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Sample records for 5-phosphate reductoisomerase mep

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

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

    2016-09-01

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

  2. 1-deoxy-d-xylulose-5-phosphate reductoisomerases and method of use

    Croteau, Rodney B. (Pullman, WA); Lange, Bernd M. (Pullman, WA)

    2001-01-01

    The present invention relates to isolated DNA sequences which code for the expression of plant 1-deoxy-D-xylulose-5-phosphate reductoisomerase protein, such as the sequence presented in SEQ ID NO:1 which encodes a 1-deoxy-D-xylulose-5-phosphate reductoisomerase protein from peppermint (Mentha x piperita). Additionally, the present invention relates to isolated plant 1-deoxy-D-xylulose-5-phosphate reductoisomerase protein. In other aspects, the present invention is directed to replicable recombinant cloning vehicles comprising a nucleic acid sequence which codes for a plant 1-deoxy-D-xylulose-5-phosphate reductoisomerase, to modified host cells transformed, transfected, infected and/or injected with a recombinant cloning vehicle and/or DNA sequence of the invention.

  3. 1-deoxy-D-xylulose-5-phosphate reductoisomerases, and methods of use

    Croteau, Rodney B. (Pullman, WA); Lange, Bernd M. (Pullman, WA)

    2002-07-16

    The present invention relates to isolated DNA sequences which code for the expression of plant 1-deoxy-D-xylulose-5-phosphate reductoisomerase protein, such as the sequence presented in SEQ ID NO:1 which encodes a 1-deoxy-D-xylulose-5-phosphate reductoisomerase protein from peppermint (Mentha x piperita). Additionally, the present invention relates to isolated plant 1-deoxy-D-xylulose-5-phosphate reductoisomerase protein. In other aspects, the present invention is directed to replicable recombinant cloning vehicles comprising a nucleic acid sequence which codes for a plant 1-deoxy-D-xylulose-5-phosphate reductoisomerase, to modified host cells transformed, transfected, infected and/or injected with a recombinant cloning vehicle and/or DNA sequence of the invention.

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

    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

  5. 1-Deoxy-d-Xylulose 5-Phosphate Reductoisomerase (IspC) from Mycobacterium tuberculosis: towards Understanding Mycobacterial Resistance to Fosmidomycin

    Dhiman, Rakesh K.; Schaeffer, Merrill L.; Bailey, Ann Marie; Testa, Charles A.; Scherman, Hataichanok; Crick, Dean C.

    2005-01-01

    1-Deoxy-d-xylulose 5-phosphate reductoisomerase (IspC) catalyzes the first committed step in the mevalonate-independent isopentenyl diphosphate biosynthetic pathway and is a potential drug target in some pathogenic bacteria. The antibiotic fosmidomycin has been shown to inhibit IspC in a number of organisms and is active against most gram-negative bacteria but not gram positives, including Mycobacterium tuberculosis, even though the mevalonate-independent pathway is the sole isopentenyl dipho...

  6. Molecular cloning, characterization and expression analysis of the gene encoding 1-deoxy-D-xylulose 5-phosphate reductoisomerase from Aquilaria sinensis (Lour.) Gilg

    Juan Liu; Yanhong Xu; Liang Liang; Jianhe Wei

    2015-06-01

    The major constituents of agarwood oils are sesquiterpenes that are obtained from isoprenoid precursors through the plastidial methylerythritol phosphate (MEP) pathway and the cytosolic mevalonate pathway. In this study, a novel full-length cDNA of 1-deoxy-D-xylulose 5-phosphate reductoisomerase (DXR), which was the second key enzyme in the plastid MEP pathway of sesquiterpenes biosynthesis was isolated from the stem of Aquilaria sinensis (Lour.) Gilg by the methods of reverse transcription polymerase chain reaction (RT-PCR) and rapid amplification of cDNA ends (RACE) technique for the first time, and named as AsDXR. The full-length cDNA of AsDXR was 1768 bp, containing a 1437 bp open reading frame (ORF) encoding a polypeptide of 478 amino acids with a molecular weight of 51.859 kD and the theoretical isoelectric point of 6.29. Comparative and bioinformatic analysis of the deduced AsDXR protein showed extensive homology with DXRs from other plant species, especially Theobroma cacao and Gossypium barbadense, and contained a conserved transit peptide for plastids, and extended pro-rich region and a highly conserved NADPH-binding motif owned by all plant DXRs. Southern blot analysis indicated that AsDXR belonged to a small gene family. Tissue expression pattern analysis revealed that AsDXR expressed strongly in root and stem, but weakly in leaf. Additionally, AsDXR expression was found to be activated by exogenous elicitor of MeJA (methyl jasmonate). The contents of three sesquiterpenes ($\\alpha$-guaiene, $\\alpha$-humulene and $\\delta$-guaiene) were significantly induced by MeJA. This study enables us to further elucidate the role of AsDXR in the biosynthesis of agarwood sesquiterpenes in A. sinensis at the molecular level.

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

    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.

    2013-01-01

    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. ...

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

    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

  9. 烟草5-磷酸脱氧木酮糖还原异构酶基因(dxr)的克隆和表达分析%Cloning and Expression Analysis of 1-deoxy-D-xylulose-5-phosphate Reductoisomerases Gene (dxr) in Nicotina tabacum

    朱晓宇; 王景; 赵二卫; 姚姗姗; 崔红

    2011-01-01

    以烟草(Nicotiana tabacum)栽培品种K326为材料,采用RT-PCR技术,克隆了萜类代谢关键酶烟草5-磷酸脱氧木酮糖还原异构酶(dxr)的cDNA片段.该基因编码区长1422 bp,编码473个氨基酸残基.利用Clustal W(1.82)和Bioedit软件,对烟草与番茄(Lycopersicon esculentum)、长春花(Catharanthus roseus)、金鱼草(A ntirrhinum majus)、薄荷(Mentha piperita)、玉米(Zea mays)、拟南芥(A rabidopsis thaliana)、念珠藻(Nostoc sp.)等物种dar基因的同源性进行分析,其氨基酸同源性分别达到93.6%、87.9%、86.3%、84.6%、84.2%、82.9%和53.5%.原核表达结果证明,该基因编码蛋白的分子量约为50 kD,与氨基酸序列估算相符合.组织表达特异性分析表明,dxr基因在烟草组织中的表达强弱为花>叶>茎>腺毛>种子>根,在花和叶片中的表达量占优势.该结果对烟草萜类代谢的分子调控和品质改良具有重要的参考价值.%Isoprenoid biosynthesis via mvalonate-independent pathway is very important to tobacco resistance and leaf quality. 1-deoxy-D-Xylulose-5-phosphate Reductoisomerases (dxr) is a key enzyme in biosynthesis of isopentenyl diphosphate, which is the precusor for monoterpenoid, diterpenoid and tetratepenoid compounds. To regulate the terpenoid metabolism pathway for tobacco improvement, some important genes such as dxr should be studied firstly. In this paper, dxr gene was cloned successfully from tobacco (Nicotiana tabacum) cultivar K326 leaf by RT-PCR. The cDNA code region was 1 422 bp long and encoding 437 amino acids. Sequence analysis by Clustal W declared that this fragment was highly homologous to dxr gene of other species. It shared 93.6% amino acid homologous to Lycopersicon esculentum, 87.9% to Catharanthus roseus, 86.3% to Antirrhinum majus,84.6% to Mentha piperita, 84.2% to Zea mays, 82.9% to Arabidopsis thaliana, and 53.5% to Nostoc sp.PCC7120. The expression vector pET21b-dxr was constructed and expressed in

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

    Mei Han

    Full Text Available 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.

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

    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

  12. Metabolic engineering of essential oil yield and composition in mint by altering expression of deoxyxylulose phosphate reductoisomerase and menthofuran synthase

    Mahmoud, Soheil S.; Croteau, Rodney B.

    2001-01-01

    Peppermint (Mentha × piperita L.) was independently transformed with a homologous sense version of the 1-deoxy-d-xylulose-5-phosphate reductoisomerase cDNA and with a homologous antisense version of the menthofuran synthase cDNA, both driven by the CaMV 35S promoter. Two groups of transgenic plants were regenerated in the reductoisomerase experiments, one of which remained normal in appearance and development; another was deficient in chlorophyll production and grew slowly. Transgenic plants ...

  13. Liberia MEP WMS

    US Agency for International Development — L-MEP WMS provides USAID, Implementing Partners, and L-MEP Staff the ability to view M view sector specific trend or base information; and answer questions about...

  14. Ketol-acid reductoisomerase enzymes and methods of use

    Govindarajan, Sridhar; Li, Yougen; Liao, Der-Ing; O'Keefe, Daniel P.; Minshull, Jeremy Stephen; Rothman, Steven Cary; Tobias, Alexander Vincent

    2015-10-27

    Provided herein are polypeptides having ketol-aid reductoisomerase activity as well as microbial host cells comprising such polypeptides. Polypeptides provided herein may be used in biosynthetic pathways, including, but not limited to, isobutanol biosynthetic pathways.

  15. Ketol-acid reductoisomerase enzymes and methods of use

    Govindarajan, Sridhar; Li, Yougen; Liao, Der-Ing; O' Keefe, Daniel P.; Minshull, Jeremy Stephen; Rothman, Steven Cary; Tobias, Alexander Vincent

    2016-07-12

    Provided herein are polypeptides having ketol-acid reductoisomerase activity as well as microbial host cells comprising such polypeptides. Polypeptides provided herein may be used in biosynthetic pathways, including, but not limited to, isobutanol biosynthetic pathways.

  16. Genetics Home Reference: pyridoxal 5'-phosphate-dependent epilepsy

    ... 5'-phosphate-dependent epilepsy pyridoxal 5'-phosphate-dependent epilepsy Enable Javascript to view the expand/collapse boxes. ... All Close All Description Pyridoxal 5'-phosphate-dependent epilepsy is a condition that involves seizures beginning soon ...

  17. Setting MEPS for electronic products

    When analysing price, performance and efficiency data for 15 consumer electronic and information and communication technology products, we found that in general price did not relate to the efficiency of the product. Prices of electronic products with comparable performance decreased over time. For products where the data allowed fitting the relationship, we found an exponential decrease in price with an average time constant of −0.30 [1/year], meaning that every year the product became 26% cheaper on average. The results imply that the classical approach of setting minimum efficiency performance standards (MEPS) by means of life cycle cost calculations cannot be applied to electronic products. Therefore, an alternative approach based on the improvement of efficiency over time and the variation in efficiency of products on the market, is presented. The concept of a policy action window can provide guidance for the decision on whether setting MEPS for a certain product is appropriate. If the (formal) procedure for setting MEPS takes longer than the policy action window, this means that the efficiency improvement will also be achieved without setting MEPS. We found short, i.e. less than three years, policy action windows for graphic cards, network attached storage products, network switches and televisions. - Highlights: • For electronic consumer products price does not relate to efficiency. • Average price decrease of selected electronic products is 26 % per year. • We give an alternative approach to life cycle cost calculations for setting MEPS. • The policy action window indicates whether setting MEPS is appropriate

  18. 21 CFR 582.5697 - Riboflavin-5-phosphate.

    2010-04-01

    ... 21 Food and Drugs 6 2010-04-01 2010-04-01 false Riboflavin-5-phosphate. 582.5697 Section 582.5697 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) ANIMAL... Supplements 1 § 582.5697 Riboflavin-5-phosphate. (a) Product. Riboflavin-5-phosphate. (b) Conditions of...

  19. Purification and characterization of ribitol-5-phosphate and xylitol-5-phosphate dehydrogenases from strains of Lactobacillus casei.

    Hausman, S Z; London, J

    1987-01-01

    A simple three-step procedure is described which yields electrophoretically homogeneous preparations of ribitol-5-phosphate dehydrogenase and xylitol-5-phosphate dehydrogenase. The former enzyme is a 115,000-molecular-weight protein composed of two subunits of identical size and is specific for its substrate, ribitol. The xylitol-5-phosphate dehydrogenase exists as a tetrameric protein with a molecular weight of 180,000; this enzyme oxidizes the phosphate esters of both xylitol and D-arabitol...

  20. Reconciling Medical Expenditure Estimates from the MEPS...

    U.S. Department of Health & Human Services — Reconciling Medical Expenditure Estimates from the MEPS and NHEA, 2007, published in Volume 2, Issue 4 of the Medicare and Medicaid Research Review, provides a...

  1. SDE Plus, SDE and MEP. Annual review 2012; SDE+, SDE en MEP. Jaarbericht 2012

    NONE

    2013-05-15

    This annual report describes the applications for SDE subsidy (renewable energy support scheme) in the period 2008-2012, the new SDE Plus which starts in 2013, and the MEP transition scheme (MEP stands for 'Environmental quality of electricity production', predecessor of SDE for the period 2003-2006) and applications from the MEP scheme [Dutch] Het jaarbericht 2012 voor de SDE+, SDE en MEP presenteert de resultaten van de regeling Stimulering Duurzame Energieproductie (SDE+ vanaf 2013 en SDE, 2008-2012) en de voorganger van de SDE, de subsidieregeling Milieukwaliteit van de Elektriciteitsproductie.

  2. Ribose 5-Phosphate Isomerase Investigations for the Undergraduate Biochemistry Laboratory

    Jewett, Kathy; Sandwick, Roger K.

    2011-01-01

    The enzyme ribose 5-phosphate isomerase (RpiA) has many features that make it attractive as a focal point of a semester-long, advanced biochemistry laboratory for undergraduate students. The protein can easily and inexpensively be isolated from spinach using traditional purification techniques. Characterization of RpiA enzyme activity can be…

  3. Ribose 5-Phosphate Isomerase B Knockdown Compromises Trypanosoma brucei Bloodstream Form Infectivity

    Inês Loureiro; Joana Faria; Christine Clayton; Sandra Macedo-Ribeiro; Nuno Santarém; Nilanjan Roy; Anabela Cordeiro-da-Siva; Joana Tavares

    2015-01-01

    Ribose 5-phosphate isomerase is an enzyme involved in the non-oxidative branch of the pentose phosphate pathway, and catalyzes the inter-conversion of D-ribose 5-phosphate and D-ribulose 5-phosphate. Trypanosomatids, including the agent of African sleeping sickness namely Trypanosoma brucei, have a type B ribose-5-phosphate isomerase. This enzyme is absent from humans, which have a structurally unrelated ribose 5-phosphate isomerase type A, and therefore has been proposed as an attractive dru...

  4. Four Thruster Microfluidic Electrospray Propulsion (MEP) Cubesat Board Demonstration Project

    National Aeronautics and Space Administration — The Cubesat Microfluidic Electrospray Propulsion (MEP) system module prototype will be designed, built and tested to demonstrate that a four MEP thruster system can...

  5. AutoCAD MEP 2009 Anpassad verktygspalett

    Johansson, Magnus; Klasa, Thomas

    2009-01-01

    Rapporten handlar om examensprojektet som utförts av Magnus Johansson och Thomas Klasa under våren 2009. Man har under åtta veckors tid arbetat med att hjälpa företaget LBElkonsult AB i Mölndal med att ta fram en specialanpassad verktygspalett i datorprogrammet AutoCAD MEP 2009. I rapporten finns de olika steg som genomförts för att nå det önskade slutresultatet. Allt från förstudier och själva arbetet i AutoCAD MEP 2009 till finjusteringarna som avslutningsvis gjordes på den blivande slutpro...

  6. Morton's foot and pyridoxal 5'-phosphate deficiency: genetically linked traits.

    Nichols, Trent W; Gaiteri, Christopher

    2014-12-01

    Vitamin B6 is an essential vitamin needed for many chemical reactions in the human body. It exists as several vitamins forms but pyridoxal 5'-phosphate (PLP) is the phosphorylated form needed for transamination, deamination, and decarboxylation. PLP is important in the production of neurotransmitters, acts as a Schiff base and is essential in the metabolism of homocysteine, a toxic amino acid involved in cardiovascular disease, stroke, thrombotic and Alzheimer's disease. This report announces the connection between a deficit of PLP with a genetically linked physical foot form known as the Morton's foot. Morton's foot has been associated with fibromyalgia/myofascial pain syndrome. Another gene mutation methylenetetrahydrofolate reductase (MTHFr) is now being recognized much commonly than previous with chronic fatigue, chronic Lyme diseases and as "the missing link" in other chronic diseases. PLP deficiency also plays a role in impaired glucose tolerance and may play a much bigger role in the obesity, diabetes, fatty liver and metabolic syndrome. Without the Schiff-base of PLP acting as an electron sink, storing electrons and dispensing them in the mitochondria, free radical damage occurs! The recognition that a phenotypical expression (Morton's foot) of a gene resulting in deficiency of an important cofactor enzyme pyridoxal 5'-phosphate will hopefully alert physicians and nutritionist to these phenomena. Supplementation with PLP, L5-MTHF, B12 and trimethylglycine should be used in those patients with hyperhomocysteinemia and/or MTHFR gene mutation. PMID:25441836

  7. Inactive mutants of human pyridoxine 5'-phosphate oxidase: a possible role for a noncatalytic pyridoxal 5'-phosphate tight binding site.

    Ghatge, Mohini S; Karve, Sayali S; David, Tanya M S; Ahmed, Mostafa H; Musayev, Faik N; Cunningham, Kendra; Schirch, Verne; Safo, Martin K

    2016-05-01

    Pyridoxal 5'-phosphate (PLP) is a cofactor for many vitamin B6-requiring enzymes that are important for the synthesis of neurotransmitters. Pyridoxine 5'-phosphate oxidase (PNPO) is one of two enzymes that produce PLP. Some 16 known mutations in human PNPO (hPNPO), including R95C and R229W, lead to deficiency of PLP in the cell and have been shown to cause neonatal epileptic encephalopathy (NEE). This disorder has no effective treatment, and is often fatal unless treated with PLP. In this study, we show that R95C hPNPO exhibits a 15-fold reduction in affinity for the FMN cofactor, a 71-fold decrease in affinity for the substrate PNP, a 4.9-fold decrease in specific activity, and a 343-fold reduction in catalytic activity, compared to the wild-type enzyme. We have reported similar findings for R229W hPNPO. This report also shows that wild-type, R95C and R229W hPNPO bind PLP tightly at a noncatalytic site and transfer it to activate an apo-B6 enzyme into the catalytically active holo-form. We also show for the first time that hPNPO forms specific interactions with several B6 enzymes with dissociation constants ranging from 0.3 to 12.3 μm. Our results suggest a possible in vivo role for the tight binding of PLP in hPNPO, whether wild-type or variant, by protecting the very reactive PLP, and transferring this PLP directly to activate apo-B6 enzymes. PMID:27419045

  8. The role of experience curves for setting MEPS for appliances

    Minimum efficiency performance standards (MEPS) are an important policy instrument to raise the efficiency of products. In most schemes the concept of life cycle costs (LCC) is used to guide setting the MEPS levels. Although a large body of literature shows that product cost is decreasing with increasing cumulative production, the experience curve, this is currently not used for setting MEPS. This article shows how to integrate the concept of the experience curve into LCC calculations for setting MEPS in the European Union and applies this to household laundry driers, refrigerator-freezers and televisions. The results indicate that for driers and refrigerator-freezers at least twice the energy savings compared to the current approach can be achieved. These products also show that energy label classes can successfully be used for setting MEPS. For televisions an experience curve is provided, showing a learning rate of 29%. However, television prices do not show a relation with energy efficiency but are to a large extent determined by the time the product is placed on the market. This suggests to policy makers that for televisions and other products with a short (re)design and market cycle timing is more important than the MEPS levels itself. - Highlights: • We integrate experience curves into life cycle cost calculations for MEPS. • For driers and refrigerators this results in at least twice the energy savings. • For flat panel televisions an experience curve is provided

  9. Molecular design, synthesis and biological activities of amidines as new ketol-acid reductoisomerase inhibitors

    Bao Lei Wang; Yong Hong Li; Jian Guo Wang; Yi Ma; Zheng Ming Li

    2008-01-01

    Diamidine (A) was identified in our in vitro bio-assay as a possible inhibitor of ketol-acid reductoisomerase (KARI) from the ACD database search based on the known three-dimensional crystal structure of KARI. An investigation on interaction of A on KARI active sites, led to the design and synthesis of 15 novel monoamidines. Some of those showed better biological activity than A on rice KARI (in vitro) and in greenhouse herbicidal tests (in vivo). The structure-biological activity relationship was investigated, which provides valuable information to further study of potential KARI inhibitors.

  10. Medical Expenditure Panel Survey (MEPS) Summary Data Tables

    U.S. Department of Health & Human Services — Summary Data Tables Data collected through MEPS are used to generate tables with frequently used summary statistics. These tables are available here for both the...

  11. Structure of ribose 5-phosphate isomerase from the probiotic bacterium Lactobacillus salivarius UCC118

    The crystal structure of ribose 5-phosphate isomerase has been determined to 1.72 Å resolution and is presented with a brief comparison to other known ribose 5-phosphate isomerase A structures. The structure of ribose 5-phosphate isomerase from the probiotic bacterium Lactobacillus salivarius UCC188 has been determined at 1.72 Å resolution. The structure was solved by molecular replacement, which identified the functional homodimer in the asymmetric unit. Despite only showing 57% sequence identity to its closest homologue, the structure adopted the typical α and β d-ribose 5-phosphate isomerase fold. Comparison to other related structures revealed high homology in the active site, allowing a model of the substrate-bound protein to be proposed. The determination of the structure was expedited by the use of in situ crystallization-plate screening on beamline I04-1 at Diamond Light Source to identify well diffracting protein crystals prior to routine cryocrystallography

  12. Structural basis for Mep2 ammonium transceptor activation by phosphorylation

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

    2016-01-01

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

  13. Preparation and Purification of Xylitol-5-Phosphate from a Cell Extract of Lactobacillus casei Cl-16

    Trahan, L.; Néron, S.; Bareil, M.

    1988-01-01

    A simple procedure which yields pure xylitol-5-phosphate is described. A cell extract of Lactobacillus casei Cl-16 from a 6-liter culture was used to synthesize up to 70 mg of xylitol-5-phosphate overnight from xylitol and phosphoenolpyruvate via a xylitol phosphoenolpyruvate:phosphotransferase system with a 53% yield. Centrifugation, filtration, precipitation as a barium salt, and ion-exchange batch chromatography permitted recovery of nearly 90% of the phosphorylated product synthesized. Th...

  14. Biosynthesis of riboflavin. Enzymatic formation of the xylene moiety from [14C]ribulose 5-phosphate.

    Nielsen, P; Neuberger, G; Floss, H G; Bacher, A

    1984-02-14

    We have studied the enzymatic formation of the xylene ring of riboflavin using cell extracts from the flavinogenic yeast Candida guilliermondii. 5-Amino-6-ribitylamino-2,4(1H,3H)-pyrimidinedione or its 5'-phosphate could serve as substrates. In addition, a pentose phosphate or pentulose phosphate was required. Experiments with [14C]ribulose 5-phosphate gave evidence for the incorporation of the ribulose carbon atoms except C-4 into the xylene ring of the vitamin. PMID:6546684

  15. Host cells and methods for producing 1-deoxyxylulose 5-phosphate (DXP) and/or a DXP derived compound

    Kirby, James; Fortman, Jeffrey L.; Nishimoto, Minobu; Keasling, Jay D.

    2016-07-05

    The present invention provides for a genetically modified host cell capable of producing 1-deoxyxylulose 5-phosphate or 1-deoxy-D-xylulose 5-phosphate (DXP) (12), and optionally one or more DXP derived compounds, comprising: (a) a mutant RibB, or functional variant thereof, capable of catalyzing xylulose 5-phosphate and/or ribulose 5-phosphate to DXP, or (b) a YajO, or functional variant thereof, and a XylB, or functional variant thereof.

  16. Financial incentive for the Environmental Quality of Electricity Production (MEP)

    With the MEP subsidy regulation 'Environmental quality of electricity production' The Dutch minister of Economic Affairs focussed mainly on reaching the policy objective of the EU according to which 9% of all electricity used in the Netherlands should be generated with sustainable sources. According to the opinion of the Dutch Court of Audits not enough attention has been paid to the coherence of the MEP regulation with other policy objectives in the area of sustainability, such as air quality and CO2 reduction. The same goes for the effectiveness and the financial management of the MEP regulation. Moreover, the study, which was carried out by request of the Dutch Lower House, demonstrates that it is still uncertain if the policy objective for 2010 will be reached. The report consists of two parts: part 1: Conclusions, recommendations and administrative reactions, and Part 2: Answering Lower House questions.(mk)

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

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

    2015-01-01

    The 1-deoxy-d-xylulose-5-phosphate reductoisomerase (DXR; EC1.1.1.267), an NADPH-dependent reductase, plays a pivotal role in the methylerythritol 4-phosphate pathway (MEP), in the conversion of 1-deoxy-d-xylulose-5-phosphate (DXP) into MEP. The sheath and leaf of citronella (Cymbopogon winterianus) accumulates large amount of terpenes and sesquiterpenes with proven medicinal value and economic uses. Thus, sequencing of full length dxr gene and its characterization seems to be a valuable reso...

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

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

    2014-02-01

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

  19. Mutations in Escherichia coli aceE and ribB Genes Allow Survival of Strains Defective in the First Step of the Isoprenoid Biosynthesis Pathway

    Jordi Perez-Gil; Eva Maria Uros; Susanna Sauret-Güeto; Maria Lois, L.; James Kirby; Minobu Nishimoto; Baidoo, Edward E. K.; Jay D Keasling; Albert Boronat; Manuel Rodriguez-Concepcion

    2012-01-01

    A functional 2-C-methyl-D-erythritol 4-phosphate (MEP) pathway is required for isoprenoid biosynthesis and hence survival in Escherichia coli and most other bacteria. In the first two steps of the pathway, MEP is produced from the central metabolic intermediates pyruvate and glyceraldehyde 3-phosphate via 1-deoxy-D-xylulose 5-phosphate (DXP) by the activity of the enzymes DXP synthase (DXS) and DXP reductoisomerase (DXR). Because the MEP pathway is absent from humans, it was proposed as a pro...

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

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

    2016-06-01

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

  1. Direct and indirect effects of RNA interference against pyridoxal kinase and pyridoxine 5'-phosphate oxidase genes in Bombyx mori.

    Huang, ShuoHao; Yao, LiLi; Zhang, JianYun; Huang, LongQuan

    2016-08-01

    Vitamin B6 comprises six interconvertible pyridine compounds (vitamers), among which pyridoxal 5'-phosphate is a coenzyme involved in a high diversity of biochemical reactions. Humans and animals obtain B6 vitamers from diet, and synthesize pyridoxal 5'-phosphate by pyridoxal kinase and pyridoxine 5'-phosphate oxidase. Currently, little is known on how pyridoxal 5'-phosphate biosynthesis is regulated, and pyridoxal 5'-phosphate is supplied to meet their requirement in terms of cofactor. Bombyx mori is a large silk-secreting insect, in which protein metabolism is most active, and the vitamin B6 demand is high. In this study, we successfully down-regulated the gene expression of pyridoxal kinase and pyridoxine 5'-phosphate oxidase by body cavity injection of synthesized double-stranded small interfering RNA to 5th instar larvae of Bombyx mori, and analyzed the gene transcription levels of pyridoxal 5'-phosphate dependent enzymes, phosphoserine aminotransferase and glutamic-oxaloacetic transaminase. Results show that the gene expression of pyridoxal kinase and pyridoxine 5'-phosphate oxidase has a greater impact on the gene transcription of enzymes using pyridoxal 5'-phosphate as a cofactor in Bombyx mori. Our study suggests that pyridoxal 5'-phosphate biosynthesis and dynamic balance may be regulated by genetic networks. PMID:27106120

  2. LuxS-independent formation of AI-2 from ribulose-5-phosphate

    Hardie Kim R

    2008-06-01

    Full Text Available Abstract Background In many bacteria, the signal molecule AI-2 is generated from its precursor S-ribosyl-L-homocysteine in a reaction catalysed by the enzyme LuxS. However, generation of AI-2-like activity has also been reported for organisms lacking the luxS gene and the existence of alternative pathways for AI-2 formation in Escherichia coli has recently been predicted by stochastic modelling. Here, we investigate the possibility that spontaneous conversion of ribulose-5-phosphate could be responsible for AI-2 generation in the absence of luxS. Results Buffered solutions of ribulose-5-phosphate, but not ribose-5-phosphate, were found to contain high levels of AI-2 activity following incubation at concentrations similar to those reported in vivo. To test whether this process contributes to AI-2 formation by bacterial cells in vivo, an improved Vibrio harveyi bioassay was used. In agreement with previous studies, culture supernatants of E. coli and Staphylococcus aureus luxS mutants were found not to contain detectable levels of AI-2 activity. However, low activities were detected in an E. coli pgi-eda-edd-luxS mutant, a strain which degrades glucose entirely via the oxidative pentose phosphate pathway, with ribulose-5-phosphate as an obligatory intermediate. Conclusion Our results suggest that LuxS-independent formation of AI-2, via spontaneous conversion of ribulose-5-phosphate, may indeed occur in vivo. It does not contribute to AI-2 formation in wildtype E. coli and S. aureus under the conditions tested, but may be responsible for the AI-2-like activities reported for other organisms lacking the luxS gene.

  3. Effect of exogenous hormones on transcription levels of pyridoxal 5'-phosphate biosynthetic enzymes in the silkworm (Bombyx mori).

    Huang, ShuoHao; Yang, HuanHuan; Yao, LiLi; Zhang, JianYun; Huang, LongQuan

    2016-01-01

    Vitamin B6 includes 6 pyridine derivatives, among which pyridoxal 5'-phosphate is a coenzyme for over 140 enzymes. Animals acquire their vitamin B6 from food. Through a salvage pathway, pyridoxal 5'-phosphate is synthesized from pyridoxal, pyridoxine or pyridoxamine, in a series of reactions catalyzed by pyridoxal kinase and pyridoxine 5'-phosphate oxidase. The regulation of pyridoxal 5'-phospahte biosynthesis and pyridoxal 5'-phospahte homeostasis are at the center of study for vitamin B6 nutrition. How pyridoxal 5'-phosphate biosynthesis is regulated by hormones has not been reported so far. Our previous studies have shown that pyridoxal 5'-phosphate level in silkworm larva displays cyclic developmental changes. In the current study, effects of exogenous juvenile hormone and molting hormone on the transcription level of genes coding for the enzymes involved in the biosynthesis of pyridoxal 5'-phospahte were examined. Results show that pyridoxal kinase and pyridoxine 5'-phosphate oxidase are regulated at the transcription level by development and are responsive to hormones. Molting hormone stimulates the expression of genes coding for pyridoxal kinase and pyridoxine 5'-phosphate oxidase, and juvenile hormone appears to work against molting hormone. Whether pyridoxal 5'-phosphate biosynthesis is regulated by hormones in general is an important issue for further studies. PMID:26780217

  4. Positive outcome following early diagnosis and treatment of pyridoxal-5'-phosphate oxidase deficiency: a case report.

    Porri, Stephanie; Fluss, Joel; Plecko, Barbara; Paschke, Eduard; Korff, Christian M; Kern, Ilse

    2014-02-01

    Pyridoxal-5'-phosphate oxidase (PNPO) deficiency is a rare autosomal recessive, vitamin-responsive metabolic disorder causing refractory neonatal seizures that respond to the administration of pyridoxal-5'-phosphate (PLP). There are currently few case studies that have documented the functional outcome in PNPO deficiency, which remains poor in the majority of cases. We present the case of a male infant born at 35 weeks gestation who promptly responded to oral administration of PLP, following resistance to common anticonvulsive therapy and to a pyridoxine trial. Neurological outcome at 21 months is favorable and illustrates the importance of standardized vitamin trials in the acute setting of "therapy-resistant" neonatal seizures. Early recognition of PNPO deficiency and appropriate intervention might be associated with a more favorable outcome than initially considered. PMID:24297574

  5. Phosphatidylinositol 5-phosphate 4-kinase γ (PI5P4Kγ), a lipid signalling enigma.

    Giudici, Maria-Luisa; Clarke, Jonathan H; Irvine, Robin F

    2016-05-01

    The phosphatidylinositol 5-phosphate 4-kinases (PI5P4Ks) are an important family of enzymes, whose physiological roles are being teased out by a variety of means. Phosphatidylinositol-5-phosphate 4-kinase γ (PI5P4Kγ) is especially intriguing as its in vitro activity is very low. Here we review what is known about this enzyme and discuss some recent advances towards an understanding of its physiology. Additionally, the effects of the ATP-competitive inhibitor I-OMe Tyrphostin AG-538 on all three mammalian PI5P4Ks was explored, including two PI5P4Kγ mutants with altered ATP- or PI5P-binding sites. The results suggest a strategy for targeting non-ATP binding sites on inositol lipid kinases. PMID:26710750

  6. Structural characterization of a ribose-5-phosphate isomerase B from the pathogenic fungus Coccidioides immitis

    Leibly David J

    2011-10-01

    Full Text Available Abstract Background Ribose-5-phosphate isomerase is an enzyme that catalyzes the interconversion of ribose-5-phosphate and ribulose-5-phosphate. This family of enzymes naturally occurs in two distinct classes, RpiA and RpiB, which play an important role in the pentose phosphate pathway and nucleotide and co-factor biogenesis. Results Although RpiB occurs predominantly in bacteria, here we report crystal structures of a putative RpiB from the pathogenic fungus Coccidioides immitis. A 1.9 Å resolution apo structure was solved by combined molecular replacement and single wavelength anomalous dispersion (SAD phasing using a crystal soaked briefly in a solution containing a high concentration of iodide ions. RpiB from C. immitis contains modest sequence and high structural homology to other known RpiB structures. A 1.8 Å resolution phosphate-bound structure demonstrates phosphate recognition and charge stabilization by a single positively charged residue whereas other members of this family use up to five positively charged residues to contact the phosphate of ribose-5-phosphate. A 1.7 Å resolution structure was obtained in which the catalytic base of C. immitis RpiB, Cys76, appears to form a weakly covalent bond with the central carbon of malonic acid with a bond distance of 2.2 Å. This interaction may mimic that formed by the suicide inhibitor iodoacetic acid with RpiB. Conclusion The C. immitis RpiB contains the same fold and similar features as other members of this class of enzymes such as a highly reactive active site cysteine residue, but utilizes a divergent phosphate recognition strategy and may recognize a different substrate altogether.

  7. It takes two to tango: defining an essential second active site in pyridoxal 5'-phosphate synthase.

    Cyril Moccand

    Full Text Available The prevalent de novo biosynthetic pathway of vitamin B6 involves only two enzymes (Pdx1 and Pdx2 that form an ornate multisubunit complex functioning as a glutamine amidotransferase. The synthase subunit, Pdx1, utilizes ribose 5-phosphate and glyceraldehyde 3-phosphate, as well as ammonia derived from the glutaminase activity of Pdx2 to directly form the cofactor vitamer, pyridoxal 5'-phosphate. Given the fact that a single enzyme performs the majority of the chemistry behind this reaction, a complicated mechanism is anticipated. Recently, the individual steps along the reaction co-ordinate are beginning to be unraveled. In particular, the binding of the pentose substrate and the first steps of the reaction have been elucidated but it is not known if the latter part of the chemistry, involving the triose sugar, takes place in the same or a disparate site. Here, we demonstrate through the use of enzyme assays, enzyme kinetics, and mutagenesis studies that indeed a second site is involved in binding the triose sugar and moreover, is the location of the final vitamin product, pyridoxal 5'-phosphate. Furthermore, we show that product release is triggered by the presence of a PLP-dependent enzyme. Finally, we provide evidence that a single arginine residue of the C terminus of Pdx1 is responsible for coordinating co-operativity in this elaborate protein machinery.

  8. Da antiga à nova Filologia: o Projecto MEP-BPEDig.

    Gonçalves, Maria Filomena; Banza, Ana Paula

    2013-01-01

    Neste artigo faz-se a descrição do projecto "Memória (Meta)linguística na Biblioteca Pública de Évora: Para uma Biblioteca Digital (MEP-BPEDig)", projecto que se centra em fontes existentes na bicentenária Biblioteca Pública de Évora. O projecto desenvolve-se no âmbito do CIDEHUS-UÉ e visa disponibilizar fontes relevantes para a História e Historiografia do Português.

  9. Did the Economic Impact of CCCTB affect the Voting Behaviour of MEPs?

    ANNELIES ROGGEMAN; ISABELLE VERLEYEN; PHILIPPE VAN CAUWENBERGE; CARINE COPPENS

    2014-01-01

    On 19 April 2012, the Members of the European Parliament (MEPs) voted on the European Commission’s proposal for a Common Consolidated Corporate Tax Base (CCCTB). We exploit a unique research setting which was created by an economic impact assessment of CCCTB that was made available to the MEPs. Using regression analysis, we investigate if the voting behaviour of MEPs was influenced by the predicted economic impact of CCCTB on their country. Our results show that, even after controlling for pa...

  10. Long-term effect of magnesium pyridoxal 5-phosphate glutamate in rabbits developing hypercholesterolemia.

    Panagiotopoulos, T; Ketelsen, U P; Schmidt, A; Heuck, C C

    1986-08-01

    36 male rabbits were fed with a diet enriched with 20 g cholesterol/kg for a period of 8 weeks under a strict daily dietary control. Magnesium pyridoxal 5-phosphate glutamate (MPPG, Sedalipid) was supplemented to the diet at different quantities. Hypercholesterolemia developed later and less pronounced in animals receiving a medium or high dose of MPPG. Microscopical analysis indicated a protective effect of MPPG on calcium deposition in the aorta. The fatty acid pattern in serum showed only minor differences and was unchanged in liver extracts in animals supplemented with MPPG. This observation suggest that MPPG may act on the mechanism of cholesterol absorption in the intestine. PMID:3778558

  11. Acetate selective fluorescent turn-on sensors derived using vitamin B6 cofactor pyridoxal-5-phosphate

    Sharma, Darshna; Kuba, Aman; Thomas, Rini; Ashok Kumar, S. K.; Kuwar, Anil; Choi, Heung-Jin; Sahoo, Suban K.

    2016-03-01

    Two new Schiff base receptors have been synthesized by condensation of pyridoxal-5-phosphate with 2-aminophenol (L1) or aniline (L2). In DMSO, the receptors showed both chromogenic and 'turn-on' fluorescence responses selectively in the presence of AcO- and F-. However, in mixed DMSO-H2O medium, the receptors showed AcO- selective 'turn-on' fluorescence without any interference from other tested anions including F-. The detection limit for AcO- was found to be 7.37 μM and 22.9 μM using the receptors L1 and L2, respectively.

  12. The MEPS server for identifying protein conformational epitopes

    Castrignanò Tiziana

    2007-03-01

    Full Text Available Abstract Background One of the most interesting problems in molecular immunology is epitope mapping, i.e. the identification of the regions of interaction between an antigen and an antibody. The solution to this problem, even if approximate, would help in designing experiments to precisely map the residues involved in the interaction and could be instrumental both in designing peptides able to mimic the interacting surface of the antigen and in understanding where immunologically important regions are located in its three-dimensional structure. From an experimental point of view, both genetically encoded and chemically synthesised peptide libraries can be used to identify sequences recognized by a given antibody. The problem then arises of which region of a folded protein the selected peptides correspond to. Results We have developed a method able to find the surface region of a protein that can be effectively mimicked by a peptide, given the structure of the protein and the maximum number of side chains deemed to be required for recognition. The method is implemented as a publicly available server. It can also find and report all peptide sequences of a specified length that can mimic the surface of a given protein and store them in a database. The immediate application of the server is the mapping of antibody epitopes, however the system is sufficiently flexible for allowing other questions to be asked, for example one can compare the peptides representing the surface of two proteins known to interact with the same macromolecule to find which is the most likely interacting region. Conclusion We believe that the MEPS server, available at http://www.caspur.it/meps, will be a useful tool for immunologists and structural and computational biologists. We plan to use it ourselves to implement a database of "surface mimicking peptides" for all proteins of known structure and proteins that can be reliably modelled by comparative modelling.

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

    2010-06-15

    ... or business model, the benefit of this integration and how the approach can expand service capability... this integration and how the approach can expand service capability and capacity of the MEP system. The....S. based manufacturing firms. NIST MEP invites proposals from eligible organizations for projects...

  14. Isotope effect studies of the pyridoxal 5'-phosphate dependent histidine decarboxylase from Morganella morganii

    The pyridoxal 5'-phosphate dependent histidine decarboxylase from Morganella morganii shows a nitrogen isotope effect k14/k15 = 0.9770 +/- 0.0021, a carbon isotope effect k12/k13 = 1.0308 +/- 0.0006, and a carbon isotope effect for L-[α-2H]histidine of 1.0333 +/- 0.0001 at pH 6.3, 370C. These results indicate that the overall decarboxylation rate is limited jointly by the rate of Schiff base interchange and by the rate of decarboxylation. Although the observed isotope effects are quite different from those for the analogous glutamate decarboxylase from Escherichia coli, the intrinsic isotope effects for the two enzymes are essentially the same. The difference in observed isotope effects occurs because of a roughly twofold difference in the partitioning of the pyridoxal 5'-phosphate-substrate Schiff base between decarboxylation and Schiff base interchange. The observed nitrogen isotope effect requires that the imine nitrogen in this Schiff base is protonated. Comparison of carbon isotope effects for deuteriated and undeuteriated substrates reveals that the deuterium isotope effect on the decarboxylation step is about 1.20; thus, in the transition state for the decarboxylation step, the carbon-carbon bond is about two-thirds broken

  15. Preliminary crystallographic analysis of two hypothetical ribose-5-phosphate isomerases from Streptococcus mutans

    Two hypothetical ribose-5-phosphate isomerases from S. mutans have been produced in E. coli and crystallized. The crystals diffracted to high resolutions suitable for crystallographic analyses. Study of the enzymes from sugar metabolic pathways may provide a better understanding of the pathogenesis of the human oral pathogen Streptococcus mutans. Bioinformatics, biochemical and crystallization methods were used to characterize and understand the function of two putative ribose-5-phosphate isomerases: SMU1234 and SMU2142. The proteins were cloned and constructed with N-terminal His tags. Protein purification was performed by Ni2+-chelating and size-exclusion chromatography. The crystals of SUM1234 diffracted to 1.9 Å resolution and belonged to space group P212121, with unit-cell parameters a = 48.97, b = 98.27, c = 101.09 Å, α = β = γ = 90°. The optimized SMU2142 crystals diffracted to 2.7 Å resolution and belonged to space group P1, with unit-cell parameters a = 53.7, b = 54.1, c = 86.5 Å, α = 74.2, β = 73.5, γ = 83.7°. Initial phasing of both proteins was attempted by molecular replacement; the structure of SMU1234 could easily be solved, but no useful results were obtained for SMU2142. Therefore, SeMet-labelled SMU2142 will be prepared for phasing

  16. Evidence for a reactive cysteine at the nucleotide binding site of spinach ribulose-5-phosphate kinase

    Ribulose-5-phosphate kinase from spinach was rapidly inactivated by N-bromoacetylethanolamine phosphate in a bimolecular fashion with a k2 of 2.0 m-1 s-1 at 20C and pH 8.0. Ribulose 5-phosphate had little effect on the rate of inactivation, whereas complete protection was afforded by ADP or ATP. The extent of incorporation as determined with 14C-labeled reagent was about 1 molar equivalent per subunit in the presence of ATP with full retention of enzymatic activity, and about 2 molar equivalents per subunit in the completely inactivated enzyme. Amino acid analyses of enzyme derivatized with 14C-labeled reagent reveal that all of the covalently incorporated reagent was associated with cysteinyl residues. Hence, two sulfhydryls are reactive, but the inactivation correlates with alkylation of one cysteinyl residue at or near the enzyme's nucleotide binding site. The kinase was also extremely sensitive to the sulfhydryl reagents 5,5'-dithiobis(2-nitrobenzoic acid) and N-ethylmaleimide. The reactive sulfhydryl groups are likely to be those generated by reduction of a disulfide during activation. 20 references, 3 figures, 2 tables

  17. Ribose 5-phosphate isomerase inhibits LC3 processing and basal autophagy.

    Heintze, Jacob; Costa, Joana R; Weber, Melanie; Ketteler, Robin

    2016-09-01

    Autophagy and cellular metabolism are tightly linked processes, but how individual metabolic enzymes regulate the process of autophagy is not well understood. This study implicates ribose-5-phosphate isomerase (RPIA), a key regulator of the pentose phosphate pathway, in the control of autophagy. We used a dual gene deletion strategy, combining shRNA-mediated knockdown studies with CRISPR/Cas9 genome editing. Knockdown of RPIA by shRNA or genomic deletion by CRISPR/Cas9 genome editing, results in an increase of ATG4B-mediated LC3 processing and in the appearance of LC3-positive autophagosomes in cells. Increased LC3 processing upon knockdown of RPIA can be reversed by treatment with the antioxidant N-acetyl cysteine. The results are consistent with a model in which RPIA suppresses autophagy and LC3 processing by modulation of redox signaling. PMID:27328773

  18. Binding and uptake of 125iodine-labelled, oxidized low density lipoprotein by macrophages: Comparison of the effects of α-tocopherol, probucol, pyridoxal-5'-phosphate and magnesium-pyridoxal-5'-phosphate-glutamate

    Specific and unspecific binding and uptake (internalization) by macrophages of 125iodine-labelled, copper-oxidized human low density lipoprotein is differently influenced by the anti-oxidants α-tocopherol (α-Toc), probucol (Prob), pyridoxal-5'-phosphate (PP) and the magnesium-pyridoxal-5'-phosphate glutamate complex (MPPG). Binding as well as internalization, mediated by the so-called 'scavenger receptor' is lower in the presence of MPPG whereas both specific binding and internalization are enhanced. The comparison of the effects in vitro allows a rating of the potentially anti-atherogenic and thus protective effects of the tested substances as follows: MPPG>PP>α-Toc>Prob. (orig.)

  19. Binding and uptake of 125iodine-labelled, oxidized low density lipoprotein by macrophages: comparison of the effects of alpha-tocopherol, probucol, pyridoxal-5'-phosphate and magnesium-pyridoxal-5'-phosphate-glutamate.

    Selmer, D; Senekowitsch-Schmidtke, R; Schneider, W; Elstner, E F

    1997-01-01

    Specific and unspecific binding and uptake (internalization) by macrophages of 125iodine-labelled, copper-oxidized human low density lipoprotein is differently influenced by the anti-oxidants alpha-tocopherol (alpha-Toc), probucol (Prob), pyridoxal-5'-phosphate (PP) and the magnesium-pyridoxal-5'-phosphate glutamate complex (MPPG). Binding as well as internalization, mediated by the so-called "scavenger receptor" is lower in the presence of MPPG whereas both specific binding and internalization are enhanced. The comparison of the effects in vitro allows a rating of the potentially anti-atherogenic and thus protective effects of the tested substances as follows: MPPG > PP > alpha-Toc > Prob. PMID:9090072

  20. Binding and uptake of {sup 125}iodine-labelled, oxidized low density lipoprotein by macrophages: Comparison of the effects of {alpha}-tocopherol, probucol, pyridoxal-5`-phosphate and magnesium-pyridoxal-5`-phosphate-glutamate

    Selmer, D. [Technische Univ. Muenchen, Freising-Weihenstephan (Germany). Lehrstuhl fuer Phytopathologie; Senekowitsch-Schmidtke, R. [Technische Univ. Muenchen (Germany). Nuklearmedizinische Klinik; Schneider, W. [Steigerwald Arzneimittel, Darmstadt (Germany); Elstner, E.F. [Technische Univ. Muenchen, Freising-Weihenstephan (Germany). Lehrstuhl fuer Phytopathologie

    1997-01-01

    Specific and unspecific binding and uptake (internalization) by macrophages of {sup 125}iodine-labelled, copper-oxidized human low density lipoprotein is differently influenced by the anti-oxidants {alpha}-tocopherol ({alpha}-Toc), probucol (Prob), pyridoxal-5`-phosphate (PP) and the magnesium-pyridoxal-5`-phosphate glutamate complex (MPPG). Binding as well as internalization, mediated by the so-called `scavenger receptor` is lower in the presence of MPPG whereas both specific binding and internalization are enhanced. The comparison of the effects in vitro allows a rating of the potentially anti-atherogenic and thus protective effects of the tested substances as follows: MPPG>PP>{alpha}-Toc>Prob. (orig.)

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

    2013-04-09

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

  2. 77 FR 12563 - Manufacturing Extension Partnership (MEP) Centers for South Dakota and Kentucky; Availability of...

    2012-03-01

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

  3. 77 FR 23462 - Proposed Information Collection; Comment Request; Manufacturing Extension Partnership (MEP...

    2012-04-19

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

  4. APPLICATION OF NEW TECHNOLOGY: MEPS AND LC-MS/MS FOR DETERMINATION OF THERAPEUTIC DRUGS

    Said, Rana

    2010-01-01

    Bioanalysis most often requires an extraction procedure to isolate the target compounds from a complex matrix. The aim of this work was to evaluate microextraction in packed syringe (MEPS) performance as a sample preparation technique by developing new analytical methods for different categories of compounds utilizing MEPS in combination with LC-MS/MS. The overall goal was to provide high throughput methods that can offer automation, on-line coupling to mass spectrometry and...

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

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

    2016-04-01

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

  6. Inhibitor design for ribonuclease A: the binding of two 5'-phosphate uridine analogues.

    Tsirkone, Vicky G; Dossi, Kyriaki; Drakou, Christina; Zographos, Spyros E; Kontou, Maria; Leonidas, Demetres D

    2009-07-01

    In the quest for the rational design of selective and potent inhibitors for members of the pancreatic ribonuclease A (RNase A) family of biomedical interest, the binding of uridine 5'-phosphate (U5P) and uridine 5'-diphosphate (UDP) to RNase A have been investigated using kinetic studies and X-ray crystallography. Both nucleotides are competitive inhibitors of the enzyme, with K(i) values of 4.0 and 0.65 mM, respectively. They bind to the active site of the enzyme by anchoring two molecules connected to each other by hydrogen bonds and van der Waals interactions. While the first of the inhibitor molecules binds with its nucleobase in the pyrimidinyl-binding subsite, the second is bound at the purine-preferring subsite. The unexpected binding of a pyrimidine at the purine-binding subsite has added new important elements to the rational design approach for the discovery of new potent inhibitors of the RNase A superfamily. PMID:19574636

  7. Structural insight for substrate tolerance to 2-deoxyribose-5-phosphate aldolase from the pathogen Streptococcus suis.

    Cao, Thinh-Phat; Kim, Joong-Su; Woo, Mi-Hee; Choi, Jin Myung; Jun, Youngsoo; Lee, Kun Ho; Lee, Sung Haeng

    2016-04-01

    2-deoxyribose-5-phosphate aldolase (DERA) is a class I aldolase that catalyzes aldol condensation of two aldehydes in the active site, which is particularly germane in drug manufacture. Structural and biochemical studies have shown that the active site of DERA is typically loosely packed and displays broader substrate specificity despite sharing conserved folding architecture with other aldolases. The most distinctive structural feature of DERA compared to other aldolases is short and flexible C-terminal region. This region is also responsible for substrate recognition. Therefore, substrate tolerance may be related to the C-terminal structural features of DERA. Here, we determined the crystal structures of full length and C-terminal truncated DERA from Streptococcus suis (SsDERA). In common, both contained the typical (α/β)8 TIM-barrel fold of class I aldolases. Surprisingly, C-terminal truncation resulting in missing the last α9 and β8 secondary elements, allowed DERA to maintain activity comparable to the fulllength enzyme. Specifically, Arg186 and Ser205 residues at the C-terminus appeared mutually supplemental or less indispensible for substrate phosphate moiety recognition. Our results suggest that DERA might adopt a shorter C-terminal region than conventional aldolases during evolution pathway, resulting in a broader range of substrate tolerance through active site flexibility. PMID:27033207

  8. Magnesium pyridoxal 5-phosphate glutamate reduces hyperlipidaemia in patients with chronic renal insufficiency.

    Kirsten, R; Heintz, B; Nelson, K; Sieberth, H G; Oremek, G; Hasford, J; Speck, U

    1988-01-01

    Chronic renal insufficiency is often accompanied by hyperlipidaemia and subsequent coronary heart disease. Two groups of 15 patients with serum creatinine greater than 2 mg/100 ml and serum cholesterol less than 250 mg/100 ml were given 3 x 50 mg magnesium pyridoxal 5-phosphate glutamate (MPPG) or placebo for 12 weeks in a double-blind, randomised study. Total cholesterol in the MPPG group (282.4 mg.100 ml-1) was lower than in the placebo group (354.3 mg.100 ml-1) after 12 weeks of treatment. Triglycerides in the MPPG group were 265.1 mg.100 ml-1 compared to 361.9 mg.100 ml-1. After 12 weeks on MPPG the LDL/HDL ratio of 3.56 was lower than in the placebo group-6.83. Side effects in the MPPG group were similar to those in the placebo group. Thus, MPPG was an effective antihyperlipidaemic agent in patients with renal insufficiency. PMID:3383985

  9. Initiation sites for human DNA replication at a putative ribulose-5-phosphate 3-epimerase gene

    Replication of the human genome requires the activation of thousands of replicons distributed along each one of the chromosomes. Each replicon contains an initiation, or origin, site, at which DNA synthesis begins. However, very little information is known about the nature and positioning of these initiation sites along human chromosomes. We have recently focused our attention to a 1.1 kb region of human chromosome 2 which functioned as an episomal origin in the yeast Saccharomyces cerevisiae. This region corresponded to the largest exon of a putative ribulose-5-phosphate-3-epimerase gene (RPE). In the present study we have used a real-time PCR-based nascent strand DNA abundance assay to map initiation sites for DNA replication in in vivo human chromosomes around a 13.4 kb region encompassing the putative RPE gene. By applying this analysis to a 1-1.4 kb nascent strand DNA fraction isolated from both normal skin fibroblasts, and the breast cell line MCF10; we have identified five initiation sites within the 13.4 kb region of chromosome 2. The initiation sites appear to map to similar positions in both cell lines and occur outside the coding regions of the putative RPE gene

  10. Radiometric measurement of phosphoribosylpyrophosphate and ribose 5-phosphate by enzymatic procedures

    Methods for the measurement of phosphoribosylpyrophosphate (PRPP) and ribose 5-phosphate (R-5-P) in tissues have been developed. The lability of these compounds during tissue extraction and the recovery of standards from tissue preparations have been examined. Enzymatic conversion of phosphoribosylpyrophosphate to [14C]AMP in the presence of labeled adenine or formation of [14C]GMP ([14C]IMP) in the presence of labeled guanine or hypoxanthine was accomplished in the first step. In the second step, the labeled product was separated from the substrate. For the measurement of R-5-P, the first step included phosphoribosylpyrophosphate synthetase, as well as the appropriate substrate and effector (ATP and Pi), in combination with adenine phosphoribosyl transferase. The product [14C]AMP was measured in three ways: (1) HPLC separation with an on-line radioisotope detector; (2) butanol extraction of the labeled base, and measurement of an aliquot of the aqueous phase in a scintillation counter; (3) filtration of the incubation mixture with chromatographic filter paper disks, which were then counted in a scintillation counter. When [14C]guanine was the substrate, HPLC separation was used because the butanol or paper separation was not adequate. Measurement of 5-125 pmol of PRPP or R-5-P gave a linear response

  11. Pyridoxal 5'-phosphate is a slow tight binding inhibitor of E. coli pyridoxal kinase.

    Mohini S Ghatge

    Full Text Available Pyridoxal 5'-phosphate (PLP is a cofactor for dozens of B(6 requiring enzymes. PLP reacts with apo-B(6 enzymes by forming an aldimine linkage with the ε-amino group of an active site lysine residue, thus yielding the catalytically active holo-B(6 enzyme. During protein turnover, the PLP is salvaged by first converting it to pyridoxal by a phosphatase and then back to PLP by pyridoxal kinase. Nonetheless, PLP poses a potential toxicity problem for the cell since its reactive 4'-aldehyde moiety forms covalent adducts with other compounds and non-B(6 proteins containing thiol or amino groups. The regulation of PLP homeostasis in the cell is thus an important, yet unresolved issue. In this report, using site-directed mutagenesis, kinetic, spectroscopic and chromatographic studies we show that pyridoxal kinase from E. coli forms a complex with the product PLP to form an inactive enzyme complex. Evidence is presented that, in the inhibited complex, PLP has formed an aldimine bond with an active site lysine residue during catalytic turnover. The rate of dissociation of PLP from the complex is very slow, being only partially released after a 2-hour incubation with PLP phosphatase. Interestingly, the inactive pyridoxal kinase•PLP complex can be partially reactivated by transferring the tightly bound PLP to an apo-B(6 enzyme. These results open new perspectives on the mechanism of regulation and role of pyridoxal kinase in the Escherichia coli cell.

  12. Effect of magnesium pyridoxal 5-phosphate glutamate on vascular reactivity in experimental hypercholesterolemia.

    Khayyal, M T; Khayyal, M A; Sharaf, H M; el-Sherbeeny, M; Okpanyi, S N; Schneider, W

    1998-01-01

    Hypercholesterolemia is known to affect the responsiveness of various blood vessels to endogenous and to exogenous vasoactive agents. Of particular interest is the increased responsiveness to vasoconstrictors, e.g., 5-hydroxy tryptamine and noradrenaline, and the decreased reactivity towards vasodilators, e.g., acetylcholine. This, together with the development of arteriosclerosis, could play an important role in the progression of many vascular complications, such as hypertension and coronary heart disease. Magnesium pyridoxal 5-phosphate glutamate (MPPG) has been shown to effectively reduce serum lipids in animals and in man, and to retard the progression of atherosclerotic lesions in experimental animals. It was therefore considered of interest to investigate the reactivity of both the aorta and the renal artery to different vasoactive substances in hypercholesterolemic rabbits under the influence of MPPG as well as the effect of such substances on the blood pressure of the anesthetized animals. The rabbits were fed a high cholesterol diet for 2 months, followed by MPPG for 1 month, while keeping the rabbits on the same diet. One batch of animals was used for blood pressure recording and testing drug effects, and another was used for testing the responsiveness of their aortae and renal arteries to the different mediators. In hypercholesterolemic rabbits, treatment with MPPG tended to normalize the increased responsiveness of the blood pressure to the vasoconstrictors: noradrenaline and angiotensin and the diminished sensitivity to histamine and acetylcholine. For the isolated arteries, however, MPPG did not significantly affect the responses to noradrenaline nor potassium chloride, but tended to normalize responses to clonidine and acetylcholine. It could be concluded from the present findings that the high cholesterol diet induces changes in vascular reactivity which are possibly related to endothelial and/or receptor sensitivity changes. Treatment with MPPG

  13. On the mechanism of Escherichia coli pyridoxal kinase inhibition by pyridoxal and pyridoxal 5'-phosphate.

    di Salvo, Martino Luigi; Nogués, Isabel; Parroni, Alessia; Tramonti, Angela; Milano, Teresa; Pascarella, Stefano; Contestabile, Roberto

    2015-09-01

    Pyridoxal 5'-phosphate (PLP), the catalytically active form of vitamin B6, plays a crucial role in several cellular processes. In most organisms, PLP is recycled from nutrients and degraded B6-enzymes in a salvage pathway that involves pyridoxal kinase (PLK), pyridoxine phosphate oxidase and phosphatase activities. Regulation of the salvage pathway is poorly understood. Escherichia coli possesses two distinct pyridoxal kinases, PLK1, which is the focus of the present work, and PLK2. From previous studies dating back to thirty years ago, pyridoxal (PL) was shown to inhibit E. coli PLK1 forming a covalent link with the enzyme. This inhibition was proposed to play a regulative role in vitamin B6 metabolism, although its details had never been clarified. Recently, we have shown that also PLP produced during PLK1 catalytic cycle acts as an inhibitor, forming a Schiff base with Lys229, without being released in the solvent. The question arises as to which is the actual inhibition mechanism by PL and PLP. In the present work, we demonstrated that also PL binds to Lys229 as a Schiff base. However, the isolated covalent PLK1-PL complex is not inactive but, in the presence of ATP, is able to catalyse the single turnover production of PLP, which binds tightly to the enzyme and is ultimately responsible for its inactivation. The inactivation mechanism mediated by Lys229 may play a physiological role in controlling cellular levels of PLP. This article is part of a Special Issue entitled: Cofactor-dependent proteins: evolution, chemical diversity and bio-applications. PMID:25655354

  14. Why Do MEPs Defect? An Analysis of Party Group Cohesion in the 5 th European Parliament

    Thorsten Faas

    2002-03-01

    Full Text Available This study analyses party group cohesion and patterns of defections of national party delegations from party group lines in the present European Parliament, using a total of 1,370 roll call votes. The study confirms previous findings according to which party groups in the EP show (surprisingly high levels of cohesion. In addition and notwithstanding that, it reveals the circumstances under which MEPs and their national delegations are more likely to defect. Among other factors, it was analysed how the nature of the candidate selection process, the electoral system, and the relationships between MEPs and their home parties influence these defections. Assuming that MEPs have three different goals (re-election, office, and policy and want to first of all secure re-election, one can theoretically expect that those MEPs whose chances of re-election are more dependent on national parties than others’ (due to their specific candidate selection process or their relationship to their home party are more willing to vote against the party group line, if a conflict between party group and national party emerges. Empirically, this is confirmed. In other words, MEPs in general are very well aware of their specific situation. They know who deserves their primary attention and they act accordingly.

  15. Overexpression, crystallization and preliminary X-ray analysis of xylulose-5-phosphate/fructose-6-phosphate phosphoketolase from Bifidobacterium breve

    Xylulose-5-phosphate/fructose-6-phosphate phosphoketolase from B. breve was overexpressed and crystallized. The crystals belonged to the tetragonal space group I422 and diffracted to beyond 1.7 Å resolution. The xylulose-5-phosphate/fructose-6-phosphate phosphoketolase gene from Bifidobacterium breve was cloned and overexpressed in Escherichia coli. The enzyme was purified to homogeneity and crystallized by the sitting-drop vapour-diffusion method. Crystals were obtained at 293 K using 0.05 mM thiamine diphosphate, 0.25 mM MgCl2, 24%(w/v) PEG 6000 and 0.1 M Bicine pH 9.0. The crystals belonged to the tetragonal space group I422, with unit-cell parameters a = b = 174.8, c = 163.8 Å, and diffracted to beyond 1.7 Å resolution

  16. Ribose-5-Phosphate Isomerase Deficiency: New Inborn Error in the Pentose Phosphate Pathway Associated with a Slowly Progressive Leukoencephalopathy

    Huck, Jojanneke H. J.; Verhoeven, Nanda M; Struys, Eduard A.; Salomons, Gajja S; Jakobs, Cornelis; van der Knaap, Marjo S.

    2004-01-01

    The present article describes the first patient with a deficiency of ribose-5-phosphate isomerase (RPI) (Enzyme Commission number 5.3.1.6) who presented with leukoencephalopathy and peripheral neuropathy. Proton magnetic resonance spectroscopy of the brain revealed highly elevated levels of the polyols ribitol and D-arabitol, which were subsequently also found in high concentrations in body fluids. Deficient activity of RPI, one of the pentose-phosphate-pathway (PPP) enzymes, was demonstrated...

  17. Review of the report 'MEP-compensation for onshore wind energy'

    The Energy research Centre of the Netherlands (ECN) studied the so-called MEP-compensation (MEP is the abbreviation for Environmental Quality of Electricity Production) for wind energy, and in particular the possible undesired side-effects of the applied full-load hours system. The Dutch Ministry of Economic Affairs requested PricewaterhouseCoopers (PwC) to assess the results of the ECN-report. The assessment was carried out in cooperation with Ecofys, which concentrated on the technical aspects and practical experiences

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

    2010-07-01

    ... determining eligibility and in conducting quality control procedures know the requirements for accurately... documentation; and Quality control. 200.89 Section 200.89 Education Regulations of the Offices of the Department...-interviewing; Eligibility documentation; and Quality control. (a) Allocation of funds under the MEP for...

  19. Markedly increased circulating pyridoxal-5'-phosphate levels in hypophosphatasia. Alkaline phosphatase acts in vitamin B6 metabolism.

    Whyte, M P; Mahuren, J D; Vrabel, L A; Coburn, S P

    1985-01-01

    Markedly increased circulating concentrations of pyridoxal-5'-phosphate (PLP) were found in each of 14 patients representing all clinical forms of hypophosphatasia, an inborn error characterized by deficient activity of the tissue nonspecific (bone/liver/kidney) isoenzyme of alkaline phosphatase (AP). The mean PLP concentration in plasma was 1174 nM (range, 214-3839 nM) in the patients and 57 +/- 26 nM (mean +/- SD) in 38 control subjects. In four affected children, urinary excretion of the P...

  20. Formation of xylitol and xylitol-5-phosphate and its impact on growth of d-xylose-utilizing Corynebacterium glutamicum strains.

    Radek, Andreas; Müller, Moritz-Fabian; Gätgens, Jochem; Eggeling, Lothar; Krumbach, Karin; Marienhagen, Jan; Noack, Stephan

    2016-08-10

    Wild-type Corynebacterium glutamicum has no endogenous metabolic activity for utilizing the lignocellulosic pentose d-xylose for cell growth. Therefore, two different engineering approaches have been pursued resulting in platform strains harbouring a functional version of either the Isomerase (ISO) or the Weimberg (WMB) pathway for d-xylose assimilation. In a previous study we found for C. glutamicum WMB by-product formation of xylitol during growth on d-xylose and speculated that the observed lower growth rates are due to the growth inhibiting effect of this compound. Based on a detailed phenotyping of the ISO, WMB and the wild-type strain of C. glutamicum, we here show that this organism has a natural capability to synthesize xylitol from d-xylose under aerobic cultivation conditions. We furthermore observed the intracellular accumulation of xylitol-5-phosphate as a result of the intracellular phosphorylation of xylitol, which was particularly pronounced in the C. glutamicum ISO strain. Interestingly, low amounts of supplemented xylitol strongly inhibit growth of this strain on d-xylose, d-glucose and d-arabitol. These findings demonstrate that xylitol is a suitable substrate of the endogenous xylulokinase (XK, encoded by xylB) and its overexpression in the ISO strain leads to a significant phosphorylation of xylitol in C. glutamicum. Therefore, in order to circumvent cytotoxicity by xylitol-5-phosphate, the WMB pathway represents an interesting alternative route for engineering C. glutamicum towards efficient d-xylose utilization. PMID:27297548

  1. Preliminary X-ray crystallographic analysis of the d-xylulose 5-phosphate phosphoketolase from Lactococcus lactis

    The expression, purification, preliminary crystallization and crystallographic analysis of phosphoketolase from L. lactis ssp. lactis (strain IL 1403) are reported. Phosphoketolases are thiamine diphosphate-dependent enzymes which play a central role in the pentose-phosphate pathway of heterofermentative lactic acid bacteria. They belong to the family of aldehyde-lyases and in the presence of phosphate ion cleave the carbon–carbon bond of the specific substrate d-xylulose 5-phosphate (or d-fructose 6-phosphate) to give acetyl phosphate and d-glyceraldehyde 3-phosphate (or d-erythrose 4-phosphate). Structural information about phosphoketolases is particularly important in order to fully understand their mechanism as well as the steric course of phosphoketolase-catalyzed reactions. Here, the purification, preliminary crystallization and crystallographic characterization of d-xylulose 5-phosphate phosphoketolase from Lactococcus lactis are reported. The presence of thiamine diphosphate during purification was essential for the enzymatic activity of the purified protein. The crystals belonged to the monoclinic space group P21. Diffraction data were obtained to a resolution of 2.2 Å

  2. Evaluating the Long-term Effect of NIST MEP Services on Establishment Performance

    Clifford A Lipscomb, Jan Youtie, Sanjay Arora, Andy Krause and Philip Shapira

    2015-01-01

    This work examines the effects of receipt of business assistance services from the Manufacturing Extension Partnership (MEP) on manufacturing establishment performance. Several measures of performance are considered: (1) change in value-added per employee (a measure of productivity); (2) change in sales per worker; (3) change in employment; and (4) establishment survival. To analyze these relationships, we merged program records from the MEP’s client and project information files with adminis...

  3. Online political communication strategies: MEPs e-representation and self-representation

    Lilleker, Darren; Koc-Michalska, Karolina

    2013-01-01

    Research into the communication strategies of legislators has a long history. The European Parliament offers an opportunity to add to understanding of how legislators prioritise styles of communication, with a comparative perspective across twenty-seven nations. Through content analysis of online communication we investigate how the Internet is used by MEPs. Our analysis assesses three communication strategies: homestyle, impression management and participatory. We find that a homestyle strat...

  4. Why Do MEPs Defect? An Analysis of Party Group Cohesion in the 5 th European Parliament

    Thorsten Faas

    2002-01-01

    This study analyses party group cohesion and patterns of defections of national party delegations from party group lines in the present European Parliament, using a total of 1,370 roll call votes. The study confirms previous findings according to which party groups in the EP show (surprisingly) high levels of cohesion. In addition and notwithstanding that, it reveals the circumstances under which MEPs and their national delegations are more likely to defect. Among other factors, it was analys...

  5. Crosstalk between MAV and MEP pathways in vitro grape plants exposed to UV-B radiation

    The synthesis of terpenoids from IPP (isopentenyl diphosphate) proceeds in plants throughout two pathways, the MVA (mevalonic acid) in cytosol and the MEP (2-C-methyl-D-erythritol 4-phosphate) in plastids. Ultraviolet-B (UV-B) radiation induced the synthesis of terpenes in in vitro grape plants according to the fluence rate. Low intensity UV-B promoted the MVA pathway while high intensity UV-B stimulated the MEP pathway. Mevastatin is known to inhibit the enzyme HMG-CoA reductase blocking terpene synthesis in cytosol. In vitro plants growing 45 days under 16 h-photoperiod (100 μmol m-2 s-1) were fed at the apex with mevastatin and then exposed to an UV-B dose administrated at two intensities: low UV-B (8.25 μW cm-2,16 h) or high UV-B (33 μW cm-2,4 h). Methanol: chloroform extracts were analyzed by GC-EIMS and compared with controls without mevastatin. Levels of γ-Sitosterol and Stigmasterol were significantly increased under low intensity UV-B in the controls. The plants treated with the inhibitor showed a significant decrease of both sterols and a decrease in the plastidial terpenes but sterols were higher under UV-B. These results suggest an IPP crosstalk between the MAV and MEP pathways under restrictive conditions. (authors)

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

    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. The Integration of MEPs from Central and Eastern Europe into the European Parliament

    Radko Hokovský

    2012-04-01

    Full Text Available This article evaluates the level of integration of Members of the European Parliament from Central and Eastern Europe in the European Parliament after the EU enlargements of 2004 and 2007. The main objective is to address the puzzle of how the European Parliament’s political groups could maintain or even increase their voting cohesion after the influx of a significantly large number of new MEPs coming from countries with different historical experience, socio-economic characteristics, and political and party systems. Three indicators of MEP integration are defined: integration into parliamentary leadership, integration into parliamentary work, and integration into voting patterns. The article uses data from the VoteWatch.eu website on MEPs’ activities and voting between the years 2004-2011, as well as data from official documents of the European Parliament and its political groups. Analysis of the data reveals that the new member states’ MEPs were significantly under-represented in parliamentary leadership and key legislative activities, despite the fact that their voting loyalty to their political groups was greater than that of their colleagues from older member states.

  8. Ribose-5-Phosphate Isomerase Deficiency: New Inborn Error in the Pentose Phosphate Pathway Associated with a Slowly Progressive Leukoencephalopathy

    Huck, Jojanneke H. J.; Verhoeven, Nanda M.; Struys, Eduard A.; Salomons, Gajja S.; Jakobs, Cornelis; van der Knaap, Marjo S.

    2004-01-01

    The present article describes the first patient with a deficiency of ribose-5-phosphate isomerase (RPI) (Enzyme Commission number 5.3.1.6) who presented with leukoencephalopathy and peripheral neuropathy. Proton magnetic resonance spectroscopy of the brain revealed highly elevated levels of the polyols ribitol and D-arabitol, which were subsequently also found in high concentrations in body fluids. Deficient activity of RPI, one of the pentose-phosphate-pathway (PPP) enzymes, was demonstrated in fibroblasts. RPI gene–sequence analysis revealed a frameshift and a missense mutation. Recently, we described a patient with liver cirrhosis and abnormal polyol levels in body fluids, related to a deficiency of transaldolase, another enzyme in the PPP. RPI is the second known inborn error in the reversible phase of the PPP, confirming that defects in pentose and polyol metabolism constitute a new area of inborn metabolic disorders. PMID:14988808

  9. Mechanism of the inhibition of mutagenicity of a benzo[a]pyrene 7,8-diol 9,10-epoxide by riboflavin 5'-phosphate.

    Wood, A.W.; Sayer, J M; Newmark, H L; Yagi, H.; Michaud, D P; Jerina, D M; Conney, A H

    1982-01-01

    Riboflavin 5'-phosphate (flavin mononucleotide; FMN) inhibits the mutagenicity of (+/-)-7 beta, 8 alpha-dihydroxy-9 alpha, 10 alpha-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene (B[a]P diol epoxide), the only known ultimate carcinogenic metabolite of benzo[a]pyrene. Coincubation of 10, 25, and 50 nmol of FMN with strain TA100 of histidine-dependent Salmonella typhimurium inhibits the mutagenicity of 0.05 nmol of the diol epoxide by 50, 70, and 90%, respectively. Ribose 5-phosphate and riboflavin sh...

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

    Gan Huang

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

  11. Radiation damage to nucleosides and nucleotides. I. An ESR study of uridine-5'-phosphate.2Na+ single crystals

    Single crystals of the disodium salt of uridine-5'-phosphate were irradiated by 4.0-MeV electrons and studied by electron spin resonance spectroscopy in the temperature range 77 to 310 K. At 77 K two resonances were observed. The dominating resonance (I) is ascribed to the electron adduct (anion) radical of the uracil base. The main hyperfine interaction observed is a coupling between the unpaired electron and the proton bonded to C6. The other resonance (II) exhibits a large g-value anisotropy and two almost isotropic doublet couplings. This resonance is probably due to a secondary alkoxy-type radical formed by a net loss of hydrogen from O3' in the sugar moiety. At 270 K three resonances were observed. One (III) is due to a radical formed by hydrogen abstraction from C/sub 5'/ in the sugar moiety, whereas the other two (IV,V) are probably hydrogen addition radicals at the uracil base. Radical IV is the dihydrouracil-5-yl radical formed by a net hydrogen addition to the C6 position, whereas radical V tentatively is ascribed to the 6-yl radical. The light-induced conversion V → IV took place at 270 K, whereas the reverse heat-induced process was not observed in the temperature range studied. Preliminary annealing studies of crystals irradiated at 77 K suggest that the radical reaction I → IV takes place at approximately 130 K and the reaction II → III at 150 K

  12. Large-Scale Domain Motions and Pyridoxal-5'-Phosphate Assisted Radical Catalysis in Coenzyme B12-Dependent Aminomutases

    Amarendra Nath Maity

    2014-02-01

    Full Text Available Lysine 5,6-aminomutase (5,6-LAM and ornithine 4,5-aminomutase (4,5-OAM are two of the rare enzymes that use assistance of two vitamins as cofactors. These enzymes employ radical generating capability of coenzyme B12 (5'-deoxyadenosylcobalamin, dAdoCbl and ability of pyridoxal-5'-phosphate (PLP, vitamin B6 to stabilize high-energy intermediates for performing challenging 1,2-amino rearrangements between adjacent carbons. A large-scale domain movement is required for interconversion between the catalytically inactive open form and the catalytically active closed form. In spite of all the similarities, these enzymes differ in substrate specificities. 4,5-OAM is highly specific for D-ornithine as a substrate while 5,6-LAM can accept D-lysine and L-β-lysine. This review focuses on recent computational, spectroscopic and structural studies of these enzymes and their implications on the related enzymes. Additionally, we also discuss the potential biosynthetic application of 5,6-LAM.

  13. Combinatorial engineering of 1-deoxy-D-xylulose 5-phosphate pathway using cross-lapping in vitro assembly (CLIVA method.

    Ruiyang Zou

    Full Text Available The ability to assemble multiple fragments of DNA into a plasmid in a single step is invaluable to studies in metabolic engineering and synthetic biology. Using phosphorothioate chemistry for high efficiency and site specific cleavage of sequences, a novel ligase independent cloning method (cross-lapping in vitro assembly, CLIVA was systematically and rationally optimized in E. coli. A series of 16 constructs combinatorially expressing genes encoding enzymes in the 1-deoxy-D-xylulose 5-phosphate (DXP pathway were assembled using multiple DNA modules. A plasmid (21.6 kb containing 16 pathway genes, was successfully assembled from 7 modules with high efficiency (2.0 x 10(3 cfu/ µg input DNA within 2 days. Overexpressions of these constructs revealed the unanticipated inhibitory effects of certain combinations of genes on the production of amorphadiene. Interestingly, the inhibitory effects were correlated to the increase in the accumulation of intracellular methylerythritol cyclodiphosphate (MEC, an intermediate metabolite in the DXP pathway. The overexpression of the iron sulfur cluster operon was found to modestly increase the production of amorphadiene. This study demonstrated the utility of CLIVA in the assembly of multiple fragments of DNA into a plasmid which enabled the rapid exploration of biological pathways.

  14. Biosynthesis of ribose-5-phosphate and erythrose-4-phosphate in archaea: a phylogenetic analysis of archaeal genomes

    Tim Soderberg

    2005-01-01

    Full Text Available A phylogenetic analysis of the genes encoding enzymes in the pentose phosphate pathway (PPP, the ribulose monophosphate (RuMP pathway, and the chorismate pathway of aromatic amino acid biosynthesis, employing data from 13 complete archaeal genomes, provides a potential explanation for the enigmatic phylogenetic patterns of the PPP genes in archaea. Genomic and biochemical evidence suggests that three archaeal species (Methanocaldococcus jannaschii, Thermoplasma acidophilum and Thermoplasma volcanium produce ribose-5-phosphate via the nonoxidative PPP (NOPPP, whereas nine species apparently lack an NOPPP but may employ a reverse RuMP pathway for pentose synthesis. One species (Halobacterium sp. NRC-1 lacks both the NOPPP and the RuMP pathway but may possess a modified oxidative PPP (OPPP, the details of which are not yet known. The presence of transketolase in several archaeal species that are missing the other two NOPPP genes can be explained by the existence of differing requirements for erythrose-4-phosphate (E4P among archaea: six species use transketolase to make E4P as a precursor to aromatic amino acids, six species apparently have an alternate biosynthetic pathway and may not require the ability to make E4P, and one species (Pyrococcus horikoshii probably does not synthesize aromatic amino acids at all.

  15. Thermophilic Thermotoga maritima ribose-5-phosphate isomerase RpiB: optimized heat treatment purification and basic characterization.

    Sun, Fangfang; Zhang, Xiao-Zhou; Myung, Suwan; Zhang, Y-H Percival

    2012-04-01

    The open reading frame TM1080 from Thermotoga maritima encoding ribose-5-phosphate isomerase type B (RpiB) was cloned and over-expressed in Escherichia coli BL21 (DE3). After optimization of cell culture conditions, more than 30% of intracellular proteins were soluble recombinant RpiB. High-purity RpiB was obtained by heat pretreatment through its optimization in buffer choice, buffer pH, as well as temperature and duration of pretreatment. This enzyme had the maximum activity at 70°C and pH 6.5-8.0. Under its suboptimal conditions (60°C and pH 7.0), k(cat) and K(m) values were 540s(-1) and 7.6mM, respectively; it had a half lifetime of 71h, resulting in its turn-over number of more than 2×10(8)mol of product per mol of enzyme. This study suggests that it is highly feasible to discover thermostable enzymes from exploding genomic DNA database of extremophiles with the desired stability suitable for in vitro synthetic biology projects and produce high-purity thermoenzymes at very low costs. PMID:22333529

  16. Orotate phosphoribosyl transferase MoPyr5 is involved in uridine 5'-phosphate synthesis and pathogenesis of Magnaporthe oryzae.

    Qi, Zhongqiang; Liu, Muxing; Dong, Yanhan; Yang, Jie; Zhang, Haifeng; Zheng, Xiaobo; Zhang, Zhengguang

    2016-04-01

    Orotate phosphoribosyl transferase (OPRTase) plays an important role in de novo and salvage pathways of nucleotide synthesis and is widely used as a screening marker in genetic transformation. However, the function of OPRTase in plant pathogens remains unclear. In this study, we characterized an ortholog of Saccharomyces cerevisiae Ura5, the OPRTase MoPyr5, from the rice blast fungus Magnaporthe oryzae. Targeted gene disruption revealed that MoPyr5 is required for mycelial growth, appressorial turgor pressure and penetration into plant tissues, invasive hyphal growth, and pathogenicity. Interestingly, the ∆Mopyr5 mutant is also involved in mycelial surface hydrophobicity. Exogenous uridine 5'-phosphate (UMP) restored vegetative growth and rescued the defect in pathogenicity on detached barley and rice leaf sheath. Collectively, our results show that MoPyr5 is an OPRTase for UMP biosynthesis in M. oryzae and indicate that UTP biosynthesis is closely linked with vegetative growth, cell wall integrity, and pathogenicity of fungus. Our results also suggest that UMP biosynthesis would be a good target for the development of novel fungicides against M. oryzae. PMID:26810198

  17. Studies on the increase of capillary permeability in rat skin under the action of pyridoxal 5' phosphate

    The activity of pyridoxal 5'-phosphate (PLP) is described on the vascular permeability response, measured in the abdominal wall of rats from the amount of extravased Evans blue labelled with radioactive iodine 125 or 131. The PLP effect is related to histamine release as it has been showed by tha use of antihistaminics. An attempt has been made in order to correlate structure and biological activity by using PLP analogs. The intact molecule of PLP seems to be the proper active substance. The critical role of calcium in histamine release is discussed in relation to our observations. In the presence of high concentrations of calcium and lantanium, PLP fails to increase the vascular permeability; magnesium does not show any influence. The calcium mobilization produced by theophylline results in inhibition of the response. The course of the reaction between PLP and histamine in vitro was followed; the synthetic cyclic product is deprived of activity and does not interfere with the intrinsic effects of PLP and histamine. (Author)

  18. Crystal Structure of 1-Deoxy-D-xylulose 5-Phosphate Synthase, A Crucial Enzyme for Isoprenoids Biosynthesis

    Xiang,S.; Usunow, G.; Busch, G.; Tong, L.

    2007-01-01

    Isopentenyl pyrophosphate (IPP) is a common precursor for the synthesis of all isoprenoids, which have important functions in living organisms. IPP is produced by the mevalonate pathway in archaea, fungi, and animals. In contrast, IPP is synthesized by a mevalonate-independent pathway in most bacteria, algae, and plant plastids. 1-Deoxy-D-xylulose 5-phosphate synthase (DXS) catalyzes the first and the rate-limiting step of the mevalonate-independent pathway and is an attractive target for the development of novel antibiotics, antimalarials, and herbicides. We report here the first structural information on DXS, from Escherichia coli and Deinococcus radiodurans, in complex with the coenzyme thiamine pyrophosphate (TPP). The structure contains three domains (I, II, and III), each of which bears homology to the equivalent domains in transketolase and the E1 subunit of pyruvate dehydrogenase. However, DXS has a novel arrangement of these domains as compared with the other enzymes, such that the active site of DXS is located at the interface of domains I and II in the same monomer, whereas that of transketolase is located at the interface of the dimer. The coenzyme TPP is mostly buried in the complex, but the C-2 atom of its thiazolium ring is exposed to a pocket that is the substrate-binding site. The structures identify residues that may have important roles in catalysis, which have been confirmed by our mutagenesis studies.

  19. Application of a Colorimetric Assay to Identify Putative Ribofuranosylaminobenzene 5'-Phosphate Synthase Genes Expressed with Activity in Escherichia coli

    Bechard Matthew E.

    2003-01-01

    Full Text Available Tetrahydromethanopterin (H4MPT is a tetrahydrofolate analog originally discovered in methanogenic archaea, but later found in other archaea and bacteria. The extent to which H4MPT occurs among living organisms is unknown. The key enzyme which distinguishes the biosynthetic pathways of H4MPT and tetrahydrofolate is ribofuranosylaminobenzene 5'-phosphate synthase (RFAP synthase. Given the importance of RFAP synthase in H4MPT biosynthesis, the identification of putative RFAP synthase genes and measurement of RFAP synthase activity would provide an indication of the presence of H4MPT in untested microorganisms. Investigation of putative archaeal RFAP synthase genes has been hampered by the tendency of the resulting proteins to form inactive inclusion bodies in Escherichia coli. The current work describes a colorimetric assay for measuring RFAP synthase activity, and two modified procedures for expressing recombinant RFAP synthase genes to produce soluble, active enzyme. By lowering the incubation temperature during expression, RFAP synthase from Archaeoglobus fulgidus was produced in E. coli and purified to homogeneity. The production of active RFAP synthase from Methanothermobacter thermautotrophicus was achieved by coexpression of the gene MTH0830 with a molecular chaperone. This is the first direct biochemical identification of a methanogen gene that codes for an active RFAP synthase.

  20. Molecular cloning and enzymological characterization of pyridoxal 5'-phosphate independent aspartate racemase from hyperthermophilic archaeon Thermococcus litoralis DSM 5473.

    Washio, Tsubasa; Kato, Shiro; Oikawa, Tadao

    2016-09-01

    We succeeded in expressing the aspartate racemase homolog gene from Thermococcus litoralis DSM 5473 in Escherichia coli Rosetta (DE3) and found that the gene encodes aspartate racemase. The aspartate racemase gene consisted of 687 bp and encoded 228 amino acid residues. The purified enzyme showed aspartate racemase activity with a specific activity of 1590 U/mg. The enzyme was a homodimer with a molecular mass of 56 kDa and did not require pyridoxal 5'-phosphate as a coenzyme. The enzyme showed aspartate racemase activity even at 95 °C, and the activation energy of the enzyme was calculated to be 51.8 kJ/mol. The enzyme was highly thermostable, and approximately 50 % of its initial activity remained even after incubation at 90 °C for 11 h. The enzyme showed a maximum activity at a pH of 7.5 and was stable between pH 6.0 and 7.0. The enzyme acted on L-cysteic acid and L-cysteine sulfinic acid in addition to D- and L-aspartic acids, and was strongly inhibited by iodoacetic acid. The site-directed mutagenesis of the enzyme showed that the essential cysteine residues were conserved as Cys83 and Cys194. D-Forms of aspartic acid, serine, alanine, and valine were contained in T. litoralis DSM 5473 cells. PMID:27438592

  1. Method of preparing highly active and thermostable preparations of liver uridin-kinase usable for enzymic synthesis of radioactive nucleoside-5'-phosphates

    A method is described of preparing a high-activity uridine kinase for the enzymic synthesis of radioactive nucleoside-5m-phosphates of the pyrimidine series. The preparation is separated from male rat liver after intraperitoneal application of 5'-azacytidine. Examples are given showing detailed procedures for the conversion of uridine and 6-azauridine to the corresponding 5'-phosphates. (L.K.)

  2. The Effect of Transient HMG-CoA Reductase and 1-Deoxy-D-Xylulose-5-Phosphate Synthase Overexpression on Terpene Production in Transgenic Tomato Fruits

    Gentry, Scott A; Gutensohn, Michael; Henry, Laura; Dudareva, Natalia

    2014-01-01

    Isoprenoids are secondary metabolites that control numerous plant functions including signaling, growth, photosynthesis, and membrane structure. The bioengineering of isoprenoid synthesis could produce plants with a variety of beneficial traits. Plants form isoprenoids using two different pathways, the mevalonate (MVA) pathway and the methylerithritol phosphate (MEP) pathway, which cooperate via metabolic cross-talk. Transgenic tomato lines expressing both the plastidic and cytosolic forms of...

  3. TNF-α-induced down-regulation of CDX2 suppresses MEP1A expression in colitis

    Coskun, Mehmet; Olsen, Anders Krüger; Holm, Thomas Lindebo;

    2012-01-01

    High levels of pro-inflammatory cytokines are linked to inflammatory bowel disease (IBD). The transcription factor Caudal-related homeobox transcription factor 2 (CDX2) plays a crucial role in differentiation of intestinal epithelium and regulates IBD-susceptibility genes, including meprin 1A (MEP1...

  4. Reduced neuronal expression of ribose-5-phosphate isomerase enhances tolerance to oxidative stress, extends lifespan, and attenuates polyglutamine toxicity in Drosophila

    Wang, Ching-Tzu; Chen, Yi-Chun; Wang, Yi-Yun; Huang, Ming-Hao; Yen, Tzu-Li; Li, Hsun; Liang, Cyong-Jhih; Sang, Tzu-Kang; Cho, Si-Chih; Yuh, Chiou-Hwa; Wang, Chao-Yung; Brummel, Theodore J.; Wang, Horng-Dar

    2011-01-01

    Aging and age-related diseases can be viewed as the result of the lifelong accumulation of stress insults. The identification of mutant strains and genes which are responsive to stress and can alter longevity profiles provides new therapeutic targets for age-related diseases. Here we reported that a Drosophila strain with reduced expression of ribose-5-phosphate isomerase (rpi), EP2456, exhibits increased resistance to oxidative stress and enhanced lifespan. In addition, the strain also displ...

  5. Expression and function of androgen receptor coactivator p44/Mep50/WDR77 in ovarian cancer.

    Martin Ligr

    Full Text Available Hormones, including estrogen and progesterone, and their receptors play an important role in the development and progression of ovarian carcinoma. Androgen, its receptor and coactivators have also been implicated in these processes. p44/Mep50/WDR77 was identified as a subunit of the methylosome complex and lately characterized as a steroid receptor coactivator that enhances androgen receptor as well as estrogen receptor-mediated transcriptional activity in a ligand-dependent manner. We previously described distinct expression and function of p44 in prostate, testis, and breast cancers. In this report, we examined the expression and function of p44 in ovarian cancer. In contrast to findings in prostate and testicular cancer and similar to breast cancer, p44 shows strong cytoplasmic localization in morphologically normal ovarian surface and fallopian tube epithelia, while nuclear p44 is observed in invasive ovarian carcinoma. We observed that p44 can serve as a coactivator of both androgen receptor (AR and estrogen receptor (ER in ovarian cells. Further, overexpression of nuclear-localized p44 stimulates proliferation and invasion in ovarian cancer cells in the presence of estrogen or androgen. These findings strongly suggest that p44 plays a role in mediating the effects of hormones during ovarian tumorigenesis.

  6. [Properties of 2,5-diamino-4-oxy-6-ribosylaminopyrimidine-5'- phosphate reductase, a enzyme of the second stage of flavinogenesis in Pichia guilliermondii yeasts].

    Logvinenko, E M; Shavlovskiĭ, G M; Zakal'skiĭ, A E; Kontorovskaia, N Iu

    1989-01-01

    2,5-Diamino-4-oxy-6-ribosylaminopyrimidine-5'-phosphate reductase has been isolated from cells of Pichia guilliermondii and subjected to 20-fold purification by treating extracts with streptomycin sulphate, frationating proteins (NH4)2SO4 at 45-75% of saturation and chromatography on blue sepharose CL-6B. The use of gel filtration through Sephadex G-150 and chromatography on DEAE-cellulose proved to be less effective for the enzyme purification. It has been established that it is 2,5-diamino-4-oxy-6-ribosylaminopyrimidine-5-phosphate but not its dephosphorylated form that is the substrate of the given reductase; Km is equal to 7.10(-5) M. The reaction proceeds in the presence of NADPH or NADH. The enzyme affinity to NADPH (Km = 4.7.10(-5) M) is approximately one order higher than that to NADPH (Km = 5.5.10(-4) M). The enzyme manifests the optimum of action at pH 7.2 and the temperature of 37 degrees C; the molecular weight is 140 kD. EDTA as well as flavins in the concentration of 1.10(-3) M exert no effect on the reductase activity. The enzyme is labile at 4 degrees C and is inactivated in the frozen state at -15 degrees C. The 2.5-diamino-4-oxy-6-ribosylaminopyrimidine-5'-phosphate reductase has been also revealed in Torulopsis candida, Debaryomyces klöckeri, Schwanniomyces occidentalis, Eremothecium ashbyii (flavinogenic species) and Candida utilis. Aspergillus nidulans, Neurospora crassa (nonflavinogenic species). The synthesis of this enzyme contrary to other enzymes of the riboflavin biosynthesis is not regulated in flavinogenic yeast by iron ions. PMID:2511652

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

    Ricardo eSalvador

    2015-10-01

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

  8. D-Ribulose 5-Phosphate 3-Epimerase: Functional and Structural Relationships to Members of the Ribulose-Phosphate Binding (beta/alpha)8-Barrel Superfamily

    The 'ribulose phosphate binding' superfamily defined by the Structural Classification of Proteins (SCOP) database is considered the result of divergent evolution from a common (β/α)8-barrel ancestor. The superfamily includes D-ribulose 5-phosphate 3-epimerase (RPE), orotidine 5'-monophosphate decarboxylase (OMPDC), and 3-keto-L-gulonate 6-phosphate decarboxylase (KGPDC), members of the OMPDC suprafamily, as well as enzymes involved in histidine and tryptophan biosynthesis that utilize phosphorylated metabolites as substrates. We now report studies of the functional and structural relationships of RPE to the members of the superfamily. As suggested by the results of crystallographic studies of the RPEs from rice and Plasmodium falciparum, the RPE from Streptococcus pyogenes is activated by Zn2+ which binds with a stoichiometry of one ion per polypeptide. Although wild type RPE has a high affinity for Zn2+ and inactive apoenzyme cannot be prepared, the affinity for Zn2+ is decreased by alanine substitutions for the two histidine residues that coordinate the Zn2+ ion (H34A and H67A); these mutant proteins can be prepared in an inactive, metal-free form and activated by exogenous Zn2+. The crystal structure of the RPE was solved at 1.8 Angstroms resolution in the presence of D-xylitol 5-phosphate, an inert analogue of the D-xylulose 5-phosphate substrate. This structure suggests that the 2,3-enediolate intermediate in the 1,1-proton transfer reaction is stabilized by bidentate coordination to the Zn2+ that also is liganded to His 34, Asp 36, His 67, and Asp 176; the carboxylate groups of the Asp residues are positioned also to function as the acid/base catalysts. Although the conformation of the bound analogue resembles those of ligands bound in the active sites of OMPDC and KGPDC, the identities of the active site residues that coordinate the essential Zn2+ and participate as acid/base catalysts are not conserved. We conclude that only the phosphate binding motif

  9. Uneconomical tops of renewable electricity options. Advice with regard to the determination of the MEP subsidies for 2004 and 2005

    On assignment of the Dutch Ministry of Economic Affairs ECN and KEMA have researched the financial gaps of renewable electricity production technologies. These form the basis for determining the level of the MEP-subsidies for different renewable electricity sources and technologies by the Ministry. This report contains an advice on the financial gaps that are to be used for determining the subsidy levels. The calculation of the financial gaps is based on an earlier report on the inputs for the calculations and on the subsequent stakeholder consultation with respect to these inputs

  10. Stability of pyridoxal-5-phosphate semicarbazone: applications in plasma vitamin B6 analysis and population surveys of vitamin B6 nutritional status.

    Ubbink, J B; Serfontein, W J; de Villiers, L S

    1985-08-01

    The determination of pyridoxal-5-phosphate (PLP) and pyridoxal (PL) in plasma requires the availability of dark room facilities, due to the photosensitivity of these vitamin B6 vitamers. The fact that the semicarbazone forms of PL and PLP are more strongly fluorescent than the underivatized B6 vitamers has been exploited in plasma analyses, but it was not previously realised that these semicarbazone forms are also very stable even under conditions that lead to rapid decomposition of free PL and PLP. The stabilisation of PLP and PL obtained in this manner is sufficient and fully adequate to meet the practical requirements of clinical field studies. We report a high-performance liquid chromatographic method for plasma PLP and PL determinations based on precolumn semicarbazone formation and fluorescence detection. The method is sensitive enough for quantitative plasma PLP determinations even in B6-deficient patients. PMID:4055950

  11. Preparation of riboflavin 5' - phosphate sodium from crude riboflavin%利用核黄素粗品制备核黄素5'-磷酸钠

    魏转; 周强; 孙文敬; 崔凤杰; 张铎; 余泗莲

    2011-01-01

    The objective of the present study was to investigate the feasibility of preparing riboflavin 5' - phosphate sodium from crude riboflavin. Riboflavin 5'-phosphate sodium was obtained with the yield of 68. 5% by reacting one mole of crude riboflavin with four moles of POC13 for 2 h at 35℃ , hydrolyzing the resulting mixture, and neutralizing with NaOH solution. The quality of obtained product met the requirements of US FCC (7 ). The phosphorylation reaction conditions of riboflavin was further optimized by one-factor-at-a-time experiment design, and results were: n ( POC13 ) / n ( VB2 ) 4. 6, n (POC13) /n (H20) 1.65, reaction time 2h, and reaction temperature 35 ± 1℃. Under the above condition, the yield of riboflavin 5'-phosphate sodium increased to above 73. 5%. This method significantly decreases production cost. In conclusion, preparation of riboflavin 5'-phosphate sodium from crude riboflavin was determined to be feasible e-conomically and technically.%探索利用核黄素粗品制备核黄素5'-磷酸钠的可行性.在吡啶和乙腈的混合溶剂中,使核黄素粗品与其4倍摩尔量的三氯氧磷35℃左右下反应2h,然后水解反应混合物,再用NaOH溶液中和,能够以68.5%以上的收率得到核黄素磷酸钠,产品质量符合美国FCC (7)要求.通过单因素试验,初步优化了核黄素5'-磷酸钠粗品磷酸化反应的条件,即:n (POCl3)/n(VB2)=4.6,n(POCl3)/n(H2O)=1.65,反应时间2h,反应温度35±1℃.在优化的磷酸化反应条件下,利用核黄素粗品制备核黄素5'-磷酸钠的收率平均可达73.5%以上,能够有效降低核黄素5'-磷酸钠的生产成本.研究结果表明,利用核黄素粗品制备核黄素5'-磷酸钠无论在经济上还是在技术上均是可行的.

  12. Phosphatidylinositol 5-phosphate 4-kinase type II beta is required for vitamin D receptor-dependent E-cadherin expression in SW480 cells

    Highlights: → We analyzed Phosphatidylinositol 5-phosphate kinase IIβ (PIPKIIβ) function in cancer. → PIPKIIβ is required for vitamin D receptor-mediated E-cadherin upregulation in SW480. → PIPKIIβ suppresses cellular motility through E-cadherin induction in SW480 cells. → Nuclear PIP2 but not plasma membrane-localized PIP2 mediates E-cadherin upregulation. -- Abstract: Numerous epidemiological data indicate that vitamin D receptor (VDR) signaling induced by its ligand or active metabolite 1α,25-dihydroxyvitamin D3 (1α,25(OH)2D3) has anti-cancer activity in several colon cancers. 1α,25(OH)2D3 induces the epithelial differentiation of SW480 colon cancer cells expressing VDR (SW480-ADH) by upregulating E-cadherin expression; however, its precise mechanism remains unknown. We found that phosphatidylinositol-5-phosphate 4-kinase type II beta (PIPKIIβ) but not PIPKIIα is required for VDR-mediated E-cadherin induction in SW480-ADH cells. The syntenin-2 postsynaptic density protein/disc large/zona occludens (PDZ) domain and pleckstrin homology domain of phospholipase C-delta1 (PLCδ1 PHD) possess high affinity for phosphatidylinositol-4,5-bisphosphate (PI(4,5)P2) mainly localized to the nucleus and plasma membrane, respectively. The expression of syntenin-2 PDZ but not PLCδ1 PHD inhibited 1α,25(OH)2D3-induced E-cadherin upregulation, suggesting that nuclear PI(4,5)P2 production mediates E-cadherin expression through PIPKIIβ in a VDR-dependent manner. PIPKIIβ is also involved in the suppression of the cell motility induced by 1α,25(OH)2D3. These results indicate that PIPKIIβ-mediated PI(4,5)P2 signaling is important for E-cadherin upregulation and inhibition of cellular motility induced by VDR activation.

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

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

    2011-01-01

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

  14. Mutations in Escherichia coli aceE and ribB genes allow survival of strains defective in the first step of the isoprenoid biosynthesis pathway.

    Jordi Perez-Gil

    Full Text Available A functional 2-C-methyl-D-erythritol 4-phosphate (MEP pathway is required for isoprenoid biosynthesis and hence survival in Escherichia coli and most other bacteria. In the first two steps of the pathway, MEP is produced from the central metabolic intermediates pyruvate and glyceraldehyde 3-phosphate via 1-deoxy-D-xylulose 5-phosphate (DXP by the activity of the enzymes DXP synthase (DXS and DXP reductoisomerase (DXR. Because the MEP pathway is absent from humans, it was proposed as a promising new target to develop new antibiotics. However, the lethal phenotype caused by the deletion of DXS or DXR was found to be suppressed with a relatively high efficiency by unidentified mutations. Here we report that several mutations in the unrelated genes aceE and ribB rescue growth of DXS-defective mutants because the encoded enzymes allowed the production of sufficient DXP in vivo. Together, this work unveils the diversity of mechanisms that can evolve in bacteria to circumvent a blockage of the first step of the MEP pathway.

  15. Study of the factors affecting the performance of microextraction by packed sorbent (MEPS) using liquid scintillation counter and liquid chromatography-tandem mass spectrometry

    Altun, Zeki [Karlstad University, Faculty of Technology and Science, SE-651 88 Karlstad (Sweden); Abdel-Rehim, Mohamed [Karlstad University, Faculty of Technology and Science, SE-651 88 Karlstad (Sweden); Clinical Pharmacology and DMPK, AstraZeneca R and D Soedertaelje, SE-151 85 Soedertaelje (Sweden)], E-mail: Mohamed.Abdel-Rehim@Astrazeneca.com

    2008-12-23

    Microextraction by packed sorbent (MEPS) is a new technique for sample preparation that can be connected on-line with LC or GC. In MEPS, approximately 1-2 mg of the solid packing material is inserted into a syringe (100-250 {mu}L) as a plug. Sample preparation takes place on the packed bed. The bed can be packed or coated to provide selective and suitable sampling conditions. The new method is very promising for extraction of drugs and metabolites from biological samples. In this paper, some factors affecting the performance of MEPS such as recovery, carry-over, leakage, washing volume and elution volume were studied using C18 and hydroxylated polystyrene-divinylbenzene copolymer (ENV+) as sorbents. Radioactively labelled bupivacaine in plasma samples was used as test analyte. For the extraction of this drug, using methanol/water 95:5 (v/v) (0.25% ammonium hydroxide) was used as elution solvent. The analyte response increased with increasing the elution volume and it was linear upp up to 100 {mu}L utilizing liquid scintillation counter. Further, for concentrating the sample, we found that MEPS may be used such that the sample can be drawn through the needle, up and down, several times. The analyte leakage increases as the volume washing increases, though higher washing volumes may also result in cleaner extracts. To eliminate analyte carry-over, the sorbents were washed first with 3 x 250 {mu}L elution solution and then with 3 x 250 {mu}L washing solution. In addition, the reproducibility measurements show relatively good relative standard deviation (RSD) % values concerning analyte recovery and analyte leakage. The present study provides an understanding of basic aspects when optimizing methods for MEPS. In this study, MEPS was used off-line with liquid scintillation counter and on-line with LC-MS/MS.

  16. D-Ribulose 5-Phosphate 3-Epimerase: Functional and Structural Relationships to Members of the Ribulose-Phosphate Binding (beta/alpha)8-Barrel Superfamily

    Akana,J.; Federov, A.; Federov, E.; Novak, W.; Babbitt, P.; Almo, S.; Gerlt, J.

    2006-01-01

    The 'ribulose phosphate binding' superfamily defined by the Structural Classification of Proteins (SCOP) database is considered the result of divergent evolution from a common ({beta}/{alpha}){sub 8}-barrel ancestor. The superfamily includes D-ribulose 5-phosphate 3-epimerase (RPE), orotidine 5'-monophosphate decarboxylase (OMPDC), and 3-keto-L-gulonate 6-phosphate decarboxylase (KGPDC), members of the OMPDC suprafamily, as well as enzymes involved in histidine and tryptophan biosynthesis that utilize phosphorylated metabolites as substrates. We now report studies of the functional and structural relationships of RPE to the members of the superfamily. As suggested by the results of crystallographic studies of the RPEs from rice and Plasmodium falciparum, the RPE from Streptococcus pyogenes is activated by Zn{sup 2+} which binds with a stoichiometry of one ion per polypeptide. Although wild type RPE has a high affinity for Zn{sup 2+} and inactive apoenzyme cannot be prepared, the affinity for Zn{sup 2+} is decreased by alanine substitutions for the two histidine residues that coordinate the Zn{sup 2+} ion (H34A and H67A); these mutant proteins can be prepared in an inactive, metal-free form and activated by exogenous Zn{sup 2+}. The crystal structure of the RPE was solved at 1.8 Angstroms resolution in the presence of D-xylitol 5-phosphate, an inert analogue of the D-xylulose 5-phosphate substrate. This structure suggests that the 2,3-enediolate intermediate in the 1,1-proton transfer reaction is stabilized by bidentate coordination to the Zn{sup 2+} that also is liganded to His 34, Asp 36, His 67, and Asp 176; the carboxylate groups of the Asp residues are positioned also to function as the acid/base catalysts. Although the conformation of the bound analogue resembles those of ligands bound in the active sites of OMPDC and KGPDC, the identities of the active site residues that coordinate the essential Zn{sup 2+} and participate as acid/base catalysts

  17. Phosphatidylinositol 5-phosphate 4-kinase type II beta is required for vitamin D receptor-dependent E-cadherin expression in SW480 cells

    Kouchi, Zen, E-mail: zkouchi@toyaku.ac.jp [Laboratory of Genome and Biosignals, Tokyo University of Pharmacy and Life Sciences, 1432-1 Horinouchi, Hachioji-city, Tokyo 192-0392 (Japan); Fujiwara, Yuki [Laboratory of Genome and Biosignals, Tokyo University of Pharmacy and Life Sciences, 1432-1 Horinouchi, Hachioji-city, Tokyo 192-0392 (Japan); Yamaguchi, Hideki [Division of Metastasis and Invasion Signaling, National Cancer Center Research Institute, 5-1-1 Tsukiji, Chuo-ku, Tokyo 104-0045 (Japan); PRESTO, Japan Science and Technology Agency, 4-1-8 Honcho, Kawaguchi-city, Saitama 332-0012 (Japan); Nakamura, Yoshikazu; Fukami, Kiyoko [Laboratory of Genome and Biosignals, Tokyo University of Pharmacy and Life Sciences, 1432-1 Horinouchi, Hachioji-city, Tokyo 192-0392 (Japan)

    2011-05-20

    Highlights: {yields} We analyzed Phosphatidylinositol 5-phosphate kinase II{beta} (PIPKII{beta}) function in cancer. {yields} PIPKII{beta} is required for vitamin D receptor-mediated E-cadherin upregulation in SW480. {yields} PIPKII{beta} suppresses cellular motility through E-cadherin induction in SW480 cells. {yields} Nuclear PIP{sub 2} but not plasma membrane-localized PIP{sub 2} mediates E-cadherin upregulation. -- Abstract: Numerous epidemiological data indicate that vitamin D receptor (VDR) signaling induced by its ligand or active metabolite 1{alpha},25-dihydroxyvitamin D{sub 3} (1{alpha},25(OH){sub 2}D{sub 3}) has anti-cancer activity in several colon cancers. 1{alpha},25(OH){sub 2}D{sub 3} induces the epithelial differentiation of SW480 colon cancer cells expressing VDR (SW480-ADH) by upregulating E-cadherin expression; however, its precise mechanism remains unknown. We found that phosphatidylinositol-5-phosphate 4-kinase type II beta (PIPKII{beta}) but not PIPKII{alpha} is required for VDR-mediated E-cadherin induction in SW480-ADH cells. The syntenin-2 postsynaptic density protein/disc large/zona occludens (PDZ) domain and pleckstrin homology domain of phospholipase C-delta1 (PLC{delta}1 PHD) possess high affinity for phosphatidylinositol-4,5-bisphosphate (PI(4,5)P{sub 2}) mainly localized to the nucleus and plasma membrane, respectively. The expression of syntenin-2 PDZ but not PLC{delta}1 PHD inhibited 1{alpha},25(OH){sub 2}D{sub 3}-induced E-cadherin upregulation, suggesting that nuclear PI(4,5)P{sub 2} production mediates E-cadherin expression through PIPKII{beta} in a VDR-dependent manner. PIPKII{beta} is also involved in the suppression of the cell motility induced by 1{alpha},25(OH){sub 2}D{sub 3}. These results indicate that PIPKII{beta}-mediated PI(4,5)P{sub 2} signaling is important for E-cadherin upregulation and inhibition of cellular motility induced by VDR activation.

  18. An insight into the sequential, structural and phylogenetic properties of banana 1-aminocyclopropane-1-carboxylate synthase 1 and study of its interaction with pyridoxal-5'-phosphate and aminoethoxyvinylglycine.

    Choudhury, Swarup Roy; Singh, Sanjay Kumar; Roy, Sujit; Sengupta, Dibyendu N

    2010-06-01

    In banana, ethylene production for ripening is accompanied by a dramatic increase in 1-aminocyclopropane-1-carboxylate (ACC) content, transcript level of Musa acuminata ACC synthase 1 (MA-ACS1) and the enzymatic activity of ACC synthase 1 at the onset of the climacteric period. MA-ACS1 catalyses the conversion of S-adenosyl-L-methionine (SAM) to ACC, the key regulatory step in ethylene biosynthesis. Multiple sequence alignments of 1-aminocyclopropane-1-carboxylate synthase (ACS) amino acid sequences based on database searches have indicated that MA-ACS1 is a highly conserved protein across the plant kingdom. This report describes an in silico analysis to provide the first important insightful information about the sequential, structural and phylogenetic characteristics of MA-ACS1. The three-dimensional structure of MA-ACS1, constructed based on homology modelling, in combination with the available data enabled a comparative mechanistic analysis of MA-ACS1 to explain the catalytic roles of the conserved and non-conserved active site residues. We have further demonstrated that, as in apple and tomato, banana- ACS1 (MA-ACS1) forms a homodimer and a complex with cofactor pyridoxal-5'-phosphate (PLP) and inhibitor aminoethoxyvinylglycine (AVG). We have also predicted that the residues from the PLP-binding pocket, essential for ligand binding, are mostly conserved across the MA-ACS1 structure and the competitive inhibitor AVG binds at a location adjacent to PLP. PMID:20689184

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

    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...

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

    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

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

    Natália Corniani

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

  2. Systems-Wide Prediction of Enzyme Promiscuity Reveals a New Underground Alternative Route for Pyridoxal 5'-Phosphate Production in E. coli.

    Matthew A Oberhardt

    2016-01-01

    Full Text Available Recent insights suggest that non-specific and/or promiscuous enzymes are common and active across life. Understanding the role of such enzymes is an important open question in biology. Here we develop a genome-wide method, PROPER, that uses a permissive PSI-BLAST approach to predict promiscuous activities of metabolic genes. Enzyme promiscuity is typically studied experimentally using multicopy suppression, in which over-expression of a promiscuous 'replacer' gene rescues lethality caused by inactivation of a 'target' gene. We use PROPER to predict multicopy suppression in Escherichia coli, achieving highly significant overlap with published cases (hypergeometric p = 4.4e-13. We then validate three novel predicted target-replacer gene pairs in new multicopy suppression experiments. We next go beyond PROPER and develop a network-based approach, GEM-PROPER, that integrates PROPER with genome-scale metabolic modeling to predict promiscuous replacements via alternative metabolic pathways. GEM-PROPER predicts a new indirect replacer (thiG for an essential enzyme (pdxB in production of pyridoxal 5'-phosphate (the active form of Vitamin B6, which we validate experimentally via multicopy suppression. We perform a structural analysis of thiG to determine its potential promiscuous active site, which we validate experimentally by inactivating the pertaining residues and showing a loss of replacer activity. Thus, this study is a successful example where a computational investigation leads to a network-based identification of an indirect promiscuous replacement of a key metabolic enzyme, which would have been extremely difficult to identify directly.

  3. Function of pyridoxal 5'-phosphate in glycogen phosphorylase: a model study using 6-fluoro-5'-deoxypyridoxal- and 5'-deoxypyridoxal-reconstituted enzymes

    A new vitamin B6 analogue, 6-fluoro-5'-deoxypyridoxal (6-FDPL), was synthesized and characterized. This analogue, as well as 6-fluoropyridoxal (6-FPAL), 6-fluoropyridoxal phosphate (6-FPLP), and 6-fluoropyridoxine, showed positive heteronuclear 1H-18F nuclear Overhauser effects between the 5'-protons and the 6-fluorine. Apophosphorylase reconstituted with 6-FDLP showed 1% of the activity of the native enzyme in the presence of phosphite. The kinetic pattern, apparent pH optimum of activity, and the activity-temperature dependency of the 6-FDPL-enzyme were virtually identical with those of phosphorylase reconstituted with the parent compound, 6-FPAL except the K/sub m/ of phosphite toward the 6-FDPL-enzyme was 9 times higher than that with the 6-FPAL-enzyme and the 6-FDPL-enzyme showed a lower V/sub max/ value. Phosphorylase reconstituted with 5'-deoxypyridoxal (DPL) also showed activity in the presence of phosphite. The kinetics and the temperature-activity dependency of this reconstituted enzyme were investigated. 19F nuclear magnetic resonance studies showed that the binding of glucose 1-phosphate to a 6-FDPL-enzyme-adenosine 5'-phosphate (AMP) complex shifted the 19F signal 0.6 ppm upfield, whereas a 2.1 ppm change was observed when the 6-FPAL-enzyme-AMP formed a complex with glucose 1-phosphate. Analysis of the activation parameters, activation enthalpy and activation entropy, of the reaction of glycogen degradation catalyzed by phosphorylase containing pyridoxal phosphate, 6-FDPL, pyridoxal, or DPL showed that modifications of the coenzyme molecule affected only the activation entropy, not the activation enthalpy. Results of this study indicate that the protein structure surrounding the coenzyme molecule, as well as the coenzyme configuration, is altered upon the binding of ligands

  4. Molecular evolution of B6 enzymes: Binding of pyridoxal-5'-phosphate and Lys41Arg substitution turn ribonuclease A into a model B6 protoenzyme

    Marra Ersilia

    2008-06-01

    Full Text Available Abstract Background The pyridoxal-5'-phosphate (PLP-dependent or vitamin B6-dependent enzymes that catalyze manifold reactions in the metabolism of amino acids belong to no fewer than four evolutionarily independent protein families. The multiple evolutionary origin and the essential mechanistic role of PLP in these enzymes argue for the cofactor having arrived on the evolutionary scene before the emergence of the respective apoenzymes and having played a dominant role in the molecular evolution of the B6 enzyme families. Here we report on an attempt to re-enact the emergence of a PLP-dependent protoenzyme. The starting protein was pancreatic ribonuclease A (RNase, in which active-site Lys41 or Lys7 readily form a covalent adduct with PLP. Results We screened the PLP adduct of wild-type RNase and two variant RNases (K7R and K41R for catalytic effects toward L- and D-amino acids. RNase(K41R-PLP, in which the cofactor is bound through an imine linkage to Lys7, qualifies for a model proto-B6 enzyme by the following criteria: (1 covalent linkage of PLP (internal aldimine; (2 catalytic activity toward amino acids that depends on formation of an imine linkage with the substrate (external aldimine; (3 adjoining binding sites for the cofactor and amino acid moiety that facilitate the transimination reaction of the internal to the external aldimine and stabilize the resulting noncovalent complex of the coenzyme-substrate adduct with the protein; (4 reaction specificity, the only detectable reactions being racemization of diverse amino acids and β-decarboxylation of L-aspartate; (5 acceleration factors for racemization and β-decarboxylation of >103 over and above that of PLP alone; (6 ribonuclease activity that is 103-fold lower than that of wild-type RNase, attenuation of a pre-existing biological activity being indispensable for the further evolution as a PLP-dependent protoenzyme. Conclusion A single amino acid substitution (Lys41Arg and covalent

  5. Map-Based Cloning of zb7 Encoding an IPP and DMAPP Synthase in the MEP Pathway of Maize

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

    2012-01-01

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

  6. The influence of magnesium-pyridoxal-5'-phosphate-glutamate in comparison with probucol, alpha-tocopherol and trolox on copper-induced oxidation of human low density lipoprotein in vitro.

    Kögl, C; Schneider, W; Elstner, E F

    1994-06-15

    Low density lipoprotein (LDL) in the presence of magnesium-pyridoxal-5'-phosphate-glutamate (MPPG), pyridoxal-5'-phosphate (PP), alpha-tocopherol, probucol or trolox is more resistant against copper-induced oxidation as control-LDL in vitro. The efficiency of the drugs is: probucol > MPPG > trolox > alpha-tocopherol > PP. LDL oxidation is determined by its increasing negative surface charge, fragmentation of apolipoprotein B-100 and changes of the fatty acid content of LDL. The protection of the drugs depends on their concentration and incubation time. Different experiments point to the fact that copper-induced oxidation of LDL in vitro starts with the binding of copper at the apolipoprotein B-100, resulting in an increasing negative surface charge and fragmentation of the apolipoprotein B-100. Afterwards a decrease of LDL-bound linoleic acid (18:2) is measurable. PMID:8031313

  7. Perinatal hypophosphatasia: tissue levels of vitamin B6 are unremarkable despite markedly increased circulating concentrations of pyridoxal-5'-phosphate. Evidence for an ectoenzyme role for tissue-nonspecific alkaline phosphatase.

    Whyte, M P; Mahuren, J D; Fedde, K. N.; Cole, F. S.; McCabe, E. R.; Coburn, S P

    1988-01-01

    "Perinatal" hypophosphatasia is the most severe form of this inborn error of metabolism, which is characterized by deficient activity of the tissue-nonspecific (liver/bone/kidney) isoenzyme of alkaline phosphatase (ALP) (TNSALP). We report that autopsy tissue from three affected subjects, which was profoundly low in ALP activity, had essentially unremarkable levels of pyridoxal-5'-phosphate (PLP), pyridoxal, and total vitamin B6 content despite markedly elevated plasma PLP levels (5,800, 14,5...

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

    Yurova, Alla

    2016-04-01

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

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

    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

  10. Dft Study on 4(5-Imidazole-Carbaldehyde-N(5-Phenylthiosemicarbazone (Imtph: Nmr Shielding Tensors, Thermodynamic Parameters, Nbo Analysis, Molecular Electrostatic Potential (Mep, Homo and Lumo Studies

    Masoome Sheikhi

    2014-03-01

    Full Text Available The density functional theory (DFT calculations at the level of B3LYP/6-31G was carried out on the structure 4(5-Imidazole-carbaldehyde-N(5-phenylthiosemicarbazone (ImTPh in gas phase using Gaussian 03. Dipole moment (Debye, energy of structure formation (HF; kcal/mol and point group, NMR parameters such as isotropic shielding (σiso and anisotropic shielding (σaniso, σ11, σ22 and σ33 obtained. Also thermodynamic properties and natural bond orbitals (NBO were calculated. Besides, the frontier molecular orbital (FMO analysis and the molecular electrostatic potential (MEP of the compound were investigated by theoretical calculations.

  11. Lipophilic prodrugs of FR900098 are antimicrobial against Francisella novicida in vivo and in vitro and show GlpT independent efficacy.

    Elizabeth S McKenney

    Full Text Available Bacteria, plants, and algae produce isoprenoids through the methylerythritol phosphate (MEP pathway, an attractive pathway for antimicrobial drug development as it is present in prokaryotes and some lower eukaryotes but absent from human cells. The first committed step of the MEP pathway is catalyzed by 1-deoxy-D-xylulose 5-phosphate reductoisomerase (DXR/MEP synthase. MEP pathway genes have been identified in many biothreat agents, including Francisella, Brucella, Bacillus, Burkholderia, and Yersinia. The importance of the MEP pathway to Francisella is demonstrated by the fact that MEP pathway mutations are lethal. We have previously established that fosmidomycin inhibits purified MEP synthase (DXR from F. tularensis LVS. FR900098, the acetyl derivative of fosmidomycin, was found to inhibit the activity of purified DXR from F. tularensis LVS (IC(50=230 nM. Fosmidomycin and FR900098 are effective against purified DXR from Mycobacterium tuberculosis as well, but have no effect on whole cells because the compounds are too polar to penetrate the thick cell wall. Fosmidomycin requires the GlpT transporter to enter cells, and this is absent in some pathogens, including M. tuberculosis. In this study, we have identified the GlpT homologs in F. novicida and tested transposon insertion mutants of glpT. We showed that FR900098 also requires GlpT for full activity against F. novicida. Thus, we synthesized several FR900098 prodrugs that have lipophilic groups to facilitate their passage through the bacterial cell wall and bypass the requirement for the GlpT transporter. One compound, that we termed "compound 1," was found to have GlpT-independent antimicrobial activity. We tested the ability of this best performing prodrug to inhibit F. novicida intracellular infection of eukaryotic cell lines and the caterpillar Galleria mellonella as an in vivo infection model. As a lipophilic GlpT-independent DXR inhibitor, compound 1 has the potential to be a broad

  12. High yield synthesis of 14C labelled intermediates of the L-type pentose pathway: octulose mono- and biphosphates, sedoheptulose 1,7-bisphosphate and D-arabinose 5-phosphate

    Methods for the enzymic synthesis, isolation, purification and analysis of the 14C labelled intermediates that are characteristic of the L-type pentose phosphate pathway are described. These are D-glycero D-ido octulose 1,8-bisphosphate and 8-monophosphate; D-glycero D-altro octulose mono- and bisphosphates; sedoheptulose 1,7-bisphosphate and D-arabinose 5-phosphate. The authenticity of each of the 14C-labelled sugar phosphates was confirmed using a variety of chromatographic methods, enzymatic analytic methods and NMR spectroscopy. The 14C labelled compounds are used in investigations involving the search for the identity of the pentose pathway in tissues in vitro, for the measurement of L-type pathway enzyme reactions and for testing the mechanistic predictions of the L-type pathway in vitro. (author)

  13. MEP incentive for onshore wind energy. Study on the robustness of the full load methodology and options for alternative differentiation of the incentive for onshore wind energy

    The proposed amendment to the Dutch Electricity Law of 1998, called 'environmental quality of electricity production' (MEP), provides operating support through a combination of feed-in tariffs and a reduced ecotax exemption. In order to prevent a free ride of windy locations, the feed-in tariff for onshore wind turbines is limited to a maximum of 18,000 full load hours. Several parties have expressed their concern that this system may possibly provide an incentive to increase the rated power in order to lengthen the period that the feed-in tariff is being received. This report reviews the risks of unintentional side effects of the proposed limitation of 18,000 full load hours. In addition, this report examines the possibilities for further differentiation of the feed-in tariff for onshore wind energy by reviewing methods of differentiation in a number of EU member states

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

    Lobayan, Rosana M; Bochicchio, Roberto C

    2015-07-01

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

  15. Slow-binding and competitive inhibition of 8-amino-7-oxopelargonate synthase, a pyridoxal-5'-phosphate-dependent enzyme involved in biotin biosynthesis, by substrate and intermediate analogs. Kinetic and binding studies.

    Ploux, O; Breyne, O; Carillon, S; Marquet, A

    1999-01-01

    8-Amino-7-oxopelargonate synthase catalyzes the first committed step of biotin biosynthesis in micro-organisms and plants. Because inhibitors of this pathway might lead to antibacterials or herbicides, we have undertaken an inhibition study on 8-amino-7-oxopelargonate synthase using six different compounds. d-Alanine, the enantiomer of the substrate of this pyridoxal-5'-phosphate-dependent enzyme was found to be a competitive inhibitor with respect to l-alanine with a Ki of 0.59 mm. The fact that this inhibition constant was four times lower than the Km for l-alanine was interpreted as the consequence of the inversion-retention stereochemistry of the catalyzed reaction. Schiff base formation between l or d-alanine and pyridoxal-5'-phosphate, in the active site of the enzyme, was studied using ultraviolet/visible spectroscopy. It was found that l and d-alanine form an external aldimine with equilibrium constants K = 4.1 mm and K = 37.8 mm, respectively. However, the equilibrium constant for d-alanine aldimine formation dramatically decreased to 1.3 mm in the presence of saturating concentration of pimeloyl-CoA, the second substrate. This result strongly suggests that the binding of pimeloyl-CoA induces a conformational change in the active site, and we propose that this new topology is complementary to d-alanine and to the putative reaction intermediate since they both have the same configuration. (+/-)-8-Amino-7-oxo-8-phosphonononaoic acid (1), the phosphonate derivative of the intermediate formed during the reaction, was our most potent inhibitor with a Ki of 7 microm. This compound behaved as a reversible slow-binding inhibitor, competitive with respect to l-alanine. Kinetic investigation showed that this slow process was best described by a one-step mechanism (mechanism A) with the following rate constants: k1 = 0.27 x 103 m-1.s-1, k2 = 1.8 s-1 and half-life for dissociation t1/2 = 6.3 min. The binding of compound 1 to the enzyme was also studied using

  16. Expression of the xylulose 5-phosphate phosphoketolase gene, xpkA, from Lactobacillus pentosus MD363 is induced by sugars that are fermented via the phosphoketolase pathway and is repressed by glucose mediated by CcpA and the mannose phosphoenolpyruvate phosphotransferase system

    Posthuma, C.C.; Bader, R.; Engelmann, R.; Postma, P.W.; Hengstenberg, W.; Pouwels, P.H.

    2002-01-01

    Purification of xylulose 5-phosphate phosphoketolase (XpkA), the central enzyme of the phosphoketolase pathway (PKP) in lactic acid bacteria, and cloning and sequence analysis of the encoding gene, xpkA, from Lactobacillus pentosus MD363 are described, xpkA encodes a 788-amino-acid protein with a ca

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

    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

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

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

    2016-09-01

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

  19. BAEP、BR及MEP联合检测在脑死亡诊断中的应用%Application of BAEP, BR and MEP combined detection for the diagnosis of brain death

    朴虎男; 朴莲荀; 杜婷婷

    2012-01-01

    目的 探讨神经电生理在脑死亡判定中的应用价值.方法 通过瞬间反射(BR)、脑干听觉诱发电位(BAEP)、运动诱发电位(MEP)三项联合检查对22例脑死亡患者进行评定,并与GCS评分结果进行比较.结果 BAEP、BR和MEP三项联合检查对脑死亡判断准确率为100%,与GCS评分比较差异显著(P<0.05).结论 BAEP、BR和MEP三项联合检测对评价脑死亡患者的脑功能状态、预测预后提供了客观可靠的依据.%Objective To investigate the diagnosis value of neuro-electrophysiology detection for brain death. Methods Combined detection of brainstem auditory evoked potentials ( BAEP) , blink reflex (BR) united motor evoked potentials ( MEP) were used to access 22 brain death patients, and then compared with the Glasgow Coma Scale (GCS) scores. Results The accuracy of BAEP, BR and MEP combined detection was 100% , and which showed significant difference compared with GCS scores. Conclusion BAEP, BR united MEP testing can provide an objective indicator not only for e-valuating brain function of brain death, but also for estimating prognosis.

  20. Clinical analysis of vitamin B(6): determination of pyridoxal 5'-phosphate and 4-pyridoxic acid in human serum by reversed-phase high-performance liquid chromatography with chlorite postcolumn derivatization.

    Rybak, Michael E; Pfeiffer, Christine M

    2004-10-15

    A reversed-phase high-performance liquid chromatography (HPLC) method with fluorometric detection was developed for the routine determination of pyridoxal 5'-phosphate (PLP) and 4-pyridoxic acid (4-PA) in serum. Chlorite postcolumn derivatization was used to oxidize PLP to a more fluorescent carboxylic acid form. Sensitivity improved fourfold for PLP using chlorite postcolumn derivatization over traditional bisulfite postcolumn derivatization. The HPLC injection cycle was 15 min, facilitating a throughput of 60 patient samples (72 injections that included standards and quality control (QC) samples) in 18.5h. Method precision was evaluated using three serum QC pools with PLP and 4-PA concentrations of 11.5-34.8 nmol/L and 10.4-21.0 nmol/L, respectively. Within-run (n=7) repeatabilities were 0.6-1.2% for PLP and 0.9-1.8% for 4-PA. Run-to-run (n=23) reproducibilities were 3.6-6.7% for PLP and 3.7-5.6% for 4-PA. Relative detection (3sigma(0)) and quantitation (10sigma(0)) limits were 0.3 and 0.9 nmol/L, respectively, for both PLP and 4-PA using a 10-microl sample injection volume. Analytical recoveries ranged from 97 to 102%. Patient-matched serum and plasma specimens (n=25) were analyzed to evaluate specimen-type bias. Of the plasma types evaluated, heparinized plasma introduced the lowest relative bias for PLP (-5.3%) and minimal bias for 4-PA (-2.3%) compared with serum. Ethylenediaminetetraacetic acid (EDTA) plasma showed the lowest bias for 4-PA (0.7%) but a relatively high bias for PLP (13.0%) due to a chromatographic interference. Human serum samples from a non-representative population subset (n=303) were commensurate with values published for other vitamin B(6) HPLC methods. These values gave geometric means of 42.4 nmol/L for PLP and 27.3 nmol/L for 4-PA. Medians for PLP and 4-PA were 40.1 and 21.8 nmol/L, respectively. The high sensitivity, precision, and throughput of this method, combined with its minimal serum specimen (150 microl) and sample injection

  1. Nuclear Polluters' Charter. Council directive 96/29/EURATOM (OJ L159 29th June 1996), the 'Basic Standards Directive'; briefing for MPs and MEPs

    The nuclear industry has huge 'back end' problems: acres of radioactive waste stacked up with no final disposal route; hundreds of thousands of tonnes of metals, glass, plastic, and concrete too 'hot' to re-use or dump. Sea dumping has been ruled out, Nirex's deep repository is back to square one, the waste mountain is growing, and hundreds of nuclear factories and power stations await decommissioning. But by May 2000 the UK and all member states are required to conform with a dangerously vague and permissive Directive, and deregulate much of this expensive, embarrassing, and harmful waste. Below certain very lax limits it will become a financial asset to be sold on the open market. What cannot be sold will be landfilled and incinerated without restriction. Ostensibly, the Directive is a 'harmonisation' of radiation exposure standards. It was promulgated by the European Commission under the Euratom Treaty of 1957. The European Parliament has no power over Euratom, and (with one exception) amendments advised by MEPs were ignored. The Directive effectively deregulates reuse, recycling, disposal, and incineration of radioactive materials below certain threshold levels. It specifically allows recycling of contaminated materials and drops a precautionary proviso used in earlier European legislation. Spokesmen from the nuclear industry, the regulators, and the Commission openly admit that there is nothing to stop hundreds of thousands of tonnes of radioactive materials from nuclear licensed sites - potentially, their entire inventory - being diluted into industrial feedstocks of recyclable materials and ending up in consumer goods, fertilisers or any product. The Commission's view is let the buyers beware if they don't want contaminated goods or raw materials. National radiation protection agencies which advise the Commission and national governments claim that there is no threat to health, according to internationally accepted radiation risk factors. But those same

  2. The effect of MEP pathway and other inhibitors on the intracellular localization of a plasma membrane-targeted, isoprenylable GFP reporter protein in tobacco BY-2 cells [v1; ref status: indexed, http://f1000r.es/yx

    Michael Hartmann

    2013-08-01

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

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

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

    2015-07-01

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

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

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

    2015-02-01

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

  5. Medical Expenditure Panel Survey (MEPS) Query Tool

    U.S. Department of Health & Human Services — MEPSnet HC Query Tool MEPSnet/Household Component provides easy access to nationally representative statistics of health care use, expenditures, sources of payment,...

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

    Morgan, Omar D.; And Others

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

  7. MEPs : ban hammer, sickle and swastika

    2005-01-01

    Europarlamendi liikmete Vytautas Landsbergise ja Jozsef Szajeri avalduse kohaselt tuleks Euroopa Liidus ettevalmistatava seaduse raames, mis keelustaks natsisümboli - haakristi - kasutamise, keelata ka kommunismi sümboolika kasutamine

  8. Results of the market consultation on technical-economical parameters of sustainable electricity options. Overview of the results of the consultation in response to the concept advice with regard to the assumptions for the ineffective top calculations to determine the MEP-subsidies for 2004 and 2005

    A study has been carried out on the financial gaps of renewable electricity production technologies. These form the basis for determining the level of the MEP-subsidies for different renewable electricity sources and technologies by the Ministry. This report provides an overview of the stakeholder reactions in several consultation meetings regarding the preliminary advice from ECN and KEMA on the techno-economic and financial assumptions for the calculation of the financial gaps. In this report it is motivated which reactions will be taken into account in the final advice on the financial gaps to the Dutch Ministry of Economic Affairs

  9. The effect of MEP pathway and other inhibitors on the intracellular localization of a plasma membrane-targeted, isoprenylable GFP reporter protein in tobacco BY-2 cells [v2; ref status: indexed, http://f1000r.es/2af

    Michael Hartmann

    2013-11-01

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

  10. Intracellular adenosine 3',5'-phosphate formation is essential for down-regulation of surface adenosine 3',5'-phosphate receptors in Dictyostelium

    Van Haastert, Peter J. M.

    1994-01-01

    Dictyostelium discoideum cells contain cell surface cyclic AMP (cAMP) receptors that bind cAMP as a first messenger and intracellular cAMP receptors that bind cAMP as a second messenger. Prolonged incubation of Dictyostelium cells with cAMP induces a sequential process of phosphorylation, sequestration and down-regulation of the surface receptors. The role of intracellular cAMP in down-regulation of surface receptors was investigated. Down-regulation of receptors does not occur under conditio...

  11. Lithuanian MEP loses his immunity / Rokas M. Tracevskis

    Tracevskis, Rokas M.

    2010-01-01

    7. septembril otsustas Euroopa Parlament parlamendisaadiku Viktor Uspaskihhi puutumatuse ära võtta, kuna teda kahtlustatakse Tööerakonna raamatupidamise võltsimises. Viktor Uspaskihhi poliitilisest tegevusest

  12. MEP parabolic hydrodynamical model for holes in silicon semiconductors

    Consistent hydrodynamical models for electron transport in semi-conductors, free of any fitting parameter, have been formulated on the basis of the maximum entropy principle in Continuum Mech. Thermodyn., 11 (1999) 307, 12 (2000) 31 for silicon and in Continuum Mech. Thermodyn., 14 (2002) 405 for GaAs. In this paper we use the same approach for studying the hole transport in Si, by considering a parabolic approximation for the valence energy band. Scattering of holes with non-polar optical phonons, acoustic phonons and impurities have been taken into account. On the basis of these results, a limiting energy-transport model and an explicit expression for the low field hole mobility have been obtained. The high field mobility is also analyzed by taking into account the influence of impurities

  13. Inhibitor design for ribonuclease A: the binding of two 5 '-phosphate uridine analogues

    Tsirkone, Vicky G.; Dossi, Kyriaki; Drakou, Christina; Zographos, Spyros E.; Kontou, Maria; Leonidas, Demetres D.

    2009-01-01

    The structure of ribonuclease A in complex with uridine 5′-phosphate and uridine 5′-diphosphate has been determined at 1.4 Å resolution in order to facilitate the rational design of selective and potent inhibitors.

  14. Hydrolysis of pyridoxal-5'-phosphate in plasma in conditions with raised alkaline phosphate.

    Anderson, B B; O'Brien, H.; Griffin, G E; Mollin, D. L.

    1980-01-01

    Hydrolysis of pyridoxal phosphate in plasma was demonstrated in patients with liver disease and other conditions with raised alkaline phosphatase, and this usually closely paralleled the alkaline phosphatase level, whether of liver or bone origin. The endogenous plasma pyridoxal phosphate was inversely related to the alkaline phosphatase, and plasma hydrolysis of pyridoxal phosphate may at least in part be responsible. Very large doses of vitamin B6 may be necessary to compensate for this hyd...

  15. Chemical modification of human muscle aldose reductase by pyridoxal 5'-phosphate

    Aldose reductase (ALR2) is a monomeric oxidoreductase (Mr, 37,000). This enzyme catalyzes the reduction of a wide variety of aliphatic and aromatic aldehydes to their corresponding alcohols. The ability to reduce D-glucose and utilize NADH distinguishes ALR2 from aldehyde reductase (ALR1) which is exclusively NADPH-dependent. As part of a study to determine active site residues critical for binding and catalysis they have investigated the behavior of ALR2 with pyridoxal phosphate (PLP). In contrast to ALR1, which is inactivated by PLP, the reaction of ALR2 with PLP results in a 2-3 fold activation with the incorporation of 1 mol of PLP/mol enzyme. However, despite a 3-fold increase in k/sub cat/, there is also a 13-14 fold increase in the Km for both coenzyme and substrate and catalytic efficiency (k/sub cat//Km) is actually decreased. Reaction of ALR2 with 3[H] PLP followed by digestion with endoproteinase Lys-C enabled the separation and purification by HPLC of a peptide containing a single pyridoxyllysine residue. The sequence of this 32 residue peptide is highly homologous with a peptide similarly obtained from pig and human ALR1 and is identical with one from pig ALR2. In all four enzymes, pig ALR1, ALR2; human ALR1, ALR2, a tetrapeptide containing the pyridoxylated lysine (I-P-K-S) shows absolute identity. Thus, despite differences in substrate and coenzyme specificity, the active site in both ALR1 and ALR2 is relatively conserved

  16. Study on combined monitoring in cervical cord operation by median nerve brainstem somatosensory evoked potential (MN-BSEP) and short train transcranial electrical stimulation motor evoked potential (STTES-MEP)%正中神经脑干躯体感觉诱发电位和经颅短串电刺激运动诱发电位联合术中监护在颈髓手术中的应用

    顾士欣; 徐启武; 陈炜; 车晓明

    2004-01-01

    目的研究在异丙酚全静脉麻醉方案下,联合应用正中神经脑干躯体感觉诱发电位(MN-BSEP)和经颅短串电刺激运动诱发电位(STTES-MEP)对颈髓手术实施术中监护的可行性.方法通过变换不同的刺激条件、记录条件、技术参数,分别摸索MN-BSEP和STTES-MEP的可靠检测方法,并按照总结得到的技术规范,选择我科15例接受颈髓手术的病人,进行术中联合监护,对照分析术前和术后脊髓功能的改变和诱发电位变化之间的关系.结果 MN-BSEP基本不受麻醉影响,术后的改变与术后病人感觉功能的转归情况相吻合;STTES-MEP与麻醉方案有关,在异丙酚麻醉下,可记录到清晰、稳定的运动诱发电位,其术后的变化与术后病人肌力的转归情况相吻合.结论选用异丙酚全静脉麻醉,联合应用MN-BSEP和STTES-MEP实现对颈髓功能的术中监护是可行的,其效果优于单纯应用体感诱发电位或运动诱发电位进行监护.

  17. Ribose-5-Phosphate Biosynthesis in Methanocaldococcus jannaschii Occurs in the Absence of a Pentose-Phosphate Pathway

    Grochowski, Laura L.; Xu, Huimin; White, Robert H.

    2005-01-01

    Recent work has raised a question as to the involvement of erythrose-4-phosphate, a product of the pentose phosphate pathway, in the metabolism of the methanogenic archaea (R. H. White, Biochemistry 43:7618-7627, 2004). To address the possible absence of erythrose-4-phosphate in Methanocaldococcus jannaschii, we have assayed cell extracts of this methanogen for the presence of this and other intermediates in the pentose phosphate pathway and have determined and compared the labeling patterns ...

  18. A homogeneous, high-throughput assay for phosphatidylinositol 5-phosphate 4-kinase with a novel, rapid substrate preparation.

    Mindy I Davis

    Full Text Available Phosphoinositide kinases regulate diverse cellular functions and are important targets for therapeutic development for diseases, such as diabetes and cancer. Preparation of the lipid substrate is crucial for the development of a robust and miniaturizable lipid kinase assay. Enzymatic assays for phosphoinositide kinases often use lipid substrates prepared from lyophilized lipid preparations by sonication, which result in variability in the liposome size from preparation to preparation. Herein, we report a homogeneous 1536-well luciferase-coupled bioluminescence assay for PI5P4Kα. The substrate preparation is novel and allows the rapid production of a DMSO-containing substrate solution without the need for lengthy liposome preparation protocols, thus enabling the scale-up of this traditionally difficult type of assay. The Z'-factor value was greater than 0.7 for the PI5P4Kα assay, indicating its suitability for high-throughput screening applications. Tyrphostin AG-82 had been identified as an inhibitor of PI5P4Kα by assessing the degree of phospho transfer of γ-(32P-ATP to PI5P; its inhibitory activity against PI5P4Kα was confirmed in the present miniaturized assay. From a pilot screen of a library of bioactive compounds, another tyrphostin, I-OMe tyrphostin AG-538 (I-OMe-AG-538, was identified as an ATP-competitive inhibitor of PI5P4Kα with an IC(50 of 1 µM, affirming the suitability of the assay for inhibitor discovery campaigns. This homogeneous assay may apply to other lipid kinases and should help in the identification of leads for this class of enzymes by enabling high-throughput screening efforts.

  19. Accessibility of different histone H3-binding domains of UHRF1 is allosterically regulated by phosphatidylinositol 5-phosphate.

    Gelato, K.; Tauber, M; Ong, M; Winter, S.; Hamada, K; Sindlinger, J.; Lemak, A.; Bultsma, Y.; Houliston, S.; Schwarzer, D; Divecha, N.; C. Arrowsmith; Fischle, W

    2014-01-01

    UHRF1 is a multidomain protein crucially linking histone H3 modification states and DNA methylation. While the interaction properties of its specific domains are well characterized, little is known about the regulation of these functionalities. We show that UHRF1 exists in distinct active states, binding either unmodified H3 or the H3 lysine 9 trimethylation (H3K9me3) modification. A polybasic region (PBR) in the C terminus blocks interaction of a tandem tudor domain (TTD) with H3K9me3 by occ...

  20. Accessibility of different histone H3-binding domains of UHRF1 is allosterically regulated by phosphatidylinositol 5-phosphate.

    Gelato, Kathy A; Tauber, Maria; Ong, Michelle S; Winter, Stefan; Hiragami-Hamada, Kyoko; Sindlinger, Julia; Lemak, Alexander; Bultsma, Yvette; Houliston, Scott; Schwarzer, Dirk; Divecha, Nullin; Arrowsmith, Cheryl H; Fischle, Wolfgang

    2014-06-19

    UHRF1 is a multidomain protein crucially linking histone H3 modification states and DNA methylation. While the interaction properties of its specific domains are well characterized, little is known about the regulation of these functionalities. We show that UHRF1 exists in distinct active states, binding either unmodified H3 or the H3 lysine 9 trimethylation (H3K9me3) modification. A polybasic region (PBR) in the C terminus blocks interaction of a tandem tudor domain (TTD) with H3K9me3 by occupying an essential peptide-binding groove. In this state the plant homeodomain (PHD) mediates interaction with the extreme N terminus of the unmodified H3 tail. Binding of the phosphatidylinositol phosphate PI5P to the PBR of UHRF1 results in a conformational rearrangement of the domains, allowing the TTD to bind H3K9me3. Our results define an allosteric mechanism controlling heterochromatin association of an essential regulatory protein of epigenetic states and identify a functional role for enigmatic nuclear phosphatidylinositol phosphates. PMID:24813945

  1. Targeting DXP synthase in human pathogens: enzyme inhibition and antimicrobial activity of butylacetylphosphonate

    Smith, Jessica M.; Warrington, Nicole V.; Vierling, Ryan J.; Kuhn, Misty L.; Anderson, Wayne F; Koppisch, Andrew T.; Freel Meyers, Caren L.

    2013-01-01

    The unique methylerythritol phosphate (MEP) pathway for isoprenoid biosynthesis is essential in most bacterial pathogens. The first enzyme in this pathway, 1-deoxy-D-xylulose 5-phosphate (DXP) synthase, catalyzes a distinct thiamin diphosphate (ThDP)-dependent reaction to form DXP from D-glyceraldehyde 3-phosphate (D-GAP) and pyruvate and represents a potential anti-infective drug target. We have previously demonstrated that the unnatural bisubstrate analog, butylacetylphosphonate (BAP), exhi...

  2. Crystal Structure Analyses of the Fosmidomycin-Target Enzyme from Plasmodium Falciparum

    Umeda, Tomonobu; Kusakabe, Yoshio; Tanaka, Nobutada

    The human malaria parasite Plasmodium falciparum is responsible for the death of more than a million people each year. Fosmidomycin has proved to be efficient in the treatment of P. falciparum malaria through the inhibition of 1-deoxy-D-xylulose 5-phosphate reductoisomerase (DXR), an enzyme of the non-mevalonate pathway of isoprenoid biosynthesis, which is absent in humans. Crystal structure analyses of P. falciparum DXR (PfDXR) revealed that (i) an intrinsic flexibility of the PfDXR molecule accounts for the induced-fit movement to accommodate the bound inhibitor in the active site, and (ii) a cis arrangement of the oxygen atoms of the hydroxamate group of the bound inhibitor is essential for tight binding of the inhibitor to the active site metal. We believe that our study will serve as a useful guide to develop more potent PfDXR inhibitors.

  3. Biosynthesis of β-carotene in engineered E. coli using the MEP and MVA pathways

    Yang, Jianming; Guo, Lizhong

    2014-01-01

    Background β-carotene is a carotenoid compound that has been widely used not only in the industrial production of pharmaceuticals but also as nutraceuticals, animal feed additives, functional cosmetics, and food colorants. Currently, more than 90% of commercial β-carotene is produced by chemical synthesis. Due to the growing public concern over food safety, the use of chemically synthesized β-carotene as food additives or functional cosmetic agents has been severely controlled in recent years...

  4. A STUDY OF FVC, PEFR AND MEP IN DIFFERENT TRIMESTERS OF PREGNANCY

    2012-01-01

    Objective : Pregnancy is characterized by profound changes in the function of virtually every regulatory system in the human body. The events in pregnancy elicit one of the best examples of selective anatomical, physiological & biochemical adaptation that occur during pregnancy & profound changes in respiratory physiology is a part of the same process. Thus this study was designed to evaluate the pulmonary function tests in 1st, 2nd and 3rd trimesters of pregnancy & compare them w...

  5. 77 FR 12041 - Applications for New Awards; Migrant Education Program (MEP) Consortium Incentive Grants Program

    2012-02-28

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

  6. 78 FR 34346 - Proposed Information Collection; Comment Request; NIST MEP Advanced Manufacturing Jobs and...

    2013-06-07

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

  7. Working at home: MEPs day-to-day practice of political representation in their constituency

    Poyet, Corentin

    2015-01-01

    International audience In early 2000s, Simon Hix and his colleagues declared EP constitutes a good laboratory to test theories and hypotheses about legislative or party behavior. However, scholars mainly focused on roll-call votes analysis allowing them to investigate voting behavior, coalitions formation as well as activities in technical committees (Hix 2001; Hix, Noury and Roland 2007; Kreppel 2007; Mammoudh and Raunio 2003; McElroy 2006). In this paper, we propose to go further to anal...

  8. OPEC – What Difference has it Made?: OIES paper: MEP 3

    Fattouh, Bassam; Mahadeva, Lavan

    2013-01-01

    The main purpose of this paper is to review the evolution of OPEC models and to link this evolution to some key events in the oil market. Our main conclusion is that OPEC’s pricing power varies over time. There are many instances in which OPEC can lose the power to limit oil price movements – either up or down. Such changes in pricing power are brought about by market conditions and can occur both in weak and tight market conditions. Because of OPEC’s varying conduct, there is no single model...

  9. The Implications of the Arab Uprisings for Oil and Gas Markets: OIES paper: MEP 2

    Fattouh, Bassam; Darbouche, Hakim

    2011-01-01

    The events that took place in the Arab world in the opening months of 2011 mark a watershed in the history of the Middle East and North Africa (MENA) region. Given the importance of MENA energy supplies in global economic terms, the political unrest witnessed by the region has caused widespread fears about the prospect of energy supply disruptions. With international oil and gas prices beginning to rise from 2010, there was serious concern among market and political actors that any further in...

  10. Energy Poverty in the Arab World: The Case of Yemen: OIES paper: MEP 1

    Fattouh, Bassam; El-Katiri, Laura

    2011-01-01

    While much of the emphasis of the literature on energy poverty is on the prevalence of the phenomenon in sub-Saharan Africa and South Asia, little has been written about energy poverty in the Arab world. Traditionally having being seen as one of the world’s most energy rich regions, the Arab world has in recent years often been overlooked as a region which suffers severely from energy poverty itself. In 2002, about 65 million people in the Arab world had no access to electricity, and an addi...

  11. MEPS 2.0? EUROPARLIAMENTARIANS TALKING TO VOTERS IN THE INTERNET ERA

    Stefano Braghiroli

    2010-09-01

    Full Text Available This paper systematically looks at the nature of MEPs’ internet-based “web tools” in the past EP legislature and at the extent to which their features reflect the complex nature of the EP environment (“Europeanization of communication”. To conduct this operation, a variety of structural and graphic features of MEPs’ websites have been identified, which have been made statistically analyzable, following a process of standardization and categorization, and were finally collected into a unique dataset. The preliminary figures obtained have been then controlled for a wide array of pluri-dimensional factors, operating both at micro-/individual-level and at macro-/country-level. Conceived as an explorative study towards clearer and more accurate understanding of MEPs’ internet-based communication styles and political strategies, our analysis aims at providing a stepping stone for further investigation in this direction.

  12. Development of MEPS-UHPLC-MS/MS multistatin methods for clinical analysis

    Vlčková, H.; Svoboda, P.; Novák, Ondřej; Solich, P.; Nováková, L.

    2016-01-01

    Roč. 8, č. 4 (2016), s. 333-349. ISSN 1757-6180 R&D Projects: GA MŠk(CZ) LO1204 Institutional support: RVO:61389030 Keywords : bioanalysis * dwell time * interconversion Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 3.003, year: 2014

  13. Airborne Survey Capacity Building of National Nuclear Safety Administration (MEP) in China

    Airborne survey is being paid more and attention in the nuclear radiation environment monitoring due to its unique advantages, especially monitoring due to its unique advantages, especially after the nuclear accident of Fukushima Japan. Thus, National Nuclear Safety Administration is strengthening to build airborne survey capacity. The administration has set up an advanced airborne survey system and established expert team. This airborne survey system here is fixed under a capable helicopter, which has a monitoring volume of 75.6 liters, independent advanced digital spectrometer and intelligent data processing functions. In this paper, a way that is applied for wireless data real-time transmission is presented, and our research works on calibration and the survey methods are also included. The airborne survey system can be widely used in the nuclear and radiation accidents monitoring and relative radiation monitoring in NORM. (author)

  14. Identification of novel HBV core-interacting partners PRMT5 and MEP50

    Lubyová, Barbora; Hodek, Jan; Prouzová, Hana; Hubálek, Martin; Weber, Jan

    Ontario: American Society for Virology, 2015. P29-03. [ASV 2015. Annual Meeting of the American Society for Virology /34./. 11.07.2015-15.07.2015, Ontario] R&D Projects: GA MŠk(CZ) LK11207 Institutional support: RVO:61388963 Keywords : HBV core protein * arginine methyltransferase * protein-protein interactions Subject RIV: EE - Microbiology, Virology

  15. Advanced In-Space Propulsion (AISP): Micro Electrospray Propulsion (MEP) Project

    National Aeronautics and Space Administration — Propulsion technology is often critical for space missions. High-value missions could be done with very small spacecraft, even CubeSats, but these...

  16. An insight into the sequential, structural and phylogenetic properties of banana 1-aminocyclopropane-1-carboxylate synthase 1 and study of its interaction with pyridoxal-5'-phosphate and aminoethoxyvinylglycine

    Swarup Roy Choudhury; Sanjay Kumar Singh; Sujit Roy; Dibyendu N Sengupta

    2010-06-01

    In banana, ethylene production for ripening is accompanied by a dramatic increase in 1-aminocyclopropane-1-carboxylate (ACC) content, transcript level of Musa acuminata ACC synthase 1 (MA-ACS1) and the enzymatic activity of ACC synthase 1 at the onset of the climacteric period. MA-ACS1 catalyses the conversion of -adenosyl-L-methionine (SAM) to ACC, the key regulatory step in ethylene biosynthesis. Multiple sequence alignments of 1-aminocyclopropane-1-carboxylate synthase (ACS) amino acid sequences based on database searches have indicated that MA-ACS1 is a highly conserved protein across the plant kingdom. This report describes an in silico analysis to provide the first important insightful information about the sequential, structural and phylogenetic characteristics of MA-ACS1. The three-dimensional structure of MA-ACS1, constructed based on homology modelling, in combination with the available data enabled a comparative mechanistic analysis of MA-ACS1 to explain the catalytic roles of the conserved and non-conserved active site residues. We have further demonstrated that, as in apple and tomato, banana-ACS1 (MA-ACS1) forms a homodimer and a complex with cofactor pyridoxal-5′-phosphate (PLP) and inhibitor aminoethoxyvinylglycine (AVG). We have also predicted that the residues from the PLP-binding pocket, essential for ligand binding, are mostly conserved across the MA-ACS1 structure and the competitive inhibitor AVG binds at a location adjacent to PLP.

  17. Vitamin B6 nutritional status and cellular availability of pyridoxal 5'-phosphate govern the function of the transsulfuration pathway's canonical reactions and hydrogen sulfide production via side reactions.

    Gregory, Jesse F; DeRatt, Barbara N; Rios-Avila, Luisa; Ralat, Maria; Stacpoole, Peter W

    2016-07-01

    The transsulfuration pathway (TS) acts in sulfur amino acid metabolism by contributing to the regulation of cellular homocysteine, cysteine production, and the generation of H2S for signaling functions. Regulation of TS pathway kinetics involves stimulation of cystathionine β-synthase (CBS) by S-adenosylmethionine (SAM) and oxidants such as H2O2, and by Michaelis-Menten principles whereby substrate concentrations affect reaction rates. Although pyridoxal phosphate (PLP) serves as coenzyme for both CBS and cystathionine γ-lyase (CSE), CSE exhibits much greater loss of activity than CBS during PLP insufficiency. Thus, cellular and plasma cystathionine concentrations increase in vitamin B6 deficiency mainly due to the bottleneck caused by reduced CSE activity. Because of the increase in cystathionine, the canonical production of cysteine (homocysteine → cystathionine → cysteine) is largely maintained even during vitamin B6 deficiency. Typical whole body transsulfuration flux in humans is 3-7 μmol/h per kg body weight. The in vivo kinetics of H2S production via side reactions of CBS and CSE in humans are unknown but they have been reported for cultured HepG2 cells. In these studies, cells exhibit a pronounced reduction in H2S production capacity and rates of lanthionine and homolanthionine synthesis in deficiency. In humans, plasma concentrations of lanthionine and homolanthionine exhibit little or no mean change due to 4-wk vitamin B6 restriction, nor do they respond to pyridoxine supplementation of subjects in chronically low-vitamin B6 status. Wide individual variation in responses of the H2S biomarkers to such perturbations of human vitamin B6 status suggests that the resulting modulation of H2S production may have physiological consequences in a subset of people. Supported by NIH grant DK072398. This paper refers to data from studies registered at clinicaltrials.gov as NCT01128244 and NCT00877812. PMID:26765812

  18. Complexation of neptunium(V) by salicylate, phthalate and citrate ligands in a pH 7.5 phosphate buffered system

    Conditional stability constants, enthalpies and entropies of complexation at pH 7.5 and ionic strength 0.1 have been determined for neptunium(V) complexes of phosphate, salicylate, phthalate and citrate. Results are given and discussed. At pH 7.5 salicylate does not form a complex with neptunium(V) due to the low charge density of the NpO2+ ion and incomplete ionization of the salicylate ion. In all cases, only 1:1 complexes were identified. (U.K.)

  19. Characterization of the TDP-d-ravidosamine biosynthetic pathway: one-pot enzymatic synthesis of TDP-d-ravidosamine from thymidine-5-phosphate and glucose-1-phosphate†

    Kharel, Madan K.; Lian, Hui; Rohr, Jürgen

    2011-01-01

    Ravidomycin V and related compounds, e.g., FE35A-B, exhibit potent anticancer activities against various cancer cell lines in the presence of visible light. The amino sugar moieties (d-ravidosamine and its analogues, respectively) in these molecules contribute to the higher potencies of ravidomycin and analogues when compared to closely related compounds with neutral or branched sugars. Within the ravidomycin V biosynthetic gene cluster, five putative genes encoding NDP-d-ravidosamine biosynt...

  20. De novo fragment-based design of inhibitors of DXS guided by spin-diffusion-based NMR spectroscopy.

    Masini, T.; Pilger, J.; Kroezen, B.; Illarionov, B.; Lottmann, P.; Fischer, M.; Griesinger, C.; Hirsch, A.

    2014-01-01

    We applied for the first time an innovative ligand-based NMR methodology (STI) to a medicinal-chemistry project aimed at the development of inhibitors for the enzyme 1-deoxy-D-xylulose-5-phosphate synthase (DXS). DXS is the first enzyme of the 2C-methyl-D-erythritol-4-phosphate (MEP) pathway, present in most bacteria (and not in humans) and responsible for the synthesis of the essential isoprenoid precursors. We designed de novo a first generation of fragments, using Deinococcus radiodurans D...

  1. The Arab Uprisings and MENA Political Instability – Implications for Oil & Gas Markets: OIES paper: MEP 8

    El-Katiri, Laura; Fattouh, Bassam; Mallinson, Richard

    2014-01-01

    The political turmoil that has swept across many parts of the Middle East and North Africa (MENA) since the beginning of the Arab Spring in December 2010 and the tightening of international sanctions against Iran in 2012 have reignited the recurring debate about energy security and the reliability of MENA as an energy supplier. In this paper, we examine the impact of the past three years of political turmoil in MENA on oil and gas markets. We argue that although many disruptions did occur and...

  2. BIM-lean synergies in the management on MEP works in public facilities of intensive use – a case study

    Clemente, José, 1720-1798; Cachadinha, Nuno

    2013-01-01

    21st Annual Conference of the International Group for Lean Construction – IGLC 21 – Fortaleza, Brazil AEC industry has been known for budget overruns and delays for a long time. One important reason for weak performances is the difficulty to visualize the production flow, and the deficient information transmission between the different stakeholders involved in a construction project. The use of 3D models plays a significant role in facilitating the implementation of Lean principles, ...

  3. Guangxi Nanguo Copper’s150,000 t/a Copper Smelting Project was Approval by the MEP

    2013-01-01

    <正>In the middle of August,the environmental impact report for 150,000 t/a copper smelting project of Guangxi Nanguo Copper Industry Co.,Ltd received formal approval from the Ministry of Environmental Protection,signaling that this project’s environmental impact

  4. Enzymes in the Mycobacterium tuberculosis MEP and CoA Pathways Targeted for Structure-Based Drug Design

    Björkelid, Christofer

    2012-01-01

    Tuberculosis, caused by the pathogenic bacteria Mycobacterium tuberculosis, is one of the most widespread and deadly infectious diseases today. Treatment of tuberculosis relies on antibiotics that were developed more than 50 years ago. These are now becoming ineffective due to the emergence of antibiotic resistant strains of the bacteria. The aim of the research in this thesis was to develop new antibiotics for tuberculosis treatment. To this end, we targeted enzymes from two essential biosyn...

  5. Estonian President and ex-MEP Toomas Ilves : new states "open to change" / Toomas Hendrik Ilves ; [interv. Kristiina Randmaa

    Ilves, Toomas Hendrik, 1953-

    2007-01-01

    President T.H. Ilvese intervjuu Euroopa Parlamendi internetiväljaandele. President räägib oma töökogemustest Euroopa Parlamendi saadikuna, Euroopa Liidu uute liikmesriikide erinevustest, Euroopa Liidu ja Venemaa suhetest, Eesti infotehnoloogilisest arengust

  6. Predicted essential proteins ofPlasmodium falciparum for potential drug targets

    Qing-Feng He; Li Deng; Qin-Ying Xu; Zheng Shao

    2012-01-01

    ABSTRACT Objective:To identify novel drug targets for treatment ofPlasmodium falciparum.Methods:LocalBLASTP were used to find the proteins non-homologous to human essential proteins as novel drug targets. Functional domains of novel drug targets were identified by InterPro and Pfam,3D structures of potential drug targets were predicated by theSWISS-MODELworkspace. Ligands and ligand-binding sites of the proteins were searched byEf-seek.Results:Three essential proteins were identified that might be considered as potential drug targets.AAN37254.1 belonged to1-deoxy-D-xylulose5-phosphate reductoisomerase,CAD50499.1 belonged to chorismate synthase,CAD51220.1 belonged toFAD binging3 family, but the function of CAD51220.1 was unknown. The3D structures, ligands and ligand-binding sites ofAAN37254.1 andCAD50499.1 were successfully predicated.Conclusions:Two of these potential drug targets are key enzymes in2-C-methyl-d-erythritol4-phosphate pathway and shikimate pathway, which are absent in humans, so these two essential proteins are good potential drug targets. The function and3D structures ofCAD50499.1 is still unknown, it still need further study.

  7. Enhanced Production of Bioactive Isoprenoid Compounds from Cell Suspension Cultures of Artemisia annua L. Using β-Cyclodextrins

    Francesca Rizzello

    2014-10-01

    Full Text Available Plant cell cultures as valuable tools for the production of specific metabolites can be greatly improved by the application of elicitors including cyclodextrins (CDs for enhancing the yields of the desired plant compounds. Here the effects of 2,6-dimethyl-β-cyclodextrins (DIMEB on the production of carotenoids and quinones from Artemisia annua L. cell suspension cultures were investigated. The addition of 50 mM DIMEB induced an early increase of intracellular carotenoid and quinone contents, which could be observed to a higher extent for lutein (10-fold, Q9 (3-fold and Q10 (2.5-fold. Real Time PCR analysis revealed that the expression of 1-deoxy-d-xylulose-5-phosphate reductoisomerase (DXR gene in DIMEB treated cell cultures after three days was 2.5-fold higher than in untreated samples, thus suggesting that the DIMEB induced increase of carotenoids and quinones could be due to the induction of the plastidial isoprenoid biosynthetic route. In addition, the DIMEB treatment induced an enhanced release of carotenoids and quinones into the culture medium of A. annua cell suspension cultures possibly due to the ability of CDs to form inclusion complexes with hydrophobic molecules.

  8. Enhanced production of bioactive isoprenoid compounds from cell suspension cultures of Artemisia annua L. using β-cyclodextrins.

    Rizzello, Francesca; De Paolis, Angelo; Durante, Miriana; Blando, Federica; Mita, Giovanni; Caretto, Sofia

    2014-01-01

    Plant cell cultures as valuable tools for the production of specific metabolites can be greatly improved by the application of elicitors including cyclodextrins (CDs) for enhancing the yields of the desired plant compounds. Here the effects of 2,6-dimethyl-β-cyclodextrins (DIMEB) on the production of carotenoids and quinones from Artemisia annua L. cell suspension cultures were investigated. The addition of 50 mM DIMEB induced an early increase of intracellular carotenoid and quinone contents, which could be observed to a higher extent for lutein (10-fold), Q9 (3-fold) and Q10 (2.5-fold). Real Time PCR analysis revealed that the expression of 1-deoxy-d-xylulose-5-phosphate reductoisomerase (DXR) gene in DIMEB treated cell cultures after three days was 2.5-fold higher than in untreated samples, thus suggesting that the DIMEB induced increase of carotenoids and quinones could be due to the induction of the plastidial isoprenoid biosynthetic route. In addition, the DIMEB treatment induced an enhanced release of carotenoids and quinones into the culture medium of A. annua cell suspension cultures possibly due to the ability of CDs to form inclusion complexes with hydrophobic molecules. PMID:25338048

  9. Enhanced Production of Bioactive Isoprenoid Compounds from Cell Suspension Cultures of Artemisia annua L. Using β-Cyclodextrins

    Rizzello, Francesca; De Paolis, Angelo; Durante, Miriana; Blando, Federica; Mita, Giovanni; Caretto, Sofia

    2014-01-01

    Plant cell cultures as valuable tools for the production of specific metabolites can be greatly improved by the application of elicitors including cyclodextrins (CDs) for enhancing the yields of the desired plant compounds. Here the effects of 2,6-dimethyl-β-cyclodextrins (DIMEB) on the production of carotenoids and quinones from Artemisia annua L. cell suspension cultures were investigated. The addition of 50 mM DIMEB induced an early increase of intracellular carotenoid and quinone contents, which could be observed to a higher extent for lutein (10-fold), Q9 (3-fold) and Q10 (2.5-fold). Real Time PCR analysis revealed that the expression of 1-deoxy-d-xylulose-5-phosphate reductoisomerase (DXR) gene in DIMEB treated cell cultures after three days was 2.5-fold higher than in untreated samples, thus suggesting that the DIMEB induced increase of carotenoids and quinones could be due to the induction of the plastidial isoprenoid biosynthetic route. In addition, the DIMEB treatment induced an enhanced release of carotenoids and quinones into the culture medium of A. annua cell suspension cultures possibly due to the ability of CDs to form inclusion complexes with hydrophobic molecules. PMID:25338048

  10. Inhibition of alanine racemase by alanine phosphonate: detection of an imine linkage to pyridoxal 5'-phosphate in the enzyme-inhibitor complex by solid-state 15N nuclear magnetic resonance

    Inhibition of alanine racemase from the Gram-positive bacterium Bacillus stearothermophilus by (1-aminoethyl)phosphonic acid (Ala-P) proceeds via a two-step reaction pathway in which reactivation occurs very slowly. In order to determine the mechanism of inhibition, the authors have recorded low-temperature, solid-state 15N NMR spectra from microcrystals of the [15N]Ala-P-enzyme complex, together with spectra of a series of model compounds that provide the requisite database for the interpretation of the 15N chemical shifts. Proton-decoupled spectra of the microcrystals exhibit a line at ∼ 150 ppm, which conclusively demonstrates the presence of a protonated Ala-P-PLP aldimine and thus clarifies the structure of the enzyme-inhibitor complex. They also report the pH dependence of Ala-P binding to alanine racemase

  11. Effects of mutagenesis of aspartic acid residues in the putative phosphoribosyl diphosphate binding site of Escherichia coli phosphoribosyl diphosphate synthetase on metal ion specificity and ribose-5-phosphate binding

    Willemoës, Martin; Nilsson, Dan; Hove-Jensen, Bjarne

    1996-01-01

    The three conserved aspartic acid residues of the 5-phospho-d-ribosyl a-1-diphosphate binding site (213-GRDCVLVDDMIDTGGT-228) of Escherichia coli phosphoribosyl diphosphate synthetase were studied by analysis of the mutant enzymes D220E, D220F, D221A, D224A, and D224S. The mutant enzymes showed a...

  12. Measuring the absolute disintegration rate of a radioactive gas with a moveable endplate discharge counter (MEP) and theoretical calculation of wall effect

    Jaffey, A.H.; Gray, J.; Bentley, W.C.; Lerner, J.L.

    1987-09-01

    A precision built moveable endplate Geiger-Mueller counter was used to measure the absolute disintegration rate of a beta-emitting radioactive gas. A Geiger-Mueller counter used for measuring gaseous radioactivity has <100% counting efficiency owing to two factors: (1) ''end effect,'' due to decreased and distorted fields at the ends where wire-insulator joints are placed, and (2) ''wall effect,'' due to non-ionization by beta particles emitted near to and heading into the wall. The end effect was evaluated by making one end of the counter movable and measuring counting rates at a number of endplate positions. Much of the wall effect was calculated theoretically, based on known data for primary ionization of electrons as a function of energy and gas composition. Corrections were then made for the ''shakeoff'' effect in beta decay and for backscattering of electrons from the counter wall. Measurements and calculations were made for a sample of /sup 85/Kr (beta energy, 0.67 MeV). The wall effect calculation is readily extendable to other beta energies.

  13. Measuring the absolute disintegration rate of a radioactive gas with a moveable endplate discharge counter (MEP) and theoretical calculation of wall effect

    A precision built moveable endplate Geiger-Mueller counter was used to measure the absolute disintegration rate of a beta-emitting radioactive gas. A Geiger-Mueller counter used for measuring gaseous radioactivity has 85Kr (beta energy, 0.67 MeV). The wall effect calculation is readily extendable to other beta energies

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

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

    2014-02-01

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

  15. Detection of biogenic amines in urine and plasma by liquid chromatography coupled to electrochemical detection HPLC-ED using microextraction in packed syringe MEPS

    Oppolzer, David Jerónimo

    2012-01-01

    Biogenic amines are neurotransmitters involved in several physiological processes. Changes and disturbs in their function are associated to several neurological disorders or diseases, as well as consumption of psychotherapeutic or psychotropic drugs. The detection of these compounds in biological fluids is therefore of clinical and neurochemical interest. The goal of this work was to develop and validate and analytical method for the detection and quantification of the biogenic...

  16. Price Reform in Kuwait’s Electricity and Water Sector – Assessing the Net Benefits in the Presence of Congestion: OIES paper: MEP 9

    Mahadeva, Lavan; Fattouh, Bassam

    2014-01-01

    Kuwait’s domestic electricity and water sector has been in disarray for several years, struggling with fast-rising demand for several decades as a result of rapid industrialization, population growth, rising living standards as well as due to the artificially low utility prices set by the government. We use a model-based methodology to compare the current pricing scheme against an alternative where consumer prices are raised to market levels and consumers are on average compensated by cash tr...

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

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

    2016-02-01

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

  18. Análisis energético de CYPECAD-MEP y CE3x en la edificación

    Alarcón Blaya, Juan Antonio

    2014-01-01

    Con el objeto de promover la eficiencia energética de los edificios, la Directiva 2002/91/CE y posteriormente la Directiva 2010/31/UE, exigía a los Estados miembros el establecimiento de un procedimiento de certificación dirigido a los edificios, que pusiese a disposición del posible comprador o inquilino, una información objetiva sobre el consumo energético de la vivienda. El requisito de la eficiencia energética es alcanzar unos niveles óptimos de rentabilidad en la viv...

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

    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

  20. Effects of Gibberellic Acid on Primary Terpenoids and △9-Tetrahydrocannabinol in Cannabis sativa at Flowering Stage

    Hakimeh Mansouri; Zahra Asrar; Mitra Mehrabani

    2009-01-01

    Plants synthesize an astonishing diversity of isoprenoids, some of which play essential roles in photosynthesis, respiration,and the regulation of growth and development. Two independent pathways for the biosynthesis of isoprenoid precursors coexist within the plant cell: the cytosolic mevalonic acid (MVA) pathway and the plastidial methylerythritol phosphate (MEP)pathway. However, little is known about the effects of plant hormones on the regulation of these pathways. In the present study we investigated the effect of gibberellic acid (GA3) on changes in the amounts of many produced terpenoids and the activity of the key enzymes, 1-deoxy-D-xylulose 5-phosphate synthase (DXS) and 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGR), in these pathways. Our results showed GA3 caused a decrease in DXS activity in both sexes that it wasaccompanied by a decrease in chlorophylls, carotenoids and △9-tetrahydrocannabinol (THC) contents and an increase in α-tocopherol content. The treated plants with GA3 showed an increase in HMGR activity. This increase in HMGR activity was followed by accumulation of stigmasterol and β-sitosterol in male and female plants and campestrol in male plants.The pattern of the changes in the amounts of sterols was exactly similar to the changes in the HMGR activity. These data suggest that GA3 can probably influence the MEP and MVA pathways oppositely, with stimulatory and inhibitory effects on the produced primary terpenoids in MVA and DXS pathways, respectively.

  1. VOC emissions of Grey poplar leaves as affected by salt stress and different N sources.

    Teuber, M; Zimmer, I; Kreuzwieser, J; Ache, P; Polle, A; Rennenberg, H; Schnitzler, J-P

    2008-01-01

    Nitrogen nutrition and salt stress experiments were performed in a greenhouse with hydroponic-cultured, salt-sensitive Grey poplar (Populus x canescens) plants to study the combined influence of different N sources (either 1 mm NO(3) (-) or NH(4)(+)) and salt (up to 75 mm NaCl) on leaf gas exchange, isoprene biosynthesis and VOC emissions. Net assimilation and transpiration proved to be highly sensitive to salt stress and were reduced by approximately 90% at leaf sodium concentrations higher than 1,800 microg Na g dry weight (dw)(-1). In contrast, emissions of isoprene and oxygenated VOC (i.e. acetaldehyde, formaldehyde and acetone) were unaffected. There was no significant effect of combinations of salt stress and N source, and neither NO(3)(-) or NH(4)(+) influenced the salt stress response in the Grey poplar leaves. Also, transcript levels of 1-deoxy-d-xylulose 5-phosphate reductoisomerase (PcDXR) and isoprene synthase (PcISPS) did not respond to the different N sources and only responded slightly to salt application, although isoprene synthase (PcISPS) activity was negatively affected at least in one of two experiments, despite high isoprene emission rates. A significant salt effect was the strong reduction of leaf dimethylallyl diphosphate (DMADP) content, probably due to restricted availability of photosynthates for DMADP biosynthesis. Further consequences of reduced photosynthetic gas exchange and maintaining VOC emissions are a very high C loss, up to 50%, from VOC emissions related to net CO(2) uptake and a strong increase in leaf internal isoprene concentrations, with maximum mean values up to 6.6 microl x l(-1). Why poplar leaves maintain VOC biosynthesis and emission under salt stress conditions, despite impaired photosynthetic CO(2) fixation, is discussed. PMID:18211549

  2. Relative expression of genes of terpene metabolism in different tissues of Artemisia annua L

    Lundgren Anneli

    2011-03-01

    Full Text Available Abstract Background Recently, Artemisia annua L. (annual or sweet wormwood has received increasing attention due to the fact that the plant produces the sesquiterpenoid endoperoxide artemisinin, which today is widely used for treatment of malaria. The plant produces relatively small amounts of artemisinin and a worldwide shortage of the drug has led to intense research in order to increase the yield of artemisinin. In order to improve our understanding of terpene metabolism in the plant and to evaluate the competition for precursors, which may influence the yield of artemisinin, we have used qPCR to estimate the expression of 14 genes of terpene metabolism in different tissues. Results The four genes of the artemisinin biosynthetic pathway (amorpha-4,11-diene synthase, amorphadiene-12-hydroxylase, artemisinic aldehyde ∆11(13 reductase and aldehyde dehydrogenase 1 showed remarkably higher expression (between ~40- to ~500-fold in flower buds and young leaves compared to other tissues (old leaves, stems, roots, hairy root cultures. Further, dihydroartemisinic aldehyde reductase showed a very high expression only in hairy root cultures. Germacrene A and caryophyllene synthase were mostly expressed in young leaves and flower buds while epi-cedrol synthase was highly expressed in old leaves. 3-Hydroxy-3-methyl-glutaryl coenzyme A reductase exhibited lower expression in old leaves compared to other tissues. Farnesyldiphosphate synthase, squalene synthase, and 1-deoxy-D-xylulose-5-phosphate reductoisomerase showed only modest variation in expression in the different tissues, while expression of 1-deoxy-D-xylulose-5-phosphate synthase was 7-8-fold higher in flower buds and young leaves compared to old leaves. Conclusions Four genes of artemisinin biosynthesis were highly expressed in flower buds and young leaves (tissues showing a high density of glandular trichomes. The expression of dihydroartemisinic aldehyde reductase has been suggested to have a

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

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

    2013-11-01

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

  4. A STUDY ON THE RELATIONSHIP BETWEEN MAGNETIC STIMULATION MEP AND THE NEUROPATHOLOGY OF THE CHRONIC COMPRESSED LESION OF CAT’S CERVICAL NERVE ROOTS AND THE SIGNIFICANCE OF ITS QUANTITATIVE DIAGNOSIS

    杨大志; 郭玉霞; 陈君长; 赵龙柱; 王坤正; 杨哲; 李旭东; 袁国莲

    1999-01-01

    ThetechniqueofmagneticstimulationMEPhasprogressedrapidlysinceitcameintobeingin1985.Nowitisconsideredtobeabletoconfirmthelesio...

  5. Los textos literarios que asigna el MEP para el tercer ciclo de la educación general básica en Costa Rica: algunas reflexiones al respecto

    Arias Orozco, Grettel

    2016-05-01

    Full Text Available En el presente artículo se detallan y analizan las principales modificaciones que presenta la lista de lecturas obligatorias propuesta en el año 2010 e implementada por el Ministerio de Educación Pública a partir del año 2011, en el Tercer Ciclo de la Educación General Básica costarricense. El estudio fue realizado a partir de la búsqueda bibliográfica de revistas especializadas, tesis de grado, libros, los Planes de Estudios de Español del 2005 y del año 2009, la lista de lecturas obligatorias implementada en el 2011, así como también gracias a entrevistas a especialistas en el área de la educación costarricense. Se concluye que las principales modificaciones responden a la dosificación, la categorización y la flexibilidad del listado de lecturas, pues la cantidad de textos y los géneros literarios que se estudian por nivel se modifican, así como también ahora se le brinda una gama de opciones al docente para que elija qué leer con su grupo estudiantil; no obstante, también se evidencia que ninguno de estos cambios ha sido debidamente fundamentado en la nueva propuesta. Finalmente, se destaca la incorporación de un corpus significativo de textos literarios que hacen referencia al personaje del presidente Juan Mora Porras, cuyo fin se vislumbra de carácter identitario e ideológico, pues su propósito es contrarrestar el desencanto político que ha aumentado en los últimos años e instaurarse como el nuevo referente del héroe nacional.

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

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

    2015-08-01

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

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

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

    2015-09-01

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

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

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

    2016-03-01

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

  9. Intraoperative monitoring study of ipsilateral motor evoked potentials in scoliosis surgery

    Lo, Y. L.; Dan, Y. F.; Tan, Y. E.; Fook-Chong, S; Tan, S. B.; Tan, C T; Raman, S.

    2006-01-01

    Ipsilateral motor evoked potentials (MEPs) in spinal cord surgery intraoperative monitoring is not well studied. We show that ipsilateral MEPs have significantly larger amplitudes and were elicited with lower stimulation intensities than contralateral MEPs. The possible underlying mechanisms are discussed based on current knowledge of corticospinal pathways. Ipsilateral MEPs may provide additional information on the integrity of descending motor tracts during spinal surgery monitoring.

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

    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

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

    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

  12. The European Parliament's Rejection of the Take-over Directive Proposal

    Steen Knudsen, Jette

    2003-01-01

    national characteristics: labour marketlegislation (national schemes to protect employees against dismissals) and corporategovernance issues. Labour market legislation can explain the UK and German MEP votesbut not the Swedish and French MEPs votes. These votes can be explained byemphasising measures...

  13. Mars Technology Project

    National Aeronautics and Space Administration — NASA’s Mars Exploration Program (MEP) calls for a series of highly ambitious missions over the next decade and beyond. The overall goals of the MEP must be...

  14. Host cells and methods for production of isobutanol

    Anthony, Larry Cameron; He, Hongxian; Huang, Lixuan Lisa; Okeefe, Daniel P.; Kruckeberg, Arthur Leo; Li, Yougen; Maggio-Hall, Lori Ann; McElvain, Jessica; Nelson, Mark J.; Patnaik, Ranjan; Rothman, Steven Cary

    2016-08-23

    Provided herein are recombinant yeast host cells and methods for their use for production of isobutanol. Yeast host cells provided comprise an isobutanol biosynthetic pathway and at least one of reduced or eliminated aldehyde dehydrogenase activity, reduced or eliminated acetolactate reductase activity; or a heterologous polynucleotide encoding a polypeptide having ketol-acid reductoisomerase activity.

  15. The Carotenogenesis Pathway via the Isoprenoid-β-carotene Interference Approach in a New Strain of Dunaliella salina Isolated from Baja California Mexico

    Luis Enrique Gutierrez-Millan

    2009-02-01

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

  16. Protein (Cyanobacteria): 410899 [PGDBj - Ortholog DB

    Full Text Available LFAIPIASFWGRYSQIWAISHYPYLNKEGSSKFLHKKNWRGFLIESIPSYAFLSFLIFILINIDISIISTPNLIIGIIVGFLPALIIPHLLARRLGGHSGDSYGASVVLVETCMLIIFSIILPAS ... ...GVLQVSVLILLLQLQWPNESMPFIAIALGLWITGGIHVDGLMDTADGIAAGPSRCIEAMKDSRIGASGIIALTINLLLQIAALFKLRLLI...1 cobalamin-5-phosphate synthase Prochlorococcus marinus subsp. marinus str. CCMP1375 MSSPNWLKDLTGAWLFYTVFPKLTRINPRFERIARFSPLIGVLI

  17. Main: 1H1Y [RPSD[Archive

    Full Text Available 1H1Y イネ Rice Oryza sativa L. D-Ribulose-5-Phosphate ... 3-Epimerase Oryza Sativa Molecule: D-Ribul ... ose-5-Phosphate ... 3-Epimerase; Chain: A, B; Engineered: Yes Isomeras ... e 5.1.3.1 (D-Ribulose-5-Phosphate ... 3-Epimerase) S.Jelakovic, G.E.Schulz S.Jelakovic, ...

  18. Main: 1H1Z [RPSD[Archive

    Full Text Available 1H1Z イネ Rice Oryza sativa L. D-Ribulose-5-Phosphate ... 3-Epimerase Oryza Sativa Molecule: D-Ribul ... ose-5-Phosphate ... 3-Epimerase; Chain: A, B; Engineered: Yes Isomeras ... e 5.1.3.1 (D-Ribulose-5-Phosphate ... 3-Epimerase) S.Jelakovic, G.E.Schulz S.Jelakovic, ...

  19. Modulation of sensory inhibition of motor evoked potentials elicited by TMS prior to movement?

    Leukel, Christian; Lundbye-Jensen, Jesper; Nielsen, Jens Bo; Gollhofer, Albert; Taube, Wolfgang

    Short latency afferent inhibition (SAI) refers to a decrement of the size of a motor evoked potential (MEP) by transcranial magnetic stimulation (TMS) after electrical stimulation of a peripheral afferent nerve (PNS) (Tokimura et al. 2000). Since SAI occurs when TMS is applied at the time of the...... ± 9 %) in FDI (the muscle involved in the motor task) compared to MEPs at rest (p < 0.01). There was no change of the MEPs in APB. There were also no changes in either the FDI or APB MEPs for the other tested ISIs prior to movement compared to rest. The main finding of this study was that the MEP...... susceptibility of corticospinal cells to TMS, which starts approximately 100 ms prior to the onset of movement (Chen et al. 1998). Thus, it is hypothesized that the modulation of the MEP prior to movement is linked to the afferent volley arriving at the sensorimotor cortex. It might be speculated that the MEP...

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

    Hideomi eItoh

    2014-08-01

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

  1. pKNOT: the protein KNOT web server

    Lai, Yan-Long; Yen, Shih-Chung; Yu, Sung-Huan; Hwang, Jenn-Kang

    2007-01-01

    Knotted proteins are more commonly observed in recent years due to the enormously growing number of structures in the Protein Data Bank (PDB). Studies show that the knot regions contribute to both ligand binding and enzyme activity in proteins such as the chromophore-binding domain of phytochrome, ketol–acid reductoisomerase or SpoU methyltransferase. However, there are still many misidentified knots published in the literature due to the absence of a convenient web tool available to the gene...

  2. Can Motor Evoked Potentials Be an Objective Parameter to Assess Extremity Function at the Acute or Subacute Stroke Stage?

    Kim, Gi-Wook; Won, Yu Hui; Park, Sung-Hee; Seo, Jeong-Hwan; Ko, Myoung-Hwan

    2015-01-01

    Objective To investigate whether motor evoked potential (MEP) amplitude ratio measurements are sufficiently objective to assess functional activities of the extremities. We also delineated the distribution between the presence or absence of MEPs and the Medical Research Council (MRC) scale for muscle strength of the extremities. Methods We enrolled 183 patients with first-ever unilateral hemiplegia after stroke. The MEP parameters were amplitude ratio (amplitude of affected side/amplitude of ...

  3. An Investigation of Groundwater Flow on a Coastal Barrier using Multi Electrode Profiling

    Poulsen, Søren Erbs; Christensen, Steen; Rasmussen, Keld Rømer;

    2008-01-01

    Preliminary geophysical and hydrogeological investigations indicate that multi-electrode profiling (MEP) can be used to monitor groundwater salinity on a coastal barrier where a shallow thin aquifer discharges to the North Sea. A monitoring system including five groups of piezometers and five MEP...

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

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

    2013-01-01

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

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

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

    2016-01-01

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

  6. An orthologue of the cor gene is involved in the exclusion of temperate lambdoid phages. Evidence that Cor inactivates FhuA receptor functions

    A new set of lambdoid phages (mEp) classified into different immunity groups was previously described. Phages mEp213, mEp237, and mEp410 were unable to grow in mEp167 lysogenic cells, presumably due to an exclusion mechanism expressed constitutively by the mEp167 repressed prophage. In this work, to analyze the exclusion phenomenon, we constructed a genomic library from mEp167 phage in a pPROEX derivative plasmid. A DNA fragment containing an open reading frame for a 77 amino acid polypeptide was selected by its ability to confer resistance to heteroimmune phage infection. This ORF shows high amino acid sequence identity with putative Cor proteins of phages HK022, φ80 and N15. Cells expressing the mEp167 cor gene from a plasmid (Cor+ phenotype) excluded 13 of 20 phages from different infection immunity groups. This exclusion was observed in both tonB- and tonB+ cells. Lambdoid mEp phages that were excluded in these cells were unable to infect cells defective in the outer membrane FhuA receptor (fhuA-). Thus, Cor-mediated exclusion was only observed in fhuA+ cells. Phage production after DNA transfection or the spontaneous induction of mEp prophage in Cor+ cells was not blocked. In addition, ferrichrome uptake, which is mediated by FhuA, was inhibited in Cor+ cells. Our results show that not only phage infection via FhuA but also a FhuA transport activity (ferrichrome uptake) are inhibited by Cor, presumably by inactivation of FhuA

  7. Francisella tularensis 2-C-methyl-D-erythritol 4-phosphate cytidylyltransferase: kinetic characterization and phosphoregulation.

    Arthur Tsang

    Full Text Available Deliberate and natural outbreaks of infectious disease, the prevalence of antibiotic resistant strains, and the ease by which antibiotic resistant bacteria can be intentionally engineered all underscore the necessity of effective vaccines and continued development of novel antimicrobial/antiviral therapeutics. Isoprenes, a group of molecules fundamentally involved in a variety of crucial biological functions, are derived from either the mevalonic acid (MVA or methylerythritol phosphate (MEP pathway. While mammals utilize the MVA pathway, many bacteria utilize the MEP pathway, highlighting the latter as an attractive target for antibiotic development. In this report we describe the cloning and characterization of Francisella tularensis MEP cytidylyltransferase, a MEP pathway enzyme and potential target for antibiotic development. Size exclusion chromatography indicates the protein exists as a dimer in solution. Enzyme assays produced an apparentK(MEP(M = 178 μM, K(CTP(M = 73 μM , k(MEP(cat = 1(s-1, k(CTP(cat = 0.8( s-1, and a k(MEP(cat/ K(MEP(M = 3.4 x 10(5 M(-1 min(-1. The enzyme exhibits a strict preference for Mg(+2 as a divalent cation and CTP as the nucleotide. Titanium dioxide chromatography-tandem mass spectrometry identified Thr141 as a site of phosphorylation. T141D and T141E site-directed mutants are catalytically inactive, suggesting a mechanism for post-translational control of metabolic flux through the F. tularensis MEP pathway. Overall, our study suggests that MEP cytidylyltransferase is an excellent target for the development of novel antibiotics against F. tularensis.

  8. Binding of Divalent Magnesium by Escherichia coli Phosphoribosyl Diphosphate Synthetase

    Willemoës, Martin; Hove-Jensen, Bjarne

    1997-01-01

    The mechanism of binding of the substrates MgATP and ribose 5-phosphate as well as Mg2+ to the enzyme 5-phospho-d-ribosyl a-1-diphosphate synthetase from Escherichia coli has been analyzed. By use of the competive inhibitors of ATP and ribose 5-phosphate binding, a,ß-methylene ATP and (+)-1-a,2-a...

  9. Binding of divalent magnesium by Escherichia coli phosphoribosyl diphosphate synthetase

    Willemoës, Martin; Hove-Jensen, Bjarne

    1997-01-01

    The mechanism of binding of the substrates Mg x ATP and ribose 5-phosphate as well as Mg2+ to the enzyme 5-phospho-D-ribosyl (alpha-1-diphosphate synthetase from Escherichia coli has been analyzed. By use of the competive inhibitors of ATP and ribose 5-phosphate binding, alpha,beta-methylene ATP ...

  10. Enzymatic synthesis of 3-deoxy-d-manno-octulosonic acid (KDO) and its application for LPS assembly.

    Wen, Liuqing; Zheng, Yuan; Li, Tiehai; Wang, Peng George

    2016-06-15

    The studies of 3-deoxy-d-manno-octulosonic acid (KDO) have been hindered due to its limited availability. Herein, an efficient enzymatic system for the facile synthesis of KDO from easy-to-get starting materials is described. In this one-pot three-enzyme (OPME) system, d-ribulose 5-phosphate, which was prepared from d-xylose, was employed as starting materials. The reaction process involves the isomerization of d-ribulose 5-phosphate to d-arabinose 5-phosphate catalyzed by d-arabinose 5-phosphate isomerase (KdsD), the aldol condensation of d-arabinose 5-phosphate and phosphoenolpyruvate (PEP) catalyzed by KDO 8-phosphate synthetase (KdsA), and the hydrolysis of KDO-8-phosphate catalyzed by KDO 8-phosphate phosphatase (KdsC). By using this OPME system, 72% isolated yield was obtained. The obtained KDO was further transferred to lipid A by KDO transferase from Escherichia coli (WaaA). PMID:27173798

  11. Nucleic and amino acid sequences relating to a novel transketolase, and methods for the expression thereof

    Croteau, Rodney Bruce (Pullman, WA); Wildung, Mark Raymond (Colfax, WA); Lange, Bernd Markus (Pullman, WA); McCaskill, David G. (Pullman, WA)

    2001-01-01

    cDNAs encoding 1-deoxyxylulose-5-phosphate synthase from peppermint (Mentha piperita) have been isolated and sequenced, and the corresponding amino acid sequences have been determined. Accordingly, isolated DNA sequences (SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7) are provided which code for the expression of 1-deoxyxylulose-5-phosphate synthase from plants. In another aspect the present invention provides for isolated, recombinant DXPS proteins, such as the proteins having the sequences set forth in SEQ ID NO:4, SEQ ID NO:6 and SEQ ID NO:8. In other aspects, replicable recombinant cloning vehicles are provided which code for plant 1-deoxyxylulose-5-phosphate synthases, or for a base sequence sufficiently complementary to at least a portion of 1-deoxyxylulose-5-phosphate synthase DNA or RNA to enable hybridization therewith. In yet other aspects, modified host cells are provided that have been transformed, transfected, infected and/or injected with a recombinant cloning vehicle and/or DNA sequence encoding a plant 1-deoxyxylulose-5-phosphate synthase. Thus, systems and methods are provided for the recombinant expression of the aforementioned recombinant 1-deoxyxylulose-5-phosphate synthase that may be used to facilitate its production, isolation and purification in significant amounts. Recombinant 1-deoxyxylulose-5-phosphate synthase may be used to obtain expression or enhanced expression of 1-deoxyxylulose-5-phosphate synthase in plants in order to enhance the production of 1-deoxyxylulose-5-phosphate, or its derivatives such as isopentenyl diphosphate (BP), or may be otherwise employed for the regulation or expression of 1-deoxyxylulose-5-phosphate synthase, or the production of its products.

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

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

    2015-05-01

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

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

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

    2016-06-01

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

  14. APPLICATION OF MAXIMUM ENTROPY PRINCIPLE METHOD TO THE STUDY OF WAVE CLIMATE STATISTICAL CHARACTERISTICS

    XU Fu-min; XUE Hong-chao

    2004-01-01

    The Maximum Entropy Principle (MEP) method is elaborated, and the corresponding probability density evaluation method for the random fluctuation system is introduced, the goal of the article is to find the best fitting method for the wave climate statistical distribution. For the first time, a kind of new maximum entropy probability distribution (MEP distribution) expression is deduced in accordance with the second order moment of a random process. Different from all the fitting methods in the past, the MEP distribution can describe the probability distribution of any random fluctuation system conveniently and reasonably. If the moments of the random signal is limited to the second order, that is, the ratio of the root-mean-square value to the mean value of the random variable is obtained from the random sample, the corresponding MEP distribution can be computed according to the deduced expression in this essay. The concept of the wave climate is introduced here, and the MEP distribution is applied to fit the probability density distributions of the significant wave height and spectral peak period. Take the Mexico Gulf as an example, three stations at different locations, depths and wind wave strengths are chosen in the half-closed gulf, the significant wave height and spectral peak period distributions at each station are fitted with the MEP distribution, the Weibull distribution and the Log-normal distribution respectively, the fitted results are compared with the field observations, the results show that the MEP distribution is the best fitting method, and the Weibull distribution is the worst one when applied to the significant wave height and spectral peak period distributions at different locations, water depths and wind wave strengths in the Gulf. The conclusion shows the feasibility and reasonability of fitting wave climate statistical distributions with the deduced MEP distributions in this essay, and furthermore proves the great potential of MEP method to

  15. Engineered microorganisms capable of producing target compounds under anaerobic conditions

    Buelter, Thomas; Meinhold, Peter; Feldman, Reid M. Renny; Hawkins, Andrew C.; Urano, Jun; Bastian, Sabine; Arnold, Frances

    2012-01-17

    The present invention is generally provides recombinant microorganisms comprising engineered metabolic pathways capable of producing C3-C5 alcohols under aerobic and anaerobic conditions. The invention further provides ketol-acid reductoisomerase enzymes which have been mutated or modified to increase their NADH-dependent activity or to switch the cofactor preference from NADPH to NADH and are expressed in the modified microorganisms. In addition, the invention provides isobutyraldehyde dehydrogenase enzymes expressed in modified microorganisms. Also provided are methods of producing beneficial metabolites under aerobic and anaerobic conditions by contacting a suitable substrate with the modified microorganisms of the present invention.

  16. Synthesis, Structure and Biological Activities of Some Novel N-(4,6-Disubstituted-pyrimidin-2-yl)-N'-(trifluoromethylphenyl)-guanidine Derivatives

    HE Feng-Qi; LIU Xing-Hai; WANG Bao-Lei; LI Yong-Hong; LI Zheng-Ming

    2008-01-01

    A novel series of guanidine derivatives were designed, synthesized and confirmed by FTIR, MS, 1H NMR and elemental analysis. The single crystal structure of 11b was determined by X-ray diffraction. Herbicidal activities of these guanidine derivatives were evaluated through barnyardgrass and rape cup tests, among which compounds lla, lib, and lie against Brassica campestris reached 71.2%, 86.7%, and 86.9% at 100 μg·mL-1 respectively. The preliminary ketol-acid reductoisomerase test showed that the synthesized compounds had weak activities.

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

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

    2007-06-01

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

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

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

    2016-01-01

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

  19. Toxoplasma gondii: mechanism of the parasitostatic action of 6-thioxanthine.

    Pfefferkorn, E R; Bzik, D J; Honsinger, C P

    2001-12-01

    In contrast to the cytocidal effect of 6-thiopurines on mammalian cells, the action of 6-thioxanthine on Toxoplasma gondii was only parasitostatic. 6-Thioxanthine was a substrate of the parasite's hypoxanthine-guanine phosphoribosyltransferase. That enzyme converted 6-thioxanthine to 6-thioxanthosine 5'-phosphate which accumulated to near millimolar concentrations within parasites incubated intracellularly in medium containing the drug. 6-Thioxanthosine 5'-phosphate was the only detectable metabolite of 6-thioxanthine. The absence of 6-thioguanine nucleotides explains the lack of a parasitocidal effect because the incorporation of 6-thiodeoxyguanosine triphosphate into DNA is the mechanism of the lethal effect of 6-thiopurines on mammalian cells. Extracellular parasites that had accumulated a high concentration of 6-thioxanthosine 5'-phosphate incorporated more labeled hypoxanthine or xanthine into their nucleotide pools than did control parasites. The basis for this increased nucleobase salvage remains unexplained. It was not due to up-regulation of hypoxanthine-guanine phosphoribosyltransferase and could not be explained by reduced use of labeled nucleotides for nucleic acid synthesis. Extracellular parasites that had accumulated a high concentration of 6-thioxanthosine 5'-phosphate used labeled hypoxanthine almost entirely to make adenine nucleotides while control parasites made both adenine and guanine nucleotides. Both extracellular parasites that had accumulated a high concentration of 6-thioxanthosine 5'-phosphate and control parasites efficiently used labeled xanthine to make guanine nucleotides. These observations suggested that inosine 5'-phosphate-dehydrogenase was inhibited while guanosine 5'-phosphate synthase was not. Assay of inosine 5'-phosphate dehydrogenase in soluble extracts of T. gondii confirmed that 6-thioxanthosine 5'-phosphate was an inhibitor. We conclude that 6-thioxanthine blocks the growth of T. gondii by a depletion a guanine

  20. Bilateral sensory deprivation of trigeminal afferent fibers on corticomotor control of human tongue musculature: A preliminary study

    Kothari, Mohit; Baad-Hansen, Lene; Svensson, Peter

    2016-01-01

    Background: Transcranial magnetic stimulation (TMS) has demonstrated changes in motor evoked potentials (MEPs) in human limb muscles following modulation of sensory afferent inputs. Objective: The aim of the present study was to determine whether bilateral local anaesthesia (LA) of the lingual...... the tongue dorsum in four different conditions: (1) immediately prior to anaesthesia (baseline), (2) during bilateral LA block of the lingual nerve, (3) after anaesthesia had subjectively subsided (recovery) and (4) 3 hrs after bilateral lingual block injection. MEPs were assessed using stimulus......–response curves in steps of 10% of motor threshold (T). Eight stimuli were given at each stimulus level. Results: The amplitudes of the tongue MEPs were significantly influenced by the stimulus intensity (P<0.001) but not by condition (P=0.186). However, post hoc tests showed that MEPS were statistically...

  1. Scout Missions and Future Exploration of the Martian Poles

    McBride, K. S.

    2003-01-01

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

  2. AN INVESTIGATION OF MULTIDIMENSIONAL ENERGY POVERTY IN PAKISTAN: A PROVINCE LEVEL ANALYSIS

    Falak Sher

    2014-01-01

    Full Text Available Present study employs Alkire and Foster’s (2007 methodology to measure Multidimensional Energy Poverty (MEP at provincial level in Pakistan. MEP Headcount has been calculated using PSLM data. Indoor pollution is found to be the largest contributor to MEP Headcount in all four provinces of Pakistan while cooking fuel is the second largest contributor. Results of MEP Headcount show that 47%, 51%, 69% and 66% of the households residing in Punjab, Sindh, Khyber Pakhtoon Khaw (KPK and Baluchistan provinces of Pakistan respectively are energy poor. Households of all the four provinces are most deprived in the dimension of indoor pollution i.e. in the range of 49% to 63% followed by cooking fuel i.e. in the range of 35% to 59%. Deprivation is least in the dimension of home appliances for all provinces except Baluchistan which is least deprived in entertainment appliances dimension.

  3. Nanoindentation Studies of TNZ and Ti2448 Biomaterials After Magnetoelectropolishing

    Hryniewicz T.

    2014-10-01

    Full Text Available This work presents the nanoindentation results of two newly developed titanium alloy biomaterials, TNZ and Ti2448, after different surface treatments. The investigations were performed on the samples, AR – as received, MP – after abrasive polishing, EP – after a standard electropolshing, and MEP – after magnetoelectropolishing. The electropolishing processes, both EP and MEP, were conducted in the same proprietary electrolyte based on concentrated sulfuric acid. The mechanical properties of the titanium alloys biomaterials demonstrated an evident dependence on the surface treatment method, with MEP samples revealing extremely different behaviour and mechanical properties. Such a different mechanical behaviour may mean completely different composition and thickness of the surface film formed on the studied samples after MEP

  4. 76 FR 22674 - Manufacturing Extension Partnership Advisory Board

    2011-04-22

    ... National Institute of Standards and Technology Manufacturing Extension Partnership Advisory Board AGENCY... announces that the Manufacturing Extension Partnership (MEP) Advisory Board, National Institute of Standards... than May 2, 2011. FOR FURTHER INFORMATION CONTACT: Karen Lellock, Manufacturing Extension...

  5. 75 FR 19941 - Manufacturing Extension Partnership Advisory Board

    2010-04-16

    ... National Institute of Standards and Technology Manufacturing Extension Partnership Advisory Board AGENCY... announces that the Manufacturing Extension Partnership (MEP) Advisory Board, National Institute of Standards..., 2010. FOR FURTHER INFORMATION CONTACT: Karen Lellock, Manufacturing Extension Partnership,...

  6. 77 FR 20790 - Manufacturing Extension Partnership Advisory Board

    2012-04-06

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

  7. 75 FR 50749 - Manufacturing Extension Partnership Advisory Board

    2010-08-17

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

  8. Present and Last Glacial Maximum climates as states of maximum entropy production

    Herbert, Corentin; Kageyama, Masa; Dubrulle, Berengere

    2011-01-01

    The Earth, like other planets with a relatively thick atmosphere, is not locally in radiative equilibrium and the transport of energy by the geophysical fluids (atmosphere and ocean) plays a fundamental role in determining its climate. Using simple energy-balance models, it was suggested a few decades ago that the meridional energy fluxes might follow a thermodynamic Maximum Entropy Production (MEP) principle. In the present study, we assess the MEP hypothesis in the framework of a minimal climate model based solely on a robust radiative scheme and the MEP principle, with no extra assumptions. Specifically, we show that by choosing an adequate radiative exchange formulation, the Net Exchange Formulation, a rigorous derivation of all the physical parameters can be performed. The MEP principle is also extended to surface energy fluxes, in addition to meridional energy fluxes. The climate model presented here is extremely fast, needs very little empirical data and does not rely on ad hoc parameterizations. We in...

  9. Master environmental plan for Fort Devens, Massachusetts

    Biang, C.A.; Peters, R.W.; Pearl, R.H.; Tsai, S.Y. (Argonne National Lab., IL (United States). Energy Systems Div.)

    1991-11-01

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

  10. Medical Expenditure Panel Survey Household Component

    U.S. Department of Health & Human Services — The Medical Expenditure Panel Survey (MEPS) Household Component (HC) collects data from a sample of families and individuals in selected communities across the...