selden, Thomas M

    2013-01-01

    Objective To estimate 2012 tax expenditures for employer-sponsored insurance (ESI) in the United States and to explore the sensitivity of estimates to assumptions regarding the incidence of employer premium contributions. Data Sources Nationally representative Medical Expenditure Panel Survey data from the 2005–2007 Household Component (MEPS-HC) and the 2009–2010 Insurance Component (MEPS IC). Study Design We use MEPS HC workers to construct synthetic workforces for MEPS IC establishments, applying the workers' marginal tax rates to the establishments' insurance premiums to compute the tax subsidy, in aggregate and by establishment characteristics. Simulation enables us to examine the sensitivity of ESI tax subsidy estimates to a range of scenarios for the within-firm incidence of employer premium contributions when workers have heterogeneous health risks and make heterogeneous plan choices. Principal Findings We simulate the total ESI tax subsidy for all active, civilian U.S. workers to be $257.4 billion in 2012. In the private sector, the subsidy disproportionately flows to workers in large establishments and establishments with predominantly high wage or full-time workforces. The estimates are remarkably robust to alternative incidence assumptions. Conclusions The aggregate value of the ESI tax subsidy and its distribution across firms can be reliably estimated using simplified incidence assumptions. PMID:23398400

  3. Maximum entropy production in environmental and ecological systems

    PubMed Central

    Kleidon, Axel; Malhi, Yadvinder; Cox, Peter M.

    2010-01-01

    The coupled biosphere–atmosphere system entails a vast range of processes at different scales, from ecosystem exchange fluxes of energy, water and carbon to the processes that drive global biogeochemical cycles, atmospheric composition and, ultimately, the planetary energy balance. These processes are generally complex with numerous interactions and feedbacks, and they are irreversible in their nature, thereby producing entropy. The proposed principle of maximum entropy production (MEP), based on statistical mechanics and information theory, states that thermodynamic processes far from thermodynamic equilibrium will adapt to steady states at which they dissipate energy and produce entropy at the maximum possible rate. This issue focuses on the latest development of applications of MEP to the biosphere–atmosphere system including aspects of the atmospheric circulation, the role of clouds, hydrology, vegetation effects, ecosystem exchange of energy and mass, biogeochemical interactions and the Gaia hypothesis. The examples shown in this special issue demonstrate the potential of MEP to contribute to improved understanding and modelling of the biosphere and the wider Earth system, and also explore limitations and constraints to the application of the MEP principle. PMID:20368247

  4. 75 FR 2551 - National Vaccine Injury Compensation Program: Revised Amount of the Average Cost of a Health...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2010-01-15

    ...: Revised Amount of the Average Cost of a Health Insurance Policy The Health Resources and Services Administration (HRSA) is publishing an updated monetary amount of the average cost of a health insurance policy...- Insurance Component (MEPS-IC) data available as the baseline for the average monthly cost of a...

  5. 76 FR 5180 - National Vaccine Injury Compensation Program: Revised Amount of the Average Cost of a Health...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2011-01-28

    ...: Revised Amount of the Average Cost of a Health Insurance Policy The Health Resources and Services Administration (HRSA) is publishing an updated monetary amount of the average cost of a health insurance policy...-- Insurance Component (MEPS-IC) data available as the baseline for the average monthly cost of a...

  6. 16 CFR 1203.17 - Impact attenuation test.

    Code of Federal Regulations, 2012 CFR

    2012-01-01

    ..., Motorcycle Helmets, 49 CFR 571.218 (S7.1.8). The center of gravity of the drop assembly shall lie within the... lb), the upper surface of which shall consist of a steel plate at least 12 mm (0.47 in.) thick and... test day) by dropping a spherical impactor onto an elastomeric test medium (MEP). The...

  7. Resistant Against De-depression: LTD-Like Plasticity in the Human Motor Cortex Induced by Spaced cTBS.

    PubMed

    Goldsworthy, Mitchell R; Müller-Dahlhaus, Florian; Ridding, Michael C; Ziemann, Ulf

    2015-07-01

    The long-term depression (LTD)-like changes in human primary motor cortex (M1) excitability induced by continuous theta burst stimulation (cTBS) are subject to reversal (i.e., de-depression) following behavioral engagement of M1, limiting its therapeutic potential under behaviorally relevant conditions. Experiments in animals suggest that the repeated, spaced application of stimulation trains may consolidate synaptic plasticity, making it resistant to reversal by physiological activity. Although there is evidence that repeated cTBS prolongs LTD-like M1 neuroplasticity in humans, whether these effects are resistant to de-depression has not been tested. We investigated whether the neuroplastic effects of paired cTBS trains were resistant to de-depression by a sustained, submaximal voluntary contraction of the hand muscles. In the absence of cTBS, voluntary contraction had no effect on motor evoked potentials (MEPs) recorded from the right first dorsal interosseous muscle. While the LTD-like MEP depression induced by a single cTBS was abolished by subsequent voluntary contraction, paired cTBS induced MEP depression that was resistant to reversal. This MEP depression was also resistant to reversal when an experimental de-depression protocol was used instead of a voluntary contraction. Our findings suggest that repeated cTBS applications consolidate LTD-like M1 neuroplasticity, which may have important implications for the clinical application of cTBS. PMID:24488942

  8. 77 FR 41902 - Special Local Regulation and Safety Zone; America's Cup Sailing Events, San Francisco, CA

    Federal Register 2010, 2011, 2012, 2013, 2014

    2012-07-17

    ... COTP Captain of the Port DHS Department of Homeland Security FR Federal Register MEP Marine Event... FR 4501. The Coast Guard provided a 90-day period for public comment on the proposed ] regulations... any regattas that may occur. The regulation will temporarily restrict vessel traffic in a portion...

  9. 32 CFR 728.73 - Applicants for enlistment or reenlistment in the Armed Forces, and applicants for enlistment in...

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ... Defense Department of Defense (Continued) DEPARTMENT OF THE NAVY PERSONNEL MEDICAL AND DENTAL CARE FOR... be determined. Use the hospitalization period only for diagnostic purposes. Do not correct... at Navy and Marine Corps facilities or MEPS may be furnished emergency medical and dental...

  10. 32 CFR 728.73 - Applicants for enlistment or reenlistment in the Armed Forces, and applicants for enlistment in...

    Code of Federal Regulations, 2011 CFR

    2011-07-01

    ... Defense Department of Defense (Continued) DEPARTMENT OF THE NAVY PERSONNEL MEDICAL AND DENTAL CARE FOR... be determined. Use the hospitalization period only for diagnostic purposes. Do not correct... at Navy and Marine Corps facilities or MEPS may be furnished emergency medical and dental...

  11. Lessons from Costa Rica

    ERIC Educational Resources Information Center

    Borthwick, Arlene; Lobo, Irina

    2005-01-01

    Costa Rica has one of the highest concentrations of computers in the Americas and is regarded as a Central American pioneer in technology development. The authors of this article describe their trip to Costa Rica, which included visits to several schools as well as to the Foundation Omar Dengo (FOD) and the Ministry of Public Education (MEP),…

  12. Motor excitability during movement preparation in Tourette syndrome.

    PubMed

    Draper, Amelia; Jude, Lucinda; Jackson, Georgina M; Jackson, Stephen R

    2015-03-01

    Tourette syndrome (TS) is a neurodevelopmental disorder characterized by the occurrence of motor and vocal tics. TS has been linked to the impaired operation of cortical-striatal-thalamic-cortical circuits that give rise to hyper-excitability of cortical motor areas, which may be exacerbated by dysfunctional intra-cortical inhibitory mechanisms. That said, many individuals gain control over their tics during adolescence and it has been suggested that this increased control arises as a result of the development of mechanisms that operate to suppress corticospinal excitability (CSE) ahead of volitional movements. Here we used single-pulse transcranial magnetic stimulation (TMS) in conjunction with a manual Go/NoGo task to investigate alterations in CSE ahead of volitional movements in a group of adolescents with TS (N = 10). Our study demonstrated that CSE, as measured by TMS-induced motor-evoked potentials (MEPs), was significantly reduced in the TS group in the period immediately preceding a finger movement. More specifically, we show that individuals with TS, unlike their age-matched controls, do not exhibit the predicted increase in mean MEP amplitude and decrease in MEP variability that immediately precede the execution of volitional movements in typically developing young adults. Finally, we report that the magnitude of the rise in MEP amplitude across the movement preparation period in TS is significantly negatively correlated with clinical measures of motor tic severity, suggesting that individuals with severe motor tics are least able to modulate motor cortical excitability. PMID:24283505

  13. The effect of speech distortion on the excitability of articulatory motor cortex.

    PubMed

    Nuttall, Helen E; Kennedy-Higgins, Daniel; Hogan, John; Devlin, Joseph T; Adank, Patti

    2016-03-01

    It has become increasingly evident that human motor circuits are active during speech perception. However, the conditions under which the motor system modulates speech perception are not clear. Two prominent accounts make distinct predictions for how listening to speech engages speech motor representations. The first account suggests that the motor system is most strongly activated when observing familiar actions (Pickering and Garrod, 2013). Conversely, Wilson and Knoblich's account asserts that motor excitability is greatest when observing less familiar, ambiguous actions (Wilson and Knoblich, 2005). We investigated these predictions using transcranial magnetic stimulation (TMS). Stimulation of the lip and hand representations in the left primary motor cortex elicited motor evoked potentials (MEPs) indexing the excitability of the underlying motor representation. MEPs for lip, but not for hand, were larger during perception of distorted speech produced using a tongue depressor, relative to naturally produced speech. Additional somatotopic facilitation yielded significantly larger MEPs during perception of lip-articulated distorted speech sounds relative to distorted tongue-articulated sounds. Critically, there was a positive correlation between MEP size and the perception of distorted speech sounds. These findings were consistent with predictions made by Wilson & Knoblich (Wilson and Knoblich, 2005), and provide direct evidence of increased motor excitability when speech perception is difficult. PMID:26732405

  14. Impaired Organization of Paired-Pulse TMS-Induced I-Waves After Human Spinal Cord Injury.

    PubMed

    Cirillo, John; Calabro, Finnegan J; Perez, Monica A

    2016-05-01

    Paired-pulse transcranial magnetic stimulation (TMS) of the human motor cortex results in consecutive facilitatory motor-evoked potential (MEP) peaks in surface electromyography in intact humans. Here, we tested the effect of an incomplete cervical spinal cord injury (SCI) on early (first) and late (second and third) MEP peaks in a resting intrinsic finger muscle. We found that all peaks had decreased amplitude in SCI subjects compared with controls. The second and third peaks were delayed with the third peak also showing an increased duration. The delay of the third peak was smaller than that seen in controls at lower stimulation intensity, suggesting lesser influence of decreased corticospinal inputs. A mathematical model showed that after SCI the third peak aberrantly contributed to spinal motoneurone recruitment, regardless on the motor unit threshold tested. Temporal and spatial aspects of the late peaks correlated with MEP size and hand motor output. Thus, early and late TMS-induced MEP peaks undergo distinct modulation after SCI, with the third peak likely reflecting a decreased ability to summate descending volleys at the spinal level. We argue that the later corticospinal inputs on the spinal cord might be crucial for recruitment of motoneurones after human SCI. PMID:25814508

  15. Microsome biocolloids for rapid drug metabolism and inhibition assessment by LC-MS

    PubMed Central

    Bajrami, Besnik; Krishnan, Sadagopan; Rusling, James F.

    2012-01-01

    Rat liver microsomes attached to nanoparticles were used for LC-MS studies of CYP3A and 2E1 enzymes in metabolism of N-nitroso compounds. Using these biocolloids, turnover rates were measured within 2 min. Inhibitor IC50 values for ketoconazole (KET) and 4-methylpyrazole (4-MEP) were estimated. PMID:19356087

  16. Alternative Methods for Defining Osteoarthritis and the Impact on Estimating Prevalence in a US Population-Based Survey

    PubMed Central

    Cisternas, Miriam G.; Murphy, Louise; Sacks, Jeffrey J.; Solomon, Daniel H.; Pasta, David J.; Helmick, Charles G.

    2015-01-01

    Objective Provide a contemporary estimate of osteoarthritis (OA) by comparing accuracy and prevalence of alternative definitions of OA. Methods The Medical Expenditure Panel Survey (MEPS) household component (HC) records respondent-reported medical conditions as open-ended responses; professional coders translate these responses into ICD-9-CM codes for the medical conditions files. Using these codes and other data from the MEPS-HC medical conditions files, we constructed three case definitions of OA and assessed them against medical provider diagnoses of ICD-9-CM 715 [osteoarthrosis and allied disorders] in a MEPS subsample. The three definitions were: 1) strict = ICD-9-CM 715; 2) expanded = ICD-9-CM 715, 716 [other and unspecified arthropathies], OR 719 [other and unspecified disorders of joint]); and 3) probable = strict OR expanded + respondent-reported prior diagnosis of OA or other arthritis excluding rheumatoid arthritis (RA). Results Sensitivity and specificity of the three definitions were: strict – 34.6% and 97.5%; expanded – 73.8% and 90.5%; and probable – 62.9% and 93.5%. Conclusion The strict definition for OA (ICD-9-CM 715) excludes many individuals with OA. The probable definition of OA has the optimal combination of sensitivity and specificity relative to the two other MEPS-based definitions and yields a national annual estimate of 30.8 million adults with OA (13.4% of US adult population) for 2008 – 2011. PMID:26315529

  17. 75 FR 55303 - Proposed Information Collection; Comment Request; National Institute of Standards and Technology...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2010-09-10

    ... Institute of Standards and Technology (NIST), Manufacturing Extension Partnership (MEP) Expanded Services Center Information and Reporting System AGENCY: National Institute of Standards and Technology (NIST..., or via e-mail at ccarbone@nist.gov . SUPPLEMENTARY INFORMATION: I. Abstract Sponsored by the...

  18. Analysis of Potential Energy Saving and CO2 Emission Reduction of Home Appliances and Commercial Equipments in China

    SciTech Connect

    Zhou, Nan; Fridley, David; McNeil, Michael; Zheng, Nina; Letschert, Virginie; Ke, Jing

    2011-04-01

    China has implemented a series of minimum energy performance standards (MEPS) for over 30 appliances, voluntary energy efficiency label for 40 products and a mandatory energy information label that covers 19 products to date. However, the impact of these programs and their savings potential has not been evaluated on a consistent basis. This paper uses modeling to estimate the energy saving and CO{sub 2} emission reduction potential of the appliances standard and labeling program for products for which standards are currently in place, under development or those proposed for development in 2010 under three scenarios that differ in the pace and stringency of MEPS development. In addition to a baseline 'Frozen Efficiency' scenario at 2009 MEPS level, the 'Continued Improvement Scenario' (CIS) reflects the likely pace of post-2009 MEPS revisions, and the likely improvement at each revision step. The 'Best Practice Scenario' (BPS) examined the potential of an achievement of international best practice efficiency in broad commercial use today in 2014. This paper concludes that under 'CIS', cumulative electricity consumption could be reduced by 9503 TWh, and annual CO{sub 2} emissions of energy used for all 37 products would be 16% lower than in the frozen efficiency scenario. Under a 'BPS' scenario for a subset of products, cumulative electricity savings would be 5450 TWh and annual CO{sub 2} emissions reduction of energy used for 11 appliances would be 35% lower.

  19. Elevated Accumulation of Parabens and their Metabolites in Marine Mammals from the United States Coastal Waters.

    PubMed

    Xue, Jingchuan; Sasaki, Nozomi; Elangovan, Madhavan; Diamond, Guthrie; Kannan, Kurunthachalam

    2015-10-20

    The widespread exposure of humans to parabens present in personal care products is well-known. Nevertheless, little is known about the accumulation of parabens in marine organisms. In this study, six parabens and four common metabolites of parabens were measured in 121 tissue samples from eight species of marine mammals collected along the coastal waters of Florida, California, Washington, and Alaska. Methyl paraben (MeP) was the predominant compound found in the majority of the marine mammal tissues analyzed, and the highest concentration found was 865 ng/g (wet weight [wet wt]) in the livers of bottlenose dolphins from Sarasota Bay, FL. 4-Hydroxybenzoic acid (4-HB) was the predominant paraben metabolite found in all tissue samples. The measured concentrations of 4-HB were on the order of hundreds to thousands of ng/g tissue, and these values are some of the highest ever reported in the literature. MeP and 4-HB concentrations showed a significant positive correlation (p < 0.05), which suggested a common source of exposure to these compounds in marine mammals. Trace concentrations of MeP and 4-HB were found in the livers of polar bears from the Chuckchi Sea and Beaufort Sea, which suggested widespread distribution of MeP and 4-HB in the oceanic environment. PMID:26379094

  20. Intraoperative monitoring during decompression of the spinal cord and spinal nerves using transcranial motor-evoked potentials: The law of twenty percent.

    PubMed

    Tanaka, Satoshi; Hirao, Jun; Oka, Hidehiro; Akimoto, Jiro; Takanashi, Junko; Yamada, Junichi

    2015-09-01

    Motor-evoked potential (MEP) monitoring was performed during 196 consecutive spinal (79 cervical and 117 lumbar) surgeries for the decompression of compressive spinal and spinal nerve diseases. MEP monitoring in spinal surgery has been considered sensitive to predict postoperative neurological recovery. In this series, transcranial stimulation consisted of trains of five pulses at a constant voltage (200-600 V). For the normalization of MEP, we recorded compound muscle action potentials (CMAP) after peripheral nerve stimulation, usually on the median nerve at the wrist 2 seconds before or after each transcranial stimulation of the motor area, for all operations. The sensitivity and specificity of MEP monitoring was 100% and 97.4%, respectively, or 96.9% with or without CMAP compensation (if the threshold of postoperative motor palsy was defined as 20% relative amplitude rate [RAR]). The mean RAR after CMAP normalization, of the most affected muscle in the patient group with excellent postoperative results (recovery rate of a Japan Orthopedic Association score of more than 50%) was significantly higher than that in the other groups (p=0.0224). All patients with an amplitude increase rate (AIR) with CMAP normalization of more than 20% achieved neurological recovery postoperatively. Our results suggest that if the RAR is more than 20%, postoperative motor palsy can be avoided in spinal surgery. If the AIR with normalization by CMAP after peripheral nerve stimulation is more than 20%, neurological recovery can be expected in spinal surgery. PMID:26142049