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

  1. 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

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

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

  3. 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

  4. 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

  5. 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

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

    PubMed

    Bitok, J Kipchirchir; Meyers, Caren Freel

    2012-10-19

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

  7. 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

  8. 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

  9. 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

  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. 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

  12. [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

  13. 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...

  14. 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

  15. 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

  16. 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

  17. 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

  18. 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

  19. 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

  20. 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

  1. 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

  2. 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

  3. 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

  4. 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

  5. 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

  6. 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

  7. 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

  8. 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

  9. 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

  10. 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

  11. [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

  12. 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

  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. 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

  15. 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

  16. 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

  17. 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

  18. 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

  19. 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

  20. 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

  1. 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

  2. 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

  3. 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

  4. 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

  5. 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

  6. 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

  7. 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

  8. 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

  9. 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

  10. 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

  11. 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

  12. 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

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

    PubMed

    Pazouki, Leila; Niinemets, Ülo

    2016-01-01

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

  14. 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

  15. 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

  16. 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

  17. 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

  18. 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

  19. 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

  20. 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

  1. 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

  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. 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

  4. 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

  5. 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

  6. 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

  7. 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

  8. 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

  9. 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

  10. 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

  11. 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

  12. 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

  13. 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

  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. 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

  17. 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

  18. 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

  19. 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

  20. 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

  1. 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

  2. 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

  3. 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

  4. 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

  5. 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

  6. 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

  7. 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.

  8. 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.

  9. 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

  10. 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

  11. 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

  12. 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

  13. 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

  14. 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%.

  15. 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

  16. 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

  17. 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

  18. 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

  19. 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

  20. 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

  1. 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

  2. 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

  3. 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

  4. 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

  5. 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

  6. 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

  7. 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

  8. 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

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

    SciTech Connect

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

    2011-09-28

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

  10. 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

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

    PubMed

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

    2016-01-01

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

  12. 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

  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. 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

  19. 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

  20. 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

  1. 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

  2. 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

  3. 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

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

    SciTech Connect

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

    2010-03-08

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

  5. 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.

  6. 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

  7. 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

  8. 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.

  9. 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

  10. 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

  11. 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

  12. 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

  13. 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.

  14. 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

  15. 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

  16. 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

  17. 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

  18. 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

  19. 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

  20. 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

  1. Engineering an isoprenoid pathway in Escherichia coli for production of 2-methyl-3-buten-2-ol: a potential biofuel.

    PubMed

    Gupta, Dinesh; Summers, Michael L; Basu, Chhandak

    2014-06-01

    2-Methyl-3-buten-2-ol (MBO) is a natural volatile 5-carbon alcohol produced by several pine species that have the potential to be used as biofuel. MBO has a high energy content making it superior to ethanol in terms of energy output, and due to its volatility and lower solubility in water, MBO is easier to recover than ethanol. Pine's MBO synthase enzyme utilizes the intermediate dimethylallyl pyrophosphate (DMAPP) produced by the methyl-erythritol-4-phosphate isoprenoid pathway for the production of MBO. In this study, we performed metabolic engineering of Escherichia coli to express an alternate mevalonate dependent pathway for production of DMAPP, along with a codon optimized Pinus sabiniana MBO synthase gene. This heterologous expressed pathway carried out the conversion of an acetyl CoA precursor to DMAPP leading to production of MBO. PMID:24271564

  2. [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

  3. 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

  4. Neurophysiology and itch pathways.

    PubMed

    Schmelz, Martin

    2015-01-01

    As we all can easily differentiate the sensations of itch and pain, the most straightforward neurophysiologic concept would consist of two specific pathways that independently encode itch and pain. Indeed, a neuronal pathway for histamine-induced itch in the peripheral and central nervous system has been described in animals and humans, and recently several non-histaminergic pathways for itch have been discovered in rodents that support a dichotomous concept differentiated into a pain and an itch pathway, with both pathways being composed of different "flavors." Numerous markers and mediators have been found that are linked to itch processing pathways. Thus, the delineation of neuronal pathways for itch from pain pathways seemingly proves that all sensory aspects of itch are based on an itch-specific neuronal pathway. However, such a concept is incomplete as itch can also be induced by the activation of the pain pathway in particular when the stimulus is applied in a highly localized spatial pattern. These opposite views reflect the old dispute between specificity and pattern theories of itch. Rather than only being of theoretic interest, this conceptual problem has key implication for the strategy to treat chronic itch as key therapeutic targets would be either itch-specific pathways or unspecific nociceptive pathways. PMID:25861773

  5. Potent Inhibitors of a Shikimate Pathway Enzyme from Mycobacterium tuberculosis

    PubMed Central

    Reichau, Sebastian; Jiao, Wanting; Walker, Scott R.; Hutton, Richard D.; Baker, Edward N.; Parker, Emily J.

    2011-01-01

    Tuberculosis remains a serious global health threat, with the emergence of multidrug-resistant strains highlighting the urgent need for novel antituberculosis drugs. The enzyme 3-deoxy-d-arabino-heptulosonate 7-phosphate synthase (DAH7PS) catalyzes the first step of the shikimate pathway for the biosynthesis of aromatic compounds. This pathway has been shown to be essential in Mycobacterium tuberculosis, the pathogen responsible for tuberculosis. DAH7PS catalyzes a condensation reaction between P-enolpyruvate and erythrose 4-phosphate to give 3-deoxy-d-arabino-heptulosonate 7-phosphate. The enzyme reaction mechanism is proposed to include a tetrahedral intermediate, which is formed by attack of an active site water on the central carbon of P-enolpyruvate during the course of the reaction. Molecular modeling of this intermediate into the active site reported in this study shows a configurational preference consistent with water attack from the re face of P-enolpyruvate. Based on this model, we designed and synthesized an inhibitor of DAH7PS that mimics this reaction intermediate. Both enantiomers of this intermediate mimic were potent inhibitors of M. tuberculosis DAH7PS, with inhibitory constants in the nanomolar range. The crystal structure of the DAH7PS-inhibitor complex was solved to 2.35 Å. Both the position of the inhibitor and the conformational changes of active site residues observed in this structure correspond closely to the predictions from the intermediate modeling. This structure also identifies a water molecule that is located in the appropriate position to attack the re face of P-enolpyruvate during the course of the reaction, allowing the catalytic mechanism for this enzyme to be clearly defined. PMID:21454647

  6. The pentose phosphate pathway of glucose metabolism. Enzyme profiles and transient and steady-state content of intermediates of alternative pathways of glucose metabolism in Krebs ascites cells

    PubMed Central

    Gumaa, K. A.; McLean, Patricia

    1969-01-01

    1. The pentose phosphate pathway in Krebs ascites cells was investigated for regulatory reactions. For comparison, the glycolytic pathway was studied simultaneously. 2. Activities of the pentose phosphate pathway enzymes were low in contrast with those of the enzymes of glycolysis. The Km values of glucose 6-phosphate dehydrogenase for both substrate and cofactor were about four times the reported upper limit for the enzyme from normal tissues. Fructose 1,6-diphosphate and NADPH competitively inhibited 6-phosphogluconate dehydrogenase. 3. About 28% of the hexokinase activity was in the particulate fraction of the cells. The soluble enzyme was inhibited by fructose 1,6-diphosphate and ribose 5-phosphate, but not by 3-phosphoglycerate. The behaviour of the partially purified soluble enzyme in vitro in a system simulating the concentrations of ATP, glucose 6-phosphate and Pi found in vivo is reported. 4. Kinetics of metabolite accumulation during the transient state after the addition of glucose to the cells indicated two phases of glucose phosphorylation, an initial rapid phase followed abruptly by a slow phase extending into the steady state. 5. Of the pentose phosphate pathway intermediates, accumulation of 6-phosphogluconate, sedoheptulose 7-phosphate and fructose 6-phosphate paralleled the accumulation of glucose 6-phosphate. Erythrose 4-phosphate reached the steady-state concentration by 2min., whereas the pentose phosphates accumulated linearly. 6. The mass-action ratios of the pentose phosphate pathway reactions were calculated. The transketolase reaction was at equilibrium by 30sec. and then progressively shifted away from equilibrium towards the steady-state ratio. The glucose 6-phosphate dehydrogenase was far from equilibrium at all times. 7. Investigation of the flux of [14C]glucose carbon confirmed the existence of an operative pentose phosphate pathway in ascites cells, contributing 1% of the total flux in control cells and 10% in cells treated with

  7. PATHWAYS - ELECTRON TUNNELING PATHWAYS IN PROTEINS

    NASA Technical Reports Server (NTRS)

    Beratan, D. N.

    1994-01-01

    The key to understanding the mechanisms of many important biological processes such as photosynthesis and respiration is a better understanding of the electron transfer processes which take place between metal atoms (and other groups) fixed within large protein molecules. Research is currently focused on the rate of electron transfer and the factors that influence it, such as protein composition and the distance between metal atoms. Current models explain the swift transfer of electrons over considerable distances by postulating bridge-mediated tunneling, or physical tunneling pathways, made up of interacting bonds in the medium around and between donor and acceptor sites. The program PATHWAYS is designed to predict the route along which electrons travel in the transfer processes. The basic strategy of PATHWAYS is to begin by recording each possible path element on a connectivity list, including in each entry which two atoms are connected and what contribution the connection would make to the overall rate if it were included in a pathway. The list begins with the bonded molecular structure (including the backbone sequence and side chain connectivity), and then adds probable hydrogen bond links and through-space contacts. Once this list is completed, the program runs a tree search from the donor to the acceptor site to find the dominant pathways. The speed and efficiency of the computer search offers an improvement over manual techniques. PATHWAYS is written in FORTRAN 77 for execution on DEC VAX series computers running VMS. The program inputs data from four data sets and one structure file. The software was written to input BIOGRAF (old format) structure files based on x-ray crystal structures and outputs ASCII files listing the best pathways and BIOGRAF vector files containing the paths. Relatively minor changes could be made in the input format statements for compatibility with other graphics software. The executable and source code are included with the

  8. Pathways from Poverty.

    ERIC Educational Resources Information Center

    Baldwin, Barbara, Ed.

    1995-01-01

    Articles in this theme issue are based on presentations at the Pathways from Poverty Workshop held in Albuquerque, New Mexico, on May 18-25, 1995. The event aimed to foster development of a network to address rural poverty issues in the Western Rural Development Center (WRDC) region. Articles report on outcomes from the Pathways from Poverty…

  9. Transformation pathways of liposomes.

    PubMed

    Hotani, H

    1984-09-01

    Liposomes undergoing transformation were observed by dark-field light microscopy in order to study the role of lipid in morphogenesis of biological vesicular structures. Liposomes were found to transform sequentially in a well-defined manner through one of several transformation pathways. A circular biconcave form was an initial shape in all the pathways and it transformed into a stable thin flexible filament or small spheres via a variety of regularly shaped vesicles which possessed geometrical symmetry. The transformation was reversible up to a certain point in each pathway. Osmotic pressure was found to be the driving force for the transformations. Biological membrane vesicles such as trypsinized red cell ghosts also transformed by similar pathways. PMID:6548263

  10. Updating the Wnt pathways

    PubMed Central

    Yu, Jia; Virshup, David M.

    2014-01-01

    In the three decades since the discovery of the Wnt1 proto-oncogene in virus-induced mouse mammary tumours, our understanding of the signalling pathways that are regulated by the Wnt proteins has progressively expanded. Wnts are involved in an complex signalling network that governs multiple biological processes and cross-talk with multiple additional signalling cascades, including the Notch, FGF (fibroblast growth factor), SHH (Sonic hedgehog), EGF (epidermal growth factor) and Hippo pathways. The Wnt signalling pathway also illustrates the link between abnormal regulation of the developmental processes and disease manifestation. Here we provide an overview of Wnt-regulated signalling cascades and highlight recent advances. We focus on new findings regarding the dedicated Wnt production and secretion pathway with potential therapeutic targets that might be beneficial for patients with Wnt-related diseases. PMID:25208913

  11. Identifying Branched Metabolic Pathways by Merging Linear Metabolic Pathways

    NASA Astrophysics Data System (ADS)

    Heath, Allison P.; Bennett, George N.; Kavraki, Lydia E.

    This paper presents a graph-based algorithm for identifying complex metabolic pathways in multi-genome scale metabolic data. These complex pathways are called branched pathways because they can arrive at a target compound through combinations of pathways that split compounds into smaller ones, work in parallel with many compounds, and join compounds into larger ones. While most previous work has focused on identifying linear metabolic pathways, branched metabolic pathways predominate in metabolic networks. Automatic identification of branched pathways has a number of important applications in areas that require deeper understanding of metabolism, such as metabolic engineering and drug target identification. Our algorithm utilizes explicit atom tracking to identify linear metabolic pathways and then merges them together into branched metabolic pathways. We provide results on two well-characterized metabolic pathways that demonstrate that this new merging approach can efficiently find biologically relevant branched metabolic pathways with complex structures.

  12. Regulation of Primary Metabolic Pathways in Oyster Mushroom Mycelia Induced by Blue Light Stimulation: Accumulation of Shikimic Acid

    PubMed Central

    Kojima, Masanobu; Kimura, Ninako; Miura, Ryuhei

    2015-01-01

    Shikimic acid is a key intermediate in the aromatic amino acid pathway as well as an important starting material for the synthesis of Tamiflu, a potent and selective inhibitor of the neuraminidase enzyme of influenza viruses A and B. Here we report that in oyster mushroom (Pleurotus ostreatus) mycelia cultivated in the dark, stimulation with blue light-emitting diodes induces the accumulation of shikimic acid. An integrated analysis of primary metabolites, gene expression and protein expression suggests that the accumulation of shikimic acid caused by blue light stimulation is due to an increase in 3-deoxy-D-arabinoheptulosonate 7-phosphate synthase (DAHPS, EC2.5.1.54), the rate-determining enzyme in the shikimic acid pathway, as well as phosphofructokinase (PFK, EC2.7.1.11) and glucose-6-phosphate dehydrogenase (G6PD, EC1.1.1.49), the rate-determining enzymes in the glycolysis and pentose phosphate pathways, respectively. This stimulation results in increased levels of phosphoenolpyruvic acid (PEP) and erythrose-4-phosphate (E4P), the starting materials of shikimic acid biosynthesis. PMID:25721093

  13. Regulation of primary metabolic pathways in oyster mushroom mycelia induced by blue light stimulation: accumulation of shikimic acid.

    PubMed

    Kojima, Masanobu; Kimura, Ninako; Miura, Ryuhei

    2015-01-01

    Shikimic acid is a key intermediate in the aromatic amino acid pathway as well as an important starting material for the synthesis of Tamiflu, a potent and selective inhibitor of the neuraminidase enzyme of influenza viruses A and B. Here we report that in oyster mushroom (Pleurotus ostreatus) mycelia cultivated in the dark, stimulation with blue light-emitting diodes induces the accumulation of shikimic acid. An integrated analysis of primary metabolites, gene expression and protein expression suggests that the accumulation of shikimic acid caused by blue light stimulation is due to an increase in 3-deoxy-D-arabinoheptulosonate 7-phosphate synthase (DAHPS, EC2.5.1.54), the rate-determining enzyme in the shikimic acid pathway, as well as phosphofructokinase (PFK, EC2.7.1.11) and glucose-6-phosphate dehydrogenase (G6PD, EC1.1.1.49), the rate-determining enzymes in the glycolysis and pentose phosphate pathways, respectively. This stimulation results in increased levels of phosphoenolpyruvic acid (PEP) and erythrose-4-phosphate (E4P), the starting materials of shikimic acid biosynthesis. PMID:25721093

  14. 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

  15. Inflammatory pathways in spondyloarthritis.

    PubMed

    Hreggvidsdottir, Hulda S; Noordenbos, Troy; Baeten, Dominique L

    2014-01-01

    Spondyloarthritis is the second most common form of chronic inflammatory arthritis and a unique hallmark of the disease is pathologic new bone formation. Several cytokine pathways have been genetically associated with ankylosing spondylitis (AS), the prototypic subtype of SpA, and additional evidence from human and animal studies support a role of these pathways in the disease. TNF has a key role in SpA as blockade significantly reduces inflammation and destruction, however the treatment does not halt new bone formation. New insights into the TNF pathway were recently obtained from an animal model specifically overexpressing the transmembrane form of TNF. This model leads to axial and peripheral new bone formation which is not seen in soluble TNF overexpression models, indicating different pathogenic roles of soluble and transmembrane TNF in arthritis development. Besides TNF, the IL-23/IL-17 axis is emerging as an important inflammatory pathway in SpA, as a SNP in the IL-23R locus has been associated with developing AS, mice overexpressing IL-23 develop SpA-like features and IL-17 blockade has been shown to be efficacious for AS patients in a phase II trial. In this review, we focus on the cytokine pathways that have recently been genetically associated with SpA, i.e. TNF, IL-1, IL-6 and IL-23/IL-17. We review the current genetic, experimental and human in vivo data available and discuss how these different pathways are involved in the pathophysiology of SpA. Additionally, we discuss how these pathways relate to the pathogenic new bone formation in SpA. PMID:23969080

  16. Clinical Pathway for Thyroidectomy.

    PubMed

    Villar del Moral, Jesús María; Soria Aledo, Víctor; Colina Alonso, Alberto; Flores Pastor, Benito; Gutiérrez Rodríguez, María Teresa; Ortega Serrano, Joaquín; Parra Hidalgo, Pedro; Ros López, Susana

    2015-05-01

    Clinical pathways are care plans applicable to patient care procedures that present variations in practice and a predictable clinical course. They are designed not as a substitute for clinical judgment, but rather as a means to improve the effectiveness and efficiency of the procedures. This clinical pathway is the result of a collaborative work of the Sections of Endocrine Surgery and Quality Management of the Spanish Association of Surgeons. It attempts to provide a framework for standardizing the performance of thyroidectomy, the most frequently performed operation in endocrine surgery. Along with the usual documents of clinical pathways (temporary matrix, variance tracking and information sheets, assessment indicators and a satisfaction questionnaire) it includes a review of the scientific evidence around different aspects of pre, intra and postoperative management. Among others, antibiotic and antithrombotic prophylaxis, preoperative preparation in hyperthyroidism, intraoperative neuromonitoring and systems for obtaining hemostasis are included, along with management of postoperative hypocalcemia. PMID:25732107

  17. Pathways to chromothripsis

    PubMed Central

    Ivkov, Robert; Bunz, Fred

    2015-01-01

    Chromothripsis is a recently recognized mode of genetic instability that generates chromosomes with strikingly large numbers of segmental re-arrangements. While the characterization of these derivative chromosomes has provided new insights into the processes by which cancer genomes can evolve, the underlying signaling events and molecular mechanisms remain unknown. In medulloblastomas, chromothripsis has been observed to occur in the context of mutational inactivation of p53 and activation of the canonical Hedgehog (Hh) pathway. Recent studies have illuminated mechanistic links between these 2 signaling pathways, including a novel PTCH1 homolog that is regulated by p53. Here, we integrate this new pathway into a hypothetical model for the catastrophic DNA breakage that appears to trigger profound chromosomal rearrangements. PMID:26178348

  18. Pathways to School Success

    ERIC Educational Resources Information Center

    University of Pittsburgh Office of Child Development, 2012

    2012-01-01

    In 2006, the University of Pittsburgh Office of Child Development began implementing a multi-year school readiness project in several area schools. Evidence from both research and the field point to several key elements that foster school readiness and create pathways to school success for all children. This paper presents components of a…

  19. Pathways to Prosperity

    ERIC Educational Resources Information Center

    Symonds, William C.

    2012-01-01

    Symonds, director of the Pathways to Prosperity Project, asserts that the current U.S. system of preparing students for successful adult lives fails too many youth. Citing data showing that only 40 percent of young Americans earn an associate's or bachelor's degree by age 27--and that many employers say they see candidates who don't have…

  20. Dexter energy transfer pathways.

    PubMed

    Skourtis, Spiros S; Liu, Chaoren; Antoniou, Panayiotis; Virshup, Aaron M; Beratan, David N

    2016-07-19

    Energy transfer with an associated spin change of the donor and acceptor, Dexter energy transfer, is critically important in solar energy harvesting assemblies, damage protection schemes of photobiology, and organometallic opto-electronic materials. Dexter transfer between chemically linked donors and acceptors is bridge mediated, presenting an enticing analogy with bridge-mediated electron and hole transfer. However, Dexter coupling pathways must convey both an electron and a hole from donor to acceptor, and this adds considerable richness to the mediation process. We dissect the bridge-mediated Dexter coupling mechanisms and formulate a theory for triplet energy transfer coupling pathways. Virtual donor-acceptor charge-transfer exciton intermediates dominate at shorter distances or higher tunneling energy gaps, whereas virtual intermediates with an electron and a hole both on the bridge (virtual bridge excitons) dominate for longer distances or lower energy gaps. The effects of virtual bridge excitons were neglected in earlier treatments. The two-particle pathway framework developed here shows how Dexter energy-transfer rates depend on donor, bridge, and acceptor energetics, as well as on orbital symmetry and quantum interference among pathways. PMID:27382185

  1. Hedgehog Pathway Inhibition.

    PubMed

    Sekulic, Aleksandar; Von Hoff, Daniel

    2016-02-25

    The hedgehog (Hh) signaling pathway is aberrantly activated in a majority of basal cell carcinomas (BCC). Vismodegib and sonidegib are targeted inhibitors of Smoothened (SMO). Both drugs are approved for use in locally advanced BCC (laBCC), with vismodegib also approved for metastatic BCC (mBCC). PMID:26919418

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

    PubMed

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

    2016-01-01

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

  3. 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

  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. Viral Reorganization of the Secretory Pathway Generates Distinct Organelles for RNA Replication

    PubMed Central

    Hsu, Nai-Yun; Ilnytska, Olha; Belov, Georgiy; Santiana, Marianita; Chen, Ying-Han; Takvorian, Peter M.; Pau, Cyrilla; van der Schaar, Hilde; Kaushik-Basu, Neerja; Balla, Tamas; Cameron, Craig E.; Ehrenfeld, Ellie; van Kuppeveld, Frank J.M.; Altan-Bonnet, Nihal

    2010-01-01

    SUMMARY Many RNA viruses remodel intracellular membranes to generate specialized sites for RNA replication. How membranes are remodeled and what properties make them conducive for replication are unknown. Here we show how RNA viruses can manipulate multiple components of the cellular secretory pathway to generate organelles specialized for replication that are distinct in protein and lipid composition from the host cell. Specific viral proteins modulate effector recruitment by Arf1 GTPase and its guanine nucleotide exchange factor GBF1, promoting preferential recruitment of phosphatidylinositol-4-kinase IIIβ (PI4KIIIβ) to membranes over coat proteins, yielding uncoated phosphatidylinositol-4-phosphate (PI4P) lipid-enriched organelles. The PI4P-rich lipid micro-environment is essential for both enteroviral and flaviviral RNA replication; PI4KIIIβ inhibition interferes with this process; and enteroviral RNA polymerases specifically bind PI4P. These findings reveal how RNA viruses can selectively exploit specific elements of the host to form specialized organelles where cellular phosphoinositide lipids are key to regulating viral RNA replication. PMID:20510927

  6. AIP Career Pathways

    NASA Astrophysics Data System (ADS)

    Palchak, Amanda

    2012-02-01

    American Institute of Physics (AIP) Career Pathways is a new project funded by the National Science Foundation. One of the goals of AIP Career Pathways is to prepare students to compete for Science, Technology, Engineering, and Mathematics (STEM) careers with a bachelor's degree in physics. In order to do so, I reviewed and compiled useful resources on finding a STEM career with a bachelor's degree in physics. These resources not only supply the job seeker with job postings in STEM careers but also provide them with information on resumes, interviewing skills, and networking. Recently at the 2011 Industrial Physics Forum, I interviewed companies in the private sector to obtain a unique perspective on what types of skills potential employers expect an applicant to posses with a bachelor's degree in physics. Ultimately, these components will be used as supplements at student career workshops held at annual Society of Physics Students Zone Meetings.

  7. Molecular pathways in dystonia

    PubMed Central

    Bragg, D. Cristopher; Armata, Ioanna A.; Nery, Flavia C.; Breakefield, Xandra O.; Sharma, Nutan

    2011-01-01

    The hereditary dystonias comprise a set of diseases defined by a common constellation of motor deficits. These disorders are most likely associated with different molecular etiologies, many of which have yet to be elucidated. Here we discuss recent advances in three forms of hereditary dystonia, DYT1, DYT6 and DYT16, which share a similar clinical picture: onset in childhood or adolescence, progressive spread of symptoms with generalized involvement of body regions and a steady state affliction without treatment. Unlike DYT1, the genes responsible for DYT6 and DYT16 have only recently been identified, with relatively little information about the function of the encoded proteins. Nevertheless, recent data suggest that these proteins may fit together within interacting pathways involved in dopaminergic signaling, transcriptional regulation, and cellular stress responses. This review focuses on these molecular pathways, highlighting potential common themes among these dystonias which may serve as areas for future research. PMID:21134457

  8. Pathways of tau fibrillization.

    PubMed

    Kuret, Jeff; Chirita, Carmen N; Congdon, Erin E; Kannanayakal, Theresa; Li, Guibin; Necula, Mihaela; Yin, Haishan; Zhong, Qi

    2005-01-01

    New methods for analyzing tau fibrillization have yielded insights into the biochemical transitions involved in the process. Here we review the parallels between the sequential progression of tau fibrillization observed macroscopically in Alzheimer's disease (AD) lesions and the pathway of tau aggregation observed in vitro with purified tau preparations. In addition, pharmacological agents for further dissection of fibrillization mechanism and lesion formation are discussed. PMID:15615636

  9. Alternative Respiratory Pathway

    PubMed Central

    Siedow, James N.; Girvin, Mark E.

    1980-01-01

    Oxygen uptake during the first hours of imbibition in intact soybean and mung bean seeds showed a marked sensitivity to potassium cyanide but was unaffected by addition of either salicylhydroxamic acid or propyl gallate. However O2 uptake by finely ground seed particles was very sensitive to the addition of either compound. The results indicated that O2 uptake in intact, imbibing seeds was associated with a cyanide-sensitive process, most probably mitochondrial mediated respiration, and not the result of the cyanide-insensitive lipoxygenase activity which was readily detectable in ground seed particles. The antioxidant propyl gallate was found to inhibit specifically alternative pathway electron transfer in isolated mung bean mitochondria. Half-maximal inhibition occurred with 2 to 5 micromolar propyl gallate. Kinetic analysis indicated that propyl gallate inhibition of the alternative pathway occurred at, or very near, the site of inhibition of the alternative pathway by salicylhydroxamic acid. A high level of lipoxygenase activity was found to be associated with washed mitochondria isolated from a variety of etiolated plant tissues. Most of this lipoxygenase activity could be eliminated from mung bean mitochondria if the mitochondria were purified on a discontinuous sucrose gradient. This indicated that the mitochondrial-associated activity was probably the result of nonspecific adsorption of lipoxygenase onto the mitochondrial membranes during isolation. PMID:16661259

  10. GEM, a member of the GRAM domain family of proteins, is part of the ABA signaling pathway

    PubMed Central

    Mauri, Nuria; Fernández-Marcos, María; Costas, Celina; Desvoyes, Bénédicte; Pichel, Antonio; Caro, Elena; Gutierrez, Crisanto

    2016-01-01

    Abscisic acid (ABA) is fundamental for plant development. Multiple factors have been identified that participate in the ABA signaling network, although a role of many proteins still await to be demonstrated. Here we have investigated the role of GEM (GL2 EXPRESSION MODULATOR), originally annotated as an ABA-responsive protein. GEM contains a GRAM domain, a feature shared with other eight Arabidopsis proteins for which we propose the name of GRE (GEM-RELATED) proteins. We found that (i) GEM expression responds to ABA, (ii) its promoter contains ABRE sites required for ABA response, and (iii) GEM expression depends on members of the ABA signaling pathway. This is consistent with the expression pattern of GEM during development in plant locations were ABA is known to play a direct role. We also found that GEM binds various phospholipids, e.g. mono and diphosphates and phosphatidic acid, suggesting a potential link of GEM with membrane-associated processes. Consistent with this, we found that the phosphoinositol-4-phosphate kinase PIP5K9 binds GEM in vivo. Finally, we demonstrated a role of GEM in seed dormancy. Together, our data led us to propose that GEM is an ABA-responsive protein that may function downstream of ABI5 as part of the ABA signaling pathway. PMID:26939893

  11. GEM, a member of the GRAM domain family of proteins, is part of the ABA signaling pathway.

    PubMed

    Mauri, Nuria; Fernández-Marcos, María; Costas, Celina; Desvoyes, Bénédicte; Pichel, Antonio; Caro, Elena; Gutierrez, Crisanto

    2016-01-01

    Abscisic acid (ABA) is fundamental for plant development. Multiple factors have been identified that participate in the ABA signaling network, although a role of many proteins still await to be demonstrated. Here we have investigated the role of GEM (GL2 EXPRESSION MODULATOR), originally annotated as an ABA-responsive protein. GEM contains a GRAM domain, a feature shared with other eight Arabidopsis proteins for which we propose the name of GRE (GEM-RELATED) proteins. We found that (i) GEM expression responds to ABA, (ii) its promoter contains ABRE sites required for ABA response, and (iii) GEM expression depends on members of the ABA signaling pathway. This is consistent with the expression pattern of GEM during development in plant locations were ABA is known to play a direct role. We also found that GEM binds various phospholipids, e.g. mono and diphosphates and phosphatidic acid, suggesting a potential link of GEM with membrane-associated processes. Consistent with this, we found that the phosphoinositol-4-phosphate kinase PIP5K9 binds GEM in vivo. Finally, we demonstrated a role of GEM in seed dormancy. Together, our data led us to propose that GEM is an ABA-responsive protein that may function downstream of ABI5 as part of the ABA signaling pathway. PMID:26939893

  12. 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

  13. Pathway and Resource Overview (Presentation)

    SciTech Connect

    Ruth, M. F.

    2009-11-16

    This presentation provides information about hydrogen pathway analysis, which is analysis of the total levelized cost (including return on investment). Well-to-wheels (WTW) energy use, and WTW emissions for hydrogen production, delivery, and distribution pathways.

  14. Assignment of function to a domain of unknown function: DUF1537 is a new kinase family in catabolic pathways for acid sugars.

    PubMed

    Zhang, Xinshuai; Carter, Michael S; Vetting, Matthew W; San Francisco, Brian; Zhao, Suwen; Al-Obaidi, Nawar F; Solbiati, Jose O; Thiaville, Jennifer J; de Crécy-Lagard, Valérie; Jacobson, Matthew P; Almo, Steven C; Gerlt, John A

    2016-07-19

    Using a large-scale "genomic enzymology" approach, we (i) assigned novel ATP-dependent four-carbon acid sugar kinase functions to members of the DUF1537 protein family (domain of unknown function; Pfam families PF07005 and PF17042) and (ii) discovered novel catabolic pathways for d-threonate, l-threonate, and d-erythronate. The experimentally determined ligand specificities of several solute binding proteins (SBPs) for TRAP (tripartite ATP-independent permease) transporters for four-carbon acids, including d-erythronate and l-erythronate, were used to constrain the substrates for the catabolic pathways that degrade the SBP ligands to intermediates in central carbon metabolism. Sequence similarity networks and genome neighborhood networks were used to identify the enzyme components of the pathways. Conserved genome neighborhoods encoded SBPs as well as permease components of the TRAP transporters, members of the DUF1537 family, and a member of the 4-hydroxy-l-threonine 4-phosphate dehydrogenase (PdxA) oxidative decarboxylase, class II aldolase, or ribulose 1,5-bisphosphate carboxylase/oxygenase, large subunit (RuBisCO) superfamily. Because the characterized substrates of members of the PdxA, class II aldolase, and RuBisCO superfamilies are phosphorylated, we postulated that the members of the DUF1537 family are novel ATP-dependent kinases that participate in catabolic pathways for four-carbon acid sugars. We determined that (i) the DUF1537/PdxA pair participates in a pathway for the conversion of d-threonate to dihydroxyacetone phosphate and CO2 and (ii) the DUF1537/class II aldolase pair participates in pathways for the conversion of d-erythronate and l-threonate (epimers at carbon-3) to dihydroxyacetone phosphate and CO2 The physiological importance of these pathways was demonstrated in vivo by phenotypic and genetic analyses. PMID:27402745

  15. Pathways with PathWhiz.

    PubMed

    Pon, Allison; Jewison, Timothy; Su, Yilu; Liang, Yongjie; Knox, Craig; Maciejewski, Adam; Wilson, Michael; Wishart, David S

    2015-07-01

    PathWhiz (http://smpdb.ca/pathwhiz) is a web server designed to create colourful, visually pleasing and biologically accurate pathway diagrams that are both machine-readable and interactive. As a web server, PathWhiz is accessible from almost any place and compatible with essentially any operating system. It also houses a public library of pathways and pathway components that can be easily viewed and expanded upon by its users. PathWhiz allows users to readily generate biologically complex pathways by using a specially designed drawing palette to quickly render metabolites (including automated structure generation), proteins (including quaternary structures, covalent modifications and cofactors), nucleic acids, membranes, subcellular structures, cells, tissues and organs. Both small-molecule and protein/gene pathways can be constructed by combining multiple pathway processes such as reactions, interactions, binding events and transport activities. PathWhiz's pathway replication and propagation functions allow for existing pathways to be used to create new pathways or for existing pathways to be automatically propagated across species. PathWhiz pathways can be saved in BioPAX, SBGN-ML and SBML data exchange formats, as well as PNG, PWML, HTML image map or SVG images that can be viewed offline or explored using PathWhiz's interactive viewer. PathWhiz has been used to generate over 700 pathway diagrams for a number of popular databases including HMDB, DrugBank and SMPDB. PMID:25934797

  16. Pathways with PathWhiz

    PubMed Central

    Pon, Allison; Jewison, Timothy; Su, Yilu; Liang, Yongjie; Knox, Craig; Maciejewski, Adam; Wilson, Michael; Wishart, David S.

    2015-01-01

    PathWhiz (http://smpdb.ca/pathwhiz) is a web server designed to create colourful, visually pleasing and biologically accurate pathway diagrams that are both machine-readable and interactive. As a web server, PathWhiz is accessible from almost any place and compatible with essentially any operating system. It also houses a public library of pathways and pathway components that can be easily viewed and expanded upon by its users. PathWhiz allows users to readily generate biologically complex pathways by using a specially designed drawing palette to quickly render metabolites (including automated structure generation), proteins (including quaternary structures, covalent modifications and cofactors), nucleic acids, membranes, subcellular structures, cells, tissues and organs. Both small-molecule and protein/gene pathways can be constructed by combining multiple pathway processes such as reactions, interactions, binding events and transport activities. PathWhiz's pathway replication and propagation functions allow for existing pathways to be used to create new pathways or for existing pathways to be automatically propagated across species. PathWhiz pathways can be saved in BioPAX, SBGN-ML and SBML data exchange formats, as well as PNG, PWML, HTML image map or SVG images that can be viewed offline or explored using PathWhiz's interactive viewer. PathWhiz has been used to generate over 700 pathway diagrams for a number of popular databases including HMDB, DrugBank and SMPDB. PMID:25934797

  17. Developmental pathways in colon cancer

    PubMed Central

    Bertrand, Fred E.; Angus, C. William; Partis, William J.; Sigounas, George

    2012-01-01

    A hallmark of cancer is reactivation/alteration of pathways that control cellular differentiation during developmental processes. Evidence indicates that WNT, Notch, BMP and Hedgehog pathways have a role in normal epithelial cell differentiation, and that alterations in these pathways accompany establishment of the tumorigenic state. Interestingly, there is recent evidence that these pathways are intertwined at the molecular level, and these nodes of intersection may provide opportunities for effective targeted therapies. This review will highlight the role of the WNT, Notch, BMP and Hedgehog pathways in colon cancer. PMID:23032367

  18. Improving Carbon Fixation Pathways

    PubMed Central

    Ducat, Daniel C.

    2012-01-01

    A recent resurgence in basic and applied research on photosynthesis has been driven in part by recognition that fulfilling future food and energy requirements will necessitate improvements in crop carbon-fixation efficiencies. Photosynthesis in traditional terrestrial crops is being reexamined in light of molecular strategies employed by photosynthetic microbes to enhance the activity of the Calvin cycle. Synthetic biology is well-situated to provide original approaches for compartmentalizing and enhancing photosynthetic reactions in a species independent manner. Furthermore, the elucidation of alternative carbon-fixation routes distinct from the Calvin cycle raises possibilities that alternative pathways and organisms can be utilized to fix atmospheric carbon dioxide into useful materials. PMID:22647231

  19. Growth hormone signaling pathways.

    PubMed

    Carter-Su, Christin; Schwartz, Jessica; Argetsinger, Lawrence S

    2016-06-01

    Over 20years ago, our laboratory showed that growth hormone (GH) signals through the GH receptor-associated tyrosine kinase JAK2. We showed that GH binding to its membrane-bound receptor enhances binding of JAK2 to the GHR, activates JAK2, and stimulates tyrosyl phosphorylation of both JAK2 and GHR. The activated JAK2/GHR complex recruits a variety of signaling proteins, thereby initiating multiple signaling pathways and cellular responses. These proteins and pathways include: 1) Stat transcription factors implicated in the expression of multiple genes, including the gene encoding insulin-like growth factor 1; 2) Shc adapter proteins that lead to activation of the grb2-SOS-Ras-Raf-MEK-ERK1,2 pathway; 3) insulin receptor substrate proteins implicated in the phosphatidylinositol-3-kinase and Akt pathway; 4) signal regulatory protein α, a transmembrane scaffold protein that recruits proteins including the tyrosine phosphatase SHP2; and 5) SH2B1, a scaffold protein that can activate JAK2 and enhance GH regulation of the actin cytoskeleton. Our recent work has focused on the function of SH2B1. We have shown that SH2B1β is recruited to and phosphorylated by JAK2 in response to GH. SH2B1 localizes to the plasma membrane, cytoplasm and focal adhesions; it also cycles through the nucleus. SH2B1 regulates the actin cytoskeleton and promotes GH-dependent motility of RAW264.7 macrophages. Mutations in SH2B1 have been found in humans exhibiting severe early-onset childhood obesity and insulin resistance. These mutations impair SH2B1 enhancement of GH-induced macrophage motility. As SH2B1 is expressed ubiquitously and is also recruited to a variety of receptor tyrosine kinases, our results raise the possibility that effects of SH2B1 on the actin cytoskeleton in various cell types, including neurons, may play a role in regulating body weight. PMID:26421979

  20. Improving carbon fixation pathways

    SciTech Connect

    Ducat, DC; Silver, PA

    2012-08-01

    A recent resurgence in basic and applied research on photosynthesis has been driven in part by recognition that fulfilling future food and energy requirements will necessitate improvements in crop carbon-fixation efficiencies. Photosynthesis in traditional terrestrial crops is being reexamined in light of molecular strategies employed by photosynthetic microbes to enhance the activity of the Calvin cycle. Synthetic biology is well-situated to provide original approaches for compartmentalizing and enhancing photosynthetic reactions in a species independent manner. Furthermore, the elucidation of alternative carbon-fixation routes distinct from the Calvin cycle raises possibilities that novel pathways and organisms can be utilized to fix atmospheric carbon dioxide into useful materials.

  1. The Reactome pathway knowledgebase.

    PubMed

    Croft, David; Mundo, Antonio Fabregat; Haw, Robin; Milacic, Marija; Weiser, Joel; Wu, Guanming; Caudy, Michael; Garapati, Phani; Gillespie, Marc; Kamdar, Maulik R; Jassal, Bijay; Jupe, Steven; Matthews, Lisa; May, Bruce; Palatnik, Stanislav; Rothfels, Karen; Shamovsky, Veronica; Song, Heeyeon; Williams, Mark; Birney, Ewan; Hermjakob, Henning; Stein, Lincoln; D'Eustachio, Peter

    2014-01-01

    Reactome (http://www.reactome.org) is a manually curated open-source open-data resource of human pathways and reactions. The current version 46 describes 7088 human proteins (34% of the predicted human proteome), participating in 6744 reactions based on data extracted from 15 107 research publications with PubMed links. The Reactome Web site and analysis tool set have been completely redesigned to increase speed, flexibility and user friendliness. The data model has been extended to support annotation of disease processes due to infectious agents and to mutation. PMID:24243840

  2. WikiPathways: capturing the full diversity of pathway knowledge

    PubMed Central

    Kutmon, Martina; Riutta, Anders; Nunes, Nuno; Hanspers, Kristina; Willighagen, Egon L.; Bohler, Anwesha; Mélius, Jonathan; Waagmeester, Andra; Sinha, Sravanthi R.; Miller, Ryan; Coort, Susan L.; Cirillo, Elisa; Smeets, Bart; Evelo, Chris T.; Pico, Alexander R.

    2016-01-01

    WikiPathways (http://www.wikipathways.org) is an open, collaborative platform for capturing and disseminating models of biological pathways for data visualization and analysis. Since our last NAR update, 4 years ago, WikiPathways has experienced massive growth in content, which continues to be contributed by hundreds of individuals each year. New aspects of the diversity and depth of the collected pathways are described from the perspective of researchers interested in using pathway information in their studies. We provide updates on extensions and services to support pathway analysis and visualization via popular standalone tools, i.e. PathVisio and Cytoscape, web applications and common programming environments. We introduce the Quick Edit feature for pathway authors and curators, in addition to new means of publishing pathways and maintaining custom pathway collections to serve specific research topics and communities. In addition to the latest milestones in our pathway collection and curation effort, we also highlight the latest means to access the content as publishable figures, as standard data files, and as linked data, including bulk and programmatic access. PMID:26481357

  3. WikiPathways: capturing the full diversity of pathway knowledge.

    PubMed

    Kutmon, Martina; Riutta, Anders; Nunes, Nuno; Hanspers, Kristina; Willighagen, Egon L; Bohler, Anwesha; Mélius, Jonathan; Waagmeester, Andra; Sinha, Sravanthi R; Miller, Ryan; Coort, Susan L; Cirillo, Elisa; Smeets, Bart; Evelo, Chris T; Pico, Alexander R

    2016-01-01

    WikiPathways (http://www.wikipathways.org) is an open, collaborative platform for capturing and disseminating models of biological pathways for data visualization and analysis. Since our last NAR update, 4 years ago, WikiPathways has experienced massive growth in content, which continues to be contributed by hundreds of individuals each year. New aspects of the diversity and depth of the collected pathways are described from the perspective of researchers interested in using pathway information in their studies. We provide updates on extensions and services to support pathway analysis and visualization via popular standalone tools, i.e. PathVisio and Cytoscape, web applications and common programming environments. We introduce the Quick Edit feature for pathway authors and curators, in addition to new means of publishing pathways and maintaining custom pathway collections to serve specific research topics and communities. In addition to the latest milestones in our pathway collection and curation effort, we also highlight the latest means to access the content as publishable figures, as standard data files, and as linked data, including bulk and programmatic access. PMID:26481357

  4. The Chordin Morphogenetic Pathway.

    PubMed

    De Robertis, Edward M; Moriyama, Yuki

    2016-01-01

    The ancestral Chordin/bone morphogenetic protein (BMP) signaling pathway that establishes dorsal-ventral (D-V) patterning in animal development is one of the best understood morphogenetic gradients, and is established by multiple proteins that interact with each other in the extracellular space-including several BMPs, Chordin, Tolloid, Ont-1, Crossveinless-2, and Sizzled. The D-V gradient is adjusted redundantly by regulating the synthesis of its components, by direct protein-protein interactions between morphogens, and by long-range diffusion. The entire embryo participates in maintaining the D-V BMP gradient, so that for each action in the dorsal side there is a reaction in the ventral side. A gradient of Chordin is formed in the extracellular matrix that separates ectoderm from endomesoderm, called Brachet's cleft in Xenopus. The Chordin/BMP pathway is self-organizing and able to scale pattern in the dorsal half of bisected embryos or in Spemann dorsal lip transplantation experiments. PMID:26970622

  5. Pathways of lateral spreading.

    PubMed

    Jacobi, U; Schanzer, S; Weigmann, H-J; Patzelt, A; Vergou, T; Sterry, W; Lademann, J

    2011-01-01

    In the case of topically applied substances, usually both lateral spreading and competitive penetration into the skin occur in parallel. In the present study, the pathways of lateral spreading were studied quantitatively and visually. The local distribution and lateral spreading of the UV filter substance butyl methoxydibenzoylmethane applied in an o/w emulsion was studied on the forearm and the back. The tape stripping procedure was used to determine the recovery rates inside and outside the area of application. The skin characteristics of transepidermal water loss, pH value, hydration of the stratum corneum and sebum rate were determined at both anatomic sites. Photography and laser scanning microscopy were used to visually investigate the lateral spreading of topically applied dyes. On the back, a preferred direction of lateral spreading parallel to the body axis was observed. This result was caused by differences in the network of furrows. The furrows functioned as a pathway for lateral spreading, whereas the follicles formed a reservoir for the topically applied substance. PMID:21455016

  6. The Reactome pathway Knowledgebase.

    PubMed

    Fabregat, Antonio; Sidiropoulos, Konstantinos; Garapati, Phani; Gillespie, Marc; Hausmann, Kerstin; Haw, Robin; Jassal, Bijay; Jupe, Steven; Korninger, Florian; McKay, Sheldon; Matthews, Lisa; May, Bruce; Milacic, Marija; Rothfels, Karen; Shamovsky, Veronica; Webber, Marissa; Weiser, Joel; Williams, Mark; Wu, Guanming; Stein, Lincoln; Hermjakob, Henning; D'Eustachio, Peter

    2016-01-01

    The Reactome Knowledgebase (www.reactome.org) provides molecular details of signal transduction, transport, DNA replication, metabolism and other cellular processes as an ordered network of molecular transformations-an extended version of a classic metabolic map, in a single consistent data model. Reactome functions both as an archive of biological processes and as a tool for discovering unexpected functional relationships in data such as gene expression pattern surveys or somatic mutation catalogues from tumour cells. Over the last two years we redeveloped major components of the Reactome web interface to improve usability, responsiveness and data visualization. A new pathway diagram viewer provides a faster, clearer interface and smooth zooming from the entire reaction network to the details of individual reactions. Tool performance for analysis of user datasets has been substantially improved, now generating detailed results for genome-wide expression datasets within seconds. The analysis module can now be accessed through a RESTFul interface, facilitating its inclusion in third party applications. A new overview module allows the visualization of analysis results on a genome-wide Reactome pathway hierarchy using a single screen page. The search interface now provides auto-completion as well as a faceted search to narrow result lists efficiently. PMID:26656494

  7. Signaling Pathways in Melanogenesis

    PubMed Central

    D’Mello, Stacey A. N.; Finlay, Graeme J.; Baguley, Bruce C.; Askarian-Amiri, Marjan E.

    2016-01-01

    Melanocytes are melanin-producing cells found in skin, hair follicles, eyes, inner ear, bones, heart and brain of humans. They arise from pluripotent neural crest cells and differentiate in response to a complex network of interacting regulatory pathways. Melanins are pigment molecules that are endogenously synthesized by melanocytes. The light absorption of melanin in skin and hair leads to photoreceptor shielding, thermoregulation, photoprotection, camouflage and display coloring. Melanins are also powerful cation chelators and may act as free radical sinks. Melanin formation is a product of complex biochemical events that starts from amino acid tyrosine and its metabolite, dopa. The types and amounts of melanin produced by melanocytes are determined genetically and are influenced by a variety of extrinsic and intrinsic factors such as hormonal changes, inflammation, age and exposure to UV light. These stimuli affect the different pathways in melanogenesis. In this review we will discuss the regulatory mechanisms involved in melanogenesis and explain how intrinsic and extrinsic factors regulate melanin production. We will also explain the regulatory roles of different proteins involved in melanogenesis. PMID:27428965

  8. Signaling Pathways in Melanogenesis.

    PubMed

    D'Mello, Stacey A N; Finlay, Graeme J; Baguley, Bruce C; Askarian-Amiri, Marjan E

    2016-01-01

    Melanocytes are melanin-producing cells found in skin, hair follicles, eyes, inner ear, bones, heart and brain of humans. They arise from pluripotent neural crest cells and differentiate in response to a complex network of interacting regulatory pathways. Melanins are pigment molecules that are endogenously synthesized by melanocytes. The light absorption of melanin in skin and hair leads to photoreceptor shielding, thermoregulation, photoprotection, camouflage and display coloring. Melanins are also powerful cation chelators and may act as free radical sinks. Melanin formation is a product of complex biochemical events that starts from amino acid tyrosine and its metabolite, dopa. The types and amounts of melanin produced by melanocytes are determined genetically and are influenced by a variety of extrinsic and intrinsic factors such as hormonal changes, inflammation, age and exposure to UV light. These stimuli affect the different pathways in melanogenesis. In this review we will discuss the regulatory mechanisms involved in melanogenesis and explain how intrinsic and extrinsic factors regulate melanin production. We will also explain the regulatory roles of different proteins involved in melanogenesis. PMID:27428965

  9. AIP's Career Pathways Project

    NASA Astrophysics Data System (ADS)

    Avila, Jose

    2014-03-01

    The American Institute of Physics (AIP) Career Pathways Project, funded by the National Science Foundation, aims to increase the number of undergraduates going into STEM careers. The main purposes of this project are to show students the professional opportunities for a STEM career, understand what departments can do to better prepare physics bachelor's degree recipients to enter the workforce, understand what students can do to better prepare themselves, and develop resources based on these findings. I was chosen by the Society of Physics Students (SPS) to be the 2013 summer intern of the AIP's Career Pathways Project. In this talk I will discuss several resources I worked on with the Statistical Research Center of the American Institute of Physics and SPS. These resources include how to write a resume and cover letter, how to perform an informational interview, common job titles for physics bachelors, how to find career information in physics and STEM, how to search and use job postings, and how to network.

  10. Arabidopsis J-Protein J20 Delivers the First Enzyme of the Plastidial Isoprenoid Pathway to Protein Quality Control[C][W

    PubMed Central

    Pulido, Pablo; Toledo-Ortiz, Gabriela; Phillips, Michael A.; Wright, Louwrance P.; Rodríguez-Concepción, Manuel

    2013-01-01

    Plastids provide plants with metabolic pathways that are unique among eukaryotes, including the methylerythritol 4-phosphate pathway for the production of isoprenoids essential for photosynthesis and plant growth. Here, we show that the first enzyme of the pathway, deoxyxylulose 5-phosphate synthase (DXS), interacts with the J-protein J20 in Arabidopsis thaliana. J-proteins typically act as adaptors that provide substrate specificity to heat shock protein 70 (Hsp70), a molecular chaperone. Immunoprecipitation experiments showed that J20 and DXS are found together in vivo and confirmed the presence of Hsp70 chaperones in DXS complexes. Mutants defective in J20 activity accumulated significantly increased levels of DXS protein (but no transcripts) and displayed reduced levels of DXS enzyme activity, indicating that loss of J20 function causes posttranscriptional accumulation of DXS in an inactive form. Furthermore, J20 promotes degradation of DXS following a heat shock. Together, our data indicate that J20 might identify unfolded or misfolded (damaged) forms of DXS and target them to the Hsp70 system for proper folding under normal conditions or degradation upon stress. PMID:24104567

  11. Summer 2014 Pathways Report

    NASA Technical Reports Server (NTRS)

    Hand, Zachary

    2014-01-01

    Over the summer I had the exciting opportunity to work for NASA at the Kennedy Space Center as a Mission Assurance Engineering intern. When I was offered a position in mission assurance for the Safety and Mission Assurance directorate's Launch Services Division, I didn't really know what I would be doing, but I knew it would be an excellent opportunity to learn and grow professionally. In this report I will provide some background information on the Launch Services Division, as well as detail my duties and accomplishments during my time as an intern. Additionally, I will relate the significance of my work experience to my current academic work and future career goals. This report contains background information on Mission Assurance Engineering, a description of my duties and accomplishments over the summer of 2014, and relates the significance of my work experience to my school work and future career goals. It is a required document for the Pathways program.

  12. Pathways of Antigen Processing

    PubMed Central

    Blum, Janice S.; Wearsch, Pamela A.; Cresswell, Peter

    2014-01-01

    T cell recognition of antigen presenting cells depends on their expression of a spectrum of peptides bound to Major Histocompatibility Complex class I (MHC-I) and class II (MHC-II) molecules. Conversion of antigens from pathogens or transformed cells into MHC-I and MHC-II-bound peptides is critical for mounting protective T cell responses, and similar processing of self proteins is necessary to establish and maintain tolerance. Cells use a variety of mechanisms to acquire protein antigens, from translation in the cytosol to variations on the theme of endocytosis, and to degrade them once acquired. In this review we highlight the aspects of MHC-I and MHC-II biosynthesis and assembly that have evolved to intersect these pathways and sample the peptides that are produced. PMID:23298205

  13. The Peroxide Pathway

    NASA Technical Reports Server (NTRS)

    McNeal, Curtis I., Jr.; Anderson, William

    1999-01-01

    NASA's current focus on technology roadmaps as a tool for guiding investment decisions leads naturally to a discussion of NASA's roadmap for peroxide propulsion system development. NASA's new Second Generation Space Transportation System roadmap calls for an integrated Reusable Upper-Stage (RUS) engine technology demonstration in the FY03/FY04 time period. Preceding this integrated demonstration are several years of component developments and subsystem technology demonstrations. NASA and the Air Force took the first steps at developing focused upper stage technologies with the initiation of the Upper Stage Flight Experiment with Orbital Sciences in December 1997. A review of this program's peroxide propulsion development is a useful first step in establishing the peroxide propulsion pathway that could lead to a RUS demonstration in 2004.

  14. Pathways Intern Work Report

    NASA Technical Reports Server (NTRS)

    Midulla, Laura P.

    2014-01-01

    For the spring of 2014, I continued my position at NASA as a Pathways Engineering Student Trainee in the Engineering and Technology Directorate (NE), in the Test, Operations & Support Software Engineering Branch of the Control and Data Systems Division. The Control and Data Systems Division provides control, monitor, checkout, processing, display, and simulation capabilities to support vehicle element testing, checkout and launch. In addition the division provides significant leadership associated with engineering processes and Information Technology Security management. My assignment during this semester was to continue to support the Engineering Information Technology (IT) Security team, providing IT Security support to the Ground Systems Development and Operations Spaceport Command & Control System and Ground Systems, NE Labs and other systems.

  15. Pathways of iron absorption.

    PubMed

    Conrad, Marcel E; Umbreit, Jay N

    2002-01-01

    Iron is vital for all living organisms but excess iron can be lethal because it facilitates free radical formation. Thus iron absorption is carefully regulated to maintain an equilibrium between absorption and body loss of iron. In countries where meat is a significant part of the diet, most body iron is derived from dietary heme because heme binds few of the dietary chelators that bind inorganic iron. Uptake of heme into enterocytes occurs as a metalloporphyrin in an endosomal process. Intracellular iron is released from heme by heme oxygenase to enter plasma as inorganic iron. Ferric iron is absorbed via a beta(3) integrin and mobilferrin pathway (IMP) which is unshared with other nutritional metals. Ferrous iron uptake is facilitated by a DMT-1 pathway which is shared with manganese. In the iron deficient gut, large quantities of both mobilferrin and DMT-1 are found in goblet cells and intraluminal mucins suggesting that they are secreted with mucin into the intestinal lumen to bind iron to facilitate uptake by the cells. In the cytoplasm, IMP and DMT associate in a large protein complex called paraferritin which serves as a ferrireductase. Paraferritin solublizes iron binding proteins and reduces iron to make iron available for production of iron containing proteins such as heme. Iron uptake by intestinal absorptive cells is regulated by the iron concentration within the cell. Except in hemochromatosis it remains in equilibrium with total body stores via transferrin receptors on the basolateral membrane of absorptive cells. Increased intracellular iron either up-regulates or satiates iron binding proteins on regulatory proteins to alter their location in the intestinal mucosa. PMID:12547224

  16. A pathway to spirituality.

    PubMed

    Shaw, Jon A

    2005-01-01

    The phenomenology of mystical experiences has been described throughout all the ages and in all religions. All mystical traditions identify some sense of union with the absolute as the ultimate spiritual goal. I assume that the pathway to both theistic and secular spirituality and our readiness to seek a solution in a psychological merger with something beyond the self evolves out of our human experience. Spirituality is one of man's strategies for dealing with the limitations of the life cycle, separation and loss, biological fragility, transience, and non-existence. Spirituality may serve as the affective component to a belief system or myth that is not rooted in scientific evidence but is lived as if it is true. Spirituality may take many forms, but I will suggest that in some instances it may serve as a reparative process in which one creates in the external world, through symbolic form, a nuance or facet of an internalized mental representation which has become lost or is no longer available to the self; or it may represent the continuity of the self-representation after death through a self-object merger. Lastly I will illustrate from the writings of two of our greatest poets, Dante Alighieri and William Wordsworth, how their poetry became interwoven with a profound spirituality. In Dante we will see the elaboration of a religious spirituality, while in the writings of Wordsworth a secular spirituality emerges interwoven with nature and belatedly his identification with "tragic man" as his mythos. PMID:16599401

  17. Pathways to legal immigration

    PubMed Central

    MASSEY, DOUGLAS S.; MALONE, NOLAN

    2010-01-01

    In this paper we use the New Immigrant Survey Pilot Study (NISP) to describe the amount and kind of experience that immigrants accumulate in the United States before they become permanent resident aliens. The NISP surveyed a representative sample of legal immigrants who acquired residence papers during July and August of 1996, yielding a completed sample of 1,135 adults. Our analysis revealed that roughly two-thirds of these newly arrived immigrants had prior experience in the United States within one of six basic categories: illegal border-crossers, visa abusers, non-resident visitors, non-resident workers, students or exchange visitors, and refugees/asylees. Each of these pathways to legal immigration was associated with a different profile with respect to nationality, social background, and economic status. Using simple earnings regressions we demonstrate how these differences can yield misleading conclusions about the process of immigrant adaptation and assimilation, even if measured effects are reasonably accurate. We suggest that social scientists should change the way they think and ask about immigrants’ arrival in the United States. PMID:20830313

  18. 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

  19. Representations of metabolic knowledge: pathways.

    PubMed

    Karp, P D; Paley, S M

    1994-01-01

    The automatic generation of drawings of metabolic pathways is a challenging problem that depends intimately on exactly what information has been recorded for each pathway, and on how that information is encoded. The chief contributions of the paper are a minimized representation for biochemical pathways called the predecessor list, and inference procedures for converting the predecessor list into a pathway-graph representation that can serve as input to a pathway-drawing algorithm. The predecessor list has several advantages over the pathway graph, including its compactness and its lack of redundancy. The conversion between the two representations can be formulated as both a constraint-satisfaction problem and a logical inference problem, whose goal is to assign directions to reactions, and to determine which are the main chemical compounds in the reaction. We describe a set of production rules that solves this inference problem. We also present heuristics for inferring whether the exterior compounds that are substrates of reactions at the periphery of a pathway are side or main compounds. These techniques were evaluated on 18 metabolic pathways from the EcoCyc knowledge base. PMID:7584392

  20. Vestibular pathways involved in cognition

    PubMed Central

    Hitier, Martin; Besnard, Stephane; Smith, Paul F.

    2014-01-01

    Recent discoveries have emphasized the role of the vestibular system in cognitive processes such as memory, spatial navigation and bodily self-consciousness. A precise understanding of the vestibular pathways involved is essential to understand the consequences of vestibular diseases for cognition, as well as develop therapeutic strategies to facilitate recovery. The knowledge of the “vestibular cortical projection areas”, defined as the cortical areas activated by vestibular stimulation, has dramatically increased over the last several years from both anatomical and functional points of view. Four major pathways have been hypothesized to transmit vestibular information to the vestibular cortex: (1) the vestibulo-thalamo-cortical pathway, which probably transmits spatial information about the environment via the parietal, entorhinal and perirhinal cortices to the hippocampus and is associated with spatial representation and self-versus object motion distinctions; (2) the pathway from the dorsal tegmental nucleus via the lateral mammillary nucleus, the anterodorsal nucleus of the thalamus to the entorhinal cortex, which transmits information for estimations of head direction; (3) the pathway via the nucleus reticularis pontis oralis, the supramammillary nucleus and the medial septum to the hippocampus, which transmits information supporting hippocampal theta rhythm and memory; and (4) a possible pathway via the cerebellum, and the ventral lateral nucleus of the thalamus (perhaps to the parietal cortex), which transmits information for spatial learning. Finally a new pathway is hypothesized via the basal ganglia, potentially involved in spatial learning and spatial memory. From these pathways, progressively emerges the anatomical network of vestibular cognition. PMID:25100954

  1. Pathways from Poverty Educational Network.

    ERIC Educational Resources Information Center

    Northeast Regional Center for Rural Development, University Park, PA.

    Pathways from Poverty is a public policy education and research initiative organized by the Rural Sociological Society's Task Force on Persistent Rural Poverty and the four regional rural development centers. This publication focuses on project efforts in the Northeast and includes three sections. The first section describes the Pathways from…

  2. MPW : the metabolic pathways database.

    SciTech Connect

    Selkov, E., Jr.; Grechkin, Y.; Mikhailova, N.; Selkov, E.; Mathematics and Computer Science; Russian Academy of Sciences

    1998-01-01

    The Metabolic Pathways Database (MPW) (www.biobase.com/emphome.html/homepage. html.pags/pathways.html) a derivative of EMP (www.biobase.com/EMP) plays a fundamental role in the technology of metabolic reconstructions from sequenced genomes under the PUMA (www.mcs.anl.gov/home/compbio/PUMA/Production/ ReconstructedMetabolism/reconstruction.html), WIT (www.mcs.anl.gov/home/compbio/WIT/wit.html ) and WIT2 (beauty.isdn.msc.anl.gov/WIT2.pub/CGI/user.cgi) systems. In October 1997, it included some 2800 pathway diagrams covering primary and secondary metabolism, membrane transport, signal transduction pathways, intracellular traffic, translation and transcription. In the current public release of MPW (beauty.isdn.mcs.anl.gov/MPW), the encoding is based on the logical structure of the pathways and is represented by the objects commonly used in electronic circuit design. This facilitates drawing and editing the diagrams and makes possible automation of the basic simulation operations such as deriving stoichiometric matrices, rate laws, and, ultimately, dynamic models of metabolic pathways. Individual pathway diagrams, automatically derived from the original ASCII records, are stored as SGML instances supplemented by relational indices. An auxiliary database of compound names and structures, encoded in the SMILES format, is maintained to unambiguously connect the pathways to the chemical structures of their intermediates.

  3. Pathway Interaction Database (PID) —

    Cancer.gov

    The National Cancer Institute (NCI) in collaboration with Nature Publishing Group has established the Pathway Interaction Database (PID) in order to provide a highly structured, curated collection of information about known biomolecular interactions and key cellular processes assembled into signaling pathways.

  4. Nematode endogenous small RNA pathways

    PubMed Central

    Hoogstrate, Suzanne W; Volkers, Rita JM; Sterken, Mark G; Kammenga, Jan E; Snoek, L Basten

    2014-01-01

    The discovery of small RNA silencing pathways has greatly extended our knowledge of gene regulation. Small RNAs have been presumed to play a role in every field of biology because they affect many biological processes via regulation of gene expression and chromatin remodeling. Most well-known examples of affected processes are development, fertility, and maintenance of genome stability. Here we review the role of the three main endogenous small RNA silencing pathways in Caenorhabditis elegans: microRNAs, endogenous small interfering RNAs, and PIWI-interacting RNAs. After providing an entry-level overview on how these pathways function, we discuss research on other nematode species providing insight into the evolution of these small RNA pathways. In understanding the differences between the endogenous small RNA pathways and their evolution, a more comprehensive picture is formed of the functions and effects of small RNAs. PMID:25340013

  5. On generalized distributions and pathways

    NASA Astrophysics Data System (ADS)

    Mathai, A. M.; Haubold, H. J.

    2008-03-01

    The scalar version of the pathway model of Mathai [A.M. Mathai, Linear Alg. Appl. 396 (2005) 317] is shown to be associated with a large number of probability models used in physics. Different families of densities are listed here, which are all connected through the pathway parameter α, generating a distributional pathway. The idea is to switch from one functional form to another through this parameter and it is shown that one can proceed from the generalized type-1 beta family to generalized type-2 beta family to generalized gamma family. It is also shown that the pathway model is available by maximizing a generalized measure of entropy, leading to an entropic pathway, covering the particularly interesting cases of Tsallis statistics [C. Tsallis, J. Stat. Phys. 52 (1988) 479] and superstatistics [C. Beck, E.G.D. Cohen, Physica A 322 (2003) 267].

  6. A local coastal adaptation pathway

    NASA Astrophysics Data System (ADS)

    Barnett, J.; Graham, S.; Mortreux, C.; Fincher, R.; Waters, E.; Hurlimann, A.

    2014-12-01

    Local governments are not adapting to sea-level rise because it is difficult to build consensus on the need for change and the best way to implement it. In theory, adaptation pathways can resolve this impasse. Adaptation pathways are a sequence of linked strategies that are triggered by a change in environmental conditions, and in which initial decisions can have low regrets and preserve options for future generations. We report on a project that sought to empirically test the relevance and feasibility of a local pathway for adapting to sea-level rise. We find that triggers of change that have social impacts are salient to local people, and developing a local adaptation pathway helps build consensus among diverse constituencies. Our results show that adaptation pathways are feasible at the local scale, offering a low-risk, low-cost way to begin the long process of adaptation to sea-level rise.

  7. Refining the quantitative pathway of the Pathways to Mathematics model.

    PubMed

    Sowinski, Carla; LeFevre, Jo-Anne; Skwarchuk, Sheri-Lynn; Kamawar, Deepthi; Bisanz, Jeffrey; Smith-Chant, Brenda

    2015-03-01

    In the current study, we adopted the Pathways to Mathematics model of LeFevre et al. (2010). In this model, there are three cognitive domains--labeled as the quantitative, linguistic, and working memory pathways--that make unique contributions to children's mathematical development. We attempted to refine the quantitative pathway by combining children's (N=141 in Grades 2 and 3) subitizing, counting, and symbolic magnitude comparison skills using principal components analysis. The quantitative pathway was examined in relation to dependent numerical measures (backward counting, arithmetic fluency, calculation, and number system knowledge) and a dependent reading measure, while simultaneously accounting for linguistic and working memory skills. Analyses controlled for processing speed, parental education, and gender. We hypothesized that the quantitative, linguistic, and working memory pathways would account for unique variance in the numerical outcomes; this was the case for backward counting and arithmetic fluency. However, only the quantitative and linguistic pathways (not working memory) accounted for unique variance in calculation and number system knowledge. Not surprisingly, only the linguistic pathway accounted for unique variance in the reading measure. These findings suggest that the relative contributions of quantitative, linguistic, and working memory skills vary depending on the specific cognitive task. PMID:25521665

  8. Pathways Intern Report

    NASA Technical Reports Server (NTRS)

    Bell, Evan A.

    2015-01-01

    During my time at NASA, I worked with the Granular Mechanics and Regolith Organization (GMRO), better known as Swamp Works. The goal of the lab is to find ways to utilize resources found after the astronaut or robot has landed on another planet or asteroid. This concept is known as in-situ resource utilization and it is critical to long term missions such as those to Mars. During my time here I worked on the Asteroid and Lava Tube Free Flyer project (ALTFF). A lava tube, such as the one shown in figure 1, is a long tear drop shaped cavern that is produced when molten lava tunnels through the surrounding rock creating large unground pathways. Before mining for resources on Mars or on asteroids, a sampling mission must be done to scout out useful resource deposits. ALTFF's goal is to provide a low cost, autonomous scout robot that can sample the surface and return to the mother ship or lander for further processing of the samples. The vehicle will be looking for water ice in the regolith that can be processed into either potable water, hydrogen and oxygen fuel, or a binder material for 3D printing. By using a low cost craft to sample, there is much less risk to the more expensive mother ship or lander. While my main task was the construction of a simulation environment to test control code in and the construction of the asteroid free flyer prototype, there were other tasks that I performed relating to the ALTFF project.

  9. Ascorbate Synthesis Pathway

    PubMed Central

    Gabbay, Kenneth H.; Bohren, Kurt M.; Morello, Roy; Bertin, Terry; Liu, Jeff; Vogel, Peter

    2010-01-01

    Using mouse gene knock-out models, we identify aldehyde reductase (EC 1.1.1.2, Akr1a4 (GR)) and aldose reductase (EC 1.1.1.21, Akr1b3 (AR)) as the enzymes responsible for conversion of d-glucuronate to l-gulonate, a key step in the ascorbate (ASC) synthesis pathway in mice. The gene knock-out (KO) mice show that the two enzymes, GR and AR, provide ∼85 and ∼15% of l-gulonate, respectively. GRKO/ARKO double knock-out mice are unable to synthesize ASC (>95% ASC deficit) and develop scurvy. The GRKO mice (∼85% ASC deficit) develop and grow normally when fed regular mouse chow (ASC content = 0) but suffer severe osteopenia and spontaneous fractures with stresses that increase ASC requirements, such as pregnancy or castration. Castration greatly increases osteoclast numbers and activity in GRKO mice and promotes increased bone loss as compared with wild-type controls and additionally induces proliferation of immature dysplastic osteoblasts likely because of an ASC-sensitive block(s) in early differentiation. ASC and the antioxidants pycnogenol and resveratrol block osteoclast proliferation and bone loss, but only ASC feeding restores osteoblast differentiation and prevents their dysplastic proliferation. This is the first in vivo demonstration of two independent roles for ASC as an antioxidant suppressing osteoclast activity and number as well as a cofactor promoting osteoblast differentiation. Although humans have lost the ability to synthesize ASC, our mouse models suggest the mechanisms by which suboptimal ASC availability facilitates the development of osteoporosis, which has important implications for human osteoporosis. PMID:20410296

  10. Enhanced levels of S-linalool by metabolic engineering of the terpenoid pathway in spike lavender leaves.

    PubMed

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

    2014-05-01

    Transgenic Lavandula latifolia plants overexpressing the linalool synthase (LIS) gene from Clarkia breweri, encoding the LIS enzyme that catalyzes the synthesis of linalool were generated. Most of these plants increased significantly their linalool content as compared to controls, especially in the youngest leaves, where a linalool increase up to a 1000% was observed. The phenotype of increased linalool content observed in young leaves was maintained in those T1 progenies that inherit the LIS transgene, although this phenotype was less evident in the flower essential oil. Cross-pollination of transgenic spike lavender plants allowed the generation of double transgenic plants containing the DXS (1-deoxy-d-xylulose-5-P synthase), coding for the first enzyme of the methyl-d-erythritol-4-phosphate pathway, and LIS genes. Both essential oil yield and linalool content in double DXS-LIS transgenic plants were lower than that of their parentals, which could be due to co-suppression effects linked to the structures of the constructs used. PMID:24685653

  11. Mechanisms of Hippo pathway regulation

    PubMed Central

    Meng, Zhipeng; Moroishi, Toshiro; Guan, Kun-Liang

    2016-01-01

    The Hippo pathway was initially identified in Drosophila melanogaster screens for tissue growth two decades ago and has been a subject extensively studied in both Drosophila and mammals in the last several years. The core of the Hippo pathway consists of a kinase cascade, transcription coactivators, and DNA-binding partners. Recent studies have expanded the Hippo pathway as a complex signaling network with >30 components. This pathway is regulated by intrinsic cell machineries, such as cell–cell contact, cell polarity, and actin cytoskeleton, as well as a wide range of signals, including cellular energy status, mechanical cues, and hormonal signals that act through G-protein-coupled receptors. The major functions of the Hippo pathway have been defined to restrict tissue growth in adults and modulate cell proliferation, differentiation, and migration in developing organs. Furthermore, dysregulation of the Hippo pathway leads to aberrant cell growth and neoplasia. In this review, we focus on recent developments in our understanding of the molecular actions of the core Hippo kinase cascade and discuss key open questions in the regulation and function of the Hippo pathway. PMID:26728553

  12. Mechanisms of Hippo pathway regulation.

    PubMed

    Meng, Zhipeng; Moroishi, Toshiro; Guan, Kun-Liang

    2016-01-01

    The Hippo pathway was initially identified in Drosophila melanogaster screens for tissue growth two decades ago and has been a subject extensively studied in both Drosophila and mammals in the last several years. The core of the Hippo pathway consists of a kinase cascade, transcription coactivators, and DNA-binding partners. Recent studies have expanded the Hippo pathway as a complex signaling network with >30 components. This pathway is regulated by intrinsic cell machineries, such as cell-cell contact, cell polarity, and actin cytoskeleton, as well as a wide range of signals, including cellular energy status, mechanical cues, and hormonal signals that act through G-protein-coupled receptors. The major functions of the Hippo pathway have been defined to restrict tissue growth in adults and modulate cell proliferation, differentiation, and migration in developing organs. Furthermore, dysregulation of the Hippo pathway leads to aberrant cell growth and neoplasia. In this review, we focus on recent developments in our understanding of the molecular actions of the core Hippo kinase cascade and discuss key open questions in the regulation and function of the Hippo pathway. PMID:26728553

  13. Signaling pathways in diabetic nephropathy.

    PubMed

    Kawanami, Daiji; Matoba, Keiichiro; Utsunomiya, Kazunori

    2016-10-01

    Diabetic nephropathy (DN) is a major cause of end-stage renal disease (ESRD), however, specific treatment for DN has not yet been elucidated. Therefore, it is critically important to understand the molecular mechanism underlying DN to develop cause-related therapeutic strategy. To date, various factors such as hemodynamic changes and metabolic pathways have been shown to be involved in the pathogenesis of DN. Excessive glucose influx activates cellular signaling pathways, including the diacylglycerol (DAG)-protein kinase C (PKC) pathway, advanced glycation end-products (AGE), polyol pathway, hexosamine pathway and oxidative stress. These factors interact with one another, thereby facilitating inflammatory processes, leading to the development of glomerulosclerosis under diabetic conditions. In addition to metabolic pathways, Rho-kinase, an effector of small-GTPase binding protein Rho, has been implicated as an important factor in the pathogenesis of DN. A number of studies have demonstrated that Rho-kinase plays key roles in the development of DN by inducing endothelial dysfunction, mesangial excessive extracellular matrix (ECM) production, podocyte abnormality, and tubulointerstitial fibrosis. In this review article, we describe our current understanding of the signaling pathways in DN. PMID:27094540

  14. Protein design for pathway engineering

    SciTech Connect

    Eriksen, DT; Lian, JZ; Zhao, HM

    2014-02-01

    Design and construction of biochemical pathways has increased the complexity of biosynthetically-produced compounds when compared to single enzyme biocatalysis. However, the coordination of multiple enzymes can introduce a complicated set of obstacles to overcome in order to achieve a high titer and yield of the desired compound. Metabolic engineering has made great strides in developing tools to optimize the flux through a target pathway, but the inherent characteristics of a particular enzyme within the pathway can still limit the productivity. Thus, judicious protein design is critical for metabolic and pathway engineering. This review will describe various strategies and examples of applying protein design to pathway engineering to optimize the flux through the pathway. The proteins can be engineered for altered substrate specificity/selectivity, increased catalytic activity, reduced mass transfer limitations through specific protein localization, and reduced substrate/product inhibition. Protein engineering can also be expanded to design biosensors to enable high through-put screening and to customize cell signaling networks. These strategies have successfully engineered pathways for significantly increased productivity of the desired product or in the production of novel compounds. (C) 2013 Elsevier Inc. All rights reserved.

  15. Protein Design for Pathway Engineering

    PubMed Central

    Eriksen, Dawn T.; Lian, Jiazhang; Zhao, Huimin

    2013-01-01

    Design and construction of biochemical pathways has increased the complexity of biosynthetically-produced compounds when compared to single enzyme biocatalysis. However, the coordination of multiple enzymes can introduce a complicated set of obstacles to overcome in order to achieve a high titer and yield of the desired compound. Metabolic engineering has made great strides in developing tools to optimize the flux through a target pathway, but the inherent characteristics of a particular enzyme within the pathway can still limit the productivity. Thus, judicious protein design is critical for metabolic and pathway engineering. This review will describe various strategies and examples of applying protein design to pathway engineering to optimize the flux through the pathway. The proteins can be engineered for altered substrate specificity/selectivity, increased catalytic activity, reduced mass transfer limitations through specific protein localization, and reduced substrate/product inhibition. Protein engineering can also be expanded to design biosensors to enable high through-put screening and to customize cell signaling networks. These strategies have successfully engineered pathways for significantly increased productivity of the desired product or in the production of novel compounds. PMID:23558037

  16. Nutrient Sensing Mechanisms and Pathways

    PubMed Central

    Efeyan, Alejo; Comb, William C.; Sabatini, David M.

    2015-01-01

    PREFACE The ability to sense and respond to fluctuations in environmental nutrient levels is a requisite for life. Nutrient scarcity is a selective pressure that has shaped the evolution of most cellular processes. Different pathways that detect intracellular and extracellular levels of sugars, amino acids and lipids, and surrogate metabolites, are then integrated and coordinated at the organismal level via hormonal signals. During food abundance, nutrient sensing pathways engage anabolism and storage, and scarcity triggers homeostatic mechanisms, like the mobilization of internal stores through mechanisms such as autophagy. Nutrient sensing pathways are commonly deregulated in human metabolic diseases. PMID:25592535

  17. INTERSECTINg Pathways in Cell Biology

    PubMed Central

    O'Bryan, John P.

    2010-01-01

    The endocytic pathway is involved in activation and inhibition of cellular signaling. Thus, defining the regulatory mechanisms that link endocytosis and cellular signaling is of interest. An emerging link between these processes is a family of proteins called intersectins (ITSNs). These multi-domain proteins serve as scaffolds in the assembly of endocytic vesicles, and also regulate components of various signaling pathways, including kinases, GTPases, and ubiquitin ligases. This review will summarize research on the role of ITSNs in regulating both endocytic and signal transduction pathways, discuss the link of ITSNs with human disease, and highlight future directions in the study of ITSNs. PMID:21156937

  18. Targeting the p53 pathway.

    PubMed

    Golubovskaya, Vita M; Cance, William G

    2013-10-01

    This article summarizes data on translational studies to target the p53 pathway in cancer. It describes the functions of the p53 and Mdm-2 signaling pathways, and discusses current therapeutic approaches to target p53 pathways, including reactivation of p53. In addition, direct interaction and colocalization of the p53 and focal adhesion kinase proteins in cancer cells have been demonstrated, and different approaches to target this interaction are reviewed. This is a broad review of p53 function as it relates to the diagnosis and treatment of a wide range of cancers. PMID:24012397

  19. THE PATHWAY OF ARSENIC METABLISM

    EPA Science Inventory

    The Pathway of Arsenic Methylation

    David J. Thomas, Experimental Toxicology Division, National Health and Environmental Effects Research Laboratory, Office of Research and Development, U.S. Environmental Protection Agency, Research Triangle Park, NC

    Understanding ...

  20. SRNL ALL-PATHWAYS APPLICATION

    SciTech Connect

    Koffman, L; Elmer Wilhite, E; Leonard Collard, L

    2007-05-29

    The Environmental Analysis and Performance Modeling group of Savannah River National Laboratory (SRNL) performs performance assessments of the Savannah River Site (SRS) low-level waste facilities to meet the requirements of DOE Order 435.1. One of the performance objectives in the DOE Order is that the radiological dose to representative members of the public shall not exceed 25 mrem in a year total effective dose equivalent from all exposure pathways, excluding radon. Analysis to meet this performance objective is generally referred to as all-pathways analysis. SRNL performs detailed transient groundwater transport analysis for the waste disposal units, which has been used as input for the groundwater part of all-pathways analysis. The desire to better integrate all-pathways analysis with the groundwater transport analysis lead to the development of a software application named the SRNL All-Pathways Application. Another requirement of DOE Order 435.1 is to assess the impact of nuclear waste disposal on water resources, which SRS has interpreted for groundwater protection as meeting the EPA regulations for radionuclides in drinking water. EPA specifies four separate criteria as part of their implementation guidance for radionuclides, which are specified as maximum contaminant levels (MCL). (1) Beta/gamma emitters have a combined dose limit of 4 mrem/year. (2) Alpha emitters have a combined concentration limit of 15 pCi/L (called gross alpha), excluding uranium and radon, but including radium-226. (3) Combined radium-226 and radium-228 have a concentration limit of 5 pCi/L. (4) Isotopes of uranium have a combined concentration limit of 30 {micro}g/L. The All-Pathways Application was designed to be an easy-to-use software application that utilizes transient concentration results from groundwater transport analysis to (1) calculate the groundwater part of all-pathways dose and to (2) evaluate the four EPA criteria for groundwater protection.

  1. HealthPathways: creating a pathway for health systems reform.

    PubMed

    Robinson, Suzanne; Varhol, Richard; Bell, Colin; Quirk, Frances; Durrington, Learne

    2015-02-01

    Inefficiencies in the co-ordination and integration of primary and secondary care services in Australia, have led to increases in waiting times, unnecessary presentations to emergency departments and issues around poor discharge of patients. HealthPathways is a program developed in Canterbury, New Zealand, that builds relationships between General Practitioners and Specialists and uses information technology so that efficiency is maximised and the right patient is given the right care at the right time. Healthpathways is being implemented by a number of Medicare Locals across Australia however, little is known about the impact HealthPathways may have in Australia. This article provides a short description of HealthPathways and considers what it may offer in the Australian context and some of the barriers and facilitators to implementation. PMID:25433515

  2. SRNL All-Pathways Application

    SciTech Connect

    Koffman, Larry D.; Wilhite, Elmer L.; Collard, Leonard B.

    2008-01-15

    The Environmental Analysis and Performance Modeling group of Savannah River National Laboratory (SRNL) performs performance assessments of the Savannah River Site (SRS) low-level waste facilities to meet the requirements of DOE Order 435.1. One of the performance objectives in the DOE Order is that the radiological dose to representative members of the public shall not exceed 25 mrem in a year total effective dose equivalent from all exposure pathways, excluding radon. Analysis to meet this performance objective is generally referred to as all-pathways analysis. SRNL performs detailed transient groundwater transport analysis for the waste disposal units, which has been used as input for the groundwater part of all-pathways analysis. The desire to better integrate all-pathways analysis with the groundwater transport analysis lead to the development of a software application named the SRNL All-Pathways Application. Another requirement of DOE Order 435.1 is to assess the impact of nuclear waste disposal on water resources, which SRS has interpreted for groundwater protection as meeting the EPA regulations for radionuclides in drinking water. EPA specifies four separate criteria as part of their implementation guidance for radionuclides, which are specified as maximum contaminant levels (MCL). 1. Beta/gamma emitters have a combined dose limit of 4 mrem/year. 2. Alpha emitters have a combined concentration limit of 15 pCi/L (called gross alpha), excluding uranium and radon, but including radium-226. 3. Combined radium-226 and radium-228 have a concentration limit of 5 pCi/L. 4. Isotopes of uranium have a combined concentration limit of 30 {mu}g/L. The All-Pathways Application was designed to be an easy-to-use software application that utilizes transient concentration results from groundwater transport analysis to (1) calculate the groundwater part of all-pathways dose and to (2) evaluate the four EPA criteria for groundwater protection. An application has been developed

  3. Coherence in electron transfer pathways.

    PubMed

    Skourtis, Spiros S; Beratan, David N; Waldeck, David H

    2011-01-01

    Central to the view of electron-transfer reactions is the idea that nuclear motion generates a transition state geometry at which the electron/hole amplitude propagates coherently from the electron donor to the electron acceptor. In the weakly coupled or nonadiabatic regime, the electron amplitude tunnels through an electronic barrier between the donor and acceptor. The structure of the barrier is determined by the covalent and noncovalent interactions of the bridge. Because the tunneling barrier depends on the nuclear coordinates of the reactants (and on the surrounding medium), the tunneling barrier is highly anisotropic, and it is useful to identify particular routes, or pathways, along which the transmission amplitude propagates. Moreover, when more than one such pathway exists, and the paths give rise to comparable transmission amplitude magnitudes, one may expect to observe quantum interferences among pathways if the propagation remains coherent. Given that the effective tunneling barrier height and width are affected by the nuclear positions, the modulation of the nuclear coordinates will lead to a modulation of the tunneling barrier and hence of the electron flow. For long distance electron transfer in biological and biomimetic systems, nuclear fluctuations, arising from flexible protein moieties and mobile water bridges, can become quite significant. We discuss experimental and theoretical results that explore the quantum interferences among coupling pathways in electron-transfer kinetics; we emphasize recent data and theories associated with the signatures of chirality and inelastic processes, which are manifested in the tunneling pathway coherence (or absence of coherence). PMID:23833692

  4. A biosynthetic pathway for anandamide

    PubMed Central

    Liu, Jie; Wang, Lei; Harvey-White, Judith; Osei-Hyiaman, Douglas; Razdan, Raj; Gong, Qian; Chan, Andrew C.; Zhou, Zhifeng; Huang, Bill X.; Kim, Hee-Yong; Kunos, George

    2006-01-01

    The endocannabinoid arachidonoyl ethanolamine (anandamide) is a lipid transmitter synthesized and released “on demand” by neurons in the brain. Anandamide is also generated by macrophages where its endotoxin (LPS)-induced synthesis has been implicated in the hypotension of septic shock and advanced liver cirrhosis. Anandamide can be generated from its membrane precursor, N-arachidonoyl phosphatidylethanolamine (NAPE) through cleavage by a phospholipase D (NAPE–PLD). Here we document a biosynthetic pathway for anandamide in mouse brain and RAW264.7 macrophages that involves the phospholipase C (PLC)-catalyzed cleavage of NAPE to generate a lipid, phosphoanandamide, which is subsequently dephosphorylated by phosphatases, including PTPN22, previously described as a protein tyrosine phosphatase. Bacterial endotoxin (LPS)-induced synthesis of anandamide in macrophages is mediated exclusively by the PLC/phosphatase pathway, which is up-regulated by LPS, whereas NAPE–PLD is down-regulated by LPS and functions as a salvage pathway of anandamide synthesis when the PLC/phosphatase pathway is compromised. Both PTPN22 and endocannabinoids have been implicated in autoimmune diseases, suggesting that the PLC/phosphatase pathway of anandamide synthesis may be a pharmacotherapeutic target. PMID:16938887

  5. The HEART Pathway Randomized Trial

    PubMed Central

    Mahler, Simon A.; Riley, Robert F.; Hiestand, Brian C.; Russell, Gregory B.; Hoekstra, James W.; Lefebvre, Cedric W.; Nicks, Bret A.; Cline, David M.; Askew, Kim L.; Elliott, Stephanie B.; Herrington, David M.; Burke, Gregory L.; Miller, Chadwick D.

    2015-01-01

    Background The HEART Pathway is a decision aid designed to identify emergency department patients with acute chest pain for early discharge. No randomized trials have compared the HEART Pathway with usual care. Methods and Results Adult emergency department patients with symptoms related to acute coronary syndrome without ST-elevation on ECG (n=282) were randomized to the HEART Pathway or usual care. In the HEART Pathway arm, emergency department providers used the HEART score, a validated decision aid, and troponin measures at 0 and 3 hours to identify patients for early discharge. Usual care was based on American College of Cardiology/American Heart Association guidelines. The primary outcome, objective cardiac testing (stress testing or angiography), and secondary outcomes, index length of stay, early discharge, and major adverse cardiac events (death, myocardial infarction, or coronary revascularization), were assessed at 30 days by phone interview and record review. Participants had a mean age of 53 years, 16% had previous myocardial infarction, and 6% (95% confidence interval, 3.6%–9.5%) had major adverse cardiac events within 30 days of randomization. Compared with usual care, use of the HEART Pathway decreased objective cardiac testing at 30 days by 12.1% (68.8% versus 56.7%; P=0.048) and length of stay by 12 hours (9.9 versus 21.9 hours; P=0.013) and increased early discharges by 21.3% (39.7% versus 18.4%; P<0.001). No patients identified for early discharge had major adverse cardiac events within 30 days. Conclusions The HEART Pathway reduces objective cardiac testing during 30 days, shortens length of stay, and increases early discharges. These important efficiency gains occurred without any patients identified for early discharge suffering MACE at 30 days. PMID:25737484

  6. Collateral Pathways in Portal Hypertension

    PubMed Central

    Sharma, Malay; Rameshbabu, Chittapuram S.

    2012-01-01

    Presence of portosystemic collateral veins (PSCV) is common in portal hypertension due to cirrhosis. Physiologically, normal portosystemic anastomoses exist which exhibit hepatofugal flow. With the development of portal hypertension, transmission of backpressure leads to increased flow in these patent normal portosystemic anastomoses. In extrahepatic portal vein obstruction collateral circulation develops in a hepatopetal direction and portoportal pathways are frequently found. The objective of this review is to illustrate the various PSCV and portoportal collateral vein pathways pertinent to portal hypertension in liver cirrhosis and EHPVO. PMID:25755456

  7. [Pathways of flowering regulation in plants].

    PubMed

    Liu, Yongping; Yang, Jing; Yang, Mingfeng

    2015-11-01

    Flowering, the floral transition from vegetative growth to reproductive growth, is induced by diverse endogenous and exogenous cues, such as photoperiod, temperature, hormones and age. Precise flowering time is critical to plant growth and evolution of species. The numerous renewal molecular and genetic results have revealed five flowering time pathways, including classical photoperiod pathway, vernalization pathway, autonomous pathway, gibberellins (GA) pathway and newly identified age pathway. These pathways take on relatively independent role, and involve extensive crosstalks and feedback loops. This review describes the complicated regulatory network of this floral transition to understand the molecular mechanism of flowering and provide references for further research in more plants. PMID:26939439

  8. SMPDB: The Small Molecule Pathway Database.

    PubMed

    Frolkis, Alex; Knox, Craig; Lim, Emilia; Jewison, Timothy; Law, Vivian; Hau, David D; Liu, Phillip; Gautam, Bijaya; Ly, Son; Guo, An Chi; Xia, Jianguo; Liang, Yongjie; Shrivastava, Savita; Wishart, David S

    2010-01-01

    The Small Molecule Pathway Database (SMPDB) is an interactive, visual database containing more than 350 small-molecule pathways found in humans. More than 2/3 of these pathways (>280) are not found in any other pathway database. SMPDB is designed specifically to support pathway elucidation and pathway discovery in clinical metabolomics, transcriptomics, proteomics and systems biology. SMPDB provides exquisitely detailed, hyperlinked diagrams of human metabolic pathways, metabolic disease pathways, metabolite signaling pathways and drug-action pathways. All SMPDB pathways include information on the relevant organs, organelles, subcellular compartments, protein cofactors, protein locations, metabolite locations, chemical structures and protein quaternary structures. Each small molecule is hyperlinked to detailed descriptions contained in the Human Metabolome Database (HMDB) or DrugBank and each protein or enzyme complex is hyperlinked to UniProt. All SMPDB pathways are accompanied with detailed descriptions, providing an overview of the pathway, condition or processes depicted in each diagram. The database is easily browsed and supports full text searching. Users may query SMPDB with lists of metabolite names, drug names, genes/protein names, SwissProt IDs, GenBank IDs, Affymetrix IDs or Agilent microarray IDs. These queries will produce lists of matching pathways and highlight the matching molecules on each of the pathway diagrams. Gene, metabolite and protein concentration data can also be visualized through SMPDB's mapping interface. All of SMPDB's images, image maps, descriptions and tables are downloadable. SMPDB is available at: http://www.smpdb.ca. PMID:19948758

  9. UniPathway: a resource for the exploration and annotation of metabolic pathways.

    PubMed

    Morgat, Anne; Coissac, Eric; Coudert, Elisabeth; Axelsen, Kristian B; Keller, Guillaume; Bairoch, Amos; Bridge, Alan; Bougueleret, Lydie; Xenarios, Ioannis; Viari, Alain

    2012-01-01

    UniPathway (http://www.unipathway.org) is a fully manually curated resource for the representation and annotation of metabolic pathways. UniPathway provides explicit representations of enzyme-catalyzed and spontaneous chemical reactions, as well as a hierarchical representation of metabolic pathways. This hierarchy uses linear subpathways as the basic building block for the assembly of larger and more complex pathways, including species-specific pathway variants. All of the pathway data in UniPathway has been extensively cross-linked to existing pathway resources such as KEGG and MetaCyc, as well as sequence resources such as the UniProt KnowledgeBase (UniProtKB), for which UniPathway provides a controlled vocabulary for pathway annotation. We introduce here the basic concepts underlying the UniPathway resource, with the aim of allowing users to fully exploit the information provided by UniPathway. PMID:22102589

  10. UniPathway: a resource for the exploration and annotation of metabolic pathways

    PubMed Central

    Morgat, Anne; Coissac, Eric; Coudert, Elisabeth; Axelsen, Kristian B.; Keller, Guillaume; Bairoch, Amos; Bridge, Alan; Bougueleret, Lydie; Xenarios, Ioannis; Viari, Alain

    2012-01-01

    UniPathway (http://www.unipathway.org) is a fully manually curated resource for the representation and annotation of metabolic pathways. UniPathway provides explicit representations of enzyme-catalyzed and spontaneous chemical reactions, as well as a hierarchical representation of metabolic pathways. This hierarchy uses linear subpathways as the basic building block for the assembly of larger and more complex pathways, including species-specific pathway variants. All of the pathway data in UniPathway has been extensively cross-linked to existing pathway resources such as KEGG and MetaCyc, as well as sequence resources such as the UniProt KnowledgeBase (UniProtKB), for which UniPathway provides a controlled vocabulary for pathway annotation. We introduce here the basic concepts underlying the UniPathway resource, with the aim of allowing users to fully exploit the information provided by UniPathway. PMID:22102589

  11. Reverse Engineering Adverse Outcome Pathways

    SciTech Connect

    Perkins, Edward; Chipman, J.K.; Edwards, Stephen; Habib, Tanwir; Falciani, Francesco; Taylor, Ronald C.; Van Aggelen, Graham; Vulpe, Chris; Antczak, Philipp; Loguinov, Alexandre

    2011-01-30

    The toxicological effects of many stressors are mediated through unknown, or poorly characterized, mechanisms of action. We describe the application of reverse engineering complex interaction networks from high dimensional omics data (gene, protein, metabolic, signaling) to characterize adverse outcome pathways (AOPs) for chemicals that disrupt the hypothalamus-pituitary-gonadal endocrine axis in fathead minnows. Gene expression changes in fathead minnow ovaries in response to 7 different chemicals, over different times, doses, and in vivo versus in vitro conditions were captured in a large data set of 868 arrays. We examined potential AOPs of the antiandrogen flutamide using two mutual information theory methods, ARACNE and CLR to infer gene regulatory networks and potential adverse outcome pathways. Representative networks from these studies were used to predict a network path from stressor to adverse outcome as a candidate AOP. The relationship of individual chemicals to an adverse outcome can be determined by following perturbations through the network in response to chemical treatment leading to the nodes associated with the adverse outcome. Identification of candidate pathways allows for formation of testable hypotheses about key biologic processes, biomarkers or alternative endpoints, which could be used to monitor an adverse outcome pathway. Finally, we identify the unique challenges facing the application of this approach in ecotoxicology, and attempt to provide a road map for the utilization of these tools. Key Words: mechanism of action, toxicology, microarray, network inference

  12. Capstone Design Project Course Pathways

    ERIC Educational Resources Information Center

    Eppes, Tom A.; Milanovic, Ivana

    2011-01-01

    Capstones are open-ended undertakings where students are expected to creatively analyze, synthesize, and apply a wide-variety of learning outcomes from prior coursework. This paper discusses the structure, approach and evolution of the capstone project pathways within our College. Specifically two programs, MET and EET, have adopted different…

  13. Pathways to poly-victimization.

    PubMed

    Finkelhor, David; Ormrod, Richard; Turner, Heather; Holt, Melissa

    2009-11-01

    Some children, whom we have labeled poly-victims, experience very high levels of victimizations of different types. This article finds support for a conceptual model suggesting that there may be four distinct pathways to becoming such a poly-victim: (a) residing in a dangerous community, (b) living in a dangerous family, (c) having a chaotic, multiproblem family environment, or (d) having emotional problems that increase risk behavior, engender antagonism, and compromise the capacity to protect oneself. It uses three waves of the Developmental Victimization Survey, a nationally representative sample of children aged 2-17 years. All four hypothesized pathways showed significant independent association with poly-victim onset. For the younger children, the symptom score representing emotional problems was the only significant predictor. For the older children, the other three pathway variables were significant predictors--dangerous communities, dangerous families, and problem families--but not symptom score. Poly-victimization onset was also disproportionately likely to occur in the year prior to children's 7th and 15th birthday, corresponding roughly to the entry into elementary school and high school. The identification of such pathways and the ages of high onset should help practitioners design programs for preventing vulnerable children from becoming poly-victims. PMID:19837972

  14. Cancer stem cell signaling pathways.

    PubMed

    Matsui, William H

    2016-09-01

    Tissue development and homeostasis are governed by the actions of stem cells. Multipotent cells are capable of self-renewal during the course of one's lifetime. The accurate and appropriate regulation of stem cell functions is absolutely critical for normal biological activity. Several key developmental or signaling pathways have been shown to play essential roles in this regulatory capacity. Specifically, the Janus-activated kinase/signal transducer and activator of transcription, Hedgehog, Wnt, Notch, phosphatidylinositol 3-kinase/phosphatase and tensin homolog, and nuclear factor-κB signaling pathways have all been shown experimentally to mediate various stem cell properties, such as self-renewal, cell fate decisions, survival, proliferation, and differentiation. Unsurprisingly, many of these crucial signaling pathways are dysregulated in cancer. Growing evidence suggests that overactive or abnormal signaling within and among these pathways may contribute to the survival of cancer stem cells (CSCs). CSCs are a relatively rare population of cancer cells capable of self-renewal, differentiation, and generation of serially transplantable heterogeneous tumors of several types of cancer. PMID:27611937

  15. Career Technical Education Pathways Initiative

    ERIC Educational Resources Information Center

    California Community Colleges, Chancellor's Office, 2013

    2013-01-01

    California's education system--the largest in the United States--is an essential resource for ensuring strong economic growth in the state. The Career Technical Education Pathways Initiative (referred to as the Initiative in this report), which became law in 2005, brings together community colleges, K-12 school districts, employers, organized…

  16. Overskilling Dynamics and Education Pathways

    ERIC Educational Resources Information Center

    Mavromaras, Kostas; McGuinness, Seamus

    2012-01-01

    This paper uses panel data and econometric methods to estimate the incidence and the dynamic properties of overskilling among employed individuals. The paper begins by asking whether there is extensive overskilling in the labour market, and whether overskilling differs by education pathway. The answer to both questions is yes. The paper continues…

  17. Signalling pathways in endometrial cancer.

    PubMed

    Markowska, Anna; Pawałowska, Monika; Lubin, Jolanta; Markowska, Janina

    2014-01-01

    Carcinogenesis is a multistage process, during which the activity of signalling pathways responsible for cell cycle regulation and division is disrupted which leads to inhibition of apoptosis and enhanced proliferation. Improper activation of Wnt/β-catenin and PI3K. Akt pathways play essential role in endometrial cancers (EC), mainly type I. Mutations in APC, axin or CTNBB1 may lead to β-catenin overactivation leading to excessive gene expression. PTEN inactivation, mutations in the PIK3CA or Akt result in increased transmission in the PI3K/Akt pathway, apoptosis inhibition, intensive cell division, mTOR excitation. In non-endometrioid cancers, key mutations include suppressor gene TP53 responsible for repairing damaged DNA or apoptosis initiation. Irregularities in gene P16, encoding a protein forming the p16-cyclinD/CDK-pRb have also been described. Understanding the complex relations between specific proteins taking part in signal transduction of the abovementioned pathways is key to research on drugs used in targeted therapy. PMID:25520571

  18. The Phenylpropanoid Pathway in Arabidopsis

    PubMed Central

    Fraser, Christopher M.; Chapple, Clint

    2011-01-01

    The phenylpropanoid pathway serves as a rich source of metabolites in plants, being required for the biosynthesis of lignin, and serving as a starting point for the production of many other important compounds, such as the flavonoids, coumarins, and lignans. In spite of the fact that the phenylpropanoids and their derivatives are sometimes classified as secondary metabolites, their relevance to plant survival has been made clear via the study of Arabidopsis and other plant species. As a model system, Arabidopsis has helped to elucidate many details of the phenylpropanoid pathway, its enzymes and intermediates, and the interconnectedness of the pathway with plant metabolism as a whole. These advances in our understanding have been made possible in large part by the relative ease with which mutations can be generated, identified, and studied in Arabidopsis. Herein, we provide an overview of the research progress that has been made in recent years, emphasizing both the genes (and gene families) associated with the phenylpropanoid pathway in Arabidopsis, and the end products that have contributed to the identification of many mutants deficient in the phenylpropanoid metabolism: the sinapate esters. PMID:22303276

  19. Multiple pathways regulate shoot branching

    PubMed Central

    Rameau, Catherine; Bertheloot, Jessica; Leduc, Nathalie; Andrieu, Bruno; Foucher, Fabrice; Sakr, Soulaiman

    2015-01-01

    Shoot branching patterns result from the spatio-temporal regulation of axillary bud outgrowth. Numerous endogenous, developmental and environmental factors are integrated at the bud and plant levels to determine numbers of growing shoots. Multiple pathways that converge to common integrators are most probably involved. We propose several pathways involving not only the classical hormones auxin, cytokinins and strigolactones, but also other signals with a strong influence on shoot branching such as gibberellins, sugars or molecular actors of plant phase transition. We also deal with recent findings about the molecular mechanisms and the pathway involved in the response to shade as an example of an environmental signal controlling branching. We propose the TEOSINTE BRANCHED1, CYCLOIDEA, PCF transcription factor TB1/BRC1 and the polar auxin transport stream in the stem as possible integrators of these pathways. We finally discuss how modeling can help to represent this highly dynamic system by articulating knowledges and hypothesis and calculating the phenotype properties they imply. PMID:25628627

  20. Rapid prototype extruded conductive pathways

    DOEpatents

    Bobbitt, III, John T.

    2016-06-21

    A process of producing electrically conductive pathways within additively manufactured parts and similar parts made by plastic extrusion nozzles. The process allows for a three-dimensional part having both conductive and non-conductive portions and allows for such parts to be manufactured in a single production step.

  1. Multiple Pathways for All Students

    ERIC Educational Resources Information Center

    Stirling, Lee Anna

    2012-01-01

    Maine has been focusing on the importance of postsecondary training. Maine's Skowhegan Area High School (SAHS) and Somerset Career and Technical Center (SCTC) have partnered in a Multiple Pathways initiative (funded by the Nellie Mae Education Foundation) to increase students' high school completion rate and to increase enrollment in postsecondary…

  2. Establishment of an alternative phosphoketolase-dependent pathway for fructose catabolism in Ralstonia eutropha H16.

    PubMed

    Fleige, Christian; Kroll, Jens; Steinbüchel, Alexander

    2011-08-01

    The β-proteobacterium Ralstonia eutropha H16 utilizes fructose and gluconate as carbon sources for heterotrophic growth exclusively via the Entner-Doudoroff pathway with its key enzyme 2-keto-3-deoxy-6-phosphogluconate (KDPG) aldolase. By deletion of the responsible gene eda, we constructed a KDPG aldolase-negative strain, which is disabled to supply pyruvate for energy metabolism from fructose or gluconate as sole carbon sources. To restore growth on fructose, an alternative pathway, similar to the fructose-6-phosphate shunt of heterofermentative bifidobacteria, was established. For this, the xfp gene from Bifidobacterium animalis, coding for a bifunctional xylulose-5-phosphate/fructose-6-phosphate phosphoketolase (Xfp; Meile et al. in J Bacteriol 183:2929-2936, 2001), was expressed in R. eutropha H16 PHB(-)4 Δeda. This Xfp catalyzes the phosphorolytic cleavage of fructose 6-phosphate to erythrose 4-phosphate and acetylphosphate as well as of xylulose 5-phosphate to glyceralaldehyde 3-phosphate and acetylphosphate. The recombinant strain showed phosphoketolase (PKT) activity on either substrate, and was able to use fructose as sole carbon source for growth, because PKT is the only enzyme that is missing in R. eutropha H16 to establish the artificial fructose-6-phosphate shunt. The Xfp-expressing strain R. eutropha H16 PHB(-)4 Δeda (pBBR1MCS-3::xfp) should be applicable for a novel variant of a plasmid addiction system to stably maintain episomally encoded genetic information during fermentative production processes. Plasmid addiction systems are often used to ensure plasmid stability in many biotechnology relevant microorganisms and processes without the need to apply external selection pressure, like the addition of antibiotics. By episomal expression of xfp in a R. eutropha H16 mutant lacking KDPG aldolase activity and cultivation in mineral salt medium with fructose as sole carbon source, the growth of this bacterium was addicted to the constructed xfp

  3. Two-Electron Transfer Pathways.

    PubMed

    Lin, Jiaxing; Balamurugan, D; Zhang, Peng; Skourtis, Spiros S; Beratan, David N

    2015-06-18

    The frontiers of electron-transfer chemistry demand that we develop theoretical frameworks to describe the delivery of multiple electrons, atoms, and ions in molecular systems. When electrons move over long distances through high barriers, where the probability for thermal population of oxidized or reduced bridge-localized states is very small, the electrons will tunnel from the donor (D) to acceptor (A), facilitated by bridge-mediated superexchange interactions. If the stable donor and acceptor redox states on D and A differ by two electrons, it is possible that the electrons will propagate coherently from D to A. While structure-function relations for single-electron superexchange in molecules are well established, strategies to manipulate the coherent flow of multiple electrons are largely unknown. In contrast to one-electron superexchange, two-electron superexchange involves both one- and two-electron virtual intermediate states, the number of virtual intermediates increases very rapidly with system size, and multiple classes of pathways interfere with one another. In the study described here, we developed simple superexchange models for two-electron transfer. We explored how the bridge structure and energetics influence multielectron superexchange, and we compared two-electron superexchange interactions to single-electron superexchange. Multielectron superexchange introduces interference between singly and doubly oxidized (or reduced) bridge virtual states, so that even simple linear donor-bridge-acceptor systems have pathway topologies that resemble those seen for one-electron superexchange through bridges with multiple parallel pathways. The simple model systems studied here exhibit a richness that is amenable to experimental exploration by manipulating the multiple pathways, pathway crosstalk, and changes in the number of donor and acceptor species. The features that emerge from these studies may assist in developing new strategies to deliver multiple

  4. Modulation of the Host Lipid Landscape to Promote RNA Virus Replication: The Picornavirus Encephalomyocarditis Virus Converges on the Pathway Used by Hepatitis C Virus

    PubMed Central

    Dorobantu, Cristina M.; Albulescu, Lucian; Harak, Christian; Feng, Qian; van Kampen, Mirjam; Strating, Jeroen R. P. M.; Gorbalenya, Alexander E.; Lohmann, Volker

    2015-01-01

    Cardioviruses, including encephalomyocarditis virus (EMCV) and the human Saffold virus, are small non-enveloped viruses belonging to the Picornaviridae, a large family of positive-sense RNA [(+)RNA] viruses. All (+)RNA viruses remodel intracellular membranes into unique structures for viral genome replication. Accumulating evidence suggests that picornaviruses from different genera use different strategies to generate viral replication organelles (ROs). For instance, enteroviruses (e.g. poliovirus, coxsackievirus, rhinovirus) rely on the Golgi-localized phosphatidylinositol 4-kinase III beta (PI4KB), while cardioviruses replicate independently of the kinase. By which mechanisms cardioviruses develop their ROs is currently unknown. Here we show that cardioviruses manipulate another PI4K, namely the ER-localized phosphatidylinositol 4-kinase III alpha (PI4KA), to generate PI4P-enriched ROs. By siRNA-mediated knockdown and pharmacological inhibition, we demonstrate that PI4KA is an essential host factor for EMCV genome replication. We reveal that the EMCV nonstructural protein 3A interacts with and is responsible for PI4KA recruitment to viral ROs. The ensuing phosphatidylinositol 4-phosphate (PI4P) proved important for the recruitment of oxysterol-binding protein (OSBP), which delivers cholesterol to EMCV ROs in a PI4P-dependent manner. PI4P lipids and cholesterol are shown to be required for the global organization of the ROs and for viral genome replication. Consistently, inhibition of OSBP expression or function efficiently blocked EMCV RNA replication. In conclusion, we describe for the first time a cellular pathway involved in the biogenesis of cardiovirus ROs. Remarkably, the same pathway was reported to promote formation of the replication sites of hepatitis C virus, a member of the Flaviviridae family, but not other picornaviruses or flaviviruses. Thus, our results highlight the convergent recruitment by distantly related (+)RNA viruses of a host lipid

  5. 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

  6. Molecular Pathways: Targeting DNA Repair Pathway Defects Enriched in Metastasis.

    PubMed

    Corcoran, Niall M; Clarkson, Michael J; Stuchbery, Ryan; Hovens, Christopher M

    2016-07-01

    The maintenance of a pristine genome, free from errors, is necessary to prevent cellular transformation and degeneration. When errors in DNA are detected, DNA damage repair (DDR) genes and their regulators are activated to effect repair. When these DDR pathways are themselves mutated or aberrantly downregulated, cancer and neurodegenerative disorders can ensue. Multiple lines of evidence now indicate, however, that defects in key regulators of DNA repair pathways are highly enriched in human metastasis specimens and hence may be a key step in the acquisition of metastasis and the ability of localized disease to disseminate. Some of the key regulators of checkpoints in the DNA damage response are the TP53 protein and the PARP enzyme family. Targeting of these pathways, especially through PARP inhibition, is now being exploited therapeutically to effect significant clinical responses in subsets of individuals, particularly in patients with ovarian cancer or prostate cancer, including cancers with a marked metastatic burden. Targeting DNA repair-deficient tumors with drugs that take advantage of the fundamental differences between normal repair-proficient cells and repair-deficient tumors offers new avenues for treating advanced disease in the future. Clin Cancer Res; 22(13); 3132-7. ©2016 AACR. PMID:27169997

  7. Reactome from a WikiPathways Perspective.

    PubMed

    Bohler, Anwesha; Wu, Guanming; Kutmon, Martina; Pradhana, Leontius Adhika; Coort, Susan L; Hanspers, Kristina; Haw, Robin; Pico, Alexander R; Evelo, Chris T

    2016-05-01

    Reactome and WikiPathways are two of the most popular freely available databases for biological pathways. Reactome pathways are centrally curated with periodic input from selected domain experts. WikiPathways is a community-based platform where pathways are created and continually curated by any interested party. The nascent collaboration between WikiPathways and Reactome illustrates the mutual benefits of combining these two approaches. We created a format converter that converts Reactome pathways to the GPML format used in WikiPathways. In addition, we developed the ComplexViz plugin for PathVisio which simplifies looking up complex components. The plugin can also score the complexes on a pathway based on a user defined criterion. This score can then be visualized on the complex nodes using the visualization options provided by the plugin. Using the merged collection of curated and converted Reactome pathways, we demonstrate improved pathway coverage of relevant biological processes for the analysis of a previously described polycystic ovary syndrome gene expression dataset. Additionally, this conversion allows researchers to visualize their data on Reactome pathways using PathVisio's advanced data visualization functionalities. WikiPathways benefits from the dedicated focus and attention provided to the content converted from Reactome and the wealth of semantic information about interactions. Reactome in turn benefits from the continuous community curation available on WikiPathways. The research community at large benefits from the availability of a larger set of pathways for analysis in PathVisio and Cytoscape. The pathway statistics results obtained from PathVisio are significantly better when using a larger set of candidate pathways for analysis. The conversion serves as a general model for integration of multiple pathway resources developed using different approaches. PMID:27203685

  8. Reactome from a WikiPathways Perspective

    PubMed Central

    Bohler, Anwesha; Wu, Guanming; Pradhana, Leontius Adhika; Hanspers, Kristina; Haw, Robin; Pico, Alexander R.

    2016-01-01

    Reactome and WikiPathways are two of the most popular freely available databases for biological pathways. Reactome pathways are centrally curated with periodic input from selected domain experts. WikiPathways is a community-based platform where pathways are created and continually curated by any interested party. The nascent collaboration between WikiPathways and Reactome illustrates the mutual benefits of combining these two approaches. We created a format converter that converts Reactome pathways to the GPML format used in WikiPathways. In addition, we developed the ComplexViz plugin for PathVisio which simplifies looking up complex components. The plugin can also score the complexes on a pathway based on a user defined criterion. This score can then be visualized on the complex nodes using the visualization options provided by the plugin. Using the merged collection of curated and converted Reactome pathways, we demonstrate improved pathway coverage of relevant biological processes for the analysis of a previously described polycystic ovary syndrome gene expression dataset. Additionally, this conversion allows researchers to visualize their data on Reactome pathways using PathVisio’s advanced data visualization functionalities. WikiPathways benefits from the dedicated focus and attention provided to the content converted from Reactome and the wealth of semantic information about interactions. Reactome in turn benefits from the continuous community curation available on WikiPathways. The research community at large benefits from the availability of a larger set of pathways for analysis in PathVisio and Cytoscape. The pathway statistics results obtained from PathVisio are significantly better when using a larger set of candidate pathways for analysis. The conversion serves as a general model for integration of multiple pathway resources developed using different approaches. PMID:27203685

  9. Pathway engineering for production of aromatics in Escherichia coli: Confirmation of stoichiometric analysis by independent modulation of AroG, TktA, and Pps activities

    SciTech Connect

    Patnaik, R.; Spitzer, R.G.; Liao, J.C.

    1995-05-20

    The synthesis of 3-deoxy-D-arabino-heptulosonate-7-phosphate (DAHP) is the first commitment of resources toward aromatics production in Escherichia coli. DAHP is produced during the condensation reaction between phosphenolpyruvate (PEP) and erythrose 4-phosphate (E4P) catalyzed by DAHP synthases (coded by aroF, aroG, and aroH). Stoichiometric analysis has shown a severe PEP limitation in the theoretical yield of DAHP production from glucose due to the phosphotransferase system (PTS) for sugar uptake. In the present study the authors confirm the predictions of the stoichiometric analysis by introducing pps, tktA, and aroG into vectors under independently controlled promoters, In glucose medium, although TktA has some positive effect on the final DAHP concentration, it has no effect on the yield (percent conversion). With Pps overexpression, the DAHP concentration produced from glucose is increased almost twofold and the yield is approaching the theoretical maximum, the final DAHP concentration and the yield are completely determined by the AroG activity. TktA and Pps play no or insignificant roles, and the yield can reach the theoretical maximum without overexpression of these two enzymes. The results shown hare are important for both rational design of metabolic pathways and industrial production of aromatics such as tryptophan, phenylalanine, indigo, quinic acid, and catechol.

  10. Importance of phosphoinositide-dependent signaling pathways in the control of gene expression in resting cells and in response to phytohormones

    PubMed Central

    Kalachova, Tetiana; Kravets, Volodymyr; Zachowski, Alain; Ruelland, Eric

    2015-01-01

    “Phosphoinositide” refers to phosphorylated forms of phosphatidylinositol, including phosphatidylinositol-4-phosphate and phosphatidylinositol-4,5-bisphosphate. Both of these molecules could be in vivo substrates of plant phospholipase C. These phosphoinositides can also be biologically active “per se,” by directly binding to proteins and thus altering their location and/or activity. The use of pharmacological agents in Arabidopsis suspension cells allowed us to identify genes whose expression was positively or negatively controlled, in the basal state, by products of phosphoinositide-dependent phospholipase C. In this basal state, it seems that no genes exhibit a phosphoinositide-dependent expression “per se.” However, many genes whose expression is altered in the presence of phospholipase C inhibitors appeared to be responsive to salicylic acid. This allowed us to show that salicylic acid acts both by increasing the phosphoinositide pool and by inhibiting the phospholipase C. In response to salicylic acid it is possible to identify genes whose expression is controlled by products of PI-PLC, but also genes whose expression is controlled by phosphoinositides “per se.” Our data highlight the importance of phosphoinositide-dependent pathways in gene expression in resting cells and in response to phytohormones. PMID:26039482

  11. Signaling on the endocytic pathway.

    PubMed

    McPherson, P S; Kay, B K; Hussain, N K

    2001-06-01

    Ligand binding to receptor tyrosine kinases and G-protein-coupled receptors initiates signal transduction events and induces receptor endocytosis via clathrin-coated pits and vesicles. While receptor-mediated endocytosis has been traditionally considered an effective mechanism to attenuate ligand-activated responses, more recent studies demonstrate that signaling continues on the endocytic pathway. In fact, certain signaling events, such as the activation of the extracellular signal-regulated kinases, appear to require endocytosis. Protein components of signal transduction cascades can assemble at clathrin coated pits and remain associated with endocytic vesicles following their dynamin-dependent release from the plasma membrane. Thus, endocytic vesicles can function as a signaling compartment distinct from the plasma membrane. These observations demonstrate that endocytosis plays an important role in the activation and propagation of signaling pathways. PMID:11389765

  12. Signaling pathways mediating alcohol effects.

    PubMed

    Ron, Dorit; Messing, Robert O

    2013-01-01

    Ethanol's effects on intracellular signaling pathways contribute to acute effects of ethanol as well as to neuroadaptive responses to repeated ethanol exposure. In this chapter we review recent discoveries that demonstrate how ethanol alters signaling pathways involving several receptor tyrosine kinases and intracellular tyrosine and serine-threonine kinases, with consequences for regulation of cell surface receptor function, gene expression, protein translation, neuronal excitability and animal behavior. We also describe recent work that demonstrates a key role for ethanol in regulating the function of scaffolding proteins that organize signaling complexes into functional units. Finally, we review recent exciting studies demonstrating ethanol modulation of DNA and histone modification and the expression of microRNAs, indicating epigenetic mechanisms by which ethanol regulates neuronal gene expression and addictive behaviors. PMID:21877259

  13. Fundamental reaction pathways during coprocessing

    SciTech Connect

    Stock, L.M.; Gatsis, J.G.

    1992-12-01

    The objective of this research was to investigate the fundamental reaction pathways in coal petroleum residuum coprocessing. Once the reaction pathways are defined, further efforts can be directed at improving those aspects of the chemistry of coprocessing that are responsible for the desired results such as high oil yields, low dihydrogen consumption, and mild reaction conditions. We decided to carry out this investigation by looking at four basic aspects of coprocessing: (1) the effect of fossil fuel materials on promoting reactions essential to coprocessing such as hydrogen atom transfer, carbon-carbon bond scission, and hydrodemethylation; (2) the effect of varied mild conditions on the coprocessing reactions; (3) determination of dihydrogen uptake and utilization under severe conditions as a function of the coal or petroleum residuum employed; and (4) the effect of varied dihydrogen pressure, temperature, and residence time on the uptake and utilization of dihydrogen and on the distribution of the coprocessed products. Accomplishments are described.

  14. Signaling Pathways in Cartilage Repair

    PubMed Central

    Mariani, Erminia; Pulsatelli, Lia; Facchini, Andrea

    2014-01-01

    In adult healthy cartilage, chondrocytes are in a quiescent phase characterized by a fine balance between anabolic and catabolic activities. In ageing, degenerative joint diseases and traumatic injuries of cartilage, a loss of homeostatic conditions and an up-regulation of catabolic pathways occur. Since cartilage differentiation and maintenance of homeostasis are finely tuned by a complex network of signaling molecules and biophysical factors, shedding light on these mechanisms appears to be extremely relevant for both the identification of pathogenic key factors, as specific therapeutic targets, and the development of biological approaches for cartilage regeneration. This review will focus on the main signaling pathways that can activate cellular and molecular processes, regulating the functional behavior of cartilage in both physiological and pathological conditions. These networks may be relevant in the crosstalk among joint compartments and increased knowledge in this field may lead to the development of more effective strategies for inducing cartilage repair. PMID:24837833

  15. Developmental pathways of motor dysfunction.

    PubMed

    Kleven, Gale A; Bellinger, Seanceray A

    2015-05-01

    Recent evidence has revealed unique patterns of behavioral development after prenatal insult similar to those outlined in studies of adult metabolic dysfunction after prenatal malnutrition. The hallmark features of this Developmental Pathway include a prenatal insult to the nervous system (environmental or genetic) followed by a period of Silent Vulnerability, where no or few functional deficits are observed, and finally emergence of later dysfunction. Possible mechanisms leading to later dysfunction from prenatal insult may include secondary or cascade effects due to the timing of prenatal insults relative to later developing structures in the brain. Methods best employed to study the mechanisms of these pathways are microgenetic and longitudinal designs that include behavioral assessment during the prenatal period of development, and animal models such as the guinea pig. PMID:25864561

  16. Fragmentation pathways of polymer ions.

    PubMed

    Wesdemiotis, Chrys; Solak, Nilüfer; Polce, Michael J; Dabney, David E; Chaicharoen, Kittisak; Katzenmeyer, Bryan C

    2011-01-01

    Tandem mass spectrometry (MS/MS) is increasingly applied to synthetic polymers to characterize chain-end or in-chain substituents, distinguish isobaric and isomeric species, and determine macromolecular connectivities and architectures. For confident structural assignments, the fragmentation mechanisms of polymer ions must be understood, as they provide guidelines on how to deduce the desired information from the fragments observed in MS/MS spectra. This article reviews the fragmentation pathways of synthetic polymer ions that have been energized to decompose via collisionally activated dissociation (CAD), the most widely used activation method in polymer analysis. The compounds discussed encompass polystyrenes, poly(2-vinyl pyridine), polyacrylates, poly(vinyl acetate), aliphatic polyester copolymers, polyethers, and poly(dimethylsiloxane). For a number of these polymers, several substitution patterns and architectures are considered, and questions regarding the ionization agent and internal energy of the dissociating precursor ions are also addressed. Competing and consecutive dissociations are evaluated in terms of the structural insight they provide about the macromolecular structure. The fragmentation pathways of the diverse array of polymer ions examined fall into three categories, viz. (1) charge-directed fragmentations, (2) charge-remote rearrangements, and (3) charge-remote fragmentations via radical intermediates. Charge-remote processes predominate. Depending on the ionizing agent and the functional groups in the polymer, the incipient fragments arising by pathways (1)-(3) may form ion-molecule complexes that survive long enough to permit inter-fragment hydrogen atom, proton, or hydride transfers. PMID:20623599

  17. Quantifying macromolecular conformational transition pathways

    NASA Astrophysics Data System (ADS)

    Seyler, Sean; Kumar, Avishek; Thorpe, Michael; Beckstein, Oliver

    2015-03-01

    Diverse classes of proteins function through large-scale conformational changes that are challenging for computer simulations. A range of fast path-sampling techniques have been used to generate transitions, but it has been difficult to compare paths from (and assess the relative strengths of) different methods. We introduce a comprehensive method (pathway similarity analysis, PSA) for quantitatively characterizing and comparing macromolecular pathways. The Hausdorff and Fréchet metrics (known from computational geometry) are used to quantify the degree of similarity between polygonal curves in configuration space. A strength of PSA is its use of the full information available from the 3 N-dimensional configuration space trajectory without requiring additional specific knowledge about the system. We compare a sample of eleven different methods for the closed-to-open transitions of the apo enzyme adenylate kinase (AdK) and also apply PSA to an ensemble of 400 AdK trajectories produced by dynamic importance sampling MD and the Geometrical Pathways algorithm. We discuss the method's potential to enhance our understanding of transition path sampling methods, validate them, and help guide future research toward deeper physical insights into conformational transitions.

  18. Identification of Metabolic Pathway Systems

    PubMed Central

    Dolatshahi, Sepideh; Voit, Eberhard O.

    2016-01-01

    The estimation of parameters in even moderately large biological systems is a significant challenge. This challenge is greatly exacerbated if the mathematical formats of appropriate process descriptions are unknown. To address this challenge, the method of dynamic flux estimation (DFE) was proposed for the analysis of metabolic time series data. Under ideal conditions, the first phase of DFE yields numerical representations of all fluxes within a metabolic pathway system, either as values at each time point or as plots against their substrates and modulators. However, this numerical result does not reveal the mathematical format of each flux. Thus, the second phase of DFE selects functional formats that are consistent with the numerical trends obtained from the first phase. While greatly facilitating metabolic data analysis, DFE is only directly applicable if the pathway system contains as many dependent variables as fluxes. Because most actual systems contain more fluxes than metabolite pools, this requirement is seldom satisfied. Auxiliary methods have been proposed to alleviate this issue, but they are not general. Here we propose strategies that extend DFE toward general, slightly underdetermined pathway systems. PMID:26904095

  19. Alcohol Effects on Stress Pathways

    PubMed Central

    Blaine, Sara K.; Milivojevic, Verica; Fox, Helen

    2016-01-01

    A significant amount of neurobiological research regarding the development of alcohol use disorders (AUDs) has focused on alcohol-related activation and long-term alterations in the mesocortical dopaminergic reward pathways. However, alcohol does not only interact with brain reward systems. Many of its acute and chronic effects may be related to allostatic adaptations in hypothalamic and extrahypothalamic stress regulation pathways. For example, acute binge intoxication is associated with hypothalamically driven increases in blood cortisol, norepinephrine, and sex steroid metabolite levels. This may contribute to the development of mesocortical sensitization to alcohol. Furthermore, chronic alcohol exposure is associated with systemic dysregulation of the hypothalamic pituitary adrenal axis, sympathetic adrenal medullary system, and sex steroid systems. This dysregulation appears to manifest as neuroendocrine tolerance. In this review, we first summarize the literature suggesting that alcohol-induced alterations in these hypothalamic systems influence craving and contribute to the development of AUDs. We note that for women, the effects of alcohol on these neuroendocrine stress regulation systems may be influenced by the rhythmic variations of hormones and steroids across the menstrual cycle. Second, we discuss how changes in these systems may indicate progression of AUDs and increased risk of relapse in both sexes. Specifically, neuroendocrine tolerance may contribute to mesocortical sensitization, which in turn may lead to decreased prefrontal inhibitory control of the dopaminergic reward and hypothalamic stress systems. Thus, pharmacological strategies that counteract alcohol-associated changes in hypothalamic and extrahypothalamic stress regulation pathways may slow the development and progression of AUDs. PMID:27254089

  20. Imbalanced kynurenine pathway in schizophrenia.

    PubMed

    Kegel, Magdalena E; Bhat, Maria; Skogh, Elisabeth; Samuelsson, Martin; Lundberg, Kristina; Dahl, Marja-Liisa; Sellgren, Carl; Schwieler, Lilly; Engberg, Göran; Schuppe-Koistinen, Ina; Erhardt, Sophie

    2014-01-01

    Several studies suggest a role for kynurenic acid (KYNA) in the pathophysiology of schizophrenia. It has been proposed that increased brain KYNA levels in schizophrenia result from a pathological shift in the kynurenine pathway toward enhanced KYNA formation, away from the other branch of the pathway leading to quinolinic acid (QUIN). Here we investigate the levels of QUIN in cerebrospinal fluid (CSF) of patients with schizophrenia and healthy controls, and relate those to CSF levels of KYNA and other kynurenine metabolites from the same individuals. CSF QUIN levels from stable outpatients treated with olanzapine (n = 22) and those of controls (n = 26) were analyzed using liquid chromatography-mass spectrometry. No difference in CSF QUIN levels between patients and controls was observed (20.6 ± 1.5 nM vs. 18.2 ± 1.1 nM, P = 0.36). CSF QUIN was positively correlated to CSF kynurenine and CSF KYNA in patients but not in controls. The CSF QUIN/KYNA ratio was lower in patients than in controls (P = 0.027). In summary, the present study offers support for an over-activated and imbalanced kynurenine pathway, favoring the production of KYNA over QUIN in patients with schizophrenia. PMID:25288889

  1. Imbalanced Kynurenine Pathway in Schizophrenia

    PubMed Central

    Kegel, Magdalena E; Bhat, Maria; Skogh, Elisabeth; Samuelsson, Martin; Lundberg, Kristina; Dahl, Marja-Liisa; Sellgren, Carl; Schwieler, Lilly; Engberg, Göran; Schuppe-Koistinen, Ina; Erhardt, Sophie

    2014-01-01

    Several studies suggest a role for kynurenic acid (KYNA) in the pathophysiology of schizophrenia. It has been proposed that increased brain KYNA levels in schizophrenia result from a pathological shift in the kynurenine pathway toward enhanced KYNA formation, away from the other branch of the pathway leading to quinolinic acid (QUIN). Here we investigate the levels of QUIN in cerebrospinal fluid (CSF) of patients with schizophrenia and healthy controls, and relate those to CSF levels of KYNA and other kynurenine metabolites from the same individuals. CSF QUIN levels from stable outpatients treated with olanzapine (n = 22) and those of controls (n = 26) were analyzed using liquid chromatography-mass spectrometry. No difference in CSF QUIN levels between patients and controls was observed (20.6 ± 1.5 nM vs. 18.2 ± 1.1 nM, P = 0.36). CSF QUIN was positively correlated to CSF kynurenine and CSF KYNA in patients but not in controls. The CSF QUIN/KYNA ratio was lower in patients than in controls (P = 0.027). In summary, the present study offers support for an over-activated and imbalanced kynurenine pathway, favoring the production of KYNA over QUIN in patients with schizophrenia. PMID:25288889

  2. 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

  3. MOLECULAR PATHWAYS: JAK/STAT PATHWAY: MUTATIONS, INHIBITORS, AND RESISTANCE

    PubMed Central

    Quintás-Cardama, Alfonso; Verstovsek, Srdan

    2016-01-01

    Aberrant activation of the JAK/STAT pathway has been reported in a variety of disease states, including inflammatory conditions, hematologic malignancies, and solid tumors. For instance, a large proportion of patients with myeloproliferative neoplasms (MPNs) carry the acquired gain-of-function JAK2 V617F somatic mutation. This knowledge has dramatically improved our understanding of the pathogenesis of MPNs and it has facilitated the development of therapeutics capable of suppressing the constitutive activation of the JAK/STAT pathway, now recognized as a common underlying biological abnormality in MPNs. Ruxolitinib is an oral JAK1 and JAK2 inhibitor that has recently been approved for the treatment of myelofibrosis and has been tested against other hematologic malignancies. A series of agents with different specificities against different members of the JAK family of proteins is currently undergoing evaluation in clinical trials for patients with MPNs, lymphoma, and solid tumors such as breast or pancreatic cancer. Despite their significant clinical activity exhibited in myelofibrosis, some patients fail to respond or progress during JAK kinase inhibitor therapy. Recent reports have shed light into the mechanisms of resistance to JAK kinase inhibitor therapy. Several approaches hold promise to overcome such resistance. PMID:23406773

  4. Secretory Pathway of Trypanosomatid Parasites

    PubMed Central

    McConville, Malcolm J.; Mullin, Kylie A.; Ilgoutz, Steven C.; Teasdale, Rohan D.

    2002-01-01

    The Trypanosomatidae comprise a large group of parasitic protozoa, some of which cause important diseases in humans. These include Trypanosoma brucei (the causative agent of African sleeping sickness and nagana in cattle), Trypanosoma cruzi (the causative agent of Chagas' disease in Central and South America), and Leishmania spp. (the causative agent of visceral and [muco]cutaneous leishmaniasis throughout the tropics and subtropics). The cell surfaces of these parasites are covered in complex protein- or carbohydrate-rich coats that are required for parasite survival and infectivity in their respective insect vectors and mammalian hosts. These molecules are assembled in the secretory pathway. Recent advances in the genetic manipulation of these parasites as well as progress with the parasite genome projects has greatly advanced our understanding of processes that underlie secretory transport in trypanosomatids. This article provides an overview of the organization of the trypanosomatid secretory pathway and connections that exist with endocytic organelles and multiple lytic and storage vacuoles. A number of the molecular components that are required for vesicular transport have been identified, as have some of the sorting signals that direct proteins to the cell surface or organelles in the endosome-vacuole system. Finally, the subcellular organization of the major glycosylation pathways in these parasites is reviewed. Studies on these highly divergent eukaryotes provide important insights into the molecular processes underlying secretory transport that arose very early in eukaryotic evolution. They also reveal unusual or novel aspects of secretory transport and protein glycosylation that may be exploited in developing new antiparasite drugs. PMID:11875130

  5. Pharmacology of intracellular signalling pathways

    PubMed Central

    Nahorski, Stefan R

    2006-01-01

    This article provides a brief and somewhat personalized review of the dramatic developments that have occurred over the last 45 years in our understanding of intracellular signalling pathways associated with G-protein-coupled receptor activation. Signalling via cyclic AMP, the phosphoinositides and Ca2+ is emphasized and these systems have already been revealed as new pharmacological targets. The therapeutic benefits of most of such targets are, however, yet to be realized, but it is certain that the discipline of pharmacology needs to widen its boundaries to meet these challenges in the future. PMID:16402119

  6. Signaling Pathways in Osteoclast Differentiation.

    PubMed

    Kim, Jung Ha; Kim, Nacksung

    2016-01-01

    Osteoclasts are multinucleated cells of hematopoietic origin that are responsible for the degradation of old bone matrix. Osteoclast differentiation and activity are controlled by two essential cytokines, macrophage colony-stimulating factor (M-CSF) and the receptor activator of nuclear factor-κB ligand (RANKL). M-CSF and RANKL bind to their respective receptors c-Fms and RANK to stimulate osteoclast differentiation through regulation of delicate signaling systems. Here, we summarize the critical or essential signaling pathways for osteoclast differentiation including M-CSF-c-Fms signaling, RANKL-RANK signaling, and costimulatory signaling for RANK. PMID:26865996

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

    NASA Astrophysics Data System (ADS)

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

    2011-06-01

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

  8. Exploring Biological Electron Transfer Pathway Dynamics with the Pathways Plugin for VMD

    PubMed Central

    Balabin, Ilya A.; Hu, Xiangqian; Beratan, David N.

    2012-01-01

    We describe the new Pathways plugin for the molecular visualization program VMD. The plugin identifies and visualizes tunneling pathways and pathway families in biomolecules and calculates relative electronic couplings. The plugin includes unique features to estimate the importance of individual atoms for mediating the coupling, to analyze the coupling sensitivity to thermal motion, and to visualize pathway fluctuations. The Pathways plugin is open source software distributed under the terms of the GNU public license. PMID:22298319

  9. Proteolysis in the secretory pathway

    SciTech Connect

    Guzowski, D.E.; Bienkowski, R.S.

    1987-05-01

    Many secretory proteins are degraded intracellularly rather than secreted, however the location of this catabolic process is not known. The authors have tested the hypothesis that the degradation occurs in the organelles of the secretory pathway. Slices of rat liver were incubated with (/sup 14/C)leucine for 3 h and then incubated under chase conditions for 30 min. The tissue was homogenized and the Golgi apparatus, smooth endoplasmic reticulum (sER) and rough endoplasmic reticulum (rER) were isolated by ultracentrifugation on a discontinuous sucrose gradient. The organelles were incubated in 0.3M sucrose-50 mM citrate (pH 4) for 8-12 h at 37 C; control samples were incubated at 4 C. Percent degradation was calculated as the amount of acid soluble radioactivity released relative to total radioactivity in the sample. Proteolysis in the organelles incubated at 37 C was as follows: Golgi: 15-25%; sER: 10-20%; rER: 10-20%. Proteolysis at 4 C was negligible in all cases. These results support the hypothesis that the compartments of the secretory pathway are capable of degrading newly synthesized secretory proteins.

  10. Model pathways in lignin thermolysis

    SciTech Connect

    Klein, M.T.; Virk, P.S.

    1981-02-01

    A fundamental description of lignin thermolysis was attempted. Analysis of the chemical topology of lignin suggested likely reaction pathways of import to lignin pyrolysis. In turn, 20 model compound pyrolysis substrates were selected to mimic the important reactive functional groups present in whole-lignin thermolysis. The more salient models were: phenethyl phenyl ether (PPE), which depicts the most prevalent lignin interunit linkage, guaiacol, model of the predominant aromatic methoxyl, and saligenol and cinnamyl alcohol, models of important propanoid side chains. Detailed pathway and kinetic analyses and determination of reaction Arrhenius parameters provided mechanistic insights into the model compound pyrolyses. Several pericyclic reaction mechanisms, hitherto not mentioned in the lignin pyrolysis literature, were suggested. In particular, PPE likely pyrolyses via a concerted retro-ene mechanism, whereas guaiacol and saligenol may respectively eliminate methane and water by concerted group transfers. A statistical interpretation of the lignin substrate coupled with the experimental model compound pyrolyses allowed simulation of whole-lignin thermolysis. The simulations were in substantial agreement with experimental pyrolyses reported in the literature in regard to overall gas, methane, carbon monoxide, individual phenols, and carbonaceous residue yields. Weight loss kinetics deduced from the time dependency of the latter yield also accorded well with the experimental literature.

  11. Combustion kinetics and reaction pathways

    SciTech Connect

    Klemm, R.B.; Sutherland, J.W.

    1993-12-01

    This project is focused on the fundamental chemistry of combustion. The overall objectives are to determine rate constants for elementary reactions and to elucidate the pathways of multichannel reactions. A multitechnique approach that features three independent experiments provides unique capabilities in performing reliable kinetic measurements over an exceptionally wide range in temperature, 300 to 2500 K. Recent kinetic work has focused on experimental studies and theoretical calculations of the methane dissociation system (CH{sub 4} + Ar {yields} CH{sub 3} + H + Ar and H + CH{sub 4} {yields} CH{sub 3} + H{sub 2}). Additionally, a discharge flow-photoionization mass spectrometer (DF-PIMS) experiment is used to determine branching fractions for multichannel reactions and to measure ionization thresholds of free radicals. Thus, these photoionization experiments generate data that are relevant to both reaction pathways studies (reaction dynamics) and fundamental thermochemical research. Two distinct advantages of performing PIMS with high intensity, tunable vacuum ultraviolet light at the National Synchrotron Light Source are high detection sensitivity and exceptional selectivity in monitoring radical species.

  12. Pathway analysis of coronary atherosclerosis.

    PubMed

    King, Jennifer Y; Ferrara, Rossella; Tabibiazar, Raymond; Spin, Joshua M; Chen, Mary M; Kuchinsky, Allan; Vailaya, Aditya; Kincaid, Robert; Tsalenko, Anya; Deng, David Xing-Fei; Connolly, Andrew; Zhang, Peng; Yang, Eugene; Watt, Clifton; Yakhini, Zohar; Ben-Dor, Amir; Adler, Annette; Bruhn, Laurakay; Tsao, Philip; Quertermous, Thomas; Ashley, Euan A

    2005-09-21

    Large-scale gene expression studies provide significant insight into genes differentially regulated in disease processes such as cancer. However, these investigations offer limited understanding of multisystem, multicellular diseases such as atherosclerosis. A systems biology approach that accounts for gene interactions, incorporates nontranscriptionally regulated genes, and integrates prior knowledge offers many advantages. We performed a comprehensive gene level assessment of coronary atherosclerosis using 51 coronary artery segments isolated from the explanted hearts of 22 cardiac transplant patients. After histological grading of vascular segments according to American Heart Association guidelines, isolated RNA was hybridized onto a customized 22-K oligonucleotide microarray, and significance analysis of microarrays and gene ontology analyses were performed to identify significant gene expression profiles. Our studies revealed that loss of differentiated smooth muscle cell gene expression is the primary expression signature of disease progression in atherosclerosis. Furthermore, we provide insight into the severe form of coronary artery disease associated with diabetes, reporting an overabundance of immune and inflammatory signals in diabetics. We present a novel approach to pathway development based on connectivity, determined by language parsing of the published literature, and ranking, determined by the significance of differentially regulated genes in the network. In doing this, we identify highly connected "nexus" genes that are attractive candidates for therapeutic targeting and followup studies. Our use of pathway techniques to study atherosclerosis as an integrated network of gene interactions expands on traditional microarray analysis methods and emphasizes the significant advantages of a systems-based approach to analyzing complex disease. PMID:15942018

  13. Metabolic pathway engineering based on metabolomics confers acetic and formic acid tolerance to a recombinant xylose-fermenting strain of Saccharomyces cerevisiae

    PubMed Central

    2011-01-01

    Background The development of novel yeast strains with increased tolerance toward inhibitors in lignocellulosic hydrolysates is highly desirable for the production of bio-ethanol. Weak organic acids such as acetic and formic acids are necessarily released during the pretreatment (i.e. solubilization and hydrolysis) of lignocelluloses, which negatively affect microbial growth and ethanol production. However, since the mode of toxicity is complicated, genetic engineering strategies addressing yeast tolerance to weak organic acids have been rare. Thus, enhanced basic research is expected to identify target genes for improved weak acid tolerance. Results In this study, the effect of acetic acid on xylose fermentation was analyzed by examining metabolite profiles in a recombinant xylose-fermenting strain of Saccharomyces cerevisiae. Metabolome analysis revealed that metabolites involved in the non-oxidative pentose phosphate pathway (PPP) [e.g. sedoheptulose-7-phosphate, ribulose-5-phosphate, ribose-5-phosphate and erythrose-4-phosphate] were significantly accumulated by the addition of acetate, indicating the possibility that acetic acid slows down the flux of the pathway. Accordingly, a gene encoding a PPP-related enzyme, transaldolase or transketolase, was overexpressed in the xylose-fermenting yeast, which successfully conferred increased ethanol productivity in the presence of acetic and formic acid. Conclusions Our metabolomic approach revealed one of the molecular events underlying the response to acetic acid and focuses attention on the non-oxidative PPP as a target for metabolic engineering. An important challenge for metabolic engineering is identification of gene targets that have material importance. This study has demonstrated that metabolomics is a powerful tool to develop rational strategies to confer tolerance to stress through genetic engineering. PMID:21219616

  14. Transneuronal pathways to the vestibulocerebellum

    NASA Technical Reports Server (NTRS)

    Kaufman, G. D.; Mustari, M. J.; Miselis, R. R.; Perachio, A. A.

    1996-01-01

    The alpha-herpes virus (pseudorabies, PRV) was used to observe central nervous system (CNS) pathways associated with the vestibulocerebellar system. Retrograde transneuronal migration of alpha-herpes virions from specific lobules of the gerbil and rat vestibulo-cerebellar cortex was detected immunohistochemically. Using a time series analysis, progression of infection along polyneuronal cerebellar afferent pathways was examined. Pressure injections of > 20 nanoliters of a 10(8) plaque forming units (pfu) per ml solution of virus were sufficient to initiate an infectious locus which resulted in labeled neurons in the inferior olivary subnuclei, vestibular nuclei, and their afferent cell groups in a progressive temporal fashion and in growing complexity with increasing incubation time. We show that climbing fibers and some other cerebellar afferent fibers transported the virus retrogradely from the cerebellum within 24 hours. One to three days after cerebellar infection discrete cell groups were labeled and appropriate laterality within crossed projections was preserved. Subsequent nuclei labeled with PRV after infection of the flocculus/paraflocculus, or nodulus/uvula, included the following: vestibular (e.g., z) and inferior olivary nuclei (e.g., dorsal cap), accessory oculomotor (e.g., Darkschewitsch n.) and accessory optic related nuclei, (e.g., the nucleus of the optic tract, and the medial terminal nucleus); noradrenergic, raphe, and reticular cell groups (e.g., locus coeruleus, dorsal raphe, raphe pontis, and the lateral reticular tract); other vestibulocerebellum sites, the periaqueductal gray, substantia nigra, hippocampus, thalamus and hypothalamus, amygdala, septal nuclei, and the frontal, cingulate, entorhinal, perirhinal, and insular cortices. However, there were differences in the resulting labeling between infection in either region. Double-labeling experiments revealed that vestibular efferent neurons are located adjacent to, but are not included

  15. PathwayMatrix: visualizing binary relationships between proteins in biological pathways

    PubMed Central

    2015-01-01

    Background Molecular activation pathways are inherently complex, and understanding relations across many biochemical reactions and reaction types is difficult. Visualizing and analyzing a pathway is a challenge due to the network size and the diversity of relations between proteins and molecules. Results In this paper, we introduce PathwayMatrix, a visualization tool that presents the binary relations between proteins in the pathway via the use of an interactive adjacency matrix. We provide filtering, lensing, clustering, and brushing and linking capabilities in order to present relevant details about proteins within a pathway. Conclusions We evaluated PathwayMatrix by conducting a series of in-depth interviews with domain experts who provided positive feedback, leading us to believe that our visualization technique could be helpful for the larger community of researchers utilizing pathway visualizations. PathwayMatrix is freely available at https://github.com/CreativeCodingLab/PathwayMatrix. PMID:26361499

  16. Oxidative biodegradation pathways of PAHs

    SciTech Connect

    Cerniglia, C.E.

    1993-12-31

    Polycyclic aromatic hydrocarbons (PAHs) constitute a class of hazardous organic chemical consisting of three of more fused benzene rings in linear, angular and cluster arrangements. PAHs mostly occur as a result of fossil fuel combustion, as by-product of industrial processing and during the cooking of foods. A catabolically diverse microbial community, consisting of bacteria, fungi and algae, metabolizes aromatic compounds. Molecular oxygen is essential for the initial hydroxylation of polycyclic aromatic hydrocarbons by microorganisms. In contrast to bacteria, filamentous fungi use hydroxylation as a prelude to detoxification rather than to catabolism and assimilation. The biochemical principles underlying the degradation of polycyclic aromatic hydrocarbons will be discussed. The oxidative pathways of polycyclic aromatic hydrocarbon catabolism will be discussed. Studies will be presented on the relationship between the chemical structure of the polycyclic aromatic hydrocarbon and the rate of polycyclic aromatic hydrocarbon biodegradation in aquatic and terrestrial ecosystems.

  17. Nonicosahedral pathways for capsid expansion

    NASA Astrophysics Data System (ADS)

    Cermelli, Paolo; Indelicato, Giuliana; Twarock, Reidun

    2013-09-01

    For a significant number of viruses a structural transition of the protein container that encapsulates the viral genome forms an important part of the life cycle and is a prerequisite for the particle becoming infectious. Despite many recent efforts the mechanism of this process is still not fully understood, and a complete characterization of the expansion pathways is still lacking. We present here a coarse-grained model that captures the essential features of the expansion process and allows us to investigate the conditions under which a viral capsid becomes unstable. Based on this model we demonstrate that the structural transitions in icosahedral viral capsids are likely to occur through a low-symmetry cascade of local expansion events spreading in a wavelike manner over the capsid surface.

  18. Asparagine Metabolic Pathways in Arabidopsis.

    PubMed

    Gaufichon, Laure; Rothstein, Steven J; Suzuki, Akira

    2016-04-01

    Inorganic nitrogen in the form of ammonium is assimilated into asparagine via multiple steps involving glutamine synthetase (GS), glutamate synthase (GOGAT), aspartate aminotransferase (AspAT) and asparagine synthetase (AS) in Arabidopsis. The asparagine amide group is liberated by the reaction catalyzed by asparaginase (ASPG) and also the amino group of asparagine is released by asparagine aminotransferase (AsnAT) for use in the biosynthesis of amino acids. Asparagine plays a primary role in nitrogen recycling, storage and transport in developing and germinating seeds, as well as in vegetative and senescence organs. A small multigene family encodes isoenzymes of each step of asparagine metabolism in Arabidopsis, except for asparagine aminotransferase encoded by a single gene. The aim of this study is to highlight the structure of the genes and encoded enzyme proteins involved in asparagine metabolic pathways; the regulation and role of different isogenes; and kinetic and physiological properties of encoded enzymes in different tissues and developmental stages. PMID:26628609

  19. The mitochondrial p53 pathway

    PubMed Central

    Vaseva, Angelina V.; Moll, Ute M.

    2010-01-01

    p53 is one of the most mutated tumor suppressors in human cancers and as such has been intensively studied for a long time. p53 is a major orchestrator of the cellular response to a broad array of stress types by regulating apoptosis, cell cycle arrest, senescence, DNA repair and genetic stability. For a long time it was thought that these functions of p53 solely rely on its function as a transcription factor, and numerous p53 target genes have been identified [1]. In the last 8 years however, a novel transcription-independent proapoptotic function mediated by the cytoplasmic pool of p53 has been revealed. p53 participates directly in the intrinsic apoptosis pathway by interacting with the multidomain members of the Bcl-2 family to induce mitochondrial outer membrane permeabilization. Our review will discuss these studies, focusing on recent advances in the field. PMID:19007744

  20. Biosynthetic pathways of ergot alkaloids.

    PubMed

    Gerhards, Nina; Neubauer, Lisa; Tudzynski, Paul; Li, Shu-Ming

    2014-01-01

    Ergot alkaloids are nitrogen-containing natural products belonging to indole alkaloids. The best known producers are fungi of the phylum Ascomycota, e.g., Claviceps, Epichloë, Penicillium and Aspergillus species. According to their structures, ergot alkaloids can be divided into three groups: clavines, lysergic acid amides and peptides (ergopeptines). All of them share the first biosynthetic steps, which lead to the formation of the tetracyclic ergoline ring system (except the simplest, tricyclic compound: chanoclavine). Different modifications on the ergoline ring by specific enzymes result in an abundance of bioactive natural products, which are used as pharmaceutical drugs or precursors thereof. From the 1950s through to recent years, most of the biosynthetic pathways have been elucidated. Gene clusters from several ergot alkaloid producers have been identified by genome mining and the functions of many of those genes have been demonstrated by knock-out experiments or biochemical investigations of the overproduced enzymes. PMID:25513893

  1. [Stress and the kynurenine pathway].

    PubMed

    Majláth, Zsófia; Vécsei, László

    2015-08-30

    The kynurenine pathway is the main route of tryptophan degradation which gives rise to several neuroactive metabolites. Kynurenic acid is an endogenous antagonist of excitatory receptors, which proved to be neuroprotective in the preclinical settings. Kynurenines have been implicated in the neuroendocrine regulatory processes. Stress induces several alterations in the kynurenine metabolism and this process may contribute to the development of stress-related pathological processes. Irritable bowel disease and gastric ulcer are well-known disorders which are related to psychiatric comorbidity and stress. In experimental conditions kynurenic acid proved to be beneficial by reducing inflammatory processes and normalizing microcirculation in the bowel. Further investigations are needed to better understand the relations of stress and the kynurenines, with the aim of developing novel therapeutic tools for stress-related pathologies. PMID:26299831

  2. Mars - Pathway to the stars

    NASA Astrophysics Data System (ADS)

    Angelo, J. A., Jr.; Buden, D.

    Mars has and will continue to play a key role in our exploration and conquest of the Solar System. Within the context of the creation of humanity's extraterrestrial civilization, the major technical features of the following Mars programs are reviewed: the Mars Geoscience/Climatology Orbiter; the Mars Aeronomy Orbiter; the Mars airplane; the Mars Penetrator Network; Mars surface rovers and mobility systems; human exploration of Mars; and permanent Martian bases and settlements. Mars properly explored and utilized opens the way to the resources of the asteroid belt and the outer planets; supports the creation of smart machines for space exploration and exploitation; and encourages the creation of autonomous niches of intelligent life within heliocentric space. All of these developments, in turn, establish the technological pathway for the first interstellar missions.

  3. Biosynthetic Pathways of Ergot Alkaloids

    PubMed Central

    Gerhards, Nina; Neubauer, Lisa; Tudzynski, Paul; Li, Shu-Ming

    2014-01-01

    Ergot alkaloids are nitrogen-containing natural products belonging to indole alkaloids. The best known producers are fungi of the phylum Ascomycota, e.g., Claviceps, Epichloë, Penicillium and Aspergillus species. According to their structures, ergot alkaloids can be divided into three groups: clavines, lysergic acid amides and peptides (ergopeptines). All of them share the first biosynthetic steps, which lead to the formation of the tetracyclic ergoline ring system (except the simplest, tricyclic compound: chanoclavine). Different modifications on the ergoline ring by specific enzymes result in an abundance of bioactive natural products, which are used as pharmaceutical drugs or precursors thereof. From the 1950s through to recent years, most of the biosynthetic pathways have been elucidated. Gene clusters from several ergot alkaloid producers have been identified by genome mining and the functions of many of those genes have been demonstrated by knock-out experiments or biochemical investigations of the overproduced enzymes. PMID:25513893

  4. Steering electrons on moving pathways.

    PubMed

    Beratan, David N; Skourtis, Spiros S; Balabin, Ilya A; Balaeff, Alexander; Keinan, Shahar; Venkatramani, Ravindra; Xiao, Dequan

    2009-10-20

    Electron transfer (ET) reactions provide a nexus among chemistry, biochemistry, and physics. These reactions underpin the "power plants" and "power grids" of bioenergetics, and they challenge us to understand how evolution manipulates structure to control ET kinetics. Ball-and-stick models for the machinery of electron transfer, however, fail to capture the rich electronic and nuclear dynamics of ET molecules: these static representations disguise, for example, the range of thermally accessible molecular conformations. The influence of structural fluctuations on electron-transfer kinetics is amplified by the exponential decay of electron tunneling probabilities with distance, as well as the delicate interference among coupling pathways. Fluctuations in the surrounding medium can also switch transport between coherent and incoherent ET mechanisms--and may gate ET so that its kinetics is limited by conformational interconversion times, rather than by the intrinsic ET time scale. Moreover, preparation of a charge-polarized donor state or of a donor state with linear or angular momentum can have profound dynamical and kinetic consequences. In this Account, we establish a vocabulary to describe how the conformational ensemble and the prepared donor state influence ET kinetics in macromolecules. This framework is helping to unravel the richness of functional biological ET pathways, which have evolved within fluctuating macromolecular structures. The conceptual framework for describing nonadiabatic ET seems disarmingly simple: compute the ensemble-averaged (mean-squared) donor-acceptor (DA) tunneling interaction, , and the Franck-Condon weighted density of states, rho(FC), to describe the rate, (2pi/variant Planck's over 2pi)rho(FC). Modern descriptions of the thermally averaged electronic coupling and of the Franck-Condon factor establish a useful predictive framework in biology, chemistry, and nanoscience. Describing the influence of geometric and

  5. A pathway to academic accreditation

    SciTech Connect

    Seitz, M.R.

    1994-09-01

    The pathways to successfully accrediting programs through a partnership with a local college can be convoluted and offer many dead ends. Those pathways can be made straighter and have fewer false starts by following a plan that has worked. Accreditation of courses and programs can add credibility and prestige to a program. The process can be facilitated by following a basic plan such as the one outlined. The discussion will track the preliminary activities that form the ground work for the beginning of the accreditation process through final approval by a college`s State Board of trustees or regents. On the road to approval, the packaging of courses for presentation, the formulation and composition of an advisory committee, the subsequent use of the advisors, presentation to the faculty committees, the presentation to the college`s governing board of trustees or regents, and final approval by the State Board are covered. An important benefit of accreditation is the formation of a partnership with the local college. Teaming with a local college to provide an accredited certificate in a field of employee training is an excellent opportunity to establish an educational partnership within the local community that will be of benefit to the participating entities. It also represents a training/retraining opportunity in direct support of the US Department of Energy`s current missions of partnership and localization. The accredited modules can be taught where appropriate by college personnel or loaned instructors from the work site. By using the company employees who are working with the topics covered in the modules, the courses are kept up-to-date.

  6. Fuel Dependence of Benzene Pathways

    SciTech Connect

    Zhang, H; Eddings, E; Sarofim, A; Westbrook, C

    2008-07-14

    The relative importance of formation pathways for benzene, an important precursor to soot formation, was determined from the simulation of 22 premixed flames for a wide range of equivalence ratios (1.0 to 3.06), fuels (C{sub 1}-C{sub 12}), and pressures (20 to 760 torr). The maximum benzene concentrations in 15 out of these flames were well reproduced within 30% of the experimental data. Fuel structural properties were found to be critical for benzene production. Cyclohexanes and C{sub 3} and C{sub 4} fuels were found to be among the most productive in benzene formation; and long-chain normal paraffins produce the least amount of benzene. Other properties, such as equivalence ratio and combustion temperatures, were also found to be important in determining the amount of benzene produced in flames. Reaction pathways for benzene formation were examined critically in four premixed flames of structurally different fuels of acetylene, n-decane, butadiene, and cyclohexane. Reactions involving precursors, such as C{sub 3} and C{sub 4} species, were examined. Combination reactions of C{sub 3} species were identified to be the major benzene formation routes with the exception of the cyclohexane flame, in which benzene is formed exclusively from cascading fuel dehydrogenation via cyclohexene and cyclohexadiene intermediates. Acetylene addition makes a minor contribution to benzene formation, except in the butadiene flame where C{sub 4}H{sub 5} radicals are produced directly from the fuel, and in the n-decane flame where C{sub 4}H{sub 5} radicals are produced from large alkyl radical decomposition and H atom abstraction from the resulting large olefins.

  7. Human stretch reflex pathways reexamined

    PubMed Central

    Yavuz, Ş. Utku; Mrachacz-Kersting, Natalie; Sebik, Oğuz; Berna Ünver, M.; Farina, Dario

    2013-01-01

    Reflex responses of tibialis anterior motor units to stretch stimuli were investigated in human subjects. Three types of stretch stimuli were applied (tap-like, ramp-and-hold, and half-sine stretch). Stimulus-induced responses in single motor units were analyzed using the classical technique, which involved building average surface electromyogram (SEMG) and peristimulus time histograms (PSTH) from the discharge times of motor units and peristimulus frequencygrams (PSF) from the instantaneous discharge rates of single motor units. With the use of SEMG and PSTH, the tap-like stretch stimulus induced five separate reflex responses, on average. With the same single motor unit data, the PSF technique indicated that the tap stimulus induced only three reflex responses. Similar to the finding using the tap-like stretch stimuli, ramp-and-hold stimuli induced several peaks and troughs in the SEMG and PSTH. The PSF analyses displayed genuine increases in discharge rates underlying the peaks but not underlying the troughs. Half-sine stretch stimuli induced a long-lasting excitation followed by a long-lasting silent period in SEMG and PSTH. The increase in the discharge rate, however, lasted for the entire duration of the stimulus and continued during the silent period. The results are discussed in the light of the fact that the discharge rate of a motoneuron has a strong positive linear association with the effective synaptic current it receives and hence represents changes in the membrane potential more directly and accurately than the other indirect measures. This study suggests that the neuronal pathway of the human stretch reflex does not include inhibitory pathways. PMID:24225537

  8. Reaction pathways of propene pyrolysis.

    PubMed

    Qu, Yena; Su, Kehe; Wang, Xin; Liu, Yan; Zeng, Qingfeng; Cheng, Laifei; Zhang, Litong

    2010-05-01

    The gas-phase reaction pathways in preparing pyrolytic carbon with propene pyrolysis have been investigated in detail with a total number of 110 transition states and 50 intermediates. The structure of the species was determined with density functional theory at B3PW91/6-311G(d,p) level. The transition states and their linked intermediates were confirmed with frequency and the intrinsic reaction coordinates analyses. The elementary reactions were explored in the pathways of both direct and the radical attacking decompositions. The energy barriers and the reaction energies were determined with accurate model chemistry method at G3(MP2) level after an examination of the nondynamic electronic correlations. The heat capacities and entropies were obtained with statistical thermodynamics. The Gibbs free energies at 298.15 K for all the reaction steps were reported. Those at any temperature can be developed with classical thermodynamics by using the fitted (as a function of temperature) heat capacities. It was found that the most favorable paths are mainly in the radical attacking chain reactions. The chain was proposed with 26 reaction steps including two steps of the initialization of the chain to produce H and CH(3) radicals. For a typical temperature (1200 K) adopted in the experiments, the highest energy barriers were found in the production of C(3) to be 203.4 and 193.7 kJ/mol. The highest energy barriers for the production of C(2) and C were found 174.1 and 181.4 kJ/mol, respectively. These results are comparable with the most recent experimental observation of the apparent activation energy 201.9 +/- 0.6 or 137 +/- 25 kJ/mol. PMID:20082392

  9. Clathrin-Independent Pathways of Endocytosis

    PubMed Central

    Mayor, Satyajit; Parton, Robert G.; Donaldson, Julie G.

    2014-01-01

    There are many pathways of endocytosis at the cell surface that apparently operate at the same time. With the advent of new molecular genetic and imaging tools, an understanding of the different ways by which a cell may endocytose cargo is increasing by leaps and bounds. In this review we explore pathways of endocytosis that occur in the absence of clathrin. These are referred to as clathrin-independent endocytosis (CIE). Here we primarily focus on those pathways that function at the small scale in which some have distinct coats (caveolae) and others function in the absence of specific coated intermediates. We follow the trafficking itineraries of the material endocytosed by these pathways and finally discuss the functional roles that these pathways play in cell and tissue physiology. It is likely that these pathways will play key roles in the regulation of plasma membrane area and tension and also control the availability of membrane during cell migration. PMID:24890511

  10. Premetazoan origin of the Hippo signaling pathway

    PubMed Central

    Sebé-Pedrós, Arnau; Zheng, Yonggang; Ruiz-Trillo, Iñaki; Pan, Duojia

    2012-01-01

    Summary Non-aggregative multicellularity requires strict control of cell number. The Hippo signaling pathway coordinates cell proliferation and apoptosis and is a central regulator of organ size in animals. Recent studies have shown the presence of key members of the Hippo pathway in non-bilaterian animals, but failed to identify this pathway outside Metazoa. Through comparative analyses of recently sequenced holozoan genomes, we show that Hippo pathway components, such as the kinases Hippo and Warts, the co-activator Yorkie and the transcription factor Scalloped, were already present in the unicellular ancestors of animals. Remarkably, functional analysis of Hippo components of the amoeboid holozoan Capsaspora owczarzaki, performed in Drosophila, demonstrate that the growth-regulatory activity of the Hippo pathway is conserved in this unicellular lineage. Our findings show that the Hippo pathway evolved well before the origin of Metazoa and highlight the importance of Hippo signaling as a key developmental mechanism pre-dating the origin of Metazoa. PMID:22832104

  11. Stochasticity in the yeast mating pathway

    NASA Astrophysics Data System (ADS)

    Wang, Hong-Li; Fu, Zheng-Ping; Xu, Xin-Hang; Ouyang, Qi

    2009-05-01

    We report stochastic simulations of the yeast mating signal transduction pathway. The effects of intrinsic and external noise, the influence of cell-to-cell difference in the pathway capacity, and noise propagation in the pathway have been examined. The stochastic temporal behaviour of the pathway is found to be robust to the influence of inherent fluctuations, and intrinsic noise propagates in the pathway in a uniform pattern when the yeasts are treated with pheromones of different stimulus strengths and of varied fluctuations. In agreement with recent experimental findings, extrinsic noise is found to play a more prominent role than intrinsic noise in the variability of proteins. The occurrence frequency for the reactions in the pathway are also examined and a more compact network is obtained by dropping most of the reactions of least occurrence.

  12. Developmental pathways: Sonic hedgehog-Patched-GLI.

    PubMed Central

    Walterhouse, D O; Yoon, J W; Iannaccone, P M

    1999-01-01

    Developmental pathways are networks of genes that act coordinately to establish the body plan. Disruptions of genes in one pathway can have effects in related pathways and may result in serious dysmorphogenesis or cancer. Environmental exposures can be associated with poor pregnancy outcomes, including dysmorphic offspring or children with a variety of diseases. An important goal of environmental science should be reduction of these poor outcomes. This will require an understanding of the genes affected by specific exposures and the consequence of alterations in these genes or their products, which in turn will require an understanding of the pathways critical in development. The ligand Sonic hedgehog, the receptors Patched and Smoothened, and the GLI family of transcription factors represent one such pathway. This pathway illustrates several operating principles important in the consideration of developmental consequences of environmental exposures to toxins. Images Figure 1 Figure 2 PMID:10064544

  13. Pathway-Based Functional Analysis of Metagenomes

    NASA Astrophysics Data System (ADS)

    Bercovici, Sivan; Sharon, Itai; Pinter, Ron Y.; Shlomi, Tomer

    Metagenomic data enables the study of microbes and viruses through their DNA as retrieved directly from the environment in which they live. Functional analysis of metagenomes explores the abundance of gene families, pathways, and systems, rather than their taxonomy. Through such analysis researchers are able to identify those functional capabilities most important to organisms in the examined environment. Recently, a statistical framework for the functional analysis of metagenomes was described that focuses on gene families. Here we describe two pathway level computational models for functional analysis that take into account important, yet unaddressed issues such as pathway size, gene length and overlap in gene content among pathways. We test our models over carefully designed simulated data and propose novel approaches for performance evaluation. Our models significantly improve over current approach with respect to pathway ranking and the computations of relative abundance of pathways in environments.

  14. Manipulating Quantum Pathways on the Fly

    NASA Astrophysics Data System (ADS)

    Rey-de-Castro, Roberto; Leghtas, Zaki; Rabitz, Herschel

    2013-05-01

    The expectation value of a quantum system observable can be written as a sum over interfering pathway amplitudes. In this Letter, we demonstrate for the fist time adaptive manipulation of quantum pathways using the Hamiltonian encoding-observable decoding (HE-OD) technique. The principles of HE-OD are illustrated for population transfer in atomic rubidium using shaped femtosecond laser pulses. The ability to manipulate multiple pathway amplitudes is of fundamental importance in all quantum control applications.

  15. Effects of PDT on the endocytic pathway

    NASA Astrophysics Data System (ADS)

    Kessel, David

    2010-02-01

    Two lines of evidence point to an early effect of photodamage on membrane trafficking. [1] Internalization of a fluorescent probe for hydrophobic membrane loci was impaired by prior photodamage. [2] Interference with the endocytic pathway by the PI-3 kinase antagonist wortmannin led to accumulation of cytoplasmic vacuoles suggesting a block in the recycling of plasma membrane components. Prior photodamage blocked this pathway so that no vacuoles were formed upon exposure of cells to wortmannin. In a murine hepatoma line, the endocytic pathway was preferentially sensitive to lysosomal photodamage. The role of photodamage to the endocytic pathway as a factor in PDT efficacy remains to be assessed.

  16. Photodegradation Pathways in Arid Ecosystems

    NASA Astrophysics Data System (ADS)

    King, J. Y.; Lin, Y.; Adair, E. C.; Brandt, L.; Carbone, M. S.

    2013-12-01

    Recent interest in improving our understanding of decomposition patterns in arid and semi-arid ecosystems and under potentially drier future conditions has led to a flurry of research related to abiotic degradation processes. Oxidation of organic matter by solar radiation (photodegradation) is one abiotic degradation process that contributes significantly to litter decomposition rates. Our meta-analysis results show that increasing solar radiation exposure corresponds to an average increase of 23% in litter mass loss rate with large variation among studies associated primarily with environmental and litter chemistry characteristics. Laboratory studies demonstrate that photodegradation results in CO2 emissions. Indirect estimates suggest that photodegradation could account for as much as 60% of ecosystem CO2 emissions from dry ecosystems, but these CO2 fluxes have not been measured in intact ecosystems. The current data suggest that photodegradation is important, not only for understanding decomposition patterns, but also for modeling organic matter turnover and ecosystem C cycling. However, the mechanisms by which photodegradation operates, along with their environmental and litter chemistry controls, are still poorly understood. Photodegradation can directly influence decomposition rates and ecosystem CO2 flux via photochemical mineralization. It can also indirectly influence biotic decomposition rates by facilitating microbial degradation through breakdown of more recalcitrant compounds into simpler substrates or by suppressing microbial activity directly. All of these pathways influence the decomposition process, but the relative importance of each is uncertain. Furthermore, a specific suite of controls regulates each of these pathways (e.g., environmental conditions such as temperature and relative humidity; physical environment such as canopy architecture and contact with soil; and litter chemistry characteristics such as lignin and cellulose content), and

  17. The pathway ontology – updates and applications

    PubMed Central

    2014-01-01

    Background The Pathway Ontology (PW) developed at the Rat Genome Database (RGD), covers all types of biological pathways, including altered and disease pathways and captures the relationships between them within the hierarchical structure of a directed acyclic graph. The ontology allows for the standardized annotation of rat, and of human and mouse genes to pathway terms. It also constitutes a vehicle for easy navigation between gene and ontology report pages, between reports and interactive pathway diagrams, between pathways directly connected within a diagram and between those that are globally related in pathway suites and suite networks. Surveys of the literature and the development of the Pathway and Disease Portals are important sources for the ongoing development of the ontology. User requests and mapping of pathways in other databases to terms in the ontology further contribute to increasing its content. Recently built automated pipelines use the mapped terms to make available the annotations generated by other groups. Results The two released pipelines – the Pathway Interaction Database (PID) Annotation Import Pipeline and the Kyoto Encyclopedia of Genes and Genomes (KEGG) Annotation Import Pipeline, make available over 7,400 and 31,000 pathway gene annotations, respectively. Building the PID pipeline lead to the addition of new terms within the signaling node, also augmented by the release of the RGD “Immune and Inflammatory Disease Portal” at that time. Building the KEGG pipeline lead to a substantial increase in the number of disease pathway terms, such as those within the ‘infectious disease pathway’ parent term category. The ‘drug pathway’ node has also seen increases in the number of terms as well as a restructuring of the node. Literature surveys, disease portal deployments and user requests have contributed and continue to contribute additional new terms across the ontology. Since first presented, the content of PW has increased by

  18. Orexin: Pathways to obesity resistance?

    PubMed Central

    Butterick, Tammy A.; Billington, Charles J.; Kotz, Catherine M.; Nixon, Joshua P.

    2016-01-01

    Obesity has increased in prevalence worldwide, attributed in part to the influences of an obesity-promoting environment and genetic factors. While obesity and overweight increasingly seem to be the norm, there remain individuals who resist obesity. We present here an overview of data supporting the idea that hypothalamic neuropeptide orexin A (OXA; hypocretin 1) may be a key component of brain mechanisms underlying obesity resistance. Prior work with models of obesity and obesity resistance in rodents has shown that increased orexin and/or orexin sensitivity is correlated with elevated spontaneous physical activity (SPA), and that orexin-induced SPA contributes to obesity resistance via increased non-exercise activity thermogenesis (NEAT). However, central hypothalamic orexin signaling mechanisms that regulate SPA remain undefined. Our ongoing studies and work of others support the hypothesis that one such mechanism may be upregulation of a hypoxia-inducible factor 1 alpha (HIF-1α)-dependent pathway, suggesting that orexin may promote obesity resistance both by increasing SPA and by influencing the metabolic state of orexin-responsive hypothalamic neurons. We discuss potential mechanisms based on both animal and in vitro pharmacological studies, in the context of elucidating potential molecular targets for obesity prevention and therapy. PMID:24005942

  19. Two pathways ensuring social harmony

    NASA Astrophysics Data System (ADS)

    Konrad, Matthias; Pamminger, Tobias; Foitzik, Susanne

    2012-08-01

    Reproductive division of labour is a characteristic trait of social insects. The dominant reproductive individual, often the queen, uses chemical communication and/or behaviour to maintain her social status. Queens of many social insects communicate their fertility status via cuticle-bound substances. As these substances usually possess a low volatility, their range in queen-worker communication is potentially limited. Here, we investigate the range and impact of behavioural and chemical queen signals on workers of the ant Temnothorax longispinosus. We compared the behaviour and ovary development of workers subjected to three different treatments: workers with direct chemical and physical contact to the queen, those solely under the influence of volatile queen substances and those entirely separated from the queen. In addition to short-ranged queen signals preventing ovary development in workers, we discovered a novel secondary pathway influencing worker behaviour. Workers with no physical contact to the queen, but exposed to volatile substances, started to develop their ovaries, but did not change their behaviour compared to workers in direct contact to the queen. In contrast, workers in queen-separated groups showed both increased ovary development and aggressive dominance interactions. We conclude that T. longispinosus queens influence worker ovary development and behaviour via two independent signals, both ensuring social harmony within the colony.

  20. Inconsistent pathways of household waste

    SciTech Connect

    Dahlen, Lisa Aberg, Helena; Lagerkvist, Anders; Berg, Per E.O.

    2009-06-15

    The aim of this study was to provide policy-makers and waste management planners with information about how recycling programs affect the quantities of specific materials recycled and disposed of. Two questions were addressed: which factors influence household waste generation and pathways? and how reliable are official waste data? Household waste flows were studied in 35 Swedish municipalities, and a wide variation in the amount of waste per capita was observed. When evaluating the effect of different waste collection policies, it was found to be important to identify site-specific factors influencing waste generation. Eleven municipal variables were investigated in an attempt to explain the variation. The amount of household waste per resident was higher in populous municipalities and when net commuting was positive. Property-close collection of dry recyclables led to increased delivery of sorted metal, plastic and paper packaging. No difference was seen in the amount of separated recyclables per capita when weight-based billing for the collection of residual waste was applied, but the amount of residual waste was lower. Sixteen sources of error in official waste statistics were identified and the results of the study emphasize the importance of reliable waste generation and composition data to underpin waste management policies.

  1. Glycosyltransferase efficiently controls phenylpropanoid pathway

    PubMed Central

    Aksamit-Stachurska, Anna; Korobczak-Sosna, Alina; Kulma, Anna; Szopa, Jan

    2008-01-01

    Background In a previous study, anthocyanin levels in potato plants were increased by manipulating genes connected with the flavonoid biosynthesis pathway. However, starch content and tuber yield were dramatically reduced in the transgenic plants, which over-expressed dihydroflavonol reductase (DFR). Results Transgenic plants over-expressing dihydroflavonol reductase (DFR) were subsequently transformed with the cDNA coding for the glycosyltransferase (UGT) of Solanum sogarandinum in order to obtain plants with a high anthocyanin content without reducing tuber yield and quality. Based on enzyme studies, the recombinant UGT is a 7-O-glycosyltransferase whose natural substrates include both anthocyanidins and flavonols such as kaempferol and quercetin. In the super-transformed plants, tuber production was much higher than in the original transgenic plants bearing only the transgene coding for DFR, and was almost the same as in the control plants. The anthocyanin level was lower than in the initial plants, but still higher than in the control plants. Unexpectedly, the super-transformed plants also produced large amounts of kaempferol, chlorogenic acid, isochlorogenic acid, sinapic acid and proanthocyanins. Conclusion In plants over-expressing both the transgene for DFR and the transgene for UGT, the synthesis of phenolic acids was diverted away from the anthocyanin branch. This represents a novel approach to manipulating phenolic acids synthesis in plants. PMID:18321380

  2. Racial discrimination & health: pathways & evidence.

    PubMed

    Ahmed, Ameena T; Mohammed, Selina A; Williams, David R

    2007-10-01

    This review provides an overview of the existing empirical research of the multiple ways by which discrimination can affect health. Institutional mechanisms of discrimination such as restricting marginalized groups to live in undesirable residential areas can have deleterious health consequences by limiting socio-economic status (SES) and creating health-damaging conditions in residential environments. Discrimination can also adversely affect health through restricting access to desirable services such as medical care and creating elevated exposure to traditional stressors such as unemployment and financial strain. Central to racism is an ideology of inferiority that can adversely affect non-dominant groups because some members of marginalized populations will accept as true the dominant society's ideology of their group's inferiority. Limited empirical research indicates that internalized racism is inversely related to health. In addition, the existence of these negative stereotypes can lead dominant group members to consciously and unconsciously discriminate against the stigmatized. An overview of the growing body of research examining the ways in which psychosocial stress generated by subjective experiences of discrimination can affect health is also provided. We review the evidence from the United States and other societies that suggest that the subjective experience of discrimination can adversely affect health and health enhancing behaviours. Advancing our understanding of the relationship between discrimination and health requires improved assessment of the phenomenon of discrimination and increased attention to identifying the psychosocial and biological pathways that may link exposure to discrimination to health status. PMID:18032807

  3. Nicotinic receptors in addiction pathways.

    PubMed

    Leslie, Frances M; Mojica, Celina Y; Reynaga, Daisy D

    2013-04-01

    Neuronal nicotinic acetylcholine receptors (nAChRs) are ligand-gated ion channels that consist of pentameric combinations of α and β subunits. These receptors are widely distributed throughout the brain and are highly expressed in addiction circuitry. The role of nAChRs in regulating neuronal activity and motivated behavior is complex and varies both in and among brain regions. The rich diversity of central nAChRs has hampered the characterization of their structure and function with use of classic pharmacological techniques. However, recent molecular approaches using null mutant mice with specific regional lentiviral re-expression, in combination with neuroanatomical and electrophysiological techniques, have allowed the elucidation of the influence of different nAChR types on neuronal circuit activity and behavior. This review will address the influence of nAChRs on limbic dopamine circuitry and the medial habenula-interpeduncular nucleus complex, which are critical mediators of reinforced behavior. Characterization of the mechanisms underlying regulation of addiction pathways by endogenous cholinergic transmission and by nicotine may lead to the identification of new therapeutic targets for treating tobacco dependence and other addictions. PMID:23247824

  4. Greater Capital Region Career Pathways Initiative

    ERIC Educational Resources Information Center

    Workforce Strategy Center, 2005

    2005-01-01

    This case study documents the activities of the Greater Capital Region Career Pathways Initiative (CPI), a partnership to develop regional, demand driven career pathways for the biotechnology sector in and around Albany, NY. This document is written for three primary audiences. First, it provides a report to the New York State Department of Labor…

  5. Implementing Guided Pathways: Tips and Tools

    ERIC Educational Resources Information Center

    Bailey, Thomas; Jaggars, Shanna Smith; Jenkins, Davis

    2015-01-01

    A growing number of community colleges and four-year universities are seeking to improve student outcomes by redesigning academic programs and student support services following the guided pathways approach. These institutions are mapping out highly structured, educationally coherent program pathways for students to follow by starting with the end…

  6. Quicker cancer care: reshaping patient pathways.

    PubMed

    Towler, Lucy

    2009-07-01

    A new pathway has been devised for patients with ovarian cancer who attend a day-care unit for chemotherapy. This pathway, which is provided by nurses and doctors, has reduced patients' waiting time for treatment. Its implementation shows, therefore, that good clinical leadership can effect positive change. PMID:19639906

  7. Diversifying Carotenoid Biosynthetic Pathways by Directed Evolution

    PubMed Central

    Umeno, Daisuke; Tobias, Alexander V.; Arnold, Frances H.

    2005-01-01

    Microorganisms and plants synthesize a diverse array of natural products, many of which have proven indispensable to human health and well-being. Although many thousands of these have been characterized, the space of possible natural products—those that could be made biosynthetically—remains largely unexplored. For decades, this space has largely been the domain of chemists, who have synthesized scores of natural product analogs and have found many with improved or novel functions. New natural products have also been made in recombinant organisms, via engineered biosynthetic pathways. Recently, methods inspired by natural evolution have begun to be applied to the search for new natural products. These methods force pathways to evolve in convenient laboratory organisms, where the products of new pathways can be identified and characterized in high-throughput screening programs. Carotenoid biosynthetic pathways have served as a convenient experimental system with which to demonstrate these ideas. Researchers have mixed, matched, and mutated carotenoid biosynthetic enzymes and screened libraries of these “evolved” pathways for the emergence of new carotenoid products. This has led to dozens of new pathway products not previously known to be made by the assembled enzymes. These new products include whole families of carotenoids built from backbones not found in nature. This review details the strategies and specific methods that have been employed to generate new carotenoid biosynthetic pathways in the laboratory. The potential application of laboratory evolution to other biosynthetic pathways is also discussed. PMID:15755953

  8. Fuel Pathway Integration Technical Team Roadmap

    SciTech Connect

    2013-06-01

    The Fuel Pathway Integration Technical Team (FPITT) supports the U.S. DRIVE Partnership (the Partnership) in the identification and evaluation of implementation scenarios for fuel cell technology pathways, including hydrogen and fuel cell electric vehicles in the transportation sector, both during a transition period and in the long term.

  9. Women's Work Pathways Across the Life Course.

    PubMed

    Damaske, Sarah; Frech, Adrianne

    2016-04-01

    Despite numerous changes in women's employment in the latter half of the twentieth century, women's employment continues to be uneven and stalled. Drawing from data on women's weekly work hours in the National Longitudinal Survey of Youth (NLSY79), we identify significant inequality in women's labor force experiences across adulthood. We find two pathways of stable full-time work for women, three pathways of part-time employment, and a pathway of unpaid labor. A majority of women follow one of the two full-time work pathways, while fewer than 10% follow a pathway of unpaid labor. Our findings provide evidence of the lasting influence of work-family conflict and early socioeconomic advantages and disadvantages on women's work pathways. Indeed, race, poverty, educational attainment, and early family characteristics significantly shaped women's work careers. Work-family opportunities and constraints also were related to women's work hours, as were a woman's gendered beliefs and expectations. We conclude that women's employment pathways are a product of both their resources and changing social environment as well as individual agency. Significantly, we point to social stratification, gender ideologies, and work-family constraints, all working in concert, as key explanations for how women are "tracked" onto work pathways from an early age. PMID:27001314

  10. Improving College System Pathways: Project Highlights Reports

    ERIC Educational Resources Information Center

    Colleges Ontario, 2008

    2008-01-01

    In 2006, Ontario's colleges received funding from the Ministry of Training, Colleges and Universities for the Improving College System Pathways Project. The project goals were to significantly increase educational pathways within and between colleges by developing a clearer understanding of student mobility within the system; to identify the scope…

  11. Modeling biochemical pathways in the gene ontology

    PubMed Central

    Hill, David P.; D’Eustachio, Peter; Berardini, Tanya Z.; Mungall, Christopher J.; Renedo, Nikolai; Blake, Judith A.

    2016-01-01

    The concept of a biological pathway, an ordered sequence of molecular transformations, is used to collect and represent molecular knowledge for a broad span of organismal biology. Representations of biomedical pathways typically are rich but idiosyncratic presentations of organized knowledge about individual pathways. Meanwhile, biomedical ontologies and associated annotation files are powerful tools that organize molecular information in a logically rigorous form to support computational analysis. The Gene Ontology (GO), representing Molecular Functions, Biological Processes and Cellular Components, incorporates many aspects of biological pathways within its ontological representations. Here we present a methodology for extending and refining the classes in the GO for more comprehensive, consistent and integrated representation of pathways, leveraging knowledge embedded in current pathway representations such as those in the Reactome Knowledgebase and MetaCyc. With carbohydrate metabolic pathways as a use case, we discuss how our representation supports the integration of variant pathway classes into a unified ontological structure that can be used for data comparison and analysis. PMID:27589964

  12. Pathways to Success for Michigan's Opportunity Youth

    ERIC Educational Resources Information Center

    American Youth Policy Forum, 2015

    2015-01-01

    Each young person must navigate his/her own pathway into and through postsecondary education and the workforce to long-term success personalized to his/her own unique needs and desires. The pathway to long-term success is often articulated as a straight road through K-12 education into postsecondary education (either academic or technical…

  13. Optic pathway degeneration in Japanese black cattle

    PubMed Central

    CHIBA, Shiori; FUNATO, Shingo; HORIUCHI, Noriyuki; MATSUMOTO, Kotaro; INOKUMA, Hisashi; FURUOKA, Hidefumi; KOBAYASHI, Yoshiyasu

    2014-01-01

    Degeneration of the optic pathway has been reported in various animal species including cattle. We experienced a case of bilateral optic tract degeneration characterized by severe gliosis in a Japanese black cattle without any obvious visual defects. To evaluate the significance, pathological nature and pathogenesis of the lesions, we examined the optic pathway in 60 cattle (41 Japanese black, 13 Holstein and 6 crossbreed) with or without ocular abnormalities. None of these animals had optic canal stenosis. Degenerative changes with severe gliosis in the optic pathway, which includes the optic nerve, optic chiasm and optic tract, were only observed in 8 Japanese black cattle with or without ocular abnormalities. Furthermore, strong immunoreactivity of glial fibrillary acidic protein was observed in the retinal stratum opticum and ganglion cell layer in all 5 cattle in which the optic pathway lesions could be examined. As etiological research, we also examined whether the concentrations of vitamin A and vitamin B12 or bovine viral diarrhea virus (BVDV) infection was associated with optic pathway degeneration. However, our results suggested that the observed optic pathway degeneration was probably not caused by these factors. These facts indicate the presence of optic pathway degeneration characterized by severe gliosis that has never been reported in cattle without bilateral compressive lesions in the optic pathway or bilateral severe retinal atrophy. PMID:25421501

  14. The Career Pathways How-To Guide

    ERIC Educational Resources Information Center

    Jenkins, Davis; Spence, Christopher

    2006-01-01

    Career pathways is a series of connected education and training programs and support services that enable individuals to secure employment within a specific industry or occupational sector, and to advance over time to successively higher levels of education and employment in that sector. Career pathways are distinct from most educational efforts…

  15. Modeling biochemical pathways in the gene ontology.

    PubMed

    Hill, David P; D'Eustachio, Peter; Berardini, Tanya Z; Mungall, Christopher J; Renedo, Nikolai; Blake, Judith A

    2016-01-01

    The concept of a biological pathway, an ordered sequence of molecular transformations, is used to collect and represent molecular knowledge for a broad span of organismal biology. Representations of biomedical pathways typically are rich but idiosyncratic presentations of organized knowledge about individual pathways. Meanwhile, biomedical ontologies and associated annotation files are powerful tools that organize molecular information in a logically rigorous form to support computational analysis. The Gene Ontology (GO), representing Molecular Functions, Biological Processes and Cellular Components, incorporates many aspects of biological pathways within its ontological representations. Here we present a methodology for extending and refining the classes in the GO for more comprehensive, consistent and integrated representation of pathways, leveraging knowledge embedded in current pathway representations such as those in the Reactome Knowledgebase and MetaCyc. With carbohydrate metabolic pathways as a use case, we discuss how our representation supports the integration of variant pathway classes into a unified ontological structure that can be used for data comparison and analysis. PMID:27589964

  16. The metabolic pathway collection: an update.

    PubMed

    Selkov, E; Galimova, M; Goryanin, I; Gretchkin, Y; Ivanova, N; Komarov, Y; Maltsev, N; Mikhailova, N; Nenashev, V; Overbeek, R; Panyushkina, E; Pronevitch, L; Selkov, E

    1997-01-01

    The Metabolic Pathway Collection from EMP is an extraction of data from the larger Enzymes and Metabolic Pathways database (EMP). This extraction has been made publicly available in the hope that others will find it useful for a variety of purposes. The original release in October 1995 contained 1814 distinct pathways. The current collection contains 2180. Metabolic reconstructions for the first completely sequenced organisms-Haemophilus influenzae,Mycoplasma genitalium,Saccharomyces cerevisiaeandMethanococcus janaschii-are all included in the current release. All of the pathways in the collections are available as ASCII files in the form generated by the main curator, Evgeni Selkov. In addition, we are offering a more structured encoding of a subset of the collection; our initial release of this subcollection includes all of the pathways inMycoplasma genitalium, and we ultimately intend to offer the entire collection in this form as well. PMID:9016500

  17. Proton Pathways in Green Fluorescence Protein

    PubMed Central

    Agmon, Noam

    2005-01-01

    Proton pathways in green fluorescent protein (GFP) are more extended than previously reported. In the x-ray data of wild-type GFP, a two-step exit pathway exists from the active site to the protein surface, controlled by a threonine switch. A proton entry pathway begins at a glutamate-lysine cluster around Glu-5, and extends all the way to the buried Glu-222 near the active site. This structural evidence suggests that GFP may function as a portable light-driven proton-pump, with proton emitted in the excited state through the switchable exit pathway, and replenished from Glu-222 and the Glu-5 entry pathway in the ground state. PMID:15681647

  18. Pathway Analysis Incorporating Protein-Protein Interaction Networks Identified Candidate Pathways for the Seven Common Diseases.

    PubMed

    Lin, Peng-Lin; Yu, Ya-Wen; Chung, Ren-Hua

    2016-01-01

    Pathway analysis has become popular as a secondary analysis strategy for genome-wide association studies (GWAS). Most of the current pathway analysis methods aggregate signals from the main effects of single nucleotide polymorphisms (SNPs) in genes within a pathway without considering the effects of gene-gene interactions. However, gene-gene interactions can also have critical effects on complex diseases. Protein-protein interaction (PPI) networks have been used to define gene pairs for the gene-gene interaction tests. Incorporating the PPI information to define gene pairs for interaction tests within pathways can increase the power for pathway-based association tests. We propose a pathway association test, which aggregates the interaction signals in PPI networks within a pathway, for GWAS with case-control samples. Gene size is properly considered in the test so that genes do not contribute more to the test statistic simply due to their size. Simulation studies were performed to verify that the method is a valid test and can have more power than other pathway association tests in the presence of gene-gene interactions within a pathway under different scenarios. We applied the test to the Wellcome Trust Case Control Consortium GWAS datasets for seven common diseases. The most significant pathway is the chaperones modulate interferon signaling pathway for Crohn's disease (p-value = 0.0003). The pathway modulates interferon gamma, which induces the JAK/STAT pathway that is involved in Crohn's disease. Several other pathways that have functional implications for the seven diseases were also identified. The proposed test based on gene-gene interaction signals in PPI networks can be used as a complementary tool to the current existing pathway analysis methods focusing on main effects of genes. An efficient software implementing the method is freely available at http://puppi.sourceforge.net. PMID:27622767

  19. Pathway-PDT: a flexible pathway analysis tool for nuclear families

    PubMed Central

    2013-01-01

    Background Pathway analysis based on Genome-Wide Association Studies (GWAS) data has become popular as a secondary analysis strategy. Although many pathway analysis tools have been developed for case–control studies, there is no tool that can use all information from raw genotypes in general nuclear families. We developed Pathway-PDT, which uses the framework of Pedigree Disequilibrium Test (PDT) for general family data, to perform pathway analysis based on raw genotypes in family-based GWAS. Results Simulation results showed that Pathway-PDT is more powerful than the p-value based method, ALIGATOR. Pathway-PDT also can be more powerful than the PLINK set-based test when analyzing general nuclear families with multiple siblings or missing parents. Additionally, Pathway-PDT has a flexible and convenient user interface, which allows users to modify their analysis parameters as well as to apply various types of gene and pathway definitions. Conclusions The Pathway-PDT method is implemented in C++ with POSIX threads and is computationally feasible for pathway analysis with large scale family GWAS datasets. The Windows binary along with Makefile and source codes for the Linux are available at https://sourceforge.net/projects/pathway-pdt/. PMID:24006871

  20. Hippo pathway regulation of gastrointestinal tissues.

    PubMed

    Yu, Fa-Xing; Meng, Zhipeng; Plouffe, Steven W; Guan, Kun-Liang

    2015-01-01

    The Hippo pathway plays a crucial role in regulating tissue homeostasis and organ size, and its deregulation is frequently observed in human cancer. Yap is the major effector of and is inhibited by the Hippo pathway. In mouse model studies, inducible Yap expression in multiple tissues results in organ overgrowth. In the liver, knockout of upstream Hippo pathway components or transgenic expression of Yap leads to liver enlargement and hepatocellular carcinoma. In the small intestine or colon, deletion of upstream Hippo pathway components also results in expansion of intestinal progenitor cells and eventual development of adenomas. Genetic deletion of Yap in the intestine does not change the intestinal structure, but Yap is essential for intestinal repair upon certain types of tissue injury. The function of the Hippo pathway has also been studied in other gastrointestinal tissues, including the pancreas and stomach. Here we provide a brief overview of the Hippo pathway and discuss the physiological and pathological functions of this tumor suppressor pathway in gastrointestinal tissues. PMID:25293527

  1. Dysregulation of the mevalonate pathway promotes transformation

    PubMed Central

    Clendening, James W.; Pandyra, Aleks; Boutros, Paul C.; Ghamrasni, Samah El; Khosravi, Fereshteh; Trentin, Grace A.; Martirosyan, Anna; Hakem, Anne; Hakem, Razqallah; Jurisica, Igor; Penn, Linda Z.

    2010-01-01

    The importance of cancer metabolism has been appreciated for many years, but the intricacies of how metabolic pathways interconnect with oncogenic signaling are not fully understood. With a clear understanding of how metabolism contributes to tumorigenesis, we will be better able to integrate the targeting of these fundamental biochemical pathways into patient care. The mevalonate (MVA) pathway, paced by its rate-limiting enzyme, hydroxymethylglutaryl coenzyme A reductase (HMGCR), is required for the generation of several fundamental end-products including cholesterol and isoprenoids. Despite years of extensive research from the perspective of cardiovascular disease, the contribution of a dysregulated MVA pathway to human cancer remains largely unexplored. We address this issue directly by showing that dysregulation of the MVA pathway, achieved by ectopic expression of either full-length HMGCR or its novel splice variant, promotes transformation. Ectopic HMGCR accentuates growth of transformed and nontransformed cells under anchorage-independent conditions or as xenografts in immunocompromised mice and, importantly, cooperates with RAS to drive the transformation of primary mouse embryonic fibroblasts cells. We further explore whether the MVA pathway may play a role in the etiology of human cancers and show that high mRNA levels of HMGCR and additional MVA pathway genes correlate with poor prognosis in a meta-analysis of six microarray datasets of primary breast cancer. Taken together, our results suggest that HMGCR is a candidate metabolic oncogene and provide a molecular rationale for further exploring the statin family of HMGCR inhibitors as anticancer agents. PMID:20696928

  2. Targeting the Apoptosis Pathway in Hematologic Malignancies

    PubMed Central

    Zaman, Shadia; Wang, Rui; Gandhi, Varsha

    2014-01-01

    Apoptosis is a cell death program that is well-orchestrated for normal tissue homeostasis and for removal of damaged, old, or infected cells. It is regulated by intrinsic and extrinsic pathways. The intrinsic pathway responds to signals such as ultraviolet radiation or DNA damage and activates “executioner” caspases through a mitochondria-dependent pathway. The extrinsic pathway is activated by death signals induced, for example, by an infection that activates the immune system or receptor-mediated pathways. The extrinsic pathway signals also cascade down to executioner caspases that cleave target proteins and lead to cell death. Strict control of cellular apoptosis is important for the hematopoietic system as it has a high turnover rate. However, the apoptosis program is often deregulated in hematologic malignancies leading to the accumulation of malignant cells. Therefore, apoptosis pathways have been identified for development of anticancer therapeutics. We review here the proteins that have been targeted for anticancer drug development in hematologic malignancies. These include BCL-2 family proteins, death ligands and receptors, inhibitor of apoptosis family proteins, and caspases. Except for caspase activators, drugs that target each of these classes of proteins have advanced into clinical trials. PMID:24295132

  3. Brain evolution by brain pathway duplication

    PubMed Central

    Chakraborty, Mukta; Jarvis, Erich D.

    2015-01-01

    Understanding the mechanisms of evolution of brain pathways for complex behaviours is still in its infancy. Making further advances requires a deeper understanding of brain homologies, novelties and analogies. It also requires an understanding of how adaptive genetic modifications lead to restructuring of the brain. Recent advances in genomic and molecular biology techniques applied to brain research have provided exciting insights into how complex behaviours are shaped by selection of novel brain pathways and functions of the nervous system. Here, we review and further develop some insights to a new hypothesis on one mechanism that may contribute to nervous system evolution, in particular by brain pathway duplication. Like gene duplication, we propose that whole brain pathways can duplicate and the duplicated pathway diverge to take on new functions. We suggest that one mechanism of brain pathway duplication could be through gene duplication, although other mechanisms are possible. We focus on brain pathways for vocal learning and spoken language in song-learning birds and humans as example systems. This view presents a new framework for future research in our understanding of brain evolution and novel behavioural traits. PMID:26554045

  4. The Wnt pathway: emerging anticancer strategies.

    PubMed

    Gupta, Aman; Verma, Anukriti; Mishra, Ashutosh K; Wadhwa, Gulshan; Sharma, Sanjeev K; Jain, Chakresh K

    2013-05-01

    The canonical Wnt cascade has emerged as a critical regulator of cancer cells. Activation of the Wnt signaling pathway has also been associated with stem cell, thus raising the possibility of its role in embryogenesis and in the proliferation of malignant cancer cells. Wnt pathway has been reported to be involved in normal physiological processes in adult animals and integrally associated with cancer cell growth and maintenance, thus has been harnessed to devise strategies for anticancer therapy. The presence or absence of some members in this pathway, such as β-catenin, Axin or APC, has been found to involve in different types of tumors in human beings. Dysregulation of the canonical Wnt/β-catenin signaling pathway, mostly by inactivating mutations of the APC tumor suppressor, or oncogenic mutations of β-catenin, has been implicated in colorectal tumorigenesis. Further, elevated levels of β-catenin protein, a hallmark of activated canonical Wnt pathway, have been significantly observed in common forms of human malignancies, indicating that activation of the Wnt pathway may play an important role in tumor development and hence could be a crucial consideration for drug development. The paper discusses the potential therapeutic and diagnostic strategies directing on Wnt pathways on the basis of recent patents and their analysis. PMID:23432158

  5. Brain evolution by brain pathway duplication.

    PubMed

    Chakraborty, Mukta; Jarvis, Erich D

    2015-12-19

    Understanding the mechanisms of evolution of brain pathways for complex behaviours is still in its infancy. Making further advances requires a deeper understanding of brain homologies, novelties and analogies. It also requires an understanding of how adaptive genetic modifications lead to restructuring of the brain. Recent advances in genomic and molecular biology techniques applied to brain research have provided exciting insights into how complex behaviours are shaped by selection of novel brain pathways and functions of the nervous system. Here, we review and further develop some insights to a new hypothesis on one mechanism that may contribute to nervous system evolution, in particular by brain pathway duplication. Like gene duplication, we propose that whole brain pathways can duplicate and the duplicated pathway diverge to take on new functions. We suggest that one mechanism of brain pathway duplication could be through gene duplication, although other mechanisms are possible. We focus on brain pathways for vocal learning and spoken language in song-learning birds and humans as example systems. This view presents a new framework for future research in our understanding of brain evolution and novel behavioural traits. PMID:26554045

  6. Driving and dementia: a clinical decision pathway

    PubMed Central

    Carter, Kirsty; Monaghan, Sophie; O'Brien, John; Teodorczuk, Andrew; Mosimann, Urs; Taylor, John-Paul

    2015-01-01

    Objective This study aimed to develop a pathway to bring together current UK legislation, good clinical practice and appropriate management strategies that could be applied across a range of healthcare settings. Methods The pathway was constructed by a multidisciplinary clinical team based in a busy Memory Assessment Service. A process of successive iteration was used to develop the pathway, with input and refinement provided via survey and small group meetings with individuals from a wide range of regional clinical networks and diverse clinical backgrounds as well as discussion with mobility centres and Forum of Mobility Centres, UK. Results We present a succinct clinical pathway for patients with dementia, which provides a decision-making framework for how health professionals across a range of disciplines deal with patients with dementia who drive. Conclusions By integrating the latest guidance from diverse roles within older people's health services and key experts in the field, the resulting pathway reflects up-to-date policy and encompasses differing perspectives and good practice. It is potentially a generalisable pathway that can be easily adaptable for use internationally, by replacing UK legislation for local regulations. A limitation of this pathway is that it does not address the concern of mild cognitive impairment and how this condition relates to driving safety. © 2014 The Authors. International Journal of Geriatric Psychiatry published by John Wiley & Sons, Ltd. PMID:24865643

  7. Predicting microbial nitrogen pathways from basic principles.

    PubMed

    van de Leemput, Ingrid A; Veraart, Annelies J; Dakos, Vasilis; de Klein, Jeroen J M; Strous, Marc; Scheffer, Marten

    2011-06-01

    Nitrogen compounds are transformed by a complicated network of competing geochemical processes or microbial pathways, each performed by a different ecological guild of microorganisms. Complete experimental unravelling of this network requires a prohibitive experimental effort. Here we present a simple model that predicts relative rates of hypothetical nitrogen pathways, based only on the stoichiometry and energy yield of the performed redox reaction, assuming competition for resources between alternative pathways. Simulating competing pathways in hypothetical freshwater and marine sediment situations, we surprisingly found that much of the variation observed in nature can simply be predicted from these basic principles. Investigating discrepancies between observations and predictions led to two important biochemical factors that may create barriers for the viability of pathways: enzymatic costs for long pathways and high ammonium activation energy. We hypothesize that some discrepancies can be explained by non-equilibrium dynamics. The model predicted a pathway that has not been discovered in nature yet: the dismutation of nitrite to the level of nitrate and dinitrogen gas. PMID:21429064

  8. Metabolic pathways of ochratoxin A.

    PubMed

    Wu, Qinghua; Dohnal, Vlastimil; Huang, Lingli; Kuča, Kamil; Wang, Xu; Chen, Guyue; Yuan, Zonghui

    2011-01-01

    Ochratoxin A (OTA) as a carcinogenic of group 2B to humans is produced by various fungi strains as Aspergillus and Penicillium. It is one of the most common contaminant in foodstuff. OTA is nephrotoxic, hepatotoxic, teratogenic, and immunotoxic and is assumed to cause Balkan Endemic Nephropathy (BEN), a chronic kidney disease in humans when it is digested in combination with mycotoxin citrinin. The metabolism affects greatly the fates and the toxicity of a mycotoxins in humans, animals, and plants. The understanding of the metabolism of mycotoxins by the organism as fungi, yeast, bacteria and enzymes would be very helpful for the control of the contamination by the mycotoxins in foods and feeds, and understanding of the biotransformation of the mycotoxin in the body of humans, animals, plants, microorganisms would be beneficial to the risk assessment of food safety. In animals and humans, OTA can be metabolized in the kidney, liver and intestines. Hydrolysis, hydroxylation, lactone-opening and conjugation are the major metabolic pathways. OTalpha (OTα) formed by the cleavage of the peptidic bond in OTA is a major metabolite not only in animals and humans, but also in microorganisms and enzyme systems. It is considered as a nontoxic product. However, the lactone-opened product (OP-OTA), found in rodents, is higher toxic than its parent, OTA.. (4R)-4-OH-OTA is the major hydroxy product in rodents, whereas the 4S isomer is the major in pigs. 10-OH-OTA is currently found only in rabbits. Furthermore, OTA can lose the chlorine on C-5 to produce ochratoxin B (OTB), and OTB is further to 4-OH-OTB and ochratoxin β (OTβ). Ochratoxin quinine/hydroquinone (OTQ/OTHQ) is the metabolite of OTA in animals. In addition, the conjugates of OTA such as hexose and pentose conjugates can be found in animals. Such more polar metabolites make OTA to eliminate faster. Currently, a debate exits on the formation of OTA-DNA adducts. Plants can metabolize OTA as well. OH-OTA methyl ester

  9. Syngas Upgrading to Hydrocarbon Fuels Technology Pathway

    SciTech Connect

    Talmadge, M.; Biddy, M.; Dutta, A.; Jones, S.; Meyer, A.

    2013-03-01

    This technology pathway case investigates the upgrading of woody biomass derived synthesis gas (syngas) to hydrocarbon biofuels. While this specific discussion focuses on the conversion of syngas via a methanol intermediate to hydrocarbon blendstocks, there are a number of alternative conversion routes for production of hydrocarbons through a wide array of intermediates from syngas. Future work will also consider the variations to this pathway to determine the most economically viable and lowest risk conversion route. Technical barriers and key research needs have been identified that should be pursued for the syngas-to-hydrocarbon pathway to be competitive with petroleum-derived gasoline-, diesel- and jet-range hydrocarbon blendstocks.

  10. Evolution of the JAK-STAT pathway.

    PubMed

    Liongue, Clifford; Ward, Alister C

    2013-01-01

    The JAK-STAT pathway represents a finely tuned orchestra capable of rapidly facilitating an exquisite symphony of responses from a complex array of extracellular signals. This review explores the evolution of the JAK-STAT pathway: the origins of the individual domains from which it is constructed, the formation of individual components from these basic building blocks, the assembly of the components into a functional pathway, and the subsequent reiteration of this basic template to fulfill a variety of roles downstream of cytokine receptors. PMID:24058787

  11. Electron Transfer Pathways in Cholesterol Synthesis.

    PubMed

    Porter, Todd D

    2015-10-01

    Cholesterol synthesis in the endoplasmic reticulum requires electron input at multiple steps and utilizes both NADH and NADPH as the electron source. Four enzymes catalyzing five steps in the pathway require electron input: squalene monooxygenase, lanosterol demethylase, sterol 4α-methyl oxidase, and sterol C5-desaturase. The electron-donor proteins for these enzymes include cytochrome P450 reductase and the cytochrome b5 pathway. Here I review the evidence for electron donor protein requirements with these enzymes, the evidence for additional electron donor pathways, and the effect of deletion of these redox enzymes on cholesterol and lipid metabolism. PMID:26344922

  12. [Hedgehog signaling pathway and human disorders].

    PubMed

    Fujii, Katsunori; Miyashita, Toshiyuki

    2009-07-01

    The hedgehog signaling pathway plays pivotal roles in embryonic development and cancer formation. This pathway in mammals consists of multiple molecules such as Sonic Hedgehog, PTCH, SMO, and GLI. Mutations of these components result in various human malformations or tumors, i.e., holoprosencephaly, Gorlin syndrome, Greig encephalopolysyndactyly, Pallister-Hall syndrome, Rubinstein-Taybi syndrome, basal cell carcinomas, and medulloblastomas. Recently, small molecules that inhibit this signaling pathway were developed, and clinically applied to cancer therapy. Thus, understanding of these molecular relationships may facilitate the development of new therapies and treatments for diseases caused by hedgehog signaling disorders. PMID:19618878

  13. Evolution of the JAK-STAT pathway

    PubMed Central

    Liongue, Clifford; Ward, Alister C.

    2013-01-01

    The JAK-STAT pathway represents a finely tuned orchestra capable of rapidly facilitating an exquisite symphony of responses from a complex array of extracellular signals. This review explores the evolution of the JAK-STAT pathway: the origins of the individual domains from which it is constructed, the formation of individual components from these basic building blocks, the assembly of the components into a functional pathway, and the subsequent reiteration of this basic template to fulfill a variety of roles downstream of cytokine receptors. PMID:24058787

  14. Identification of disturbed pathways in heart failure based on Gibbs sampling and pathway enrichment analysis.

    PubMed

    Chen, P; Guo, L H; Guo, Y K; Qu, Z J; Gao, Y; Qiu, H

    2016-01-01

    We identified disturbed pathways in heart failure (HF) based on Gibbs sampling combined with pathway enrichment analysis. A total of 396 Markov chains (MCs) (gene count >5) were obtained. After Gibbs sampling, six differentially expressed molecular functions (DEMFs) (possibility ≥0.8) were obtained. As statistical analysis was performed on the number of individual differentially expressed genes (DEGs), we found that there were 137 DEGs with frequency of occurrence ≥2 in the DEMFs. Pathway enrichment analysis showed that these 137 DEGs were enriched in eight significant pathways under the condition of P < 0.001. The five most significant pathways were: the calcium signaling pathway (P = 9.08E-19), arrhythmogenic right ventricular cardiomyopathy (P = 5.66E-13), cardiac muscle contraction (P = 8.04E-13), hypertrophic cardiomyopathy (P = 2.55E-12), and dilated cardiomyopathy (P = 7.30E-12). In conclusion, this novel method for identifying significant pathways in HF based on Gibbs sampling combined with pathway enrichment analysis was suitable. We predict that several altered pathways (such as the calcium signaling pathway and dilated cardiomyopathy) may play important roles in HF and are potentially novel predictive and prognostic markers for HF. PMID:27173293

  15. Genetic dissection of cardiac growth control pathways

    NASA Technical Reports Server (NTRS)

    MacLellan, W. R.; Schneider, M. D.

    2000-01-01

    Cardiac muscle cells exhibit two related but distinct modes of growth that are highly regulated during development and disease. Cardiac myocytes rapidly proliferate during fetal life but exit the cell cycle irreversibly soon after birth, following which the predominant form of growth shifts from hyperplastic to hypertrophic. Much research has focused on identifying the candidate mitogens, hypertrophic agonists, and signaling pathways that mediate these processes in isolated cells. What drives the proliferative growth of embryonic myocardium in vivo and the mechanisms by which adult cardiac myocytes hypertrophy in vivo are less clear. Efforts to answer these questions have benefited from rapid progress made in techniques to manipulate the murine genome. Complementary technologies for gain- and loss-of-function now permit a mutational analysis of these growth control pathways in vivo in the intact heart. These studies have confirmed the importance of suspected pathways, have implicated unexpected pathways as well, and have led to new paradigms for the control of cardiac growth.

  16. Signalling pathways: jack of all cascades.

    PubMed

    Cahill, M A; Janknecht, R; Nordheim, A

    1996-01-01

    The transcription factors that bind the c-fos promoter element SRE are targeted by multiple, independent signalling cascades; the identities of these signalling pathways and their modes of activation are being elucidated. PMID:8805215

  17. Adverse Outcome Pathways: From Definition to Application

    EPA Science Inventory

    A challenge for both human health and ecological toxicologists is the transparent application of mechanistic (e.g., molecular, biochemical, histological) data to risk assessments. The adverse outcome pathway (AOP) is a conceptual framework designed to meet this need. Specifical...

  18. Computing folding pathways between RNA secondary structures.

    PubMed

    Dotu, Ivan; Lorenz, William A; Van Hentenryck, Pascal; Clote, Peter

    2010-03-01

    Given an RNA sequence and two designated secondary structures A, B, we describe a new algorithm that computes a nearly optimal folding pathway from A to B. The algorithm, RNAtabupath, employs a tabu semi-greedy heuristic, known to be an effective search strategy in combinatorial optimization. Folding pathways, sometimes called routes or trajectories, are computed by RNAtabupath in a fraction of the time required by the barriers program of Vienna RNA Package. We benchmark RNAtabupath with other algorithms to compute low energy folding pathways between experimentally known structures of several conformational switches. The RNApathfinder web server, source code for algorithms to compute and analyze pathways and supplementary data are available at http://bioinformatics.bc.edu/clotelab/RNApathfinder. PMID:20044352

  19. The Pentose Phosphate Pathway in Parasitic Trypanosomatids.

    PubMed

    Kovářová, Julie; Barrett, Michael P

    2016-08-01

    Parasitic trypanosomatids cause important diseases. Dissecting the biochemistry of these organisms offers a means of discovering targets against which inhibitors may be designed and developed as drugs. The pentose phosphate pathway is a key route of glucose metabolism in most organisms, providing NADPH for use as a cellular reductant and various carbohydrate intermediates used in cellular metabolism. The pathway and its enzymes have been studied in Trypanosoma brucei, Trypanosoma cruzi, and various Leishmania species. Its functions in these parasites are becoming clear. Some enzymes of the pathway are essential to the parasites and have structural features distinguishing them from their mammalian counterparts, and this has stimulated several programs of inhibitor discovery with a view to targeting the pathway with new drugs. PMID:27174163

  20. Function and Regulation in MAPK Signaling Pathways

    PubMed Central

    Chen, Raymond E.; Thorner, Jeremy

    2007-01-01

    Signaling pathways that activate different mitogen-activated protein kinases (MAPKs) elicit many of the responses that are evoked in cells by changes in certain environmental conditions and upon exposure to a variety of hormonal and other stimuli. These pathways were first elucidated in the unicellular eukaryote Saccharomyces cerevisiae (budding yeast). Studies of MAPK pathways in this organism continue to be especially informative in revealing the molecular mechanisms by which MAPK cascades operate, propagate signals, modulate cellular processes, and are controlled by regulatory factors both internal to and external to the pathways. Here we highlight recent advances and new insights about MAPK-based signaling that have been made through studies in yeast, which provide lessons directly applicable to, and that enhance our understanding of, MAPK-mediated signaling in mammalian cells. PMID:17604854

  1. Cholangiocarcinoma: Molecular Pathways and Therapeutic Opportunities

    PubMed Central

    Rizvi, Sumera; Borad, Mitesh J.; Patel, Tushar; Gores, Gregory J.

    2015-01-01

    Cholangiocarcinoma (CCA) is an aggressive biliary tract malignancy with limited treatment options and low survival rates. Currently, there are no curative medical therapies for CCA. Recent advances have enhanced our understanding of the genetic basis of this disease, and elucidated therapeutically relevant targets. Therapeutic efforts in development are directed at several key pathways due to genetic aberrations including receptor tyrosine kinase pathways, mutant IDH enzymes, the PI3K-AKT-mTOR pathway, and chromatin remodeling networks. A highly desmoplastic, hypovascular stroma is characteristic of CCAs and recent work has highlighted the importance of targeting this pathway via stromal myofibroblast depletion. Future efforts should concentrate on combination therapies with action against the cancer cell and the surrounding tumor stroma. As the mutational landscape of CCA is being illuminated, molecular profiling of patient tumors will enable identification of specific mutations and the opportunity to offer directed, personalized treatment options. PMID:25369307

  2. Virus Budding and the ESCRT Pathway

    PubMed Central

    Votteler, Jörg; Sundquist, Wesley I.

    2013-01-01

    Enveloped viruses escape infected cells by budding through limiting membranes. In the decade since the discovery that the Human Immunodeficiency Virus (HIV) recruits cellular ESCRT (endosomal sorting complexes required for transport) machinery to facilitate viral budding, this pathway has emerged as the major escape route for enveloped viruses. In cells, the ESCRT pathway catalyzes the analogous membrane fission events required for the abscission stage of cytokinesis and for a series of “reverse topology” vesiculation events. Studies of enveloped virus budding are therefore providing insights into the complex cellular mechanisms of cell division and membrane protein trafficking (and vice versa). Here, we review how viruses mimic cellular recruiting signals to usurp the ESCRT pathway, discuss mechanistic models for ESCRT pathway functions, and highlight important research frontiers. PMID:24034610

  3. Modularized TGFbeta-Smad Signaling Pathway

    NASA Technical Reports Server (NTRS)

    Li, Yongfeng; Wang, M.; Carra, C.; Cucinotta, F. A.

    2011-01-01

    The Transforming Growth Factor beta (TGFbeta) signaling pathway is a prominent regulatory signaling pathway controlling various important cellular processes. It can be induced by several factors, including ionizing radiation. It is regulated by Smads in a negative feedback loop through promoting increases in the regulatory Smads in the cell nucleus, and subsequent expression of inhibitory Smad, Smad7 to form a ubiquitin ligase with Smurf targeting active TGF receptors for degradation. In this work, we proposed a mathematical model to study the radiation-induced Smad-regulated TGF signaling pathway. By modularization, we are able to analyze each module (subsystem) and recover the nonlinear dynamics of the entire network system. Meanwhile the excitability, a common feature observed in the biological systems, along the TGF signaling pathway is discussed by mathematical analysis and numerical simulation.

  4. Advances in Targeting Signal Transduction Pathways

    PubMed Central

    McCubrey, James A.; Steelman, Linda S.; Chappell, William H.; Sun, Lin; Davis, Nicole M.; Abrams, Stephen L.; Franklin, Richard A.; Cocco, Lucio; Evangelisti, Camilla; Chiarini, Francesca; Martelli, Alberto M.; Libra, Massimo; Candido, Saverio; Ligresti, Giovanni; Malaponte, Grazia; Mazzarino, Maria C.; Fagone, Paolo; Donia, Marco; Nicoletti, Ferdinando; Polesel, Jerry; Talamini, Renato; Bäsecke, Jörg; Mijatovic, Sanja; Maksimovic-Ivanic, Danijela; Milella, Michele; Tafuri, Agostino; Dulińska-Litewka, Joanna; Laidler, Piotr; D'Assoro, Antonio B.; Drobot, Lyudmyla; Umezawa, Kazuo; Montalto, Giuseppe; Cervello, Melchiorre; Demidenko, Zoya N.

    2012-01-01

    Over the past few years, significant advances have occurred in both our understanding of the complexity of signal transduction pathways as well as the isolation of specific inhibitors which target key components in those pathways. Furthermore critical information is being accrued regarding how genetic mutations can affect the sensitivity of various types of patients to targeted therapy. Finally, genetic mechanisms responsible for the development of resistance after targeted therapy are being discovered which may allow the creation of alternative therapies to overcome resistance. This review will discuss some of the highlights over the past few years on the roles of key signaling pathways in various diseases, the targeting of signal transduction pathways and the genetic mechanisms governing sensitivity and resistance to targeted therapies. PMID:23455493

  5. Biochemical pathways in seed oil synthesis.

    PubMed

    Bates, Philip D; Stymne, Sten; Ohlrogge, John

    2013-06-01

    Oil produced in plant seeds is utilized as a major source of calories for human nutrition, as feedstocks for non-food uses such as soaps and polymers, and can serve as a high-energy biofuel. The biochemical pathways leading to oil (triacylglycerol) synthesis in seeds involve multiple subcellular organelles, requiring extensive lipid trafficking. Phosphatidylcholine plays a central role in these pathways as a substrate for acyl modifications and likely as a carrier for the trafficking of acyl groups between organelles and membrane subdomains. Although much has been clarified regarding the enzymes and pathways responsible for acyl-group flux, there are still major gaps in our understanding. These include the identity of several key enzymes, how flux between alternative pathways is controlled and the specialized cell biology leading to biogenesis of oil bodies that store up to 80% of carbon in seeds. PMID:23529069

  6. Targeting Apoptosis Signaling Pathways for Anticancer Therapy

    PubMed Central

    Fulda, Simone

    2011-01-01

    Treatment approaches for cancer, for example chemotherapy, radiotherapy, or immunotherapy, primarily act by inducing cell death in cancer cells. Consequently, the inability to trigger cell death pathways or alternatively, evasion of cancer cells to the induction of cell death pathways can result in resistance of cancers to current treatment protocols. Therefore, in order to overcome treatment resistance a better understanding of the underlying mechanisms that regulate cell death and survival pathways in cancers and in response to cancer therapy is necessary to develop molecular-targeted therapies. This strategy should lead to more effective and individualized treatment strategies that selectively target deregulated signaling pathways in a tumor type- and patient-specific manner. PMID:22655234

  7. Cholangiocarcinoma: molecular pathways and therapeutic opportunities.

    PubMed

    Rizvi, Sumera; Borad, Mitesh J; Patel, Tushar; Gores, Gregory J

    2014-11-01

    Cholangiocarcinoma (CCA) is an aggressive biliary tract malignancy with limited treatment options and low survival rates. Currently, there are no curative medical therapies for CCA. Recent advances have enhanced our understanding of the genetic basis of this disease, and elucidated therapeutically relevant targets. Therapeutic efforts in development are directed at several key pathways due to genetic aberrations including receptor tyrosine kinase pathways, mutant IDH enzymes, the PI3K-AKT-mTOR pathway, and chromatin remodeling networks. A highly desmoplastic, hypovascular stroma is characteristic of CCAs and recent work has highlighted the importance of targeting this pathway via stromal myofibroblast depletion. Future efforts should concentrate on combination therapies with action against the cancer cell and the surrounding tumor stroma. As the mutational landscape of CCA is being illuminated, molecular profiling of patient tumors will enable identification of specific mutations and the opportunity to offer directed, personalized treatment options. PMID:25369307

  8. Targeting RTK Signaling Pathways in Cancer

    PubMed Central

    Regad, Tarik

    2015-01-01

    The RAS/MAP kinase and the RAS/PI3K/AKT pathways play a key role in the regulation of proliferation, differentiation and survival. The induction of these pathways depends on Receptor Tyrosine Kinases (RTKs) that are activated upon ligand binding. In cancer, constitutive and aberrant activations of components of those pathways result in increased proliferation, survival and metastasis. For instance, mutations affecting RTKs, Ras, B-Raf, PI3K and AKT are common in perpetuating the malignancy of several types of cancers and from different tissue origins. Therefore, these signaling pathways became prime targets for cancer therapy. This review aims to provide an overview about the most frequently encountered mutations, the pathogenesis that results from such mutations and the known therapeutic strategies developed to counteract their aberrant functions. PMID:26404379

  9. Minimal distortion pathways in polyhedral rearrangements.

    PubMed

    Casanova, David; Cirera, Jordi; Llunell, Miquel; Alemany, Pere; Avnir, David; Alvarez, Santiago

    2004-02-18

    A definition of minimum distortion paths between two polyhedra in terms of continuous shape measures (CShM) is presented. A general analytical expression deduced for such pathways makes use of one parameter, the minimum distortion constant, that can be easily obtained through the CShM methodology and is herein tabulated for pairs of polyhedra having four to eight vertexes. The work presented here also allows us to obtain representative model molecular structures along the interconversion pathways. Several commonly used polytopal rearrangement pathways are shown to be in fact minimum distortion pathways: the spread path leading from the tetrahedron to the square, the Berry pseudorotation that interconverts a square pyramid and a trigonal bipyramid, and the Bailar twist for the interconversion of the octahedron and the trigonal prism. Examples of applications to the analysis of the stereochemistries of several families of metal complexes are presented. PMID:14871107

  10. The casein kinases Yck1p and Yck2p act in the secretory pathway, in part, by regulating the Rab exchange factor Sec2p

    PubMed Central

    Stalder, Danièle; Novick, Peter J.

    2016-01-01

    Sec2p is a guanine nucleotide exchange factor that activates Sec4p, the final Rab GTPase of the yeast secretory pathway. Sec2p is recruited to secretory vesicles by the upstream Rab Ypt32p acting in concert with phosphatidylinositol-4-phosphate (PI(4)P). Sec2p also binds to the Sec4p effector Sec15p, yet Ypt32p and Sec15p compete against each other for binding to Sec2p. We report here that the redundant casein kinases Yck1p and Yck2p phosphorylate sites within the Ypt32p/Sec15p binding region and in doing so promote binding to Sec15p and inhibit binding to Ypt32p. We show that Yck2p binds to the autoinhibitory domain of Sec2p, adjacent to the PI(4)P binding site, and that addition of PI(4)P inhibits Sec2p phosphorylation by Yck2p. Loss of Yck1p and Yck2p function leads to accumulation of an intracellular pool of the secreted glucanase Bgl2p, as well as to accumulation of Golgi-related structures in the cytoplasm. We propose that Sec2p is phosphorylated after it has been recruited to secretory vesicles and the level of PI(4)P has been reduced. This promotes Sec2p function by stimulating its interaction with Sec15p. Finally, Sec2p is dephosphorylated very late in the exocytic reaction to facilitate recycling. PMID:26700316

  11. Pathway-based analysis of breast cancer

    PubMed Central

    Song, Dong; Cui, Miao; Zhao, Gang; Fan, Zhimin; Nolan, Katherine; Yang, Ying; Lee, Peng; Ye, Fei; Zhang, David Y

    2014-01-01

    Introduction: Although HER2 and ER pathways are predominant pathways altered in breast cancer, it is now well accepted that many other signaling pathways are also involved in the pathogenesis of breast cancer. The understanding of these additional pathways may assist in identifying new therapeutic approaches for breast cancer. Methods: 13 invasive ductal carcinoma tissues and 5 benign breast tissues were analyzed for the mRNA expression level of 1243 cancer pathway-related genes using SmartChip (WaferGen, CA), a real-time PCR-base method. In addition, the levels of 131 cancer pathway-related proteins and phosphoproteins in 33 paired breast cancers were measured using our innovative Protein Pathway Array. Results: Out of 1,243 mRNAs, 68.7% (854) were detected in breast cancer and 395 mRNAs were statistically significant (fold change >2) between benign and cancer tissues. Of these mRNAs, 105 only expressed in breast cancer tissues and 33 mRNAs only expressed in normal breast tissues. Out of 131 proteins and phosphoproteins, 68% (89) were detected in cancer tissues and 57 proteins were significantly differentiated between tumor and normal tissues. Interestingly, only 3 genes (CDK6, Vimentin and SLUG) showed decreases in both protein and mRNA. Six proteins (BCL6, CCNE1, PCNA, PDK1, SRC and XIAP) were differentially expressed between tumor and normal tissues but no differences were observed at mRNA levels. Analyses of mRNA and protein data using Ingenuity Pathway Analysis showed more than 15 pathways were altered in breast cancer and 6 of which were shared between mRNAs and proteins, including p53, IL17, HGF, NGF, PTEN and PI3K/AKT pathways. Conclusions: There is a broad dysregulation of various pathways in breast cancer both at protein levels and mRNA levels. It is important to note that mRNA expression does not correlate with protein level, suggesting different regulation mechanisms between proteins and mRNAs. PMID:24936222

  12. A more flexible lipoprotein sorting pathway.

    PubMed

    Chahales, Peter; Thanassi, David G

    2015-05-01

    Lipoprotein biogenesis in Gram-negative bacteria occurs by a conserved pathway, each step of which is considered essential. In contrast to this model, LoVullo and colleagues demonstrate that the N-acyl transferase Lnt is not required in Francisella tularensis or Neisseria gonorrhoeae. This suggests the existence of a more flexible lipoprotein pathway, likely due to a modified Lol transporter complex, and raises the possibility that pathogens may regulate lipoprotein processing to modulate interactions with the host. PMID:25755190

  13. Pathway Model and Nonextensive Statistical Mechanics

    NASA Astrophysics Data System (ADS)

    Mathai, A. M.; Haubold, H. J.; Tsallis, C.

    2015-12-01

    The established technique of eliminating upper or lower parameters in a general hypergeometric series is profitably exploited to create pathways among confluent hypergeometric functions, binomial functions, Bessel functions, and exponential series. One such pathway, from the mathematical statistics point of view, results in distributions which naturally emerge within nonextensive statistical mechanics and Beck-Cohen superstatistics, as pursued in generalizations of Boltzmann-Gibbs statistics.

  14. Determination of Ligand Pathways in Globins

    PubMed Central

    Salter, Mallory D.; Blouin, George C.; Soman, Jayashree; Singleton, Eileen W.; Dewilde, Sylvia; Moens, Luc; Pesce, Alessandra; Nardini, Marco; Bolognesi, Martino; Olson, John S.

    2012-01-01

    Although molecular dynamics simulations suggest multiple interior pathways for O2 entry into and exit from globins, most experiments indicate well defined single pathways. In 2001, we highlighted the effects of large-to-small amino acid replacements on rates for ligand entry and exit onto the three-dimensional structure of sperm whale myoglobin. The resultant map argued strongly for ligand movement through a short channel from the heme iron to solvent that is gated by the distal histidine (His-64(E7)) near the solvent edge of the porphyrin ring. In this work, we have applied the same mutagenesis mapping strategy to the neuronal mini-hemoglobin from Cerebratulus lacteus (CerHb), which has a large internal tunnel from the heme iron to the C-terminal ends of the E and H helices, a direction that is 180° opposite to the E7 channel. Detailed comparisons of the new CerHb map with expanded results for Mb show unambiguously that the dominant (>90%) ligand pathway in CerHb is through the internal tunnel, and the major (>75%) ligand pathway in Mb is through the E7 gate. These results demonstrate that: 1) mutagenesis mapping can identify internal pathways when they exist; 2) molecular dynamics simulations need to be refined to address discrepancies with experimental observations; and 3) alternative pathways have evolved in globins to meet specific physiological demands. PMID:22859299

  15. The ectodysplasin pathway: from diseases to adaptations.

    PubMed

    Sadier, Alexa; Viriot, Laurent; Pantalacci, Sophie; Laudet, Vincent

    2014-01-01

    The ectodysplasin (EDA) pathway, which is active during the development of ectodermal organs, including teeth, hairs, feathers, and mammary glands, and which is crucial for fine-tuning the developmental network controlling the number, size, and density of these structures, was discovered by studying human patients affected by anhidrotic/hypohidrotic ectodermal dysplasia. It comprises three main gene products: EDA, a ligand that belongs to the tumor necrosis factor (TNF)-α family, EDAR, a receptor related to the TNFα receptors, and EDARADD, a specific adaptor. This core pathway relies on downstream NF-κB pathway activation to regulate target genes. The pathway has recently been found to be associated with specific adaptations in natural populations: the magnitude of armor plates in sticklebacks and the hair structure in Asian human populations. Thus, despite its role in human disease, the EDA pathway is a 'hopeful pathway' that could allow adaptive changes in ectodermal appendages which, as specialized interfaces with the environment, are considered hot-spots of morphological evolution. PMID:24070496

  16. The ectodysplasin pathway in feather tract development.

    PubMed

    Houghton, Leslie; Lindon, Catherine; Morgan, Bruce A

    2005-03-01

    The ectodysplasin pathway, comprising the ligand ectodysplasin, its receptor Edar and a dedicated death domain adaptor protein Edaradd, plays an important role in epidermal organ formation in mammals. Mutations in the genes encoding these proteins cause dysplasia or absence of teeth, sweat glands and hair follicles. However, the relative position of this pathway in the regulatory hierarchy directing follicle formation remains unclear. In this work, the chicken orthologs of Eda, Edar and Edaradd were cloned to exploit the temporal precision of the feather tract system in order to study the role of the ectodysplasin pathway. We find that these genes are expressed in a similar pattern during feather and hair development, with the notable difference that the ligand Eda, which is expressed in the epidermis of the mouse, is expressed in the dermis of the feather tract. Contrary to conclusions reached from the analysis of mutant mice, we find that localization of Edar expression to the nascent placode is coincident or subsequent to the local expression of other markers of placodal differentiation, and not an upstream event in tract patterning. Furthermore, forced expression of BMP and activated beta-catenin demonstrate that local expression of Edar is dictated by the interaction between these two pathways. These results suggest that activation of the ectodysplasin pathway may be permissive for activating signals to overcome signals that inhibit placode formation, but the function of this pathway in the specification of follicle initiation lies downstream of other patterning events. PMID:15673574

  17. MetaPathways: a modular pipeline for constructing pathway/genome databases from environmental sequence information

    PubMed Central

    2013-01-01

    Background A central challenge to understanding the ecological and biogeochemical roles of microorganisms in natural and human engineered ecosystems is the reconstruction of metabolic interaction networks from environmental sequence information. The dominant paradigm in metabolic reconstruction is to assign functional annotations using BLAST. Functional annotations are then projected onto symbolic representations of metabolism in the form of KEGG pathways or SEED subsystems. Results Here we present MetaPathways, an open source pipeline for pathway inference that uses the PathoLogic algorithm to map functional annotations onto the MetaCyc collection of reactions and pathways, and construct environmental Pathway/Genome Databases (ePGDBs) compatible with the editing and navigation features of Pathway Tools. The pipeline accepts assembled or unassembled nucleotide sequences, performs quality assessment and control, predicts and annotates noncoding genes and open reading frames, and produces inputs to PathoLogic. In addition to constructing ePGDBs, MetaPathways uses MLTreeMap to build phylogenetic trees for selected taxonomic anchor and functional gene markers, converts General Feature Format (GFF) files into concatenated GenBank files for ePGDB construction based on third-party annotations, and generates useful file formats including Sequin files for direct GenBank submission and gene feature tables summarizing annotations, MLTreeMap trees, and ePGDB pathway coverage summaries for statistical comparisons. Conclusions MetaPathways provides users with a modular annotation and analysis pipeline for predicting metabolic interaction networks from environmental sequence information using an alternative to KEGG pathways and SEED subsystems mapping. It is extensible to genomic and transcriptomic datasets from a wide range of sequencing platforms, and generates useful data products for microbial community structure and function analysis. The MetaPathways software package

  18. Subpathway Analysis based on Signaling-Pathway Impact Analysis of Signaling Pathway

    PubMed Central

    Li, Xianbin; Shen, Liangzhong; Shang, Xuequn; Liu, Wenbin

    2015-01-01

    Pathway analysis is a common approach to gain insight from biological experiments. Signaling-pathway impact analysis (SPIA) is one such method and combines both the classical enrichment analysis and the actual perturbation on a given pathway. Because this method focuses on a single pathway, its resolution generally is not very high because the differentially expressed genes may be enriched in a local region of the pathway. In the present work, to identify cancer-related pathways, we incorporated a recent subpathway analysis method into the SPIA method to form the “sub-SPIA method.” The original subpathway analysis uses the k-clique structure to define a subpathway. However, it is not sufficiently flexible to capture subpathways with complex structure and usually results in many overlapping subpathways. We therefore propose using the minimal-spanning-tree structure to find a subpathway. We apply this approach to colorectal cancer and lung cancer datasets, and our results show that sub-SPIA can identify many significant pathways associated with each specific cancer that other methods miss. Based on the entire pathway network in the Kyoto Encyclopedia of Genes and Genomes, we find that the pathways identified by sub-SPIA not only have the largest average degree, but also are more closely connected than those identified by other methods. This result suggests that the abnormality signal propagating through them might be responsible for the specific cancer or disease. PMID:26207919

  19. Neural pathways for visual speech perception

    PubMed Central

    Bernstein, Lynne E.; Liebenthal, Einat

    2014-01-01

    This paper examines the questions, what levels of speech can be perceived visually, and how is visual speech represented by the brain? Review of the literature leads to the conclusions that every level of psycholinguistic speech structure (i.e., phonetic features, phonemes, syllables, words, and prosody) can be perceived visually, although individuals differ in their abilities to do so; and that there are visual modality-specific representations of speech qua speech in higher-level vision brain areas. That is, the visual system represents the modal patterns of visual speech. The suggestion that the auditory speech pathway receives and represents visual speech is examined in light of neuroimaging evidence on the auditory speech pathways. We outline the generally agreed-upon organization of the visual ventral and dorsal pathways and examine several types of visual processing that might be related to speech through those pathways, specifically, face and body, orthography, and sign language processing. In this context, we examine the visual speech processing literature, which reveals widespread diverse patterns of activity in posterior temporal cortices in response to visual speech stimuli. We outline a model of the visual and auditory speech pathways and make several suggestions: (1) The visual perception of speech relies on visual pathway representations of speech qua speech. (2) A proposed site of these representations, the temporal visual speech area (TVSA) has been demonstrated in posterior temporal cortex, ventral and posterior to multisensory posterior superior temporal sulcus (pSTS). (3) Given that visual speech has dynamic and configural features, its representations in feedforward visual pathways are expected to integrate these features, possibly in TVSA. PMID:25520611

  20. Pathways of Transport Protein Evolution: Recent Advances

    PubMed Central

    Lam, Vincent H.; Lee, Jong-Hoon; Silverio, Abe; Chan, Henry; Gomolplitinant, Kenny M.; Povolotsky, Tatyana L.; Orlova, Ekaterina; Sun, Eric I.; Welliver, Carl H.; Saier, Milton H.

    2014-01-01

    We herein report recent advances in our understanding of transport protein evolution. The Drug-Metabolite Transporter (DMT) superfamily (TC# 2.A.7) arose from a 2TMS precursor to give 4TMS proteins which then added one and duplicated to give 10. The proposed pathway is 2 –> 4 –> 5 –> 10. This superfamily provides a rare example where all proposed topological intermediates in this evolutionary pathway have been identified in current protein databases. Another family, the Oligopeptide Transporter (OPT) family (TC# 2.A.67), also started with a 2 TMS peptide precursor, but it followed the pathway: Only 16 and 17 TMS OPT family members have been identified in current databases. The TRIC family of K+ channels, characterized in animals, arose via the pathway: where the seventh TMS was added c-terminally to the 6 TMS precursor that resulted from a 3 TMS duplication. Surprisingly, animal TRIC channels proved to have numerous 7 TMS homologues in prokaryotes, none of which had been identified previously. We found that two families of integral membrane proteins gave rise to multiple current topological types. Members of the SdpC killer factor immunity protein family, SdpI (TC# 9.A.32) probably arose via the pathway: while members of the Heme Handling Protein (HHP) Family (TC# 9.B.14) arose via the pathway: Predictions are also made for an evolutionary pathway giving rise to the seven topological types of P-type ATPases so far identified in the P-ATPase superfamily. Finally, the ubiquitous CDF carriers (TC# 1.A.4) of 6TMSs probably gave rise to CRAC channels of 4TMSs by loss of the first two TMSs an unusual example of retroevolution. PMID:21194372

  1. Embryonic myogenesis pathways in muscle regeneration.

    PubMed

    Zhao, Po; Hoffman, Eric P

    2004-02-01

    Embryonic myogenesis involves the staged induction of myogenic regulatory factors and positional cues that dictate cell determination, proliferation, and differentiation into adult muscle. Muscle is able to regenerate after damage, and muscle regeneration is generally thought to recapitulate myogenesis during embryogenesis. There has been considerable progress in the delineation of myogenesis pathways during embryogenesis, but it is not known whether the same signaling pathways are relevant to muscle regeneration in adults. Here, we defined the subset of embryogenesis pathways induced in muscle regeneration using a 27 time-point in vivo muscle regeneration series. The embryonic Wnt (Wnt1, 3a, 7a, 11), Shh pathway, and the BMP (BMP2, 4, 7) pathway were not induced during muscle regeneration. Moreover, antagonists of Wnt signaling, sFRP1, sFRP2, and sFRP4 (secreted frizzled-related proteins) were significantly up-regulated, suggesting active inhibition of the Wnt pathway. The pro-differentiation FGFR4 pathway was transiently expressed at day 3, commensurate with expression of MyoD, Myogenin, Myf5, and Pax7. Protein verification studies showed fibroblast growth factor receptor 4 (FGFR4) protein to be strongly expressed in differentiating myoblasts and newly formed myotubes. We present evidence that FGF6 is likely the key ligand for FGFR4 during muscle regeneration, and further suggest that FGF6 is released from necrotic myofibers where it is then sequestered by basal laminae. We also confirmed activation of Notch1 in the regenerating muscle. Finally, known MyoD coactivators (MEF2A, p/CIP, TCF12) and repressors (Twist, Id2) were strongly induced at appropriate time points. Taken together, our results suggest that embryonic positional signals (Wnt, Shh, and BMP) are not induced in postnatal muscle regeneration, whereas cell-autonomous factors (Pax7, MRFs, FGFR4) involving muscle precursor proliferation and differentiation are recapitulated by muscle regeneration. PMID

  2. Alternative isoleucine synthesis pathway in cyanobacterial species.

    PubMed

    Wu, Bing; Zhang, Baichen; Feng, Xueyang; Rubens, Jacob R; Huang, Rick; Hicks, Leslie M; Pakrasi, Himadri B; Tang, Yinjie J

    2010-02-01

    Cyanothece sp. ATCC 51142 is an aerobic N(2)-fixing and hydrogen-producing cyanobacterium. Isotopomer analysis of its amino acids revealed an identical labelling profile for leucine and isoleucine when Cyanothece 51142 was grown mixotrophically using 2-(13)C-labelled glycerol as the main carbon source. This indicated that Cyanothece 51142 employs the atypical alternative citramalate pathway for isoleucine synthesis, with pyruvate and acetyl-CoA as precursors. Utilization of the citramalate pathway was confirmed by an enzyme assay and LC-MS/MS analysis. Furthermore, the genome sequence of Cyanothece 51142 shows that the gene encoding the key enzyme (threonine ammonia-lyase) in the normal isoleucine pathway is missing. Instead, the cce_0248 gene in Cyanothece 51142 exhibits 53 % identity to the gene encoding citramalate synthase (CimA, GSU1798) from Geobacter sulfurreducens. Reverse-transcription PCR indicated that the cce_0248 gene is expressed and its transcriptional level is lower in medium with isoleucine than in isoleucine-free medium. Additionally, a blast search for citramalate synthase and threonine ammonia-lyase implies that this alternative isoleucine synthesis pathway may be present in other cyanobacteria, such as Cyanothece and Synechococcus. This suggests that the pathway is more widespread than originally thought, as previous identifications of the citramalate pathway are limited to mostly anaerobic bacteria or archaea. Furthermore, this discovery opens the possibility that such autrotrophic micro-organisms may be engineered for robust butanol and propanol production from 2-ketobutyrate, which is an intermediate in the isoleucine biosynthesis pathway. PMID:19875435

  3. Targeting the Hedgehog Pathway in Pediatric Medulloblastoma

    PubMed Central

    Huang, Sherri Y.; Yang, Jer-Yen

    2015-01-01

    Medulloblastoma (MB), a primitive neuroectomal tumor of the cerebellum, is the most common malignant pediatric brain tumor. The cause of MB is largely unknown, but aberrant activation of Hedgehog (Hh) pathway is responsible for ~30% of MB. Despite aggressive treatment with surgical resection, radiation and chemotherapy, 70%–80% of pediatric medulloblastoma cases can be controlled, but most treated patients suffer devastating side effects. Therefore, developing a new effective treatment strategy is urgently needed. Hh signaling controls transcription of target genes by regulating activities of the three Glioma-associated oncogene (Gli1-3) transcription factors. In this review, we will focus on current clinical treatment options of MB and discuss mechanisms of drug resistance. In addition, we will describe current known molecular pathways which crosstalk with the Hedgehog pathway both in the context of medulloblastoma and non-medulloblastoma cancer development. Finally, we will introduce post-translational modifications that modulate Gli1 activity and summarize the positive and negative regulations of the Hh/Gli1 pathway. Towards developing novel combination therapies for medulloblastoma treatment, current information on interacting pathways and direct regulation of Hh signaling should prove critical. PMID:26512695

  4. Leptin signalling pathways in hypothalamic neurons.

    PubMed

    Kwon, Obin; Kim, Ki Woo; Kim, Min-Seon

    2016-04-01

    Leptin is the most critical hormone in the homeostatic regulation of energy balance among those so far discovered. Leptin primarily acts on the neurons of the mediobasal part of hypothalamus to regulate food intake, thermogenesis, and the blood glucose level. In the hypothalamic neurons, leptin binding to the long form leptin receptors on the plasma membrane initiates multiple signaling cascades. The signaling pathways known to mediate the actions of leptin include JAK-STAT signaling, PI3K-Akt-FoxO1 signaling, SHP2-ERK signaling, AMPK signaling, and mTOR-S6K signaling. Recent evidence suggests that leptin signaling in hypothalamic neurons is also linked to primary cilia function. On the other hand, signaling molecules/pathways mitigating leptin actions in hypothalamic neurons have been extensively investigated in an effort to treat leptin resistance observed in obesity. These include SOCS3, tyrosine phosphatase PTP1B, and inflammatory signaling pathways such as IKK-NFκB and JNK signaling, and ER stress-mitochondrial signaling. In this review, we discuss leptin signaling pathways in the hypothalamus, with a particular focus on the most recently discovered pathways. PMID:26786898

  5. Infectious Entry Pathway of Enterovirus B Species

    PubMed Central

    Marjomäki, Varpu; Turkki, Paula; Huttunen, Moona

    2015-01-01

    Enterovirus B species (EV-B) are responsible for a vast number of mild and serious acute infections. They are also suspected of remaining in the body, where they cause persistent infections contributing to chronic diseases such as type I diabetes. Recent studies of the infectious entry pathway of these viruses revealed remarkable similarities, including non-clathrin entry of large endosomes originating from the plasma membrane invaginations. Many cellular factors regulating the efficient entry have recently been associated with macropinocytic uptake, such as Rac1, serine/threonine p21-activated kinase (Pak1), actin, Na/H exchanger, phospholipace C (PLC) and protein kinase Cα (PKCα). Another characteristic feature is the entry of these viruses to neutral endosomes, independence of endosomal acidification and low association with acidic lysosomes. The biogenesis of neutral multivesicular bodies is crucial for their infection, at least for echovirus 1 (E1) and coxsackievirus A9 (CVA9). These pathways are triggered by the virus binding to their receptors on the plasma membrane, and they are not efficiently recycled like other cellular pathways used by circulating receptors. Therefore, the best “markers” of these pathways may be the viruses and often their receptors. A deeper understanding of this pathway and associated endosomes is crucial in elucidating the mechanisms of enterovirus uncoating and genome release from the endosomes to start efficient replication. PMID:26690201

  6. Unraveling tissue regeneration pathways using chemical genetics.

    PubMed

    Mathew, Lijoy K; Sengupta, Sumitra; Kawakami, Atsushi; Andreasen, Eric A; Löhr, Christiane V; Loynes, Catherine A; Renshaw, Stephen A; Peterson, Randall T; Tanguay, Robert L

    2007-11-30

    Identifying the molecular pathways that are required for regeneration remains one of the great challenges of regenerative medicine. Although genetic mutations have been useful for identifying some molecular pathways, small molecule probes of regenerative pathways might offer some advantages, including the ability to disrupt pathway function with precise temporal control. However, a vertebrate regeneration model amenable to rapid throughput small molecule screening is not currently available. We report here the development of a zebrafish early life stage fin regeneration model and its use in screening for small molecules that modulate tissue regeneration. By screening 2000 biologically active small molecules, we identified 17 that specifically inhibited regeneration. These compounds include a cluster of glucocorticoids, and we demonstrate that transient activation of the glucocorticoid receptor is sufficient to block regeneration, but only if activation occurs during wound healing/blastema formation. In addition, knockdown of the glucocorticoid receptor restores regenerative capability to nonregenerative, glucocorticoid-exposed zebrafish. To test whether the classical anti-inflammatory action of glucocorticoids is responsible for blocking regeneration, we prevented acute inflammation following amputation by antisense repression of the Pu.1 gene. Although loss of Pu.1 prevents the inflammatory response, regeneration is not affected. Collectively, these results indicate that signaling from exogenous glucocorticoids impairs blastema formation and limits regenerative capacity through an acute inflammation-independent mechanism. These studies also demonstrate the feasibility of exploiting chemical genetics to define the pathways that govern vertebrate regeneration. PMID:17848559

  7. The noncanonical NF-κB pathway

    PubMed Central

    Sun, Shao-Cong

    2012-01-01

    Summary The noncanonical nuclear factor-κB (NF-κB) signaling pathway mediates activation of the p52/RelB NF-κB complex and, thereby, regulates specific immunological processes. This NF-κB pathway relies on the inducible processing of NF-κB2 precursor protein, p100, as opposed to the degradation of IκBα in the canonical NF-κB pathway. A central signaling component of the noncanonical NF-κB pathway is NF-κB-inducing kinase (NIK), which functions together with a downstream kinase, inhibitor of NF-κB kinase α (IKKα), to induce phosphorylation-dependent ubiquitination and processing of p100. Under normal conditions, NIK is targeted for continuous degradation by a tumor necrosis factor (TNF) receptor-associated factor-3 (TRAF3)-dependent E3 ubiquitin ligase. In response to signals mediated by a subset of TNF receptor superfamily members, NIK becomes stabilized as a result of TRAF3 degradation, leading to the activation of noncanonical NF-κB. This review discusses both the historical perspectives and the recent progress in the regulation and biological function of the noncanonical NF-κB pathway. PMID:22435551

  8. Molecular Pathways: Targeting the PI3K Pathway in Cancer-BET Inhibitors to the Rescue.

    PubMed

    Stratikopoulos, Elias E; Parsons, Ramon E

    2016-06-01

    The PI3K signaling pathway is a complex and tightly regulated network that is critical for many physiologic processes, such as cell growth, proliferation, metabolism, and survival. Aberrant activation of this pathway can occur through mutation of almost any of its major nodes and has been implicated in a number of human diseases, including cancer. The high frequency of mutations in this pathway in multiple types of cancer has led to the development of small-molecule inhibitors of PI3K, several of which are currently in clinical trials. However, several feedback mechanisms either within the PI3K pathway or in compensatory pathways can render tumor cells resistant to therapy. Recently, targeting proteins of the bromodomain and extraterminal (BET) family of epigenetic readers of histone acetylation has been shown to effectively block adaptive signaling response of cancer cells to inhibitors of the PI3K pathway, which at least in some cases can restore sensitivity. BET inhibitors also enforce blockade of the MAPK, JAK/STAT, and ER pathways, suggesting they may be a rational combinatorial partner for divergent oncogenic signals that are subject to homeostatic regulation. Here, we review the PI3K pathway as a target for cancer therapy and discuss the potential use of BET inhibition to enhance the clinical efficacy of PI3K inhibitors. Clin Cancer Res; 22(11); 2605-10. ©2016 AACR. PMID:27250929

  9. Environmental Management Technologies. Curriculum Pathways, Pathway Narratives, Competency Documentation Sheets, and Program Application.

    ERIC Educational Resources Information Center

    Columbus State Community Coll., OH.

    This report presents information on the systemic changes that have occurred and will occur in environmental management technologies curriculum in participating secondary and postsecondary institutions as a result of the installation of tech prep pathways in the Heart of Ohio Tech Prep Consortium. Part I contains the curriculum pathways and pathway…

  10. Plant phytotoxicity: A self-regulating pathway

    SciTech Connect

    Not Available

    1989-07-01

    During the session on regulating sludge utilization, held at BioCycle's 19th Annual National conference on Composting and Recycling, a participant asked one of the speakers, Dr. Alan Rubin of the US EPA's Office of Water Regulations and Standards, why the plant phytotoxicity pathway should be the most limiting, especially when there is no concern about human or animal health. The question related specifically to copper being the most limiting metal concentration limit for many sludge composting and land application programs under the proposed Part 503 regulations. And the most limiting pathway for copper is Pathway 7, sludge-soil-plant phytotoxicity. Rubin responded that the regulation is supposed to protect both human health and the environment, e.g. plants and animals other than humans.

  11. Remodeling of Calcium Entry Pathways in Cancer.

    PubMed

    Villalobos, Carlos; Sobradillo, Diego; Hernández-Morales, Miriam; Núñez, Lucía

    2016-01-01

    Ca(2+) entry pathways play important roles in control of many cellular functions, including long-term proliferation, migration and cell death. In recent years, it is becoming increasingly clear that, in some types of tumors, remodeling of Ca(2+) entry pathways could contribute to cancer hallmarks such as excessive proliferation, cell migration and invasion as well as resistance to cell death or survival. In this chapter we briefly review findings related to remodeling of Ca(2+) entry pathways in cancer with emphasis on the mechanisms that contribute to increased store-operated Ca(2+) entry (SOCE) and store-operated currents (SOCs) in colorectal cancer cells. Finally, since SOCE appears critically involved in colon tumorogenesis, the inhibition of SOCE by aspirin and other NSAIDs and its possible contribution to colon cancer chemoprevention is reviewed. PMID:27161240

  12. Reconstructing biochemical pathways from time course data.

    PubMed

    Srividhya, Jeyaraman; Crampin, Edmund J; McSharry, Patrick E; Schnell, Santiago

    2007-03-01

    Time series data on biochemical reactions reveal transient behavior, away from chemical equilibrium, and contain information on the dynamic interactions among reacting components. However, this information can be difficult to extract using conventional analysis techniques. We present a new method to infer biochemical pathway mechanisms from time course data using a global nonlinear modeling technique to identify the elementary reaction steps which constitute the pathway. The method involves the generation of a complete dictionary of polynomial basis functions based on the law of mass action. Using these basis functions, there are two approaches to model construction, namely the general to specific and the specific to general approach. We demonstrate that our new methodology reconstructs the chemical reaction steps and connectivity of the glycolytic pathway of Lactococcus lactis from time course experimental data. PMID:17370261

  13. mitochondrial pathway for biosynthesis of lipid mediators

    PubMed Central

    Tyurina, Yulia Y.; Poloyac, Samuel M.; Tyurin, Vladimir A.; Kapralov, Alexander A.; Jiang, Jianfei; Anthonymuthu, Tamil Selvan; Kapralova, Valentina I.; Vikulina, Anna S.; Jung, Mi-Yeon; Epperly, Michael W.; Mohammadyani, Dariush; Klein-Seetharaman, Judith; Jackson, Travis C.; Kochanek, Patrick M.; Pitt, Bruce R.; Greenberger, Joel S.; Vladimirov, Yury A.; Bayır, Hülya; Kagan, Valerian E.

    2014-01-01

    The central role of mitochondria in metabolic pathways and in cell death mechanisms requires sophisticated signaling systems. Essential in this signaling process is an array of lipid mediators derived from polyunsaturated fatty acids. However, the molecular machinery for the production of oxygenated polyunsaturated fatty acids is localized in the cytosol and their biosynthesis has not been identified in mitochondria. Here we report that a range of diversified polyunsaturated molecular species derived from a mitochondria-specific phospholipid, cardiolipin, are oxidized by the intermembrane space hemoprotein, cytochrome c. We show that an assortment of oxygenated cardiolipin species undergoes phospholipase A2-catalyzed hydrolysis thus generating multiple oxygenated fatty acids, including well known lipid mediators. This represents a new biosynthetic pathway for lipid mediators. We demonstrate that this pathway including oxidation of polyunsaturated cardiolipins and accumulation of their hydrolysis products – oxygenated linoleic, arachidonic acids and monolyso-cardiolipins – is activated in vivo after acute tissue injury. PMID:24848241

  14. Multiple Pathways Linking Racism to Health Outcomes

    PubMed Central

    Harrell, Camara Jules P.; Burford, Tanisha I.; Cage, Brandi N.; Nelson, Travette McNair; Shearon, Sheronda; Thompson, Adrian; Green, Steven

    2012-01-01

    This commentary discusses advances in the conceptual understanding of racism and selected research findings in the social neurosciences. The traditional stress and coping model holds that racism constitutes a source of aversive experiences that, when perceived by the individual, eventually lead to poor health outcomes. Current evidence points to additional psychophysiological pathways linking facets of racist environments with physiological reactions that contribute to disease. The alternative pathways emphasize prenatal experiences, subcortical emotional neural circuits, conscious and preconscious emotion regulation, perseverative cognitions, and negative affective states stemming from racist cognitive schemata. Recognition of these pathways challenges change agents to use an array of cognitive and self-controlling interventions in mitigating racism’s impact. Additionally, it charges policy makers to develop strategies that eliminate deep-seated structural aspects of racism in society. PMID:22518195

  15. Kynurenine pathway and disease: an overview.

    PubMed

    Pérez-De La Cruz, Verónica; Königsberg, Mina; Santamaría, Abel

    2007-12-01

    Kynurenine pathway is gaining more and more attention every day in biomedical research since this catabolic route for tryptophan decomposition is not only implicated in different neurological disorders, but also possesses neuroactive metabolites with different biological properties, such as pro-oxidant and antioxidant regulators. Thus, the intensive research on this metabolic pathway is helping us to understand those mechanisms underlying neurodegenerative events during the occurrence of pathological process in the central nervous system (CNS), thereby allowing the design of potential therapies for those disorders involving excitotoxic, oxidative and inflammatory components. Here we intend to provide a brief overview on the relevance of this route for several CNS disorders, and discuss recent information on the different biological properties of the neuroactive metabolites of this pathway and their significance for further research. PMID:18220779

  16. Research Resources for Nuclear Receptor Signaling Pathways.

    PubMed

    McKenna, Neil J

    2016-08-01

    Nuclear receptor (NR) signaling pathways impact cellular function in a broad variety of tissues in both normal physiology and disease states. The complex tissue-specific biology of these pathways is an enduring impediment to the development of clinical NR small-molecule modulators that combine therapeutically desirable effects in specific target tissues with suppression of off-target effects in other tissues. Supporting the important primary research in this area is a variety of web-based resources that assist researchers in gaining an appreciation of the molecular determinants of the pharmacology of a NR pathway in a given tissue. In this study, selected representative examples of these tools are reviewed, along with discussions on how current and future generations of tools might optimally adapt to the future of NR signaling research. PMID:27216565

  17. Evolutionary algorithm for metabolic pathways synthesis.

    PubMed

    Gerard, Matias F; Stegmayer, Georgina; Milone, Diego H

    2016-06-01

    Metabolic pathway building is an active field of research, necessary to understand and manipulate the metabolism of organisms. There are different approaches, mainly based on classical search methods, to find linear sequences of reactions linking two compounds. However, an important limitation of these methods is the exponential increase of search trees when a large number of compounds and reactions is considered. Besides, such models do not take into account all substrates for each reaction during the search, leading to solutions that lack biological feasibility in many cases. This work proposes a new evolutionary algorithm that allows searching not only linear, but also branched metabolic pathways, formed by feasible reactions that relate multiple compounds simultaneously. Tests performed using several sets of reactions show that this algorithm is able to find feasible linear and branched metabolic pathways. PMID:27080162

  18. Modelling and Decision Support of Clinical Pathways

    NASA Astrophysics Data System (ADS)

    Gabriel, Roland; Lux, Thomas

    The German health care market is under a rapid rate of change, forcing especially hospitals to provide high-quality services at low costs. Appropriate measures for more effective and efficient service provision are process orientation and decision support by information technology of clinical pathway of a patient. The essential requirements are adequate modelling of clinical pathways as well as usage of adequate systems, which are capable of assisting the complete path of a patient within a hospital, and preferably also outside of it, in a digital way. To fulfil these specifications the authors present a suitable concept, which meets the challenges of well-structured clinical pathways as well as rather poorly structured diagnostic and therapeutic decisions, by interplay of process-oriented and knowledge-based hospital information systems.

  19. A C35 Carotenoid Biosynthetic Pathway

    PubMed Central

    Umeno, Daisuke; Arnold, Frances H.

    2003-01-01

    Upon coexpression with Erwinia geranylgeranyldiphosphate (GGDP) synthase in Escherichia coli, C30 carotenoid synthase CrtM from Staphylococcus aureus produces novel carotenoids with the asymmetrical C35 backbone. The products of condensation of farnesyldiphosphate and GDP, C35 structures comprise 40 to 60% of total carotenoid accumulated. Carotene desaturases and carotene cyclases from C40 or C30 pathways accepted and converted the C35 substrate, thus creating a C35 carotenoid biosynthetic pathway in E. coli. Directed evolution to modulate desaturase step number, together with combinatorial expression of the desaturase variants with lycopene cyclases, allowed us to produce at least 10 compounds not previously described. This result highlights the plastic and expansible nature of carotenoid pathways and illustrates how combinatorial biosynthesis coupled with directed evolution can rapidly access diverse chemical structures. PMID:12788765

  20. Coinhibitory Pathways in Immunotherapy for Cancer.

    PubMed

    Baumeister, Susanne H; Freeman, Gordon J; Dranoff, Glenn; Sharpe, Arlene H

    2016-05-20

    The immune system is capable of recognizing tumors and eliminates many early malignant cells. However, tumors evolve to evade immune attack, and the tumor microenvironment is immunosuppressive. Immune responses are regulated by a number of immunological checkpoints that promote protective immunity and maintain tolerance. T cell coinhibitory pathways restrict the strength and duration of immune responses, thereby limiting immune-mediated tissue damage, controlling resolution of inflammation, and maintaining tolerance to prevent autoimmunity. Tumors exploit these coinhibitory pathways to evade immune eradication. Blockade of the PD-1 and CTLA-4 checkpoints is proving to be an effective and durable cancer immunotherapy in a subset of patients with a variety of tumor types, and additional combinations are further improving response rates. In this review we discuss the immunoregulatory functions of coinhibitory pathways and their translation to effective immunotherapies for cancer. PMID:26927206

  1. Crosstalk of the Wnt/β-catenin pathway with other pathways in cancer cells

    PubMed Central

    Morris, Saint-Aaron L.; Huang, Suyun

    2016-01-01

    Many cancers have similar aberrations in various signaling cascades with crucial roles in cellular proliferation, differentiation, and morphogenesis. Dysregulation of signal cascades that play integral roles during early cellular development is well known to be a central feature of many malignancies. One such signaling cascade is the Wnt/β-catenin pathway, which has a profound effect on stem cell proliferation, migration, and differentiation. This pathway is dysregulated in numerous cell types, underscoring its global oncogenetic potential. This review highlights regulators and downstream effectors of this receptor cascade and addresses the increasingly apparent crosstalk of Wnt with other tumorigenic signaling pathways. As understanding of the genetic and epigenetic changes unique to these malignancies increases, identifying the regulatory mechanisms unique to the Wnt/β-catenin pathway and similarly aberrant receptor pathways will be imperative. PMID:27081668

  2. Geoscience Academic Provenance: A Comparison of Undergraduate Students' Pathways to Faculty Pathways

    NASA Astrophysics Data System (ADS)

    Houlton, H. R.; Keane, C. M.; Wilson, C. E.

    2012-12-01

    Most Science, Technology, Engineering and Mathematics (STEM) disciplines have a direct recruiting method of high school science courses to supply their undergraduate majors. However, recruitment and retention of students into geoscience academic programs, who will be the future workforce, remains an important issue. The geoscience community is reaching a critical point in its ability to supply enough geoscientists to meet the current and near-future demand. Previous work done by Houlton (2010) determined that undergraduate geoscience majors follow distinct pathways when pursuing their degree and career. These pathways are comprised of students' interests, experiences, goals and career aspirations, which are depicted in six pathway steps. Three population groups were determined from the original 17 participants, which exhibited differences in pathway trajectories. Continued data collection efforts developed and refined the pathway framework. As part of an informal workshop activity, data were collected from 27 participants who are underrepresented minority early-career and future faculty in the geosciences. In addition, 20 geoscience departments' Heads and Chairs participated in an online survey about their pathway trajectories. Pathways were determined from each of these new sample populations and compared against the original geoscience undergraduate student participants. Several pathway components consistently spanned across sample populations. Identification of these themes have illuminated broad geoscience-related interests, experiences and aspirations that can be used to broadly impact recruitment and retention initiatives for our discipline. Furthermore, fundamental differences between participants' ages, stages in career and racial/ethnic backgrounds have exhibited subtle nuances in their geoscience pathway trajectories. In particular, those who've had research experiences, who think "creativity" is an important aspect of a geoscience career and those who

  3. Display of cardiac activation pathways with echocardiography

    NASA Astrophysics Data System (ADS)

    Olstad, Bjoern; Brodin, Lars A.; Berg, Sevald

    1997-05-01

    The study of cardiac activation dynamics is an important factor in the characterization of the cardiac function. One such example is the localization of WPW-pathways inside the myocardium. Accurate localization of these pathways can be used to determine if the patient should be treated with catheter techniques or surgical techniques. This paper analyzes the temporal information in tissue velocity imaging with both qualitative and quantitative methods. The clinical experiments indicate that echocardiography can become an alternative technique for non-invasive electrophysiology in these kinds of applications.

  4. UV signaling pathways within the skin

    PubMed Central

    Chen, Hongxiang; Weng, Qing Yu; Fisher, David E.

    2014-01-01

    The effects of UVR on the skin include tanning, carcinogenesis, immunomodulation, and synthesis of vitamin D, among others. Melanocortin 1 receptor polymorphisms correlate with skin pigmentation, UV sensitivity, and skin cancer risk. This article reviews pathways through which UVR induces cutaneous stress and the pigmentation response. Modulators of the UV tanning pathway include sunscreen agents, MC1R activators, adenylate cyclase activators, phosphodiesterase 4D3 inhibitors, T oligos, and MITF regulators such as histone deacetylase (HDAC)-inhibitors. UVR, as one of the most ubiquitous carcinogens, represents both a challenge and enormous opportunity in skin cancer prevention. PMID:24759085

  5. Recent progress on MAP kinase pathway inhibitors.

    PubMed

    Uehling, David E; Harris, Philip A

    2015-10-01

    The RAS-RAF-MEK-ERK, or ERK signaling pathway propagates signals through an intracellular signal transduction cascade. Since approximately one third of human cancers are impacted by mutations in the ERK signaling pathway, intensive efforts to develop drugs targeting members of this cascade are ongoing. While efforts to develop drugs aimed at inhibiting RAS are still at an early stage, substantial progress in discovering clinical drugs targeting RAF, MEK, and ERK have been made. This review will highlight the recent progress in this area. PMID:26298497

  6. Can we safely target the WNT pathway?

    PubMed Central

    Kahn, Michael

    2015-01-01

    WNT–β-catenin signalling is involved in a multitude of developmental processes and the maintenance of adult tissue homeostasis by regulating cell proliferation, differentiation, migration, genetic stability and apoptosis, as well as by maintaining adult stem cells in a pluripotent state. Not surprisingly, aberrant regulation of this pathway is therefore associated with a variety of diseases, including cancer, fibrosis and neurodegeneration. Despite this knowledge, therapeutic agents specifically targeting the WNT pathway have only recently entered clinical trials and none has yet been approved. This Review examines the problems and potential solutions to this vexing situation and attempts to bring them into perspective. PMID:24981364

  7. Multiple oxygen entry pathways in globin proteins revealed by intrinsic pathway identification method

    NASA Astrophysics Data System (ADS)

    Takayanagi, Masayoshi; Kurisaki, Ikuo; Nagaoka, Masataka

    2015-12-01

    Each subunit of human hemoglobin (HbA) stores an oxygen molecule (O2) in the binding site (BS) cavity near the heme group. The BS is buried in the interior of the subunit so that there is a debate over the O2 entry pathways from solvent to the BS; histidine gate or multiple pathways. To elucidate the O2 entry pathways, we executed ensemble molecular dynamics (MD) simulations of T-state tetramer HbA in high concentration O2 solvent to simulate spontaneous O2 entry from solvent into the BS. By analyzing 128 independent 8 ns MD trajectories by intrinsic pathway identification by clustering (IPIC) method, we found 141 and 425 O2 entry events into the BS of the α and β subunits, respectively. In both subunits, we found that multiple O2 entry pathways through inside cavities play a significant role for O2 entry process of HbA. The rate constants of O2 entry estimated from the MD trajectories correspond to the experimentally observed values. In addition, by analyzing monomer myoglobin, we verified that the high O2 concentration condition can reproduce the ratios of each multiple pathway in the one-tenth lower O2 concentration condition. These indicate the validity of the multiple pathways obtained in our MD simulations.

  8. PathwayExplorer: web service for visualizing high-throughput expression data on biological pathways.

    PubMed

    Mlecnik, Bernhard; Scheideler, Marcel; Hackl, Hubert; Hartler, Jürgen; Sanchez-Cabo, Fatima; Trajanoski, Zlatko

    2005-07-01

    While generation of high-throughput expression data is becoming routine, the fast, easy, and systematic presentation and analysis of these data in a biological context is still an obstacle. To address this need, we have developed PathwayExplorer, which maps expression profiles of genes or proteins simultaneously onto major, currently available regulatory, metabolic and cellular pathways from KEGG, BioCarta and GenMAPP. PathwayExplorer is a platform-independent web server application with an optional standalone Java application using a SOAP (simple object access protocol) interface. Mapped pathways are ranked for the easy selection of the pathway of interest, displaying all available genes of this pathway with their expression profiles in a selectable and intuitive color code. Pathway maps produced can be downloaded as PNG, JPG or as high-resolution vector graphics SVG. The web service is freely available at https://pathwayexplorer.genome.tugraz.at; the standalone client can be downloaded at http://genome.tugraz.at. PMID:15980551

  9. Final report on the Pathway Analysis Task

    SciTech Connect

    Whicker, F.W.; Kirchner, T.B.

    1993-04-01

    The Pathway Analysis Task constituted one of several multi-laboratory efforts to estimate radiation doses to people, considering all important pathways of exposure, from the testing of nuclear devices at the Nevada Test Site (NTS). The primary goal of the Pathway Analysis Task was to predict radionuclide ingestion by residents of Utah, Nevada, and portions of seven other adjoining western states following radioactive fallout deposition from individual events at the NTS. This report provides comprehensive documentation of the activities and accomplishments of Colorado State University`s Pathway Analysis Task during the entire period of support (1979--91). The history of the project will be summarized, indicating the principal dates and milestones, personnel involved, subcontractors, and budget information. Accomplishments, both primary and auxiliary, will be summarized with general results rather than technical details being emphasized. This will also serve as a guide to the reports and open literature publications produced, where the methodological details and specific results are documented. Selected examples of results on internal dose estimates are provided in this report because the data have not been published elsewhere.

  10. The Intracellular Trafficking Pathway of Transferrin

    PubMed Central

    Mayle, Kristine M.; Le, Alexander M.; Kamei, Daniel T.

    2011-01-01

    Background Transferrin (Tf) is an iron-binding protein that facilitates iron-uptake in cells. Iron-loaded Tf first binds to the Tf receptor (TfR) and enters the cell through clathrin-mediated endocytosis. Inside the cell, Tf is trafficked to early endosomes, delivers iron, and then is subsequently directed to recycling endosomes to be taken back to the cell surface. Scope of Review We aim to review the various methods and techniques that researchers have employed for elucidating the Tf trafficking pathway and the cell-machinery components involved. These experimental methods can be categorized as microscopy, radioactivity, and surface plasmon resonance (SPR). Major Conclusions Qualitative experiments, such as total internal reflectance fluorescence (TIRF), electron, laser-scanning confocal, and spinning-disk confocal microscopy, have been utilized to determine the roles of key components in the Tf trafficking pathway. These techniques allow temporal resolution and are useful for imaging Tf endocytosis and recycling, which occur on the order of seconds to minutes. Additionally, radiolabeling and SPR methods, when combined with mathematical modeling, have enabled researchers to estimate quantitative kinetic parameters and equilibrium constants associated with Tf binding and trafficking. General Significance Both qualitative and quantitative data can be used to analyze the Tf trafficking pathway. The valuable information that is obtained about the Tf trafficking pathway can then be combined with mathematical models to identify design criteria to improve the ability of Tf to deliver anticancer drugs. PMID:21968002

  11. Instructional Partnerships: A Pathway to Leadership

    ERIC Educational Resources Information Center

    Moreillon, Judi, Ed.; Ballard, Susan, Ed.

    2013-01-01

    In this Best of "Knowledge Quest" monograph, the editors have collected seminal articles to support pre-service and in-service school librarians in developing and strengthening the instructional partner role. "Instructional Partnerships: A Pathway to Leadership" provides readers with background knowledge, research-based…

  12. Strategic approaches to adverse outcome pathway development

    EPA Science Inventory

    Adverse outcome pathways (AOPs) are conceptual frameworks for organizing biological and toxicological knowledge in a manner that supports extrapolation of data pertaining to the initiation or early progression of toxicity to an apical adverse outcome that occurs at a level of org...

  13. Connecticut Postsecondary Pathways for Opportunity Youth

    ERIC Educational Resources Information Center

    American Youth Policy Forum, 2015

    2015-01-01

    Pathways to Postsecondary Opportunities are the range of options created across education institutions, training providers, and community-­based organizations so that each and every young person can access the necessary and personally relevant credentials, skills, and training beyond the completion of a secondary credential that will propel…

  14. Syngas Upgrading to Hydrocarbon Fuels Technology Pathway

    SciTech Connect

    Talmadge, M.; Biddy, Mary J.; Dutta, Abhijit; Jones, Susanne B.; Meyer, Pimphan A.

    2013-03-31

    In support of the Bioenergy Technologies Office, the National Renewable Energy Laboratory (NREL) and the Pacific Northwest National Laboratory (PNNL) are undertaking studies of biomass conversion technologies to hydrocarbon fuels to identify barriers and target research toward reducing conversion costs. Process designs and preliminary economic estimates for each of these pathway cases were developed using rigorous modeling tools (Aspen Plus and Chemcad). These analyses incorporated the best information available at the time of development, including data from recent pilot and bench-scale demonstrations, collaborative industrial and academic partners, and published literature and patents. This pathway case investigates the upgrading of biomass derived synthesis gas (‘syngas’) to hydrocarbon biofuels. While this specific discussion focuses on the conversion of syngas via a methanol intermediate to hydrocarbon blendstocks, there are a number of alternative conversion routes for production of hydrocarbons through a wide array of intermediates from syngas. Future work will also consider the variations to this pathway to determine the most economically viable and risk adverse conversion route. Technical barriers and key research needs have been identified that should be pursued for the syngas to hydrocarbon pathway to be competitive with petroleum-derived gasoline, diesel and jet range blendstocks.

  15. Disentangling Adolescent Pathways of Sexual Risk Taking

    ERIC Educational Resources Information Center

    Brookmeyer, Kathryn A.; Henrich, Christopher C.

    2009-01-01

    Using data from the National Longitudinal Survey of Youth, the authors aimed to describe the pathways of risk within sexual risk taking, alcohol use, and delinquency, and then identify how the trajectory of sexual risk is linked to alcohol use and delinquency. Risk trajectories were measured with adolescents aged 15-24 years (N = 1,778). Using…

  16. [The medicine use pathway in paediatrics].

    PubMed

    Didelot, Nicolas

    2016-01-01

    The medicine use pathway is a process which is constantly evolving in order to comply with intangible rules. As in other therapeutic fields, the drug regimen in paediatrics must tolerate no error and must be able to detect all warning signs, however minor, in order to optimise this approach. PMID:27177481

  17. The adverse outcome pathway knowledge base

    EPA Science Inventory

    The rapid advancement of the Adverse Outcome Pathway (AOP) framework has been paralleled by the development of tools to store, analyse, and explore AOPs. The AOP Knowledge Base (AOP-KB) project has brought three independently developed platforms (Effectopedia, AOP-Wiki, and AOP-X...

  18. Using biological pathway data with paxtools.

    PubMed

    Demir, Emek; Babur, Ozgün; Rodchenkov, Igor; Aksoy, Bülent Arman; Fukuda, Ken I; Gross, Benjamin; Sümer, Onur Selçuk; Bader, Gary D; Sander, Chris

    2013-01-01

    A rapidly growing corpus of formal, computable pathway information can be used to answer important biological questions including finding non-trivial connections between cellular processes, identifying significantly altered portions of the cellular network in a disease state and building predictive models that can be used for precision medicine. Due to its complexity and fragmented nature, however, working with pathway data is still difficult. We present Paxtools, a Java library that contains algorithms, software components and converters for biological pathways represented in the standard BioPAX language. Paxtools allows scientists to focus on their scientific problem by removing technical barriers to access and analyse pathway information. Paxtools can run on any platform that has a Java Runtime Environment and was tested on most modern operating systems. Paxtools is open source and is available under the Lesser GNU public license (LGPL), which allows users to freely use the code in their software systems with a requirement for attribution. Source code for the current release (4.2.0) can be found in Software S1. A detailed manual for obtaining and using Paxtools can be found in Protocol S1. The latest sources and release bundles can be obtained from biopax.org/paxtools. PMID:24068901

  19. Alternative Certification Pathways: Filling a Gap?

    ERIC Educational Resources Information Center

    Ludlow, Carlyn

    2013-01-01

    The purpose of this article is to examine the proliferation of alternative certification pathways through an analysis of the role and history of teacher certification and supply followed by a synthesis of national, regional, and state research studies on alternative routes to certification programs and a review of studies conducted on well-known…

  20. Air Research Program: Key Pathways research track

    EPA Science Inventory

    The pathways research track applies animal, cellular, and human studies to discern whether there is a common molecular mechanism (e.g. production of oxidative stress, phosphatase inhibition, disruption of iron homeostasis) through which air pollutants induce toxicity of air pollu...

  1. Response Ability Pathways: A Curriculum for Connecting

    ERIC Educational Resources Information Center

    Koehler, Nancy; Seger, Vikki

    2005-01-01

    This article describes a new training curriculum for educators, youth workers, and mentors which draws from research and best practices in positive youth development and positive behavior support. Response Ability Pathways or RAP focuses on three practical interventions: connect to others for support, clarify challenging problems, and restore…

  2. Career Technical Education Pathways Initiative Annual Report

    ERIC Educational Resources Information Center

    California Community Colleges, Chancellor's Office, 2014

    2014-01-01

    California's education system--the largest in the United States--is an essential resource for ensuring strong economic growth in the state. The Career Technical Education Pathways Initiative (the Initiative) became law in 2005 with Senate Bills 70 and 1133 and provided more than $380 million over eight years to improve career technical education…

  3. Student Age and the Collegiate Pathway

    ERIC Educational Resources Information Center

    Hurwitz, Michael; Smith, Jonathan; Howell, Jessica S.

    2015-01-01

    Using a rich data set of all SAT test takers from the 2004 through 2008 high school graduation cohorts, we investigate the impact of state-specific school age-of-entry laws on students' pathways into and through college. We document that these laws do not impact the probability that a student takes the SAT; however, we find strong evidence…

  4. Play as a Pathway of Behavior

    ERIC Educational Resources Information Center

    Henricks, Thomas S.

    2011-01-01

    Seeking to understand play as part of a more general theory of human relationships, the author defines play as one of four fundamental categories of behavior, the others being work, ritual, and communitas. He discusses how each of these behaviors is organized as a "pathway" that offers distinctive opportunities for experiencing life and for…

  5. ELUCIDATING THE PATHWAY FOR ARSENIC METHYLATION

    EPA Science Inventory

    Enzymatically-catalyzed methylation of arsenic is part of a metabolic pathway that converts inorganic arsenic into methylated products. Hence, in humans chronically exposed to inorganic arsenic, methyl and dimethyl arsenic account for most of the arsenic that is excreted in the ...

  6. Evolution-guided optimization of biosynthetic pathways

    PubMed Central

    Raman, Srivatsan; Rogers, Jameson K.; Taylor, Noah D.; Church, George M.

    2014-01-01

    Engineering biosynthetic pathways for chemical production requires extensive optimization of the host cellular metabolic machinery. Because it is challenging to specify a priori an optimal design, metabolic engineers often need to construct and evaluate a large number of variants of the pathway. We report a general strategy that combines targeted genome-wide mutagenesis to generate pathway variants with evolution to enrich for rare high producers. We convert the intracellular presence of the target chemical into a fitness advantage for the cell by using a sensor domain responsive to the chemical to control a reporter gene necessary for survival under selective conditions. Because artificial selection tends to amplify unproductive cheaters, we devised a negative selection scheme to eliminate cheaters while preserving library diversity. This scheme allows us to perform multiple rounds of evolution (addressing ∼109 cells per round) with minimal carryover of cheaters after each round. Based on candidate genes identified by flux balance analysis, we used targeted genome-wide mutagenesis to vary the expression of pathway genes involved in the production of naringenin and glucaric acid. Through up to four rounds of evolution, we increased production of naringenin and glucaric acid by 36- and 22-fold, respectively. Naringenin production (61 mg/L) from glucose was more than double the previous highest titer reported. Whole-genome sequencing of evolved strains revealed additional untargeted mutations that likely benefit production, suggesting new routes for optimization. PMID:25453111

  7. Vitamins and aging: pathways to NAD+ synthesis.

    PubMed

    Denu, John M

    2007-05-01

    Recent genetic evidence reveals additional salvage pathways for NAD(+) synthesis. In this issue, Belenky et al. (2007) report that nicotinamide riboside, a new NAD(+) precursor, regulates Sir2 deacetylase activity and life span in yeast. The ability of nicotinamide riboside to enhance life span does not depend on calorie restriction. PMID:17482537

  8. Molecular aspects of the endocytic pathway.

    PubMed Central

    Clague, M J

    1998-01-01

    Observation of the flow of material along the endocytic pathway has lead to the description of the basic architecture of the pathway and provided insight into the relationship between compartments. Significant advances have been made in the study of endocytic transport steps at the molecular level, of which studies of cargo selection, vesicle budding and membrane fusion events comprise the major part. Progress in this area has been driven by two approaches, yeast genetics and in vitro or cell-free assays, which reconstitute particular transport steps and allow biochemical manipulation. The complex protein machineries that control vesicle budding and fusion are significantly conserved between the secretory and endocytic pathways such that proteins that regulate particular steps are often part of a larger family of proteins which exercise a conserved function at other locations within the cell. Well characterized examples include vesicle coat proteins, rabs (small GTPases) and soluble N-ethylmaleimide-sensitive fusion protein (NSF) attachment protein (SNAP) receptors (SNAREs). Intracompartmental pH, lipid composition and cytoskeletal organization have also been identified as important determinants of the orderly flow of material within the endocytic pathway. PMID:9820800

  9. An evolutionarily conserved pathway controls proteasome homeostasis.

    PubMed

    Rousseau, Adrien; Bertolotti, Anne

    2016-08-11

    The proteasome is essential for the selective degradation of most cellular proteins, but how cells maintain adequate amounts of proteasome is unclear. Here we show that there is an evolutionarily conserved signalling pathway controlling proteasome homeostasis. Central to this pathway is TORC1, the inhibition of which induced all known yeast 19S regulatory particle assembly-chaperones (RACs), as well as proteasome subunits. Downstream of TORC1 inhibition, the yeast mitogen-activated protein kinase, Mpk1, acts to increase the supply of RACs and proteasome subunits under challenging conditions in order to maintain proteasomal degradation and cell viability. This adaptive pathway was evolutionarily conserved, with mTOR and ERK5 controlling the levels of the four mammalian RACs and proteasome abundance. Thus, the central growth and stress controllers, TORC1 and Mpk1/ERK5, endow cells with a rapid and vital adaptive response to adjust proteasome abundance in response to the rising needs of cells. Enhancing this pathway may be a useful therapeutic approach for diseases resulting from impaired proteasomal degradation. PMID:27462806

  10. Students' Perspectives of an EAP Pathway Program

    ERIC Educational Resources Information Center

    Dooey, Patricia

    2010-01-01

    Increasing numbers of overseas students are applying to study at universities in Australia. Many students who meet all of the university's academic entry requirements except English language proficiency are offered pathway programs which prepare them for their tertiary studies. To date, much of the research relating to international students…

  11. Alternative Pathways to Apprenticeships. Good Practice Guide

    ERIC Educational Resources Information Center

    National Centre for Vocational Education Research (NCVER), 2015

    2015-01-01

    Apprenticeships are changing. The increasing proportions of people entering apprenticeships at various levels of ability and backgrounds are stimulating demand for alternative pathways to completions. This good practice guide assembles the key findings for education practitioners and workplace supervisors from three related research reports on…

  12. Rubric for Linked Learning Pathway Certification

    ERIC Educational Resources Information Center

    LaPlante, Arlene; Stearns, Roman

    2010-01-01

    This rubric was created to help pathway teams as they work together to develop and improve a comprehensive program of study. Specifically, the rubric can serve as a tool for: (1) Visioning; (2) Self-assessment; (3) Planning; and (4) Quality review. ConnectEd designed this rubric to be used in coordination with the Certification Criteria for Linked…

  13. [Nutritional pathway for autologous stem cell transplantation].

    PubMed

    Aoyama, Takashi; Imataki, Osamu; Inoue, Naomi; Katsumata, Mina; Katsuta, Tomoko; Kataoka, Tomomi; Yoshida, Takashi; Mochizuki, Takahiro; Motokawa, Satoshi; Tamai, Yotaro; Hagiwara, Shotaro; Kawakami, Kimihiro

    2007-08-01

    We developed a nutritional pathway for autologous stem cell transplantation (SCT) to be applied in our transplantation unit. We performed autologous SCT for 37 patients with malignant lymphoma and multiple myeloma during from April 2003 to July 2005. For 10 of them who underwent SCT since 2005,we intervened with nutritional support using our original nutritional pathway,to monitor the clinical course of SCT from the aspect of dietetics with a dietician making assessments of the individual nutrition status. From comparing the 2 groups with (n=27) or without (n=10) the nutritional pathway, oral intake at day 14 was significantly increased from 1,038 kcal to 1,440 kcal,and at discharge developed from 1,167 kcal to 1,446 kcal without statistical significance. Patients whose body weight decreased more than 5% were reduced from 52%(14/27) to 10%(1/10),and 3 days reduction of the CVC insertion period was observed after the intervention. Although the long-term clinical outcome was not fully evaluated, the efficacy of nutritional pathway for autologous SCT was suggested. PMID:17687206

  14. Adverse outcome pathway (AOP) development and evaluation

    EPA Science Inventory

    The Adverse Outcome Pathway provides a construct for assembling mechanistic information at different levels of biological organization in a form designed to support regulatory decision making. In particular, it frames the link between molecular and cellular events that can be mea...

  15. On the origin of metabolic pathways

    NASA Technical Reports Server (NTRS)

    Lazcano, A.; Miller, S. L.; Bada, J. L. (Principal Investigator)

    1999-01-01

    The heterotrophic theory of the origin of life is the only proposal available with experimental support. This comes from the ease of prebiotic synthesis under strongly reducing conditions. The prebiotic synthesis of organic compounds by reduction of CO(2) to monomers used by the first organisms would also be considered an heterotrophic origin. Autotrophy means that the first organisms biosynthesized their cell constituents as well as assembling them. Prebiotic synthetic pathways are all different from the biosynthetic pathways of the last common ancestor (LCA). The steps leading to the origin of the metabolic pathways are closer to prebiotic chemistry than to those in the LCA. There may have been different biosynthetic routes between the prebiotic and the LCAs that played an early role in metabolism but have disappeared from extant organisms. The semienzymatic theory of the origin of metabolism proposed here is similar to the Horowitz hypothesis but includes the use of compounds leaking from preexisting pathways as well as prebiotic compounds from the environment.

  16. Pathways to Aggression in Children and Adolescents

    ERIC Educational Resources Information Center

    Watson, Malcolm W.; Fischer, Kurt W.; Andreas, Jasmina Burdzovic; Smith, Kevin W.

    2004-01-01

    In this article, Malcolm Watson, Kurt Fischer, Jasmina Burdzovic Andreas, and Kevin Smith describe and compare two approaches to assessing risk factors that lead to aggression in children. The first, the severe risks approach, focuses on how risk factors form a pathway that leads to aggressive behavior. Within this approach, an inhibited…

  17. Whole Algae Hydrothermal Liquefaction Technology Pathway

    SciTech Connect

    Biddy, M.; Davis, R.; Jones, S.

    2013-03-01

    This technology pathway case investigates the feasibility of using whole wet microalgae as a feedstock for conversion via hydrothermal liquefaction. Technical barriers and key research needs have been assessed in order for the hydrothermal liquefaction of microalgae to be competitive with petroleum-derived gasoline-, diesel-, and jet-range hydrocarbon blendstocks.

  18. Pathway Analysis: State of the Art

    PubMed Central

    García-Campos, Miguel A.; Espinal-Enríquez, Jesús; Hernández-Lemus, Enrique

    2015-01-01

    Pathway analysis is a set of widely used tools for research in life sciences intended to give meaning to high-throughput biological data. The methodology of these tools settles in the gathering and usage of knowledge that comprise biomolecular functioning, coupled with statistical testing and other algorithms. Despite their wide employment, pathway analysis foundations and overall background may not be fully understood, leading to misinterpretation of analysis results. This review attempts to comprise the fundamental knowledge to take into consideration when using pathway analysis as a hypothesis generation tool. We discuss the key elements that are part of these methodologies, their capabilities and current deficiencies. We also present an overview of current and all-time popular methods, highlighting different classes across them. In doing so, we show the exploding diversity of methods that pathway analysis encompasses, point out commonly overlooked caveats, and direct attention to a potential new class of methods that attempt to zoom the analysis scope to the sample scale. PMID:26733877

  19. Science Learning Pathways for Young Children

    ERIC Educational Resources Information Center

    Gelman, Rochel; Brenneman, Kimberly

    2004-01-01

    Preschool Pathways to Science (PrePS[C]) is a science and math program for pre-K children that has been developed by a team of developmental psychologists in full collaboration with preschool directors, teachers and other staff. The PrePS[C] approach is rooted in domain-specific theories of development, theories that assume that different areas of…

  20. Macropinocytosis: a pathway to protozoan infection.

    PubMed

    de Carvalho, Tecia M U; Barrias, Emile S; de Souza, Wanderley

    2015-01-01

    Among the various endocytic mechanisms in mammalian cells, macropinocytosis involves internalization of large amounts of plasma membrane together with extracellular medium, leading to macropinosome formation. These structures are formed when plasma membrane ruffles are assembled after actin filament rearrangement. In dendritic cells, macropinocytosis has been reported to play a role in antigen presentation. Several intracellular pathogens are internalized by host cells via multiple endocytic pathways and macropinocytosis has been described as an important entry site for various organisms. Some bacteria, such as Legionella pneumophila, as well as various viruses, use this pathway to penetrate and subvert host cells. Some protozoa, which are larger than bacteria and virus, can also use this pathway to invade host cells. As macropinocytosis is characterized by the formation of large uncoated vacuoles and is triggered by various signaling pathways, which is similar to what occurs during the formation of the majority of parasitophorous vacuoles, it is believed that this phenomenon may be more widely used by parasites than is currently appreciated. Here we review protozoa host cell invasion via macropinocytosis. PMID:25914647

  1. Dorsal and Ventral Pathways for Prosody.

    PubMed

    Sammler, Daniela; Grosbras, Marie-Hélène; Anwander, Alfred; Bestelmeyer, Patricia E G; Belin, Pascal

    2015-12-01

    Our vocal tone--the prosody--contributes a lot to the meaning of speech beyond the actual words. Indeed, the hesitant tone of a "yes" may be more telling than its affirmative lexical meaning. The human brain contains dorsal and ventral processing streams in the left hemisphere that underlie core linguistic abilities such as phonology, syntax, and semantics. Whether or not prosody--a reportedly right-hemispheric faculty--involves analogous processing streams is a matter of debate. Functional connectivity studies on prosody leave no doubt about the existence of such streams, but opinions diverge on whether information travels along dorsal or ventral pathways. Here we show, with a novel paradigm using audio morphing combined with multimodal neuroimaging and brain stimulation, that prosody perception takes dual routes along dorsal and ventral pathways in the right hemisphere. In experiment 1, categorization of speech stimuli that gradually varied in their prosodic pitch contour (between statement and question) involved (1) an auditory ventral pathway along the superior temporal lobe and (2) auditory-motor dorsal pathways connecting posterior temporal and inferior frontal/premotor areas. In experiment 2, inhibitory stimulation of right premotor cortex as a key node of the dorsal stream decreased participants' performance in prosody categorization, arguing for a motor involvement in prosody perception. These data draw a dual-stream picture of prosodic processing that parallels the established left-hemispheric multi-stream architecture of language, but with relative rightward asymmetry. PMID:26549262

  2. Developmental Pathways in Boys' Disruptive Delinquent Behavior.

    ERIC Educational Resources Information Center

    Kelley, Barbara Tatem; Loeber, Rolf; Keenan, Kate; DeLamatre, Mary

    1997-01-01

    Boys may follow various developmental pathways that lead to increasingly disruptive and delinquent behavior. To most parents, teachers, youth workers, mental health professionals, and juvenile justice practitioners, the development of disruptive and delinquent behavior in boys may appear erratic and unpredictable. This bulletin summarizes…

  3. Integrating Alternative Educational Pathways: Challenges and Issues

    ERIC Educational Resources Information Center

    Brewer, Ann M.

    2008-01-01

    The paper examines the issue of educational pathways, including a brief overview of the higher education regulatory framework and market forces in Australia, particularly as recent policy reforms and political aspirations affect them. It highlights the key challenges and outlines a potential model for integrating vocational and higher educational…

  4. Pathway Analysis Software: Annotation Errors and Solutions

    PubMed Central

    Henderson-MacLennan, Nicole K.; Papp, Jeanette C.; Talbot, C. Conover; McCabe, Edward R.B.; Presson, Angela P.

    2010-01-01

    Genetic databases contain a variety of annotation errors that often go unnoticed due to the large size of modern genetic data sets. Interpretation of these data sets requires bioinformatics tools that may contribute to this problem. While providing gene symbol annotations for identifiers (IDs) such as microarray probeset, RefSeq, GenBank and Entrez Gene is seemingly trivial, the accuracy is fundamental to any subsequent conclusions. We examine gene symbol annotations and results from three commercial pathway analysis software (PAS) packages: Ingenuity Pathways Analysis, GeneGO and Pathway Studio. We compare gene symbol annotations and canonical pathway results over time and among different input ID types. We find that PAS results can be affected by variation in gene symbol annotations across software releases and the input ID type analyzed. As a result, we offer suggestions for using commercial PAS and reporting microarray results to improve research quality. We propose a wiki type website to facilitate communication of bioinformatics software problems within the scientific community. PMID:20663702

  5. Precursors of Young Women's Family Formation Pathways

    ERIC Educational Resources Information Center

    Amato, Paul R.; Landale, Nancy S.; Havasevich-Brooks, Tara C.; Booth, Alan; Eggebeen, David J.; Schoen, Robert; McHale, Susan M.

    2008-01-01

    We used latent class analysis to create family formation pathways for women between the ages of 18 and 23. Input variables included cohabitation, marriage, parenthood, full-time employment, and attending school. Data (n = 2,290) came from Waves I and III of the National Longitudinal Study of Adolescent Health (Add Health). The analysis revealed…

  6. Negotiating Assignment Pathways: Students and Academic Assignments

    ERIC Educational Resources Information Center

    McDowell, Liz

    2008-01-01

    Existing research identifies that students' approaches to assignments are related to their general approaches to study. It is suggested that students need to better understand the requirements of assignments and acquire new concepts such as "argument". This fine-grained study proposes four qualitatively distinct assignment pathways: gathering,…

  7. Pathways to Mathematics College Readiness in Maine

    ERIC Educational Resources Information Center

    Silvernail, David L; Batista, Ida A.; Sloan, James E.; Stump, Erika K.; Johnson, Amy F.

    2014-01-01

    The goal of this study was to examine the pathways to being college ready in mathematics. Students who enter high school already having demonstrated mathematics proficiency on a standardized test in the 8th grade have already taken a significant step towards being college ready. The best scenario is to enter high school proficient in mathematics…

  8. Pathways to Childlessness: A Life Course Perspective

    ERIC Educational Resources Information Center

    Hagestad, Gunhild O.; Call, Vaughn R. A.

    2007-01-01

    In this article life history data from the U.S. National Survey of Families and Households (NSFH), and the Dutch survey on Older Adults' Living Arrangements and Social Networks (NESTOR-LSN) are used to shed light on the various pathways leading to and associated with childlessness, and the proportions of men and women who have followed a…

  9. Pathways for Learning from 3D Technology

    ERIC Educational Resources Information Center

    Carrier, L. Mark; Rab, Saira S.; Rosen, Larry D.; Vasquez, Ludivina; Cheever, Nancy A.

    2012-01-01

    The purpose of this study was to find out if 3D stereoscopic presentation of information in a movie format changes a viewer's experience of the movie content. Four possible pathways from 3D presentation to memory and learning were considered: a direct connection based on cognitive neuroscience research; a connection through "immersion" in that 3D…

  10. Pathways to Relationship Aggression between Adult Partners

    ERIC Educational Resources Information Center

    Busby, Dean M.; Holman, Thomas B.; Walker, Eric

    2008-01-01

    In this study, the pathways to adult aggression beginning in the family of origin (FOO) and continuing through adult relationships were investigated. With a sample of 30,600 individuals, a comprehensive model was evaluated that included the unique influences of violent victimization in the family, witnessing parental violence, perpetrating…

  11. The Ran Pathway in Drosophila melanogaster Mitosis

    PubMed Central

    Chen, Jack W. C.; Barker, Amy R.; Wakefield, James G.

    2015-01-01

    Over the last two decades, the small GTPase Ran has emerged as a central regulator of both mitosis and meiosis, particularly in the generation, maintenance, and regulation of the microtubule (MT)-based bipolar spindle. Ran-regulated pathways in mitosis bear many similarities to the well-characterized functions of Ran in nuclear transport and, as with transport, the majority of these mitotic effects are mediated through affecting the physical interaction between karyopherins and Spindle Assembly Factors (SAFs)—a loose term describing proteins or protein complexes involved in spindle assembly through promoting nucleation, stabilization, and/or depolymerization of MTs, through anchoring MTs to specific structures such as centrosomes, chromatin or kinetochores, or through sliding MTs along each other to generate the force required to achieve bipolarity. As such, the Ran-mediated pathway represents a crucial functional module within the wider spindle assembly landscape. Research into mitosis using the model organism Drosophila melanogaster has contributed substantially to our understanding of centrosome and spindle function. However, in comparison to mammalian systems, very little is known about the contribution of Ran-mediated pathways in Drosophila mitosis. This article sets out to summarize our understanding of the roles of the Ran pathway components in Drosophila mitosis, focusing on the syncytial blastoderm embryo, arguing that it can provide important insights into the conserved functions on Ran during spindle formation. PMID:26636083

  12. Career Pathways: Education with a Purpose

    ERIC Educational Resources Information Center

    Hull, Dan M.

    2004-01-01

    Hot off the press comes the guide to the next generation of education reform. Dan Hull and some of the nation's leading practitioners and educational leaders show how to remake high schools to improve academic outcomes, prepare students for today's high-skills workplace, and motivate them to learn because they see a pathway to their future.…

  13. Regulatory pathways in the European Union.

    PubMed

    Kohler, Manuela

    2011-01-01

    In principle, there are three defined procedures to obtain approval for a medicinal product in the European Union. As discussed in this overview of the procedures, the decision on which regulatory pathway to use will depend on the nature of the active substance, the target indication(s), the history of product and/or the marketing strategy. PMID:21487236

  14. MDRC Research on Career Pathways. Issue Brief

    ERIC Educational Resources Information Center

    Kazis, Richard

    2016-01-01

    As postsecondary credentials have become increasingly important to accessing higher-quality employment, a growing number of education and workforce programs are implementing "career pathways" approaches to help both youth and adults prepare for further education and better jobs. In recent years, the Manpower Demonstration Research…

  15. Macropinocytosis: a pathway to protozoan infection

    PubMed Central

    de Carvalho, Tecia M. U.; Barrias, Emile S.; de Souza, Wanderley

    2015-01-01

    Among the various endocytic mechanisms in mammalian cells, macropinocytosis involves internalization of large amounts of plasma membrane together with extracellular medium, leading to macropinosome formation. These structures are formed when plasma membrane ruffles are assembled after actin filament rearrangement. In dendritic cells, macropinocytosis has been reported to play a role in antigen presentation. Several intracellular pathogens are internalized by host cells via multiple endocytic pathways and macropinocytosis has been described as an important entry site for various organisms. Some bacteria, such as Legionella pneumophila, as well as various viruses, use this pathway to penetrate and subvert host cells. Some protozoa, which are larger than bacteria and virus, can also use this pathway to invade host cells. As macropinocytosis is characterized by the formation of large uncoated vacuoles and is triggered by various signaling pathways, which is similar to what occurs during the formation of the majority of parasitophorous vacuoles, it is believed that this phenomenon may be more widely used by parasites than is currently appreciated. Here we review protozoa host cell invasion via macropinocytosis. PMID:25914647

  16. Pathway Analyses Implicate Glial Cells in Schizophrenia

    PubMed Central

    Duncan, Laramie E.; Holmans, Peter A.; Lee, Phil H.; O'Dushlaine, Colm T.; Kirby, Andrew W.; Smoller, Jordan W.; Öngür, Dost; Cohen, Bruce M.

    2014-01-01

    Background The quest to understand the neurobiology of schizophrenia and bipolar disorder is ongoing with multiple lines of evidence indicating abnormalities of glia, mitochondria, and glutamate in both disorders. Despite high heritability estimates of 81% for schizophrenia and 75% for bipolar disorder, compelling links between findings from neurobiological studies, and findings from large-scale genetic analyses, are only beginning to emerge. Method Ten publically available gene sets (pathways) related to glia, mitochondria, and glutamate were tested for association to schizophrenia and bipolar disorder using MAGENTA as the primary analysis method. To determine the robustness of associations, secondary analyses were performed with: ALIGATOR, INRICH, and Set Screen. Data from the Psychiatric Genomics Consortium (PGC) were used for all analyses. There were 1,068,286 SNP-level p-values for schizophrenia (9,394 cases/12,462 controls), and 2,088,878 SNP-level p-values for bipolar disorder (7,481 cases/9,250 controls). Results The Glia-Oligodendrocyte pathway was associated with schizophrenia, after correction for multiple tests, according to primary analysis (MAGENTA p = 0.0005, 75% requirement for individual gene significance) and also achieved nominal levels of significance with INRICH (p = 0.0057) and ALIGATOR (p = 0.022). For bipolar disorder, Set Screen yielded nominally and method-wide significant associations to all three glial pathways, with strongest association to the Glia-Astrocyte pathway (p = 0.002). Conclusions Consistent with findings of white matter abnormalities in schizophrenia by other methods of study, the Glia-Oligodendrocyte pathway was associated with schizophrenia in our genomic study. These findings suggest that the abnormalities of myelination observed in schizophrenia are at least in part due to inherited factors, contrasted with the alternative of purely environmental causes (e.g. medication effects or lifestyle). While not

  17. Human performance evaluation of a pathway HMD

    NASA Astrophysics Data System (ADS)

    Lorenz, Bernd; Tobben, Helmut; Schmerwitz, Sven

    2005-05-01

    Head-up displays (HUD) and helmet (or head)-mounted displays (HMD) aim at reducing the pilot's visual scanning cost in support of concurrent monitoring of both instrument information (near domain) and the outside environment (far domain). An HMD used in combination with a head tracker enables the assessment of the pilot"s head direction in real time allowing symbologies to remain spatially linked to elements of the outside environment. The paper examines the potential added benefits of improved flight path tracking to be expected by displaying symbologies of a virtual 3D perspective pathway plus predictor information on an HMD. Results of a high-fidelity flight-simulation experiment are reported that involved a series of curved approaches supported with such a pathway HMD. The study used a monocular retinal-scanning HMD and involved 18 pilots. Dependent human performance data were derived from flight path tracking measures, subjective measures of mental workload and situation awareness and pilot reactions in response to an unexpected rare event in the outside scene (intruding aircraft on the active runway for the intended landing). Comparison with a standard head-down ILS baseline condition revealed a mix of performance costs and benefits, which is consistent with most of the human factors literature on the general use of HUDs and of HUDs used in combination with pathway guidance: The pathway HMD promoted substantially better flight path tracking but caused also a delayed response to the unexpected event. This effect points to some disadvantages of HUDs referred to as 'attention capture', which may become exaggerated by the additional use of pathway guidance symbology.

  18. Origin and evolution of metabolic pathways

    NASA Astrophysics Data System (ADS)

    Fani, Renato; Fondi, Marco

    2009-03-01

    The emergence and evolution of metabolic pathways represented a crucial step in molecular and cellular evolution. In fact, the exhaustion of the prebiotic supply of amino acids and other compounds that were likely present in the ancestral environment, imposed an important selective pressure, favoring those primordial heterotrophic cells which became capable of synthesizing those molecules. Thus, the emergence of metabolic pathways allowed primitive organisms to become increasingly less-dependent on exogenous sources of organic compounds. Comparative analyses of genes and genomes from organisms belonging to Archaea, Bacteria and Eukarya revealed that, during evolution, different forces and molecular mechanisms might have driven the shaping of genomes and the arisal of new metabolic abilities. Among these gene elongations, gene and operon duplications undoubtedly played a major role since they can lead to the (immediate) appearance of new genetic material that, in turn, might undergo evolutionary divergence giving rise to new genes coding for new metabolic abilities. Gene duplication has been invoked in the different schemes proposed to explain why and how the extant metabolic pathways have arisen and shaped. Both the analysis of completely sequenced genomes and directed evolution experiments strongly support one of them, i.e. the patchwork hypothesis, according to which metabolic pathways have been assembled through the recruitment of primitive enzymes that could react with a wide range of chemically related substrates. However, the analysis of the structure and organization of genes belonging to ancient metabolic pathways, such as histidine biosynthesis and nitrogen fixation, suggested that other different hypothesis, i.e. the retrograde hypothesis or the semi-enzymatic theory, may account for the arisal of some metabolic routes.

  19. e-Science and biological pathway semantics

    PubMed Central

    Luciano, Joanne S; Stevens, Robert D

    2007-01-01

    Background The development of e-Science presents a major set of opportunities and challenges for the future progress of biological and life scientific research. Major new tools are required and corresponding demands are placed on the high-throughput data generated and used in these processes. Nowhere is the demand greater than in the semantic integration of these data. Semantic Web tools and technologies afford the chance to achieve this semantic integration. Since pathway knowledge is central to much of the scientific research today it is a good test-bed for semantic integration. Within the context of biological pathways, the BioPAX initiative, part of a broader movement towards the standardization and integration of life science databases, forms a necessary prerequisite for its successful application of e-Science in health care and life science research. This paper examines whether BioPAX, an effort to overcome the barrier of disparate and heterogeneous pathway data sources, addresses the needs of e-Science. Results We demonstrate how BioPAX pathway data can be used to ask and answer some useful biological questions. We find that BioPAX comes close to meeting a broad range of e-Science needs, but certain semantic weaknesses mean that these goals are missed. We make a series of recommendations for re-modeling some aspects of BioPAX to better meet these needs. Conclusion Once these semantic weaknesses are addressed, it will be possible to integrate pathway information in a manner that would be useful in e-Science. PMID:17493286

  20. Care pathways for dementia: current perspectives.

    PubMed

    Samsi, Kritika; Manthorpe, Jill

    2014-01-01

    Uncertainty appears to typify the experience of living with dementia. With an uncertain illness trajectory and unpredictable levels of deterioration and stability in symptoms, people with a diagnosis of dementia may live with uncertainty and anxiety and find it hard to make plans or decisions for their future. People with memory problems and caregivers seeking a diagnosis of dementia may also potentially find themselves navigating a labyrinth-like maze of services, practitioners, assessments, and memory tests, with limited understanding of test scores and little information about what support is available. In this context of uncertainty, the apparent clarity and certainty of a "dementia care pathway" may be attractive. However, the term "dementia care pathway" has multiple and overlapping meanings, which can potentially give rise to further confusion if these are ill-defined or a false consensus is presumed. This review distinguishes four meanings: 1) a mechanism for the management and containment of uncertainty and confusion, useful for the professional as well as the person with dementia; 2) a manual for sequencing care activities; 3) a guide to consumers, indicating eligibility for care activities, or a guide to self-management for dementia dyads, indicating the appropriateness of care activities; and 4) a manual for "walking with" the person. Examples of these approaches are presented from UK dementia services with illustrations of existing care pathways and associated time points, specifically focusing on: 1) early symptom identification and first service encounters, 2) assessment process, 3) diagnostic disclosure, 4) postdiagnostic support, and 5) appropriate interventions. We review the evidence around these themes, as well as discuss service pathways and referral routes used by some services in England and internationally. We conclude that the attraction of the term "care pathway" is seductive, but caution is needed in taking shared understandings for

  1. Ribitol Catabolic Pathway in Klebsiella aerogenes

    PubMed Central

    Charnetzky, W. T.; Mortlock, R. P.

    1974-01-01

    In Klebsiella aerogenes W70, there is an inducible pathway for the catabolism of ribitol consisting of at least two enzymes, ribitol dehydrogenase (RDH) and d-ribulokinase (DRK). These two enzymes are coordinately controlled and induced in response to d-ribulose, an intermediate of the pathway. Whereas wild-type K. aerogenes W70 are unable to utilize xylitol as a carbon and energy source, mutants constitutive for the ribitol pathway are able to utilize RDH to oxidize the unusual pentitol, xylitol, to d-xylulose. These mutants are able to grow on xylitol, presumably by utilization of the d-xylulose produced. Mutants constitutive for l-fucose isomerase can utilize the isomerase to convert d-arabinose to d-ribulose. In the presence of d-ribulose, RDH and DRK are induced, and such mutants are thus able to phosphorylate the d-ribulose by using the DRK of the ribitol pathway. Derivatives of an l-fucose isomerase-constitutive mutant were plated on d-arabinose, ribitol, and xylitol to select and identify mutations in the ribitol pathway. Using the transducing phage PW52, we were able to demonstrate genetic linkage of the loci involved. Three-point crosses, using constitutive mutants as donors and RDH−, DRK− double mutants as recipients and selecting for DRK+ transductants on d-arabinose, resulted in DRK+RDH+-constitutive, DRK+RDH+-inducible, and DRK+RDH−-inducible transductants but no detectable DRK+RDH− constitutive transductants, data consistent with the order rbtC-rbtD-rbtK, where rbtC is a control site and rbtD and rbtK correspond to the sites for the sites for the enzymes RDH and DRK, respectively. PMID:4366025

  2. The BioPAX community standard for pathway

    SciTech Connect

    Syed, Mustafa H

    2010-01-01

    Biological Pathway Exchange (BioPAX) is a standard language to represent biological pathways at the molecular and cellular level and to facilitate the exchange of pathway data. The rapid growth of the volume of pathway data has spurred the development of databases and computational tools to aid interpretation; however, use of these data is hampered by the current fragmentation of pathway information across many databases with incompatible formats. BioPAX, which was created through a community process, solves this problem by making pathway data substantially easier to collect, index, interpret and share. BioPAX can represent metabolic and signaling pathways, molecular and genetic interactions and gene regulation networks. Using BioPAX, millions of interactions, organized into thousands of pathways, from many organisms are available from a growing number of databases. This large amount of pathway data in a computable form will support visualization, analysis and biological discovery.

  3. PSFC: a Pathway Signal Flow Calculator App for Cytoscape

    PubMed Central

    Nersisyan, Lilit; Johnson, Graham; Riel-Mehan, Megan; Pico, Alexander; Arakelyan, Arsen

    2015-01-01

    Cell signaling pathways are sequences of biochemical reactions that propagate an input signal, such as a hormone binding to a cell-surface receptor, into the cell to trigger a reactive process. Assessment of pathway activities is crucial for determining which pathways play roles in disease versus normal conditions. To date various pathway flow/perturbation assessment tools are available, however they are constrained to specific algorithms and specific data types. There are no accepted standards for evaluation of pathway activities or simulation of flow propagation events in pathways, and the results of different software are difficult to compare. Here we present Pathway Signal Flow Calculator (PSFC), a Cytoscape app for calculation of a pathway signal flow based on the pathway topology and node input data. The app provides a rich framework for customization of different signal flow algorithms to allow users to apply various approaches within a single computational framework. PMID:26834984

  4. Minimal metabolic pathway structure is consistent with associated biomolecular interactions

    PubMed Central

    Bordbar, Aarash; Nagarajan, Harish; Lewis, Nathan E; Latif, Haythem; Ebrahim, Ali; Federowicz, Stephen; Schellenberger, Jan; Palsson, Bernhard O

    2014-01-01

    Pathways are a universal paradigm for functionally describing cellular processes. Even though advances in high-throughput data generation have transformed biology, the core of our biological understanding, and hence data interpretation, is still predicated on human-defined pathways. Here, we introduce an unbiased, pathway structure for genome-scale metabolic networks defined based on principles of parsimony that do not mimic canonical human-defined textbook pathways. Instead, these minimal pathways better describe multiple independent pathway-associated biomolecular interaction datasets suggesting a functional organization for metabolism based on parsimonious use of cellular components. We use the inherent predictive capability of these pathways to experimentally discover novel transcriptional regulatory interactions in Escherichia coli metabolism for three transcription factors, effectively doubling the known regulatory roles for Nac and MntR. This study suggests an underlying and fundamental principle in the evolutionary selection of pathway structures; namely, that pathways may be minimal, independent, and segregated. PMID:24987116

  5. Minimal metabolic pathway structure is consistent with associated biomolecular interactions.

    PubMed

    Bordbar, Aarash; Nagarajan, Harish; Lewis, Nathan E; Latif, Haythem; Ebrahim, Ali; Federowicz, Stephen; Schellenberger, Jan; Palsson, Bernhard O

    2014-01-01

    Pathways are a universal paradigm for functionally describing cellular processes. Even though advances in high-throughput data generation have transformed biology, the core of our biological understanding, and hence data interpretation, is still predicated on human-defined pathways. Here, we introduce an unbiased, pathway structure for genome-scale metabolic networks defined based on principles of parsimony that do not mimic canonical human-defined textbook pathways. Instead, these minimal pathways better describe multiple independent pathway-associated biomolecular interaction datasets suggesting a functional organization for metabolism based on parsimonious use of cellular components. We use the inherent predictive capability of these pathways to experimentally discover novel transcriptional regulatory interactions in Escherichia coli metabolism for three transcription factors, effectively doubling the known regulatory roles for Nac and MntR. This study suggests an underlying and fundamental principle in the evolutionary selection of pathway structures; namely, that pathways may be minimal, independent, and segregated. PMID:24987116

  6. Robust de novo pathway enrichment with KeyPathwayMiner 5.

    PubMed

    Alcaraz, Nicolas; List, Markus; Dissing-Hansen, Martin; Rehmsmeier, Marc; Tan, Qihua; Mollenhauer, Jan; Ditzel, Henrik J; Baumbach, Jan

    2016-01-01

    Identifying functional modules or novel active pathways, recently termed de novo pathway enrichment, is a computational systems biology challenge that has gained much attention during the last decade. Given a large biological interaction network, KeyPathwayMiner extracts connected subnetworks that are enriched for differentially active entities from a series of molecular profiles encoded as binary indicator matrices. Since interaction networks constantly evolve, an important question is how robust the extracted results are when the network is modified. We enable users to study this effect through several network perturbation techniques and over a range of perturbation degrees. In addition, users may now provide a gold-standard set to determine how enriched extracted pathways are with relevant genes compared to randomized versions of the original network. PMID:27540470

  7. Robust de novo pathway enrichment with KeyPathwayMiner 5

    PubMed Central

    Alcaraz, Nicolas; List, Markus; Dissing-Hansen, Martin; Rehmsmeier, Marc; Tan, Qihua; Mollenhauer, Jan; Ditzel, Henrik J.; Baumbach, Jan

    2016-01-01

    Identifying functional modules or novel active pathways, recently termed de novo pathway enrichment, is a computational systems biology challenge that has gained much attention during the last decade. Given a large biological interaction network, KeyPathwayMiner extracts connected subnetworks that are enriched for differentially active entities from a series of molecular profiles encoded as binary indicator matrices. Since interaction networks constantly evolve, an important question is how robust the extracted results are when the network is modified. We enable users to study this effect through several network perturbation techniques and over a range of perturbation degrees. In addition, users may now provide a gold-standard set to determine how enriched extracted pathways are with relevant genes compared to randomized versions of the original network. PMID:27540470

  8. Hypoxia signaling pathways in cancer metabolism: the importance of co-selecting interconnected physiological pathways

    PubMed Central

    2014-01-01

    Both tumor hypoxia and dysregulated metabolism are classical features of cancer. Recent analyses have revealed complex interconnections between oncogenic activation, hypoxia signaling systems and metabolic pathways that are dysregulated in cancer. These studies have demonstrated that rather than responding simply to error signals arising from energy depletion or tumor hypoxia, metabolic and hypoxia signaling pathways are also directly connected to oncogenic signaling mechanisms at many points. This review will summarize current understanding of the role of hypoxia inducible factor (HIF) in these networks. It will also discuss the role of these interconnected pathways in generating the cancer phenotype; in particular, the implications of switching massive pathways that are physiologically 'hard-wired’ to oncogenic mechanisms driving cancer. PMID:24491179

  9. Chemical Shifts to Metabolic Pathways: Identifying Metabolic Pathways Directly from a Single 2D NMR Spectrum.

    PubMed

    Dubey, Abhinav; Rangarajan, Annapoorni; Pal, Debnath; Atreya, Hanudatta S

    2015-12-15

    Identifying cellular processes in terms of metabolic pathways is one of the avowed goals of metabolomics studies. Currently, this is done after relevant metabolites are identified to allow their mapping onto specific pathways. This task is daunting due to the complex nature of cellular processes and the difficulty in establishing the identity of individual metabolites. We propose here a new method: ChemSMP (Chemical Shifts to Metabolic Pathways), which facilitates rapid analysis by identifying the active metabolic pathways directly from chemical shifts obtained from a single two-dimensional (2D) [(13)C-(1)H] correlation NMR spectrum without the need for identification and assignment of individual metabolites. ChemSMP uses a novel indexing and scoring system comprised of a "uniqueness score" and a "coverage score". Our method is demonstrated on metabolic pathways data from the Small Molecule Pathway Database (SMPDB) and chemical shifts from the Human Metabolome Database (HMDB). Benchmarks show that ChemSMP has a positive prediction rate of >90% in the presence of decluttered data and can sustain the same at 60-70% even in the presence of noise, such as deletions of peaks and chemical shift deviations. The method tested on NMR data acquired for a mixture of 20 amino acids shows a success rate of 93% in correct recovery of pathways. When used on data obtained from the cell lysate of an unexplored oncogenic cell line, it revealed active metabolic pathways responsible for regulating energy homeostasis of cancer cells. Our unique tool is thus expected to significantly enhance analysis of NMR-based metabolomics data by reducing existing impediments. PMID:26556218

  10. Genome-Wide Pathway Analysis Identifies Genetic Pathways Associated with Psoriasis.

    PubMed

    Aterido, Adrià; Julià, Antonio; Ferrándiz, Carlos; Puig, Lluís; Fonseca, Eduardo; Fernández-López, Emilia; Dauden, Esteban; Sánchez-Carazo, José Luís; López-Estebaranz, José Luís; Moreno-Ramírez, David; Vanaclocha, Francisco; Herrera, Enrique; de la Cueva, Pablo; Dand, Nick; Palau, Núria; Alonso, Arnald; López-Lasanta, María; Tortosa, Raül; García-Montero, Andrés; Codó, Laia; Gelpí, Josep Lluís; Bertranpetit, Jaume; Absher, Devin; Capon, Francesca; Myers, Richard M; Barker, Jonathan N; Marsal, Sara

    2016-03-01

    Psoriasis is a chronic inflammatory disease with a complex genetic architecture. To date, the psoriasis heritability is only partially explained. However, there is increasing evidence that the missing heritability in psoriasis could be explained by multiple genetic variants of low effect size from common genetic pathways. The objective of this study was to identify new genetic variation associated with psoriasis risk at the pathway level. We genotyped 598,258 single nucleotide polymorphisms in a discovery cohort of 2,281 case-control individuals from Spain. We performed a genome-wide pathway analysis using 1,053 reference biological pathways. A total of 14 genetic pathways (PFDR ≤ 2.55 × 10(-2)) were found to be significantly associated with psoriasis risk. Using an independent validation cohort of 7,353 individuals from the UK, a total of 6 genetic pathways were significantly replicated (PFDR ≤ 3.46 × 10(-2)). We found genetic pathways that had not been previously associated with psoriasis risk such as retinol metabolism (Pcombined = 1.84 × 10(-4)), the transport of inorganic ions and amino acids (Pcombined = 1.57 × 10(-7)), and post-translational protein modification (Pcombined = 1.57 × 10(-7)). In the latter pathway, MGAT5 showed a strong network centrality, and its association with psoriasis risk was further validated in an additional case-control cohort of 3,429 individuals (P < 0.05). These findings provide insights into the biological mechanisms associated with psoriasis susceptibility. PMID:26743605

  11. The Cardiopulmonary Effects of Ambient Air Pollution and Mechanistic Pathways: A Comparative Hierarchical Pathway Analysis

    PubMed Central

    Thomas, Duncan C.; Zhang, Junfeng; Kipen, Howard M.; Rich, David Q.; Zhu, Tong; Huang, Wei; Hu, Min; Wang, Guangfa; Wang, Yuedan; Zhu, Ping; Lu, Shou-En; Ohman-Strickland, Pamela; Diehl, Scott R.; Eckel, Sandrah P.

    2014-01-01

    Previous studies have investigated the associations between exposure to ambient air pollution and biomarkers of physiological pathways, yet little has been done on the comparison across biomarkers of different pathways to establish the temporal pattern of biological response. In the current study, we aim to compare the relative temporal patterns in responses of candidate pathways to different pollutants. Four biomarkers of pulmonary inflammation and oxidative stress, five biomarkers of systemic inflammation and oxidative stress, ten parameters of autonomic function, and three biomarkers of hemostasis were repeatedly measured in 125 young adults, along with daily concentrations of ambient CO, PM2.5, NO2, SO2, EC, OC, and sulfate, before, during, and after the Beijing Olympics. We used a two-stage modeling approach, including Stage I models to estimate the association between each biomarker and pollutant over each of 7 lags, and Stage II mixed-effect models to describe temporal patterns in the associations when grouping the biomarkers into the four physiological pathways. Our results show that candidate pathway groupings of biomarkers explained a significant amount of variation in the associations for each pollutant, and the temporal patterns of the biomarker-pollutant-lag associations varied across candidate pathways (p<0.0001) and were not linear (from lag 0 to lag 3: p = 0.0629, from lag 3 to lag 6: p = 0.0005). These findings suggest that, among this healthy young adult population, the pulmonary inflammation and oxidative stress pathway is the first to respond to ambient air pollution exposure (within 24 hours) and the hemostasis pathway responds gradually over a 2–3 day period. The initial pulmonary response may contribute to the more gradual systemic changes that likely ultimately involve the cardiovascular system. PMID:25502951

  12. Chemical pathway analysis of the Martian atmosphere: CO2-formation pathways

    NASA Astrophysics Data System (ADS)

    Stock, Joachim W.; Boxe, Christopher S.; Lehmann, Ralph; Grenfell, J. Lee; Patzer, A. Beate C.; Rauer, Heike; Yung, Yuk L.

    2012-05-01

    The chemical composition of a planetary atmosphere plays an important role for atmospheric structure, stability, and evolution. Potentially complex interactions between chemical species do not often allow for an easy understanding of the underlying chemical mechanisms governing the atmospheric composition. In particular, trace species can affect the abundance of major species by acting in catalytic cycles. On Mars, such cycles even control the abundance of its main atmospheric constituent CO2. The identification of catalytic cycles (or more generally chemical pathways) by hand is quite demanding. Hence, the application of computer algorithms is beneficial in order to analyze complex chemical reaction networks. Here, we have performed the first automated quantified chemical pathways analysis of the Martian atmosphere with respect to CO2-production in a given reaction system. For this, we applied the Pathway Analysis Program (PAP) to output data from the Caltech/JPL photochemical Mars model. All dominant chemical pathways directly related to the global CO2-production have been quantified as a function of height up to 86 km. We quantitatively show that CO2-production is dominated by chemical pathways involving HOx and Ox. In addition, we find that NOx in combination with HOx and Ox exhibits a non-negligible contribution to CO2-production, especially in Mars' lower atmosphere. This study reveals that only a small number of chemical pathways contribute significantly to the atmospheric abundance of CO2 on Mars; their contributions to CO2-production vary considerably with altitude. This analysis also endorses the importance of transport processes in governing CO2-stability in the Martian atmosphere. Lastly, we identify a previously unknown chemical pathway involving HOx, Ox, and HO2-photodissociation, contributing 8% towards global CO2-production by chemical pathways using recommended up-to-date values for reaction rate coefficients.

  13. Construction and engineering of large biochemical pathways via DNA assembler

    PubMed Central

    Shao, Zengyi; Zhao, Huimin

    2015-01-01

    Summary DNA assembler enables rapid construction and engineering of biochemical pathways in a one-step fashion by exploitation of the in vivo homologous recombination mechanism in Saccharomyces cerevisiae. It has many applications in pathway engineering, metabolic engineering, combinatorial biology, and synthetic biology. Here we use two examples including the zeaxanthin biosynthetic pathway and the aureothin biosynthetic gene cluster to describe the key steps in the construction of pathways containing multiple genes using the DNA assembler approach. Methods for construct design, pathway assembly, pathway confirmation, and functional analysis are shown. The protocol for fine genetic modifications such as site-directed mutagenesis for engineering the aureothin gene cluster is also illustrated. PMID:23996442

  14. Pathway Tools version 13.0: integrated software for pathway/genome informatics and systems biology

    PubMed Central

    Paley, Suzanne M.; Krummenacker, Markus; Latendresse, Mario; Dale, Joseph M.; Lee, Thomas J.; Kaipa, Pallavi; Gilham, Fred; Spaulding, Aaron; Popescu, Liviu; Altman, Tomer; Paulsen, Ian; Keseler, Ingrid M.; Caspi, Ron

    2010-01-01

    Pathway Tools is a production-quality software environment for creating a type of model-organism database called a Pathway/Genome Database (PGDB). A PGDB such as EcoCyc integrates the evolving understanding of the genes, proteins, metabolic network and regulatory network of an organism. This article provides an overview of Pathway Tools capabilities. The software performs multiple computational inferences including prediction of metabolic pathways, prediction of metabolic pathway hole fillers and prediction of operons. It enables interactive editing of PGDBs by DB curators. It supports web publishing of PGDBs, and provides a large number of query and visualization tools. The software also supports comparative analyses of PGDBs, and provides several systems biology analyses of PGDBs including reachability analysis of metabolic networks, and interactive tracing of metabolites through a metabolic network. More than 800 PGDBs have been created using Pathway Tools by scientists around the world, many of which are curated DBs for important model organisms. Those PGDBs can be exchanged using a peer-to-peer DB sharing system called the PGDB Registry. PMID:19955237

  15. Yeast Pathway Kit: A Method for Metabolic Pathway Assembly with Automatically Simulated Executable Documentation.

    PubMed

    Pereira, Filipa; Azevedo, Flávio; Parachin, Nadia Skorupa; Hahn-Hägerdal, Bärbel; Gorwa-Grauslund, Marie F; Johansson, Björn

    2016-05-20

    We have developed the Yeast Pathway Kit (YPK) for rational and random metabolic pathway assembly in Saccharomyces cerevisiae using reusable and redistributable genetic elements. Genetic elements are cloned in a suicide vector in a rapid process that omits PCR product purification. Single-gene expression cassettes are assembled in vivo using genetic elements that are both promoters and terminators (TP). Cassettes sharing genetic elements are assembled by recombination into multigene pathways. A wide selection of prefabricated TP elements makes assembly both rapid and inexpensive. An innovative software tool automatically produces detailed self-contained executable documentation in the form of pydna code in the narrative Jupyter notebook format to facilitate planning and sharing YPK projects. A d-xylose catabolic pathway was created using YPK with four or eight genes that resulted in one of the highest growth rates reported on d-xylose (0.18 h(-1)) for recombinant S. cerevisiae without adaptation. The two-step assembly of single-gene expression cassettes into multigene pathways may improve the yield of correct pathways at the cost of adding overall complexity, which is offset by the supplied software tool. PMID:26916955

  16. Protein folding: independent unrelated pathways or predetermined pathway with optional errors.

    PubMed

    Bédard, Sabrina; Krishna, Mallela M G; Mayne, Leland; Englander, S Walter

    2008-05-20

    The observation of heterogeneous protein folding kinetics has been widely interpreted in terms of multiple independent unrelated pathways (IUP model), both experimentally and in theoretical calculations. However, direct structural information on folding intermediates and their properties now indicates that all of a protein population folds through essentially the same stepwise pathway, determined by cooperative native-like foldon units and the way that the foldons fit together in the native protein. It is essential to decide between these fundamentally different folding mechanisms. This article shows, contrary to previous supposition, that the heterogeneous folding kinetics observed for the staphylococcal nuclease protein (SNase) does not require alternative parallel pathways. SNase folding kinetics can be fit equally well by a single predetermined pathway that allows for optional misfolding errors, which are known to occur ubiquitously in protein folding. Structural, kinetic, and thermodynamic information for the folding intermediates and pathways of many proteins is consistent with the predetermined pathway-optional error (PPOE) model but contrary to the properties implied in IUP models. PMID:18480257

  17. WholePathwayScope: a comprehensive pathway-based analysis tool for high-throughput data

    PubMed Central

    Yi, Ming; Horton, Jay D; Cohen, Jonathan C; Hobbs, Helen H; Stephens, Robert M

    2006-01-01

    Background Analysis of High Throughput (HTP) Data such as microarray and proteomics data has provided a powerful methodology to study patterns of gene regulation at genome scale. A major unresolved problem in the post-genomic era is to assemble the large amounts of data generated into a meaningful biological context. We have developed a comprehensive software tool, WholePathwayScope (WPS), for deriving biological insights from analysis of HTP data. Result WPS extracts gene lists with shared biological themes through color cue templates. WPS statistically evaluates global functional category enrichment of gene lists and pathway-level pattern enrichment of data. WPS incorporates well-known biological pathways from KEGG (Kyoto Encyclopedia of Genes and Genomes) and Biocarta, GO (Gene Ontology) terms as well as user-defined pathways or relevant gene clusters or groups, and explores gene-term relationships within the derived gene-term association networks (GTANs). WPS simultaneously compares multiple datasets within biological contexts either as pathways or as association networks. WPS also integrates Genetic Association Database and Partial MedGene Database for disease-association information. We have used this program to analyze and compare microarray and proteomics datasets derived from a variety of biological systems. Application examples demonstrated the capacity of WPS to significantly facilitate the analysis of HTP data for integrative discovery. Conclusion This tool represents a pathway-based platform for discovery integration to maximize analysis power. The tool is freely available at . PMID:16423281

  18. Molecular neurodegeneration: basic biology and disease pathways.

    PubMed

    Vassar, Robert; Zheng, Hui

    2014-01-01

    The field of neurodegeneration research has been advancing rapidly over the past few years, and has provided intriguing new insights into the normal physiological functions and pathogenic roles of a wide range of molecules associated with several devastating neurodegenerative disorders, including Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis, frontotemporal dementia, Huntington's disease, and Down syndrome. Recent developments have also facilitated initial efforts to translate preclinical discoveries toward novel therapeutic approaches and clinical trials in humans. These recent developments are reviewed in the current Review Series on "Molecular Neurodegeneration: Basic Biology and Disease Pathways" in a number of state-of-the-art manuscripts that cover themes presented at the Third International Conference on Molecular Neurodegeneration: "Basic biology and disease pathways" held in Cannes, France, September, 2013. PMID:25248568

  19. Neurobiological Pathways Linking Socioeconomic Position and Health

    PubMed Central

    Gianaros, Peter J.; Manuck, Stephen B.

    2010-01-01

    Across individuals, risk for poor health varies inversely with socioeconomic position (SEP). The pathways by which SEP affects health have been viewed from many epidemiological perspectives. Central to these perspectives is the notion that socioeconomic health disparities arise from an interplay between nested, recursive, and cumulative environmental, social, familial, psychological, behavioral, and physiological processes that unfold over the life span. Epidemiological perspectives on socioeconomic health disparities, however, have not yet formally integrated emerging findings from neuropharmacological, molecular genetic, and neuroimaging studies demonstrating that indicators of SEP relate to patterns of brain neurotransmission, brain morphology, and brain functionality implicated in the etiology of chronic medical conditions and psychological disorders. Here, we survey these emerging findings and consider how future neurobiological studies in this area can enhance our understanding of the pathways by which different dimensions of SEP become embodied by the brain to influence health throughout life. PMID:20498294

  20. A Pathway Idea in Model Building

    NASA Astrophysics Data System (ADS)

    Mathai, A. M.; Haubold, H. J.

    2014-01-01

    The pathway idea is a way of going from one family of functions to another family of functions and yet another family of functions through a parameter in the mode l so that a switching mechanism is introduced into the model through a parameter. The advantage of the idea is that the model can cover the ideal or stable situation in a physical situation as well as cover the unstable neighborhoods or move from unstable neighborhoods to the stable situation. The basic idea is illustrated for the real scalar case here and its connections to topics in astrophysics and non-extens ive statistical mechanics, namely superstatistics and Tsallis statistics, Mittag-Leffler models, hypergeometric functions and generalized special functions such as the H-function etc are pointed out. The pathway idea is available for the real and complex rectangular matrix variate cases but only the real scalar case is illustrated here.

  1. The pentose phosphate pathway and cancer

    PubMed Central

    Patra, Krushna C.; Hay, Nissim

    2015-01-01

    The pentose phosphate pathway (PPP), which branches from glycolysis at the first committed step of glucose metabolism, is required for the synthesis of ribonucleotides and is a major source of NADPH. NADPH is required for and consumed during fatty acid synthesis and the scavenging of reactive oxygen species. Therefore, the PPP plays a pivotal role in helping glycolytic cancer cells to meet their anabolic demands and combat oxidative stress. Recently, several neoplastic lesions were shown to have evolved to facilitate the flux of glucose into the pentose phosphate pathway. This review summarizes the fundamental functions of the PPP, its regulation in cancer cells, and its importance in cancer cell metabolism and survival. PMID:25037503

  2. PML Surfs into HIPPO Tumor Suppressor Pathway

    PubMed Central

    Strano, Sabrina; Fausti, Francesca; Di Agostino, Silvia; Sudol, Marius; Blandino, Giovanni

    2013-01-01

    Growth arrest, inhibition of cell proliferation, apoptosis, senescence, and differentiation are the most characterized effects of a given tumor suppressor response. It is becoming increasingly clear that tumor suppression results from the integrated and synergistic activities of different pathways. This implies that tumor suppression includes linear, as well as lateral, crosstalk signaling. The latter may happen through the concomitant involvement of common nodal proteins. Here, we discuss the role of Promyelocytic leukemia protein (PML) in functional cross-talks with the HIPPO and the p53 family tumor suppressor pathways. PML, in addition to its own anti-tumor activity, contributes to the assembly of an integrated and superior network that may be necessary for the maximization of the tumor suppressor response to diverse oncogenic insults. PMID:23459691

  3. An evolutionary approach for searching metabolic pathways.

    PubMed

    Gerard, Matias F; Stegmayer, Georgina; Milone, Diego H

    2013-11-01

    Searching metabolic pathways that relate two compounds is a common task in bioinformatics. This is of particular interest when trying, for example, to discover metabolic relations among compounds clustered with a data mining technique. Search strategies find sequences to relate two or more states (compounds) using an appropriate set of transitions (reactions). Evolutionary algorithms carry out the search guided by a fitness function and explore multiple candidate solutions using stochastic operators. In this work we propose an evolutionary algorithm for searching metabolic pathways between two compounds. The operators and fitness function employed are described and the effect of mutation rate is studied. Performance of this algorithm is compared with two classical search strategies. Source code and dataset are available at http://sourceforge.net/projects/sourcesinc/files/eamp/ PMID:24209916

  4. Pathways of major histocompatibility complex allorecognition

    PubMed Central

    Afzali, Behdad; Lombardi, Giovanna; Lechler, Robert I.

    2013-01-01

    Purpose of review Here, we review the pathways of allorecognition and their potential relevance to the balance between regulatory and effector responses following transplantation. Recent findings Transplantation between nonidentical members of the same species elicits an immune response that manifests as graft rejection or persistence. Presentation of foreign antigen to recipient T cells can occur via three nonmutually exclusive routes, the direct, indirect and semi-direct pathways. Allospecific T cells can have effector or regulatory functions, and the relative proportions of the two populations activated following alloantigen presentation are two of the factors that determine the clinical outcome. Regulatory T cells have been the subject of significant research, and there is now greater understanding of their recruitment and function in the context of allorecognition. Summary A greater understanding of the mechanisms underlying allorecognition may be fundamental to appreciating how these different populations are recruited and could in turn inform novel strategies for immunomodulation. PMID:18685342

  5. Respiratory electron transfer pathways in plant mitochondria

    PubMed Central

    Schertl, Peter; Braun, Hans-Peter

    2014-01-01

    The respiratory electron transport chain (ETC) couples electron transfer from organic substrates onto molecular oxygen with proton translocation across the inner mitochondrial membrane. The resulting proton gradient is used by the ATP synthase complex for ATP formation. In plants, the ETC is especially intricate. Besides the “classical” oxidoreductase complexes (complex I–IV) and the mobile electron transporters cytochrome c and ubiquinone, it comprises numerous “alternative oxidoreductases.” Furthermore, several dehydrogenases localized in the mitochondrial matrix and the mitochondrial intermembrane space directly or indirectly provide electrons for the ETC. Entry of electrons into the system occurs via numerous pathways which are dynamically regulated in response to the metabolic state of a plant cell as well as environmental factors. This mini review aims to summarize recent findings on respiratory electron transfer pathways in plants and on the involved components and supramolecular assemblies. PMID:24808901

  6. Development of the Retina and Optic Pathway

    PubMed Central

    Reese, Benjamin E.

    2010-01-01

    Our understanding of the development of the retina and visual pathways has seen enormous advances during the past twenty-five years. New imaging technologies, coupled with advances in molecular biology, have permitted a fuller appreciation of the histotypical events associated with proliferation, fate determination, migration, differentiation, pathway navigation, target innervation, synaptogenesis and cell death, and in many instances, in understanding the genetic, molecular, cellular and activity-dependent mechanisms underlying those developmental changes. The present review considers those advances associated with the lineal relationships between retinal nerve cells, the production of retinal nerve cell diversity, the migration, patterning and differentiation of different types of retinal nerve cells, the determinants of the decussation pattern at the optic chiasm, the formation of the retinotopic map, and the establishment of ocular domains within the thalamus. PMID:20647017

  7. Obesity-Induced Hypertension: Brain Signaling Pathways.

    PubMed

    do Carmo, Jussara M; da Silva, Alexandre A; Wang, Zhen; Fang, Taolin; Aberdein, Nicola; de Lara Rodriguez, Cecilia E P; Hall, John E

    2016-07-01

    Obesity greatly increases the risk for cardiovascular, metabolic, and renal diseases and is one of the most significant and preventable causes of increased blood pressure (BP) in patients with essential hypertension. This review highlights recent advances in our understanding of central nervous system (CNS) signaling pathways that contribute to the etiology and pathogenesis of obesity-induced hypertension. We discuss the role of excess adiposity and activation of the brain leptin-melanocortin system in causing increased sympathetic activity in obesity. In addition, we highlight other potential brain mechanisms by which increased weight gain modulates metabolic and cardiovascular functions. Unraveling the CNS mechanisms responsible for increased sympathetic activation and hypertension and how circulating hormones activate brain signaling pathways to control BP offer potentially important therapeutic targets for obesity and hypertension. PMID:27262997

  8. Studying lipids involved in the endosomal pathway.

    PubMed

    Bissig, Christin; Johnson, Shem; Gruenberg, Jean

    2012-01-01

    Endosomes along the degradation pathway exhibit a multivesicular appearance and differ in their lipid compositions. Association of proteins to specific membrane lipids and presumably also lipid-lipid interactions contribute to the formation of functional membrane platforms that regulate endosome biogenesis and function. This chapter provides a brief review of the functions of endosomal lipids in the degradation pathway, a discussion of techniques that allow studying lipid-based mechanisms and a selection of step-by-step protocols for in vivo and in vitro methods commonly used to study lipid roles in endocytosis. The techniques described here have been used to elucidate the function of the late endosomal lipid lysobisphosphatidic acid and allow the monitoring of lipid distribution, levels and dynamics, as well as the characterization of lipid-binding partners. PMID:22325596

  9. Conservation of small RNA pathways in platypus.

    PubMed

    Murchison, Elizabeth P; Kheradpour, Pouya; Sachidanandam, Ravi; Smith, Carly; Hodges, Emily; Xuan, Zhenyu; Kellis, Manolis; Grützner, Frank; Stark, Alexander; Hannon, Gregory J

    2008-06-01

    Small RNA pathways play evolutionarily conserved roles in gene regulation and defense from parasitic nucleic acids. The character and expression patterns of small RNAs show conservation throughout animal lineages, but specific animal clades also show variations on these recurring themes, including species-specific small RNAs. The monotremes, with only platypus and four species of echidna as extant members, represent the basal branch of the mammalian lineage. Here, we examine the small RNA pathways of monotremes by deep sequencing of six platypus and echidna tissues. We find that highly conserved microRNA species display their signature tissue-specific expression patterns. In addition, we find a large rapidly evolving cluster of microRNAs on platypus chromosome X1, which is unique to monotremes. Platypus and echidna testes contain a robust Piwi-interacting (piRNA) system, which appears to be participating in ongoing transposon defense. PMID:18463306

  10. Whole Algae Hydrothermal Liquefaction Technology Pathway

    SciTech Connect

    Biddy, Mary J.; Davis, Ryan; Jones, Susanne B.; Zhu, Yunhua

    2013-03-31

    In support of the Bioenergy Technologies Office, the National Renewable Energy Laboratory (NREL) and the Pacific Northwest National Laboratory (PNNL) are undertaking studies of biomass conversion technologies to hydrocarbon fuels to identify barriers and target research toward reducing conversion costs. Process designs and preliminary economic estimates for each of these pathway cases were developed using rigorous modeling tools (Aspen Plus and Chemcad). These analyses incorporated the best information available at the time of development, including data from recent pilot and bench-scale demonstrations, collaborative industrial and academic partners, and published literature and patents. This pathway case investigates the feasibility of using whole wet microalgae as a feedstock for conversion via hydrothermal liquefaction. Technical barriers and key research needs have been assessed in order for the hydrothermal liquefaction of microalgae to be competitive with petroleum-derived gasoline, diesel and jet range blendstocks.

  11. The JAK-STAT Pathway at Twenty

    PubMed Central

    Stark, George R.; Darnell, James E.

    2014-01-01

    We look back on the discoveries that the tyrosine kinases TYK2 and JAK1 and the transcription factors STAT1, STAT2, and IRF9 are required for the cellular response to type I interferons. This initial description of the JAK-STAT pathway led quickly to additional discoveries that type II interferons and many other cytokines signal through similar mechanisms. This well-understood pathway now serves as a paradigm showing how information from protein-protein contacts at the cell surface can be conveyed directly to genes in the nucleus. We also review recent work on the STAT proteins showing the importance of several different posttranslational modifications, including serine phosphorylation, acetylation, methylation, and sumoylation. These remarkably proficient proteins also provide noncanonical functions in transcriptional regulation and they also function in mitochondrial respiration and chromatin organization in ways that may not involve transcription at all. PMID:22520844

  12. Finding pathways between distant local minima.

    PubMed

    Carr, Joanne M; Trygubenko, Semen A; Wales, David J

    2005-06-15

    We report a new algorithm for constructing pathways between local minima that involve a large number of intervening transition states on the potential energy surface. A significant improvement in efficiency has been achieved by changing the strategy for choosing successive pairs of local minima that serve as endpoints for the next search. We employ Dijkstra's algorithm [E. W. Dijkstra, Numer. Math. 1, 269 (1959)] to identify the "shortest" path corresponding to missing connections within an evolving database of local minima and the transition states that connect them. The metric employed to determine the shortest missing connection is a function of the minimized Euclidean distance. We present applications to the formation of buckminsterfullerene and to the folding of various biomolecules: the B1 domain of protein G, tryptophan zippers, and the villin headpiece subdomain. The corresponding pathways contain up to 163 transition states and will be used in future discrete path sampling calculations. PMID:16008483

  13. Finding pathways between distant local minima

    NASA Astrophysics Data System (ADS)

    Carr, Joanne M.; Trygubenko, Semen A.; Wales, David J.

    2005-06-01

    We report a new algorithm for constructing pathways between local minima that involve a large number of intervening transition states on the potential energy surface. A significant improvement in efficiency has been achieved by changing the strategy for choosing successive pairs of local minima that serve as endpoints for the next search. We employ Dijkstra's algorithm [E. W. Dijkstra, Numer. Math. 1, 269 (1959)] to identify the "shortest" path corresponding to missing connections within an evolving database of local minima and the transition states that connect them. The metric employed to determine the shortest missing connection is a function of the minimized Euclidean distance. We present applications to the formation of buckminsterfullerene and to the folding of various biomolecules: the B1 domain of protein G, tryptophan zippers, and the villin headpiece subdomain. The corresponding pathways contain up to 163 transition states and will be used in future discrete path sampling calculations.

  14. Fragmentation pathways of ethylene after core ionization

    NASA Astrophysics Data System (ADS)

    Gaire, B.; Bocharova, I.; Sturm, F. P.; Gehrken, N.; Haxton, D. J.; Belkacem, A.; Weber, Th.; Zohrabi, M.; Ben-Itzhak, I.; Gatton, A.; Williams, J.; Reedy, D.; Nook, C.; Landers, A.; Gassert, H.; Zeller, S.; Voigtsberger, J.; Jahnke, T.; Doerner, R.

    2014-05-01

    We have measured the Auger electrons in coincidence with the recoil ions, resulting from the core ionization of ethylene molecules, by employing the COLd Target Recoil Ion Momentum Spectroscopy (COLTRIMS) method. The Auger-electron and recoil-ion energy maps are used to identify the fragmentation pathways and they are compared to the valence photo-double-ionization of ethylene. The dicationic electronic states favored by the propensity rules are identified and their role on the fragmentation pathways is discussed. The molecular-frame Auger electron angular distribution provides further insight into the breakup of this molecule after core ionization. Supported by the Director, Office of Science, Office of Basic Energy Sciences, and by the Division of Chemical Sciences, Geosciences, and Biosciences of the U.S. Department of Energy at LBNL under Contract No. DE-AC02-05CH11231.

  15. Biocatalytic Pathway Selection in Transient Tripeptide Nanostructures.

    PubMed

    Pappas, Charalampos G; Sasselli, Ivan R; Ulijn, Rein V

    2015-07-01

    Structural adaption in living systems is achieved by competing catalytic pathways that drive assembly and disassembly of molecular components under the influence of chemical fuels. We report on a simple mimic of such a system that displays transient, sequence-dependent formation of supramolecular nanostructures based on biocatalytic formation and hydrolysis of self-assembling tripeptides. The systems are catalyzed by α-chymotrypsin and driven by hydrolysis of dipeptide aspartyl-phenylalanine-methyl ester (the sweetener aspartame, DF-OMe). We observed switch-like pathway selection, with the kinetics and consequent lifetime of transient nanostructures controlled by the peptide sequence. In direct competition, kinetic (rather than thermodynamic) component selection is observed. PMID:26014441

  16. Optogenetic control of intracellular signaling pathways

    PubMed Central

    Zhang, Kai; Cui, Bianxiao

    2014-01-01

    Cells employ a plethora of signaling pathways to make their life-and-death decisions. Extensive genetic, biochemical, and physiological studies have led to the accumulation of knowledge about signaling components and their interactions within signaling networks. These conventional approaches, though useful, lack the ability to control the spatial and temporal aspects of signaling processes. The recently emerged optogenetic tools open up exciting opportunities by enabling signaling regulation with superior temporal and spatial resolution, easy delivery, rapid reversibility, fewer off-target side effects, and the ability to dissect complex signaling networks. Here we review recent achievements in using light to control intracellular signaling pathways, and discuss future prospects for the field, including integration of new genetic approaches into optogenetics. PMID:25529484

  17. Gene set enrichment; a problem of pathways

    PubMed Central

    Meaburn, Emma L.; Schalkwyk, Leonard C.

    2010-01-01

    Gene Set Enrichment (GSE) is a computational technique which determines whether a priori defined set of genes show statistically significant differential expression between two phenotypes. Currently, the gene sets used for GSE are derived from annotation or pathway databases, which often contain computationally based and unrepresentative data. Here, we propose a novel approach for the generation of comprehensive and biologically derived gene sets, deriving sets through the application of machine learning techniques to gene expression data. These gene sets can be produced for specific tissues, developmental stages or environments. They provide a powerful and functionally meaningful way in which to mine genomewide association and next generation sequencing data in order to identify disease-associated variants and pathways. PMID:20861160

  18. Targeting Signaling Transduction Pathways in Bladder Cancer.

    PubMed

    Abbosh, Phillip H; McConkey, David J; Plimack, Elizabeth R

    2015-12-01

    Systemic therapy for urothelial carcinoma (UC) of the bladder has largely revolved around cytotoxic chemotherapy regimens. However, several recent clinical trials have explored the roles of targeted therapies which specifically inhibit signal transduction pathways. Simultaneously, a rationale for such therapies has come to the forefront of management of this disease because an overabundance of signaling pathways are genetically deranged as a result of point mutation or copy number alteration (CNA) as identified by several recent next generation sequencing (NGS) studies. Importantly, these derangements are found in all stages of disease, and therefore targeted therapies hold promise as a next step in the evolution of the medical management of both localized and metastatic UCC. We review the rationale for and progress in studying inhibition of signal transduction as a means of treatment of UCC. PMID:26472299

  19. Emerging Common Molecular Pathways for Primary Dystonia

    PubMed Central

    LeDoux, Mark S; Dauer, William T; Warner, Thomas T

    2013-01-01

    Background The dystonias are a group of hyperkinetic movement disorders whose principal cause is neuron dysfunction at one or more interconnected nodes of the motor system. The study of genes and proteins which cause familial dystonia provides critical information about the cellular pathways involved in this dysfunction which disrupts the motor pathways at systems level. In recent years study of the increasing number of DYT genes has implicated a number of cell functions which appear to be involved in the pathogenesis of dystonia. Methods Review of literature published in English language publications available on Pubmed relating to the genetics and cellular pathology of dystonia Results and Conclusions Numerous potential pathogenetic mechanisms have been identified. We describe those which fall into three emerging thematic groups: cell cycle and transcriptional regulation in the nucleus, endoplasmic reticulum and nuclear envelope function, and control of synaptic function. PMID:23893453

  20. Circadian light-input pathways in Drosophila.

    PubMed

    Yoshii, Taishi; Hermann-Luibl, Christiane; Helfrich-Förster, Charlotte

    2016-01-01

    Light is the most important environmental cue to entrain the circadian clock in most animals. In the fruit fly Drosophila melanogaster, the light entrainment mechanisms of the clock have been well-studied. The Drosophila brain contains approximately 150 neurons that rhythmically express circadian clock genes. These neurons are called "clock neurons" and control behavioral activity rhythms. Many clock neurons express the Cryptochrome (CRY) protein, which is sensitive to UV and blue light, and thus enables clock neurons deep in the brain to directly perceive light. In addition to the CRY protein, external photoreceptors in the Drosophila eyes play an important role in circadian light-input pathways. Recent studies have provided new insights into the mechanisms that integrate these light inputs into the circadian network of the brain. In this review, we will summarize the current knowledge on the light entrainment pathways in the Drosophila circadian clock. PMID:27066180

  1. Online treatment compliance checking for clinical pathways.

    PubMed

    Huang, Zhengxing; Bao, Yurong; Dong, Wei; Lu, Xudong; Duan, Huilong

    2014-10-01

    Compliance checking for clinical pathways (CPs) is getting increasing attention in health-care organizations due to stricter requirements for cost control and treatment excellence. Many compliance measures have been proposed for treatment behavior inspection in CPs. However, most of them look at aggregated data seen from an external perspective, e.g. length of stay, cost, infection rate, etc., which may provide only a posterior impression of the overall conformance with the established CPs such that in-depth and in near real time checking on the compliance of the essential/critical treatment behaviors of CPs is limited. To provide clinicians real time insights into violations of the established CP specification and support online compliance checking, this article presents a semantic rule-based CP compliance checking system. In detail, we construct a CP ontology (CPO) model to provide a formal grounding of CP compliance checking. Using the proposed CPO, domain treatment constraints are modeled into Semantic Web Rule Language (SWRL) rules to specify the underlying treatment behaviors and their quantified temporal structure in a CP. The established SWRL rules are integrated with the CP workflow such that a series of applicable compliance checking and evaluation can be reminded and recommended during the pathway execution. The proposed approach can, therefore, provides a comprehensive compliance checking service as a paralleling activity to the patient treatment journey of a CP rather than an afterthought. The proposed approach is illustrated with a case study on the unstable angina clinical pathway implemented in the Cardiology Department of a Chinese hospital. The results demonstrate that the approach, as a feasible solution to provide near real time conformance checking of CPs, not only enables clinicians to uncover non-compliant treatment behaviors, but also empowers clinicians with the capability to make informed decisions when dealing with treatment compliance

  2. [Combining clinical pathway and patient education approaches].

    PubMed

    Bonnabel, Laurence; Huteau, Marie-Ève; Filhol, Nathalie; Clottes, Edwige; Massin, Julie; Quenet, François; Stoebner-Delbarre, Anne

    2016-01-01

    The integration of the therapeutic education of the patient into a clinical pathway approach helps to optimise nursing practice. Despite some limits, this method allows the position of the caregiver to evolve, going beyond the required methodological framework. It results in the emergence of several new educational facets which are essential for the patient and enable them to become a player in their own care. PMID:26743372

  3. Insulin signaling pathways in lepidopteran ecdysone secretion

    PubMed Central

    Smith, Wendy A.; Lamattina, Anthony; Collins, McKensie

    2014-01-01

    Molting and metamorphosis are stimulated by the secretion of ecdysteroid hormones from the prothoracic glands. Insulin-like hormones have been found to enhance prothoracic gland activity, providing a mechanism to link molting to nutritional state. In silk moths (Bombyx mori), the prothoracic glands are directly stimulated by insulin and the insulin-like hormone bombyxin. Further, in Bombyx, the neuropeptide prothoracicotropic hormone (PTTH) appears to act at least in part through the insulin-signaling pathway. In the prothoracic glands of Manduca sexta, while insulin stimulates the phosphorylation of the insulin receptor and Akt, neither insulin nor bombyxin II stimulate ecdysone secretion. Involvement of the insulin-signaling pathway in Manduca prothoracic glands was explored using two inhibitors of phosphatidylinositol-3-kinase (PI3K), LY294002 and wortmannin. PI3K inhibitors block the phosphorylation of Akt and 4EBP but have no effect on ecdysone secretion, or on the phosphorylation of the MAPkinase, ERK. Inhibitors that block phosphorylation of ERK, including the MEK inhibitor U0126, and high doses of the RSK inhibitor SL0101, effectively inhibit ecdysone secretion. The results highlight differences between the two lepidopteran insects most commonly used to directly study ecdysteroid secretion. In Bombyx, the PTTH and insulin-signaling pathways intersect; both insulin and PTTH enhance the phosphorylation of Akt and stimulate ecdysteroid secretion, and inhibition of PI3K reduces ecdysteroid secretion. By contrast, in Manduca, the action of PTTH is distinct from insulin. The results highlight species differences in the roles of translational regulators such as 4EBP, and members of the MAPkinase pathway such as ERK and RSK, in the regulation of insect ecdysone secretion, and in the impact of nutritionally-sensitive hormones such as insulin in the control of ecdysone secretion and molting. PMID:24550835

  4. Folding pathways of the Tetrahymena ribozyme

    PubMed Central

    Mitchell, David; Russell, Rick

    2014-01-01

    Like many structured RNAs, the Tetrahymena group I intron ribozyme folds through multiple pathways and intermediates. Under standard conditions in vitro, a small fraction reaches the native state (N) with kobs ≈ 0.6 min–1, while the remainder forms a long-lived misfolded conformation (M) thought to differ in topology. These alternative outcomes reflect a pathway that branches late in folding, after disruption of a trapped intermediate (Itrap). Here, we use catalytic activity to probe the folding transitions from Itrap to the native and misfolded states. We show that mutations predicted to weaken the core helix P3 do not increase the rate of folding from Itrap but they increase the fraction that reaches the native state rather than forming the misfolded state. Thus, P3 is disrupted during folding to the native state but not to the misfolded state, and P3 disruption occurs after the rate-limiting step. Interestingly, P3-strengthening mutants also increase native folding. Additional experiments show that these mutants are rapidly committed to folding to the native state, although they reach the native state with approximately the same rate constant as the wild-type ribozyme (~1 min–1). Thus, the P3-strengthening mutants populate a distinct pathway that includes at least one intermediate but avoids the M state, most likely because P3 and the correct topology are formed early. Our results highlight multiple pathways in RNA folding and illustrate how kinetic competitions between rapid events can have long-lasting effects because the ‘choice’ is enforced by energy barriers that grow larger as folding progresses. PMID:24747051

  5. The sensory transduction pathways in bacterial chemotaxis

    NASA Technical Reports Server (NTRS)

    Taylor, Barry L.

    1989-01-01

    Bacterial chemotaxis is a useful model for investigating in molecular detail the behavioral response of cells to changes in their environment. Peritrichously flagellated bacteria such as coli and typhimurium swim by rotating helical flagella in a counterclockwise direction. If flagellar rotation is briefly reversed, the bacteria tumble and change the direction of swimming. The bacteria continuously sample the environment and use a temporal sensing mechanism to compare the present and immediate past environments. Bacteria respond to a broad range of stimuli including changes in temperature, oxygen concentration, pH and osmotic strength. Bacteria are attracted to potential sources of nutrition such as sugars and amino acids and are repelled by other chemicals. In the methylation-dependent pathways for sensory transduction and adaptation in E. coli and S. typhimurium, chemoeffectors bind to transducing proteins that span the plasma membrane. The transducing proteins are postulated to control the rate of autophosphorylation of the CheA protein, which in turn phosphorylates the CheY protein. The phospho-CheY protein binds to the switch on the flagellar motor and is the signal for clockwise rotation of the motor. Adaptation to an attractant is achieved by increasing methylation of the transducing protein until the attractant stimulus is cancelled. Responses to oxygen and certain sugars involve methylation-independent pathways in which adaption occurs without methylation of a transducing protein. Taxis toward oxygen is mediated by the electron transport system and changes in the proton motive force. Recent studies have shown that the methylation-independent pathway converges with the methylation-dependent pathway at or before the CheA protein.

  6. GPCR signaling along the endocytic pathway

    PubMed Central

    Irannejad, Roshanak; von Zastrow, Mark

    2016-01-01

    Many G protein-coupled receptors (GPCRs) internalize after agonist-induced activation. While endocytosis has long been associated with homeostatic attenuation of cellular responsiveness, accumulating evidence from study of a wide range of eukaryotes reveals that the endocytic pathway also contributes to generating receptor-initiated signals themselves. Here we review recent progress in this area, discussing primarily but not exclusively GPCR signaling in mammalian cells. PMID:24680436

  7. Modulation of neurotrophic signaling pathways by polyphenols

    PubMed Central

    Moosavi, Fatemeh; Hosseini, Razieh; Saso, Luciano; Firuzi, Omidreza

    2016-01-01

    Polyphenols are an important class of phytochemicals, and several lines of evidence have demonstrated their beneficial effects in the context of a number of pathologies including neurodegenerative disorders such as Alzheimer’s and Parkinson’s disease. In this report, we review the studies on the effects of polyphenols on neuronal survival, growth, proliferation and differentiation, and the signaling pathways involved in these neurotrophic actions. Several polyphenols including flavonoids such as baicalein, daidzein, luteolin, and nobiletin as well as nonflavonoid polyphenols such as auraptene, carnosic acid, curcuminoids, and hydroxycinnamic acid derivatives including caffeic acid phentyl ester enhance neuronal survival and promote neurite outgrowth in vitro, a hallmark of neuronal differentiation. Assessment of underlying mechanisms, especially in PC12 neuronal-like cells, reveals that direct agonistic effect on tropomyosin receptor kinase (Trk) receptors, the main receptors of neurotrophic factors including nerve growth factor (NGF) and brain-derived neurotrophic factor (BDNF) explains the action of few polyphenols such as 7,8-dihydroxyflavone. However, several other polyphenolic compounds activate extracellular signal-regulated kinase (ERK) and phosphoinositide 3-kinase (PI3K)/Akt pathways. Increased expression of neurotrophic factors in vitro and in vivo is the mechanism of neurotrophic action of flavonoids such as scutellarin, daidzein, genistein, and fisetin, while compounds like apigenin and ferulic acid increase cyclic adenosine monophosphate response element-binding protein (CREB) phosphorylation. Finally, the antioxidant activity of polyphenols reflected in the activation of Nrf2 pathway and the consequent upregulation of detoxification enzymes such as heme oxygenase-1 as well as the contribution of these effects to the neurotrophic activity have also been discussed. In conclusion, a better understanding of the neurotrophic effects of polyphenols and

  8. Pathway network inference from gene expression data

    PubMed Central

    2014-01-01

    Background The development of high-throughput omics technologies enabled genome-wide measurements of the activity of cellular elements and provides the analytical resources for the progress of the Systems Biology discipline. Analysis and interpretation of gene expression data has evolved from the gene to the pathway and interaction level, i.e. from the detection of differentially expressed genes, to the establishment of gene interaction networks and the identification of enriched functional categories. Still, the understanding of biological systems requires a further level of analysis that addresses the characterization of the interaction between functional modules. Results We present a novel computational methodology to study the functional interconnections among the molecular elements of a biological system. The PANA approach uses high-throughput genomics measurements and a functional annotation scheme to extract an activity profile from each functional block -or pathway- followed by machine-learning methods to infer the relationships between these functional profiles. The result is a global, interconnected network of pathways that represents the functional cross-talk within the molecular system. We have applied this approach to describe the functional transcriptional connections during the yeast cell cycle and to identify pathways that change their connectivity in a disease condition using an Alzheimer example. Conclusions PANA is a useful tool to deepen in our understanding of the functional interdependences that operate within complex biological systems. We show the approach is algorithmically consistent and the inferred network is well supported by the available functional data. The method allows the dissection of the molecular basis of the functional connections and we describe the different regulatory mechanisms that explain the network's topology obtained for the yeast cell cycle data. PMID:25032889

  9. Pathway and network analysis of cancer genomes.

    PubMed

    2015-07-01

    Genomic information on tumors from 50 cancer types cataloged by the International Cancer Genome Consortium (ICGC) shows that only a few well-studied driver genes are frequently mutated, in contrast to many infrequently mutated genes that may also contribute to tumor biology. Hence there has been large interest in developing pathway and network analysis methods that group genes and illuminate the processes involved. We provide an overview of these analysis techniques and show where they guide mechanistic and translational investigations. PMID:26125594

  10. Pathway and Network Analysis of Cancer Genomes

    PubMed Central

    Haider, Syed; Wu, Guanming; Shibata, Tatsuhiro; Vazquez, Miguel; Mustonen, Ville; Gonzalez-Perez, Abel; Pearson, John; Sander, Chris; Raphael, Benjamin J.; Marks, Debora S.; Ouellette, B.F. Francis; Valencia, Alfonso; Bader, Gary D.; Boutros, Paul C.; Stuart, Joshua M.; Linding, Rune; Lopez-Bigas, Nuria; Stein, Lincoln D.

    2016-01-01

    Genomic information on tumors from 50 cancer types catalogued by The International Cancer Genome Consortium (ICGC) shows that only few well-studied driver genes are frequently mutated, in contrast to many infrequently mutated genes that may also contribute to tumor biology. Hence there has been large interest in developing pathway and network analysis methods that group genes and illuminate the processes involved. We provide an overview of these analysis techniques and show where they guide mechanistic and translational investigations. PMID:26125594

  11. Nonlinear fitness landscape of a molecular pathway.

    PubMed

    Perfeito, Lilia; Ghozzi, Stéphane; Berg, Johannes; Schnetz, Karin; Lässig, Michael

    2011-07-01

    Genes are regulated because their expression involves a fitness cost to the organism. The production of proteins by transcription and translation is a well-known cost factor, but the enzymatic activity of the proteins produced can also reduce fitness, depending on the internal state and the environment of the cell. Here, we map the fitness costs of a key metabolic network, the lactose utilization pathway in Escherichia coli. We measure the growth of several regulatory lac operon mutants in different environments inducing expression of the lac genes. We find a strikingly nonlinear fitness landscape, which depends on the production rate and on the activity rate of the lac proteins. A simple fitness model of the lac pathway, based on elementary biophysical processes, predicts the growth rate of all observed strains. The nonlinearity of fitness is explained by a feedback loop: production and activity of the lac proteins reduce growth, but growth also affects the density of these molecules. This nonlinearity has important consequences for molecular function and evolution. It generates a cliff in the fitness landscape, beyond which populations cannot maintain growth. In viable populations, there is an expression barrier of the lac genes, which cannot be exceeded in any stationary growth process. Furthermore, the nonlinearity determines how the fitness of operon mutants depends on the inducer environment. We argue that fitness nonlinearities, expression barriers, and gene-environment interactions are generic features of fitness landscapes for metabolic pathways, and we discuss their implications for the evolution of regulation. PMID:21814515

  12. Targeting the histidine pathway in Mycobacterium tuberculosis.

    PubMed

    Lunardi, Juleane; Nunes, José Eduardo S; Bizarro, Cristiano V; Basso, Luiz Augusto; Santos, Diógenes Santiago; Machado, Pablo

    2013-01-01

    Worldwide, tuberculosis is the leading cause of morbidity and mortality due to a single bacterial pathogen, Mycobacterium tuberculosis (Mtb). The increasing prevalence of this disease, the emergence of multi-, extensively, and totally drug-resistant strains, complicated by co-infection with the human immunodeficiency virus, and the length of tuberculosis chemotherapy have led to an urgent and continued need for the development of new and more effective antitubercular drugs. Within this context, the L-histidine biosynthetic pathway, which converts 5-phosphoribosyl 1-pyrophosphate to L-histidine in ten enzymatic steps, has been reported as a promising target of antimicrobial agents. This pathway is found in bacteria, archaebacteria, lower eukaryotes, and plants but is absent in mammals, making these enzymes highly attractive targets for the drug design of new antimycobacterial compounds with selective toxicity. Moreover, the biosynthesis of L-histidine has been described as essential for Mtb growth in vitro. Accordingly, a comprehensive overview of Mycobacterium tuberculosis histidine pathway enzymes as attractive targets for the development of new antimycobacterial agents is provided, mainly summarizing the previously reported inhibition data for Mtb or orthologous proteins. PMID:24111909

  13. Inferring differentiation pathways from gene expression

    PubMed Central

    Costa, Ivan G.; Roepcke, Stefan; Hafemeister, Christoph; Schliep, Alexander

    2008-01-01

    Motivation: The regulation of proliferation and differentiation of embryonic and adult stem cells into mature cells is central to developmental biology. Gene expression measured in distinguishable developmental stages helps to elucidate underlying molecular processes. In previous work we showed that functional gene modules, which act distinctly in the course of development, can be represented by a mixture of trees. In general, the similarities in the gene expression programs of cell populations reflect the similarities in the differentiation path. Results: We propose a novel model for gene expression profiles and an unsupervised learning method to estimate developmental similarity and infer differentiation pathways. We assess the performance of our model on simulated data and compare it with favorable results to related methods. We also infer differentiation pathways and predict functional modules in gene expression data of lymphoid development. Conclusions: We demonstrate for the first time how, in principal, the incorporation of structural knowledge about the dependence structure helps to reveal differentiation pathways and potentially relevant functional gene modules from microarray datasets. Our method applies in any area of developmental biology where it is possible to obtain cells of distinguishable differentiation stages. Availability: The implementation of our method (GPL license), data and additional results are available at http://algorithmics.molgen.mpg.de/Supplements/InfDif/ Contact: filho@molgen.mpg.de, schliep@molgen.mpg.de Supplementary information: Supplementary data is available at Bioinformatics online. PMID:18586709

  14. Pathway and Network Analysis in Proteomics

    PubMed Central

    Wu, Xiaogang; Hasan, Mohammad Al; Chen, Jake Yue

    2014-01-01

    Proteomics is inherently a systems science that studies not only measured protein and their expressions in a cell, but also the interplay of proteins, protein complexes, signaling pathways, and network modules. There is a rapid accumulation of Proteomics data in recent years. However, Proteomics data are highly variable, with results being sensitive to data preparation methods, sample condition, instrument types, and analytical method. To address this challenge in Proteomics data analysis, we review common approaches developed to incorporate biological function and network topological information. We categorize existing tools into four categories: tools with basic functional information and little topological features (e.g., GO category analysis), tools with rich functional information and little topological features (e.g., GSEA), tools with basic functional information and rich topological features (e.g., Cytoscape), and tools with rich functional information and rich topological features (e.g., PathwayExpress). We review the general application potential of these tools to Proteomics. In addition, we also review tools that can achieve automated learning of pathway modules and features, and tools that help perform integrated network visual analytics. PMID:24911777

  15. The nature of protein folding pathways.

    PubMed

    Englander, S Walter; Mayne, Leland

    2014-11-11

    How do proteins fold, and why do they fold in that way? This Perspective integrates earlier and more recent advances over the 50-y history of the protein folding problem, emphasizing unambiguously clear structural information. Experimental results show that, contrary to prior belief, proteins are multistate rather than two-state objects. They are composed of separately cooperative foldon building blocks that can be seen to repeatedly unfold and refold as units even under native conditions. Similarly, foldons are lost as units when proteins are destabilized to produce partially unfolded equilibrium molten globules. In kinetic folding, the inherently cooperative nature of foldons predisposes the thermally driven amino acid-level search to form an initial foldon and subsequent foldons in later assisted searches. The small size of foldon units, ∼ 20 residues, resolves the Levinthal time-scale search problem. These microscopic-level search processes can be identified with the disordered multitrack search envisioned in the "new view" model for protein folding. Emergent macroscopic foldon-foldon interactions then collectively provide the structural guidance and free energy bias for the ordered addition of foldons in a stepwise pathway that sequentially builds the native protein. These conclusions reconcile the seemingly opposed new view and defined pathway models; the two models account for different stages of the protein folding process. Additionally, these observations answer the "how" and the "why" questions. The protein folding pathway depends on the same foldon units and foldon-foldon interactions that construct the native structure. PMID:25326421

  16. The kynurenine pathway and neurodegenerative disease.

    PubMed

    Maddison, Daniel C; Giorgini, Flaviano

    2015-04-01

    Neuroactive metabolites of the kynurenine pathway (KP) of tryptophan degradation have been closely linked to the pathogenesis of several neurodegenerative diseases. Tryptophan is an essential amino acid required for protein synthesis, and in higher eukaryotes is also converted into the key neurotransmitters serotonin and tryptamine. However, in mammals >95% of tryptophan is metabolized through the KP, ultimately leading to the production of nicotinamide adenosine dinucleotide (NAD(+)). A number of the pathway metabolites are neuroactive; e.g. can modulate activity of several glutamate receptors and generate/scavenge free radicals. Imbalances in absolute and relative levels of KP metabolites have been strongly associated with neurodegenerative disorders including Huntington's, Alzheimer's, and Parkinson's diseases. The KP has also been implicated in the pathogenesis of other brain disorders (e.g. schizophrenia, bipolar disorder), as well as several cancers and autoimmune disorders such as HIV. Pharmacological and genetic manipulation of the KP has been shown to ameliorate neurodegenerative phenotypes in a number of model organisms, suggesting that it could prove to be a viable target for the treatment of such diseases. Here, we provide an overview of the KP, its role in neurodegeneration and the current strategies for therapeutic targeting of the pathway. PMID:25773161

  17. Modeling Protein Expression and Protein Signaling Pathways

    PubMed Central

    Telesca, Donatello; Müller, Peter; Kornblau, Steven M.; Suchard, Marc A.; Ji, Yuan

    2015-01-01

    High-throughput functional proteomic technologies provide a way to quantify the expression of proteins of interest. Statistical inference centers on identifying the activation state of proteins and their patterns of molecular interaction formalized as dependence structure. Inference on dependence structure is particularly important when proteins are selected because they are part of a common molecular pathway. In that case, inference on dependence structure reveals properties of the underlying pathway. We propose a probability model that represents molecular interactions at the level of hidden binary latent variables that can be interpreted as indicators for active versus inactive states of the proteins. The proposed approach exploits available expert knowledge about the target pathway to define an informative prior on the hidden conditional dependence structure. An important feature of this prior is that it provides an instrument to explicitly anchor the model space to a set of interactions of interest, favoring a local search approach to model determination. We apply our model to reverse-phase protein array data from a study on acute myeloid leukemia. Our inference identifies relevant subpathways in relation to the unfolding of the biological process under study. PMID:26246646

  18. Linking multiple pathogenic pathways in Alzheimer's disease.

    PubMed

    Bou Khalil, Rami; Khoury, Elie; Koussa, Salam

    2016-06-22

    Alzheimer's disease (AD) is a chronic neurodegenerative disorder presenting as progressive cognitive decline with dementia that does not, to this day, benefit from any disease-modifying drug. Multiple etiologic pathways have been explored and demonstrate promising solutions. For example, iron ion chelators, such as deferoxamine, are a potential therapeutic solution around which future studies are being directed. Another promising domain is related to thrombin inhibitors. In this minireview, a common pathophysiological pathway is suggested for the pathogenesis of AD to prove that all these mechanisms converge onto the same cascade of neuroinflammatory events. This common pathway is initiated by the presence of vascular risk factors that induce brain tissue hypoxia, which leads to endothelial cell activation. However, the ensuing hypoxia stimulates the production and release of reactive oxygen species and pro-inflammatory proteins. Furthermore, the endothelial activation may become excessive and dysfunctional in predisposed individuals, leading to thrombin activation and iron ion decompartmentalization. The oxidative stress that results from these modifications in the neurovascular unit will eventually lead to neuronal and glial cell death, ultimately leading to the development of AD. Hence, future research in this field should focus on conducting trials with combinations of potentially efficient treatments, such as the combination of intranasal deferoxamine and direct thrombin inhibitors. PMID:27354962

  19. Role of Hedgehog Signaling Pathway in NASH.

    PubMed

    Verdelho Machado, Mariana; Diehl, Anna Mae

    2016-01-01

    Non-alcoholic fatty liver disease (NAFLD) is the number one cause of chronic liver disease in the Western world. Although only a minority of patients will ultimately develop end-stage liver disease, it is not yet possible to efficiently predict who will progress and, most importantly, effective treatments are still unavailable. Better understanding of the pathophysiology of this disease is necessary to improve the clinical management of NAFLD patients. Epidemiological data indicate that NAFLD prognosis is determined by an individual's response to lipotoxic injury, rather than either the severity of exposure to lipotoxins, or the intensity of liver injury. The liver responds to injury with a synchronized wound-healing response. When this response is abnormal, it leads to pathological scarring, resulting in progressive fibrosis and cirrhosis, rather than repair. The hedgehog pathway is a crucial player in the wound-healing response. In this review, we summarize the pre-clinical and clinical evidence, which demonstrate the role of hedgehog pathway dysregulation in NAFLD pathogenesis, and the preliminary data that place the hedgehog pathway as a potential target for the treatment of this disease. PMID:27258259

  20. Alternative pathway for atmospheric particles growth.

    PubMed

    Monge, Maria Eugenia; Rosenørn, Thomas; Favez, Olivier; Müller, Markus; Adler, Gabriela; Abo Riziq, Ali; Rudich, Yinon; Herrmann, Hartmut; George, Christian; D'Anna, Barbara

    2012-05-01

    Credible climate change predictions require reliable fundamental scientific knowledge of the underlying processes. Despite extensive observational data accumulated to date, atmospheric aerosols still pose key uncertainties in the understanding of Earth's radiative balance due to direct interaction with radiation and because they modify clouds' properties. Specifically, major gaps exist in the understanding of the physicochemical pathways that lead to aerosol growth in the atmosphere and to changes in their properties while in the atmosphere. Traditionally, the driving forces for particle growth are attributed to condensation of low vapor pressure species following atmospheric oxidation of volatile compounds by gaseous oxidants. The current study presents experimental evidence of an unaccounted-for new photoinduced pathway for particle growth. We show that heterogeneous reactions activated by light can lead to fast uptake of noncondensable Volatile Organic Compounds (VOCs) at the surface of particles when only traces of a photosensitizer are present in the seed aerosol. Under such conditions, size and mass increase; changes in the chemical composition of the aerosol are also observed upon exposure to volatile organic compounds such as terpenes and near-UV irradiation. Experimentally determined growth rate values match field observations, suggesting that this photochemical process can provide a new, unaccounted-for pathway for atmospheric particle growth and should be considered by models. PMID:22517749